Annals of tropical medicine and parasitology

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ANNALS OF TROPICAL MEDICINE
AND PARASITOLOGY

@

(THE UNIVERSITY OF LIVERPOOL

ANNALS

ee OF

TROPICAL MEDICINE AND
PARASITOLOGY

ISSUED BY THE

LIVERPOOL SCHOOL OF [TROPICAL MEDICINE

VOLUME V
(April 20, 1911, to February 26, 1912)

With twenty-five plates, thirty-six figures in text, fifty-seven charts,
and three maps

LIVERPOOL :
AT THE UNIVERSITY PRESS, 57 ASHTON STREET.

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CONTENTS

No. I

McCarrison, Roser.

Further Experimental Researches on the Etiology of Endemic Goitre.
Plates I, UU.

Carter, Captain R. Markuam.

Non-ulcerating Oriental Sore: the Cultural Characteristics of the Parasite as
compared with a New similar Parasite in Erthesina fullo (Thumb), a Penta-

tomid Bug. Plates III, IV

Critien, A.

Infantile Leishmaniasis (Marda tal Biccia) in Malta.

THomson, Davip.

I.—A Research into the Production, Life and Death of Crescents in Malignant

Tertian Malaria, in Treated and Untreated Cases, by an Enumerative
Method
1].—The Leucocytes in Malarial Fever: a Method of Diagnosing Malaria

long after it is apparently cured

Boyce, Sir Rupert.

Note upon Yellow Fever in the Black Race and its bearing upon the Question

of the Endemicity of Yellow Fever in West Africa. ‘Two diagrams

FantuaM, H. B.

On the Amoebae Parasitic in the Human Intestine, with Remarks on the Life-

Cycle of Entamoeba coli in Cultures

NewstTeAp, Roser.
Some Further Observations on the ‘Tsetse-fly, described in these Annals as

Glossina grossa, etc.

Korke, Visunu T.

On the Correlation between Trypanosomes, Leucocytes, Coagulation ‘Time,
Haemoglobin and Specific Gravity of Blood

PAGE

™N

>)

J/

103

127

CONTENTS
No. 2

In Memoriam—Professor Sir Rubert Boyce, F.R.S.. With portrait

Gassi, Prorrssor UMBERTO.

Note on Tropical Diseases in Southern Italy

Newsteab, R.

The Papataci Flies (Phlebotomus) of the Maltese Islands. Plates V-VII

McCarrison, Roserr.
The Experimental Transmission of Goitre from Man to Animals. Plates
VIlI-xX
Navss, Ratpo W.; and Yorke, WARRINGTON.

Reducing Action of Trypanosomes on Haemoglobin

SrePHENs, J. W. W.

The Anti-Malarial Operations at Ismailia. ‘Two Maps

Newsteap, R.; and Carter, Henry F.

On some New Species of African Mosquitos (Culicidae). Plate XI ...

Topp, Joun L.; and Worsacy, S. B.

The Diagnosis and Distribution of Human ‘Trypanosomiasis in the Colony and
Protectorate of the Gambia. One Map

Yorke, WarrincTon ; and Nauss, Rateu W.

The Mechanism of the Production of Suppression of Urine in Blackwater
Fever. Plates XII-XIII

Simpson, G. C. E.; and Ebr, E. S.

On the Relation of the Organic Phosphorus Content of Various Diets to
Diseases of Nutrition, particularly Beri-Beri

PAGE

139

187
199

215

233

245

287

a

=. = aA

CONTENTS
No; 3

Ross, Proressor Sir Ronatp ; and Storr, WaAtrrER.
Tables of Statistical Error ...

Sripetin, Dr. Haratp.
Notes on some Blood Parasites in Reptiles. Plates XIV-XV ...
Ross, Sir Ronatp ; and Epir, E. S.

Some experiments on Larvicides

GranaM, W. M.
An Investigation of the Effects produced upon the Excretion of Urinary Pig-
ments by Salts of Quinine. Plate XVI

Yorke, WARRINGTON.
The Passage of Haemoglobin through the Kidneys. Plate XVII

Ross, Sir Ronatp ; and Tuomson, Davin.
Pseudo-Relapses in Cases of Malarial Fever during Continuous Quinine
Treatment
Yorke, Warrincton ; and Bracktock, B.

The Trypanosomes found in ‘Two Horses Naturally Infected in the Gambia.
Plate XVIII

Wisz, K. S.
An Examination of the City of Georgetown, British Guiana, for the Breeding
Places of Mosquitos

Srannus, Hucu S.; and Yorke, WARRINGTON.
A Case of Human Trypanosomiasis in Nyasaland with a Note on the Pathogenic
Agent. Plate XIX

McCarrison, Roserr.
A Second Series of Experiments dealing with the Transmission of Goitre from

Man to Animals. Plates XX-XXII ...

Tuomson, Davin.
A New Blood-Counting Pipette, for estimating the numbers of Leucocytes

and Blood Parasites per cubic millimetre
Fantuam, H. B.

Some Researches on the Life-Cycle of Spirochaetes

Srepuens, J. W. W.
“Desmogonius desmogonius, a New Species and Genus of Monostome Flukes.

Plate XXITI

409

443

453

471

479

497

CONTENTS

No. 4
PAGE
Serpetin, Dr. HARraLp
Notes on some Blood-Parasites in Man and Mammals. Plate XXIV Lath, S02
Patron, Captain W. S.; and Crace, Captain F, W.
The Genus Pristirhynchomyia, Brunetti, 1910. Plate XXV ... Ri. oe BOD
Patron, Captain W. S.; and Cracc, Caprain F. W.
The Life History of Philaematomyia insignis, Austen ere = al i ee
Bracktock, B.
The Measurements of a Thousand Examples of Trypanosoma vivax weet gee
Tomson, JoHn Gorpon.
Enumerative Studies on T. brucei in Rats and Guinea-Pigs, and a Comparison
with T. rhodesiense and T. gambiense se ed i, ei ug yg.

Biacxtock, B.

A Note on the Measurements of Trypanosoma vivax in Rabbits and White Rats 537

Ross, Srr Ronatp ; and Tuomson, Davin.

A Case of Malarial Fever, showing a True Parasitic Relapse, during Vigorous

2!) Lote

and Continuous Quinine Treatment.

INDEX

PAGE
Inpex or Auruors.. ill
GENERAL INDEX.. - SA ili
INDEX OF GENERA, “SPECIES A AND VARIETIES NEW TO ) ScteNCE Rcbhag Amae ts vil

INDEX OF AUTHORS
PAGE | PAGE

Blacklock, B.. Whe ab 20s haz | Ross Gir. ; and Rdiey Br'o:.+ 20.4, tse 305

Blacklock, B. ; and Yorke, Wee gag |) sRoss, oir Sand Stott WW. .. c2..60.2 847

- | SE Su) 3 ie 103 Ross, Sir R. ; and Thomson, D. ...409, 539

omer, HH; F.; Newstead) Rics.....: 233 Seidelin, Harald ...... ee 501

Gutter, R. Mareen TRY ake 15 | sunpson,G, C. BE. ; and Edie, E She ieosabe Vie

Cragg, F. W.; and Patton, WS + 509, 515 | Stannus, H.S.; and Yorke, W. ...... 443

Crtien, As >... pr IY Stephens, J. W. W.. HT. BIS AT

Edie, E. S. ; San Ross Sir RR ees) 465 | Stott esand Ross, Re oe 347

Edie, E. ae and Simpson, GC. E....°313. | Thomson, David. 37, 83, 471

Bamemimots. B. 7.4.0... MLDS AYO) «| Thomson, be and Ross, ‘Sit R.. ..409, 539

ERI Cette... 135 (| (homson, J. Glen. 531

Graham, W. M................0..0-. 39f | Todd, J. L.; and Wolbach, S. B.... 245

PM VIGNE Do... 5 sc se eee ese=- 127 Wise, K. S. NAG

McCarrison, Robert....... -¥,. 187, 453 Wolbach, S. ‘B.: “and Todd, Je alts . 245

Nauss, R. W. ; and Yorke, we ...199, 287 Yorke, W. arrington see .«+ 401

Newstead, Robert.. EA. 125. B20) | Yorke, W.; and Blacklock, ‘B.. 1» 413

Newstead, R.; and ‘Carter, H. jae 233 Yorke, W.; and Nauss, R. W.. 199, 287

Patton, W. S.: and Cragg, F. W ., 509, 515 Yorke, W.; and Stannus, H. Steaeee 443

GENERAL INDEX
PAGE PAGE

Amoebae parasitic in the human Blacklock, B., and Yorke, W., Try-

intestine ..... III panosomes of naturally infected

Amoeboid parasites i in blood- d-plasma HOESCS e PAD ic dessee at ev een sen sn ce 4E3

of Lizard . . 380 Blackwater Fever er, ., Kidney in ...402 ¢t seq.

Anophelines, Ave Larval ' measures at y ae Leucocytes in...... 93

Tomaiiarlei.: 44: 1220 <3 Urine, Mechanism

Pe Breeding places ahs at of Production of

ASMAaLiaeUAY. ...i5.45. OA suppression of,
Argus persicus, Spirochaetes in ......... 485 MOTE cc ctsaes we ZO
Auto- gales in human trypano- Blood-counting Pipette.. or . 471
somiasis ....... 260 Blood, in Trypanosome infections, 127, 200

Bacillus bulgaricus ii in the treatment of ,, Reducing action of Trypano-
BOLLS, 4.50.00 UA semiwdys: te somes on haemoglobin ...... 200
Barbados, Yellow Fever itt.........:..... 106 Boyce, Sir R. In Memoriam ......... 133

Beri- Beri, Phosphorus, Organic, con- Boyce, Sir R. Yellow fever in the
tent of diet and .....:... 313 cit pO A de Oe Bee 6°,

a Rice, polished and un- Cancrum oris in Infantile Leish-
polished in.. 314 et seq. RAMNGNG AA). Dead ond Whe vers be ube edb ebin LD

Blacklock, B., Measurements of T. Cater,.H. F., “and Newstead, R.
vivax . Se se GABP RST New African Mosquitos............... 233

PAGE

Carter, R. M. Non-ulcerating
oriental sore; cultural character-
istics Of parasite ...-...+ece seer

Cellia arnoldi. .........c0 ener

pseudosquamosa, N. SP.
ys Squamosa, Var. arnoldt ....++0 +--+:

Cragg, F. W., and Patton, W. S. The
Genus Pristirhbynchomyia ...++++++

Cragg, F. W., and Patton, W.S. Life
history of Philaematomyia insignis...

Critien, A. Infantile Leishmaniasis
in Malta 2i......creces de -meeeee

Dengue Fever in Italy .....----+1-00

Desmogonius desmogonius, N. 8-5
N. SP. ...2e000s ae 2 ee

Dysentery, Amoebae Of .....-.+-:++- 299+

Edie, E. S., and Ross, Sir R. Experi-
ments on larvicides ....--e ee eeres

Edie, E. S., and Simpson, Gac. &
Organic phosphorus content of diet
and BerisBerh... fof eae ree ven ee

Entamoeba sp. (Noc) ee ae: se
plea AR hceas ss S25

., Life-cycle of, in cul-

FUEES fbb fapen ste eanees =

sf ., Leucin, Skatol and
Tyrosin in Culture

media of oe
OMENS 2 eo <0 vee - E13, TE;
histolytica.........--113, 119,
” Widslia Too ct abe, 11D,
- nipponica . “TRS, 200;
7 phagocytoides «..-.- 114, 116,
5 tetragend..........--114, 116,
tropicalis _.112, 116,

undulans

Enumerative Methods aeons
Blood-count-
ing Pipette
os Studies of crescents in
malignant tertian

gna baraa casiaup eet -f¢be

$s Studies on T. brucei ...
Erthesina fullo...ccsvecececiserennre
Parasites from intestinal
sract bli .. 4... aame--9's
Fantham, H. B. On the Amoebae
parasitic in the human intestine...
Fantham, H. B. Life-cycle of Spiro-
CHACCES |. a clin cel neo kaleiss = one eo ne hae eens
Filaria imperatoris 2.0.00 c00 ce verses

” ”

29 9

479
ihe

PAGE
Gabbi, U. Tropical diseases in
Southern Italy. ......-0:ceeeseereeeeeres 135
Gambia, Horses naturally infected
with Trypanosomes in ee
Georgetown, Mosquito breeding
places in os aoe

Gland palpation and puncture in
human trypanosomiasis. ......-.257 ¢t ¢q.
Glossina grossa, Synonym of Gl. nigro-

ee

cs morsitans, 1. rhodesiense
probably transmitted by... 449
, NigrofUsca, Ni. SP. --eeeeeeeeres 125
.. palpalis, var. Wellmamt .....- 125
Goitre, Earthworms in the spread of... 187

,, Endemic, Experimental _re-
searches on Etiology 1
z a treatment of......... 12
,, Experimental, Carbonates_ of
Lime, Mag-
nesium, and
Sodium, in... 458
et Seq.
- a4 transmission
from man to
animals, 187, 453
Graham, W.M. Salts of quinine and
the excretion of urinary pigments...
Haemoglobin, Reducing action of
Trypanosomes on ore

Haemoglobinuria, Experimental eae 404

sgt

39 bP
Suppression of

urine “’

Haemogregarina imperatoris, TSP. +--+
Ismailia, Anopheline breeding places 221
,, Anti-malarial operations at 215
Italy, Southern, Tropical diseases ofee
Kala-azar, Infantile .....- .. eae

o 5 so aM [tally .. ee 136
Kidneys, Blackwater Fever, suppres-
sion of urine in......402 ef 5ég.
“4 Haemoglobin, passage of, 2
through «an « a» oeehela en
‘ Piroplasma canis ..++ 403, 406
Korke, V. T. Correlation between

trypanosomes, leucocytes, coagula-
tion time, haemoglobin and specific
gravity of blood «1.0... seers
Lactic ferments in the treatment of
BOUETE seo cece ce neer eee eeteereen see metas 12

127

PAGE
Larvicides. . bovis tp aed eer nee abee Seng
Leishmania “infantum in infantile
Leishmaniasis .......... ..44, 136
Leishmaniasis, Infantile, ocras
Ors td). 2.. 39
a Fe clinical pic-
Pures 775-4 38
t. y in ltaly?.<3)t 136
x 43 int’ Malktavys: 1°37
Leucocyte counts in blackwater fever 93
Pa 4 mialatiay 11. ee 65, 83
$3 5 oriental sore ... 20

Leucocytes ingest malarial parasites, 72, 85
Leucocytic extracts in Malaria ...... 86
McCarrison, R. Experimental re-
searches on the Etiology of Endemic
SSOMUTE. ...5..-+.. I
McCarrison, BR. Experimental tt trans-

mission of Goitre... ect yey Le
Malaria, Diagnosis by leucocytes ...... 83
a Ismailia, anti-malarial opera-
tions at Aa xcen LG
,, Latent, diagnosis of Ul es 89
= Leucocyte counts in ......... 65
Been EGUCOCYTES ID. os.csch soc. ores 83
fe) Leucocytic extracts in ...... 86
» Pilocarpine injections in...... 87
re Quinine and excretion of
urinary pigments 391

>

“ Quinine Prophylaxis...... 228, 398
»» Quinine Prophylaxis and cure 73
. Quinine treatment, pseudo-

relapses during 409

;. Quinine treatment, true par-
asitic relapse during ...... 539

“ Relapses, pseudo, during

quinine treatment ......... 409

. Relapses, true parasitic, dur-
ing quinine treatment 539
ee Wrtan i 5.¥ . 507

Malarial parasites, ingested by leuco-
5 a eS Oy

- » Methylene Blue,

the effect of, on
Crescents..st..67
Production, life and

” 9
death of crescents 57
™ s,s Quinine, effect of,
on crescent pro-
duction....,...i00 7 &
Malta, Infantile Leishmaniasis in...... 37

PAGE
Mediterranean Fever in Italy......... 135
Mercuric chloride as larvicide ......... 388

Methylene blue, the effect of, on
malarialierescentgieayites..<....0c. 67

Mtkrofilaria. See Filaria.
Mosquitos, African, new species of ... 233
4 Breeding places in ee
town . 435
= Larvicides 385
Molluscs, Lamellibranch, Spirochaete
of.. ..488 et seq.
Nauss, R. W. 3 “age vee W. Action
of trypanosomes on haemoglobin... 199
Nauss, R. W., and Yorke, W. Sup-
pression of urine in Blackwater fever 287
Neocellia christyi, n. sp.. TERS SSS
Newstead, R. Papataci Pie of
Maltese PAS 55 MERE < cwnvaned an) BOO
Newstead, R. The Tsetse-fly
described as Glossina grossa, etc.... 125
Newstead, R., and Carter, H. F. New
African Mosquitos NE waSe 2293

Nyasaland, ‘Trypanosomiasis, “human,

cases of, in 443
Oriental Sore, Italy 137
# 5, non-ulcerating, eae

characteristics of
parasite Of Wa. e (ES
Ornithodorus moubata, eet, in 485
Papataci Fever in Italy ......... 137
sau 2 Plves of Maltese Islands 139
Paraplasma subflavigenum, n. sp. ....+. 504
Patton, W.S., and Cragg, F.W. ‘The
Genus Pristirhynchomyia ........+++ 509
Patton, W.S., and Cragg, F. W. Life
history of Philaematomyta insignis... 515
Petroleum’ as larvicide %. 2 hii... 22 385
Philaematomyia gurnet, 0. sp. 513
7 insignis, Life history
OES .o 53 A ead G15
ij HARARE 2 octal oo! GIZ
Phlebotomus, Bionomics of ...140 et seq.
a Morphology of Genus
153 ¢t seq.
a Prophylactic Measures
148 et seq.
Phlebotomus minutus , 169
3 nigerrimus, N. Sp. 168
99 papatasit 174
99 pernictosus, n. sp. 172

Pilocarpine injections in Malaria...... 87

PAGE

Piroplasma canis, Kidney in 403, 406
Plasmodium sp., monkey naturally in-
fected with .......00
falciparum, Biology of
sexual form of........... 57

Polyneuritis, Experimental, in animals
314 et seq.

yeast, Pro-

595

tective

and cura-

tive effect

ai. 0...
321 et Seq.
Potassium cyanide as larvicide ......... 388
Pristirbynchomyia, Genus .. sige ¥5OO
Pyretophorus distinctus, n. sp: gba ene a4:

4 - var. inelano:
costa, N. var.... 236

Quinine, Effect of, on crescent ll
duction bazashe “39 (60

», Malaria, pseudo- relapses in,
during treatment with ... 409
Malarial Prophylaxis, 73, 228, 398

, Urinary Pigments, Effect

produced upon excretion

of, by . 391

Reedomyia simulans, n. sp. ....- . 240

Reptiles, Blood parasites of ............ 371
Ross, Sir R., and Edie, E.$. Experi-

ments on larvicides —.........:1-00++ 385
Ross, Sir R., and Stott, W. ‘Tables of

Statistical EPEOT a anh ss 347

Ross, Sir R., and Thontson,. D.
Pseudo-Relapses in malaria during
quinine treatment . sees 409

Ross, Sir R., and ‘Thomson, D.
True parasitic relapse in Malarial
Fever during quinine treatment...

539

Sanitas-Okol as larvicide ............... 386
Schaumann, H. Beri-Beri ...313 et seq.
Seidelin, H. Blood parasites in man
and. masnmals 25 gcd sith scree hOl
Seidelin, H. Blood parasites in
reptiles .. a7

Simpson, G. C. E., and Edie, E. S.
Organic phosphorus content of diet

asd Beri-Ben . 35... inate cee Gl F
Sleeping Sickness. See ‘Trypano-
somiasis, Human.
Spirochaeta anodontae, Spores of ...... 489
a balbianit, Spores of ...... 489

PAGE

Spirochaeta duttoni, Lite-cycle of, 480 et seq.

st marchouxt, Life-cycle of
480 et Seq.
5 recurrentis, Life-cycle of...
480 et seq.
3 solenis, N. Sp.......480, 488, 493
Spirochaetes, Life-cycle of . 479

Stannus, H. §., and Yorke, W. Case
of human Wide in Nyasa-

land . - 443

Statistical Error, ‘Table of. a 347
Stephens, J. W. Anti-malarial
operations at eee 215

Stephens, J.W.W. New species and
genus of monostome flukes. . uns 407
Stott, W., and Ross, Sir R. "Fables of
Statistical Ertor :.)aeee ee
Strychnine as larvicide ............:.000
Suez-Canal, Anti-malarial operations
at Ismailia . ...0...). .2c).sunes eee ae
Thomson, D. _ Blood-counting
PIPCtte os n0.ssecssnnnese abies seeeeenn
Thomson, D. Leucocytes in malarial
fever; method of diagnosing
MA ALIA sss. so.s vo.cien sie Coe
Thomson, D. ‘The production, life,
and death of crescents in malignant
tertian malaria........ shod sas eae
Thomson, D., Aer Ross,, Sir ie
Pseudo-Relapses in malaria during
quinine treatment . lads on ea
Thomson, D., and ‘Ross, Sin
True parasitic relapse in Malarial
Fever during quinine treat-
MCN Lc evcecvcccececcceccccccssccccers
Thomson, J.G. Enumerative studies
ODT. BRUCE iciiad kisi de plde see eee
Thyroid gland, Goitre ...............4 2b Seq.
Experimental
189 ¢ét seq.
454 et Seq.
Todd, J. L., and Wolbach, S. B.
Trypanosomiasis i in the Gambia .
Trematodes, Desmogonius desmogonns,
1.2.) D.Sple pees - 497
T. brucei, Enumerative studies on . . 531
I. dimorphoty todresljeas0<ss0anen 415 et Seq.
T. lewisi, Rats naturally infected with 506
T. rhodesiense in Nyasaland .. - 443
T. vivax ass ats et Seq.
5 aes Measurements a Eosaclts 521. 537

- 347
389

99 99 2)

- 245

PAGE
Trypanosomes, Correlation between
‘Trypanosomes, Leucocytes, coagula-
tion time, haemoglobin and specific
gravity of blood . sks Ease L27.
Trypanosomes, Horses ‘naturally ‘in-
fected with
ry pss 1.5 413
re Reducing action of, on
haemo-
slobin. a+: 199
Trypanosomiasis, Human, Auto-
agglutina-
{OTe 70200
Case of, in
Nyasaland 443
Diagnosis
and —dis-
tribution
in the
Gambia ... 245
Gland pal-
pation and
puncture
257 et seq.
Immunity 282
Preventive
measures
for the
Gambia ...
283 et seq.
Pulse and

bP) ”

bb) 93

bb] 9

PAGE

‘Tsetse-flies, Glossina grossa snyonym
yony.

of Gl. nigrofusca 125
nigrofusca, N. sp. 125

2 ”
a » palpalis, var.
: Wellmant ...... 125
Urinary Pigments, Quinine and excre-
ENOMOE see Besansek. SOE
West Africa, “Yellow Fever in ......... 103
Wise, K. 7 Mosquito oe
places in Georgetown... ABE

Wolbach, S. B., and Todd, cle te
Trypanosomiasis in the Gambia ... 245
Yeast, Protective and curative effect
of, in experimental polyneuritis...

321 et seq.
Yellow Fever, Barbados ............... 106
b » Black Race. ; 103

cf ae Endemicity _ in Wee
AETIGA oc.) savas: gee
Jo Immunity ..... TO4 et seq.

>
Yorke, W. Passage of haemoglobin

through the Kidneys 4o1
Yorke, W., and Blacklock, B. Try-

panosomes of naturally infected

UNS CME a tanec soa es. vrianmesas AEG
Yorke, W., and Nauss, R. W. Action

of trypanosomes on haemoglobin... 199
Yorke, W., and Nauss, R. W. Sup-

pression of urine in Blackwater fever 287
Yorke, W., and Stannus, H.S. Case

of human trypanosomiasis in Nyasa-

PO rece pharina circ Buws jo ocseeec ties ee MAG
DererualVAtIaTIA IM “Ss ic csveccrewese ss §OY

INDEX OF GENERA, SPECIES AND VARIETIES NEW TO SCIENCE

PAGE
Cellia pseudosquamosa —..iceeeceeeeeenees 236
RE IIL. « os.5.10'0.0i vies ois Mee sssviewee'ss vs AQF

a desmogonius te. Saeed 4s 407,
NGS TALTOPUSCE x0. vies ceo s,odeesones 125
Haemogregarina he Ne a shh ara Sy 374
MEME COTESEYT — . . cdsna yas ssceaumysss 238
Paraplasma subflavigenum ....ccceceus. 504.

Philaematomyia gurnet ........000000 +. 513

PAGE

Phlebotomus nigerrimus 0 ..0.0c.0c00 0+. 168
5 . .

fe POTAUIOSES. soci vcorsisvess LZ

Pyretophorus distinctus .....scscwees 234

melano-
costa, 230

Spirochaeta solenis cites bi 493

Reedomyia stmulans ...ccse ee .. 240

5 5 var.

4 rs
fbots
. aTtL
rts 4 =
mn novuled
; ,
Y tshit
; ; j owed
Me ' +
= (nid 15D
aiuitdt * :
<* ‘ <
ao! eB veecdl wth sent seatint
* : ‘ ie ii —
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r "4 ,
a vs eo .
d ; 45
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Pt, F pe
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af mae?
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Pa t i >
bi tyinieets 3 —. : a
; ol apht

- Py Saal a a. 1 ot slay e Apion hss = 4

Aerie Sue Lifton vel Fe ished AO LOUD Y a
e . re + fs iekel beaters soithie 4
. $4) Lo tee tee SE caine

iF iT atthe. Saal on Laks MY

: Ei od pas Ptail 4 te
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Volume V April, 1911 No. 1

ANNALS

OF

TROPICAL MEDICINE AND
PARASITOLOGY

ISSUED BY

THe LIVERPOOL SCHOOL OF TROPICAL MEDICINE

Editor

Prorrssor RONALD ROSS, Major I.M.S. (Rer.), D.P.H., F.R.C.S.,
Discs slr. C.B.

In Collaboration with

J. W. W. STEPHENS, M.D., Cantas., D.P.H.
R. NEWSTEAD, M.Sc., A.L.S., F.E.S., Elon. F.R.H.S5.
J. L. TGDD, BA, M.D, C.M. McGill, D.Sc., Liv.
H. WOLFERSTAN THOMAS, M.D., C.M. McGill.

ANTON BREINL, M.U.Dr.
Proressor Sir RUBERT BOYCE, M.B., F.R.S.

Gua AWVIDICGAHM: aa 1108

C. Tinling & Co., Ltd.
Printers to the University Press of Liverpool
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PURTHEK EXPERIMENT AB RE:
pe eto ON THE, ETIOLOGY
OF ENDEMIC GOITRE*

BY

ROBERT McCARRISON, M.D., R.U.I., M.R.C.P
(EOnD:), 1.M.S.

AGENCY SURGEON, GILGIT
(Recezved for publication 25 November, 1910)

OBJECT OF THE RESEARCH

In a communication which I had the honour to make to the
Royal Society, on the 26th November, 1908, I described the results
of my experimental researches on the etiology of goitre up to that
date, as follows :—

1. Goitre can be experimentally produced in man by the
administration of the matter in suspension separated by filtration
from waters which are known to be goitre-producing.

2. Goitre cannot be so produced when the suspended matter
is boiled.

3. The disease is due, therefore, not to the mineral but to the
living component of the suspended matter, in other words, to a
living organism of disease.

4. The incubation period of experimentally produced goitre is
thirteen to fifteen days.

5. Goitre can be cured by the administration of intestinal
antiseptics. It is possible, therefore, that the organism which is
the cause of the disease is parasitic in the human intestine.

These conclusions were based on the results of experimental
observations carried out on man during the years 1907-1908. It has
been the object of the present research to test their accuracy by

*Read in abstract before the Royal Society, 2 February, 1911.

2

experimentation on a larger number of men. My investigations
may be summarised as follows :—

I. The experimental administration to man of suspended
matter, separated by filtration and sedimentation from goitre-
producing waters.

II. The experimental administration to man of suspended
matter from the same source, which had been subjected to dozing
for at least ten minutes.

III. The effect of the administration of filtered goitre-
producing water to man under experimental conditions.

IV. The action exercised by the lactic ferments when applied
to the treatment of goitre.

V. The effect on dogs of the administration of extracts from
faeces of goitrous individuals.

I and II. THE EXPERIMENTAL ADMINISTRATION TO MAN
OF THE (2) UNTREATED AND (2) BOILED SUSPENDED
MATTER OF GOITRE-PRODUCING WATERS

Experiment G.—Carried out during October and November,
1909.

Thirteen healthy young men, aged between eighteen and
twenty-four years, volunteered for this experiment. All were
new-comers to the district, and were in every respect perfectly
normal. During the whole course of the experiment the men lived
under the strictest guard. Their diet was chiefly vegetable. They
were kept at hard exercise, and were not permitted to handle the
soil, or to drink or eat anything whatsoever except what was
prescribed. The hygienic conditions of life under which they lived
were excellent. They were encamped on a non-goitrous site, and
were provided with water for drinking, bathing, and other domestic
purposes, from the Bermis spring, which is non-goitre producing,
but which, as an additional precaution, was boiled. In no case
was the restraint imposed upon them broken through.

Kashrote Village water was used for the purposes of this
experiment. Goitre prevails in this village to the extent of 45 per
cent. The water flows through a narrow irrigation channel, which
receives the drainings from cultivated land, and is subjected to
much contamination both from human and animal sources. This

3

water, which at the time of collection was purposely made muddy
by agitation, was brought daily from the village to the camp on
the hilly ground above, a distance of about one and a half miles. It
was allowed to stand in suitable vessels and to deposit its sediment.
Four ounces of this sediment were administered to each subject
before the morning and evening meals for thirty days.

The results were as follows :—

1. Nine individuals showed no change in the thyroid gland
which could be detected by physical examination.

2. In two other cases a uniform swelling of the organ was
observed on the tenth day of the experiment. The swelling gave
rise to feelings of discomfort, and complaint was made of throbbing
in the neck and the tightness of collar bands which had previously
fitted well. The measurement of the neck increased by one
centimetre in one case, by one and a half in the other. The
enlargement persisted up to the twentieth day of observation and
then gradually disappeared; the gland was observed to have
regained its normal size on the thirtieth day, when the experiment
was concluded.

3. In the two remaining cases a more marked reaction on the
part of the thyroid gland was observed. In both these cases the
right lobe was chiefly affected, and the swelling was accompanied
with feelings of discomfort, throbbing in the neck and tightness of
the neck band. The measurement of the neck increased one and a
half centimetres in one case, and slightly over one centimetre in the
other. The enlargement made its appearance between the tenth and
fifteenth days of the experiment in each of these cases, and persisted
up to the thirtieth day, when the experiment was terminated.

Unfortunately, no photographic records of these cases could be
obtained owing to the fact that I had just returned from Europe,
and my materials for obtaining such records had not reached me.
The thyroid enlargements observed in the two latter cases resembled
very closely that shown in Figs. 2 and 9.

EXPERIMENT H.—Carried out during October and November,
1909.

The conditions of this experiment were precisely similar to
those detailed in the previous one. In this case, however, the
thirteen individuals who volunteered for it were given the same

4

Kashrote water sediment whkzch had been previously boiled for ten
minutes. In none of these individuals could any reaction on the
part of the thyroid gland be detected. The experiment, which
lasted thirty days, was carried out concurrently with Experiment G,
and formed a very suitable control to it.

EXPERIMENT I.—Carried out from 15th March to oth May, igIo.

The conditions of this experiment were precisely similar to
those of G and H, with the exception that in this case the
goitre-producing water of Kashrote was filtered through a
Berkefeld house-filter. The deposit on the candle was washed off in
distilled water, and a quantity of this dark grey mixture, equal to
about two ounces, was given to each subject in milk before the
morning and evening meals. Ten individuals volunteered for this
experiment. Their average age was twenty-two. All were enjoying
excellent health and were in every respect normal.

Of these individuals four showed no reaction whatever on the
part of the thyroid gland during the fifty-five days the experiment
lasted. One developed a slight degree of enteritis on the fifteenth
day when the experiment in his case was discontinued. The
following is the more detailed account of the alterations observed
in the remaining five cases :—

1. G.M.—15.3.—The thyroid gland was normal. The measurement of the neck
on this date, when the experiment commenced, was thirty-three
and a half centimetres. The subject was photographed.
(Fig. 1.)

31.3—The measurement of the neck was thirty-four and a half
centimetres. The right lobe of the thyroid gland was slightly
enlarged. There was distinct fullness of the neck on that side.
During the act of deglutition the right lobe of the organ was
felt to pass under the fingers as a rounded boss. The subject
complained of tightness of the shirt collar and of throbbing in
the neck.

6.4—There was no marked change to be recorded. The subject
complained of the same symptoms as at the previous
examination. The outline of the gland was readily seen and
felt. The measurement of the neck was _ thirty-four
centimetres.

10.4—There was no appreciable change on physical examination. The
measurement of the neck was thirty-four and a _ half
centimetres. The subject complained of inability to button his
collar band.

15.4—The thyroid gland was noticeably enlarged. The enlargement was
especially well seen during the act of swallowing. The
measurement of the neck was thirty-five centimetres. The
subject was photographed. (Fig. 2.)

5

21.4—The measurement of the neck was thirty-four and a_ half
centimetres.

1.5—The thyroid enlargement was not so evident. The neck measured
thirty-four centimetres.

g-5—The thyroid gland was undoubtedly smaller on this date than on
the 15th April. The measurement of the neck was thirty-four
centimetres. The experiment was discontinued and the subject
was given thymol, ten grains night and morning. No trace
cf enlargement of the thyroid gland was observable one month
later.

2. S.A.—15.3.—The thyroid gland was normal. The measurement of the neck was

thirty-three centimetres. The subject was photographed.
(Fig..3:

31.3.—There was slight enlargement of the left lobe of the thyroid gland
which was clearly seen and readily felt when the subject
swallowed. The measurement of the neck was thirty-three and
a half centimetres.

6.4.—The thyroid gland was noticeably enlarged especially on the left
side. The enlargement was well seen on swallowing. The
subject complained : “‘ that the neck of his shirt had been very
tight for the last five or six days; and that he could only button
it by pulling the collar band up above the swelling; that he
slept a great deal; that he was light-headed; that there was
much throbbing in the neck.’”” The measurement was thirty-four
and a quarter centimetres.

10.4.—No appreciable change was observed since the date of the previous
note. The neck measured thirty-four and a half centimetres.

15.4.—The thyroid gland showed well-marked enlargement over its whole
extent, but especially of the left lobe. The neck measured
thirty-five centimetres. The subject was photographed.
(Fig. 4.) He had been quite unable to button the neck of his
shirt, which previously fitted well, and had tied the button-holes
together with cord. (Fig. 5.)

21.4.—The neck measured thirty-five centimetres. There was no change
since date of previous note.

1.5.—The neck measured thirty-four and a half centimetres.

g-5.—The neck measured thirty-four and a quarter centimetres, but
looked decidedly smaller than on the 15th April. The subject
was noticed to be somewhat anaemic. The experiment was
discontinued and the subject placed under treatment by thymol
and tonics. No trace of enlargement could be detected one
month later.

3. A.M.—15.3.—The thyroid gland was normal. The neck measured thirty-four

centimetres. The subject was photographed. (Fig. 6.)

31.3.—There was considerable enlargement of the gland in the region
of the isthmus, which could be felt as a rounded boss under the
finger, and was noticeable on swallowing. The neck measured
thirty-four and a half centimetres.

6.4.—The right side of the neck was fuller than at the previous
examination. The swelling of the isthmus was more marked,
and, on digital examination was found to be dumb-bell-shaped.
The left side of the dumb-bell was larger than the right.
The swelling of the isthmus was weil seen on swallowing, but it
partially disappeared behind the sternum when the subject was
at rest. The subject complained of tightness of his collar and
throbbing in the neck. Measurement showed the neck to be
thirty-five centimetres in circumference.

6

15.4.—The neck measured thirty-five and a half centimetres and the
swelling of the thyroid and especially of the isthmus was
more noticeable than at the last examination.

21.4.—There was no change to be found on physical examination. The
subject was photographed. (Fig. 7.)

31.4.—The neck measured thirty-four and a half centimetres, and the
thyroid appeared to be slightly smaller.

9.5.—There was no noticeable alteration since the day of the last
examination. The neck measured thirty-four and a half
centimetres. The experiment was discontinued and the subject
placed under treatment.

4. A.D.—15.3.—The thyroid gland was normal. The circumferential measurement
of the neck was thirty-four centimetres. The subject was
photographed. (Fig. 8.)
31.3-—The neck measured thirty-five centimetres. There was some
fullness under the sterno-mastoids, and the whole outline of
the gland was readily seen when the subject swallowed. The
subject did not complain of any symptoms.

6.4.—The neck measured thirty-five and a half centimetres. There
was no appreciable difference from the appearances observed
at the date of the last note.

15.4.—The neck measured thirty-five and a half centimetres. The
subject complained of no symptoms though the gland was seen
to be undoubtedly, but slightly, enlarged. The subject was
photographed. (Fig. 9.)

g-5.—The neck measured thirty-five and a half centimetres. There was
no further change to be recorded. The experiment was
discontinued and the subject placed on treatment by thymol.
No trace of enlargement could be found fifteen days later.

5. F.A.—In this case the original measurement of the neck was thirty-two and a
half centimetres. On the twentieth day of the experiment the
subject complained of a sense of fullness in the neck and of
tightness of his collar band. The neck then measured
thirty-three and a half centimetres. There was some fullness
under the sterno-mastoids, and the outline of the gland was
weil marked and easily palpable. No further enlargement
occurred, and on the 15th April the gland was found to
have returned to its normal size.

EXPERIMENT J.—Carried out from 15th March to oth April,
IQIO.

The conditions of this experiment were in all respects similar to
those of Experiment I, with the exception that the doled residue
was given instead of the untreated residue. Ten individuals, of
whom the average age was twenty-two, volunteered for the
experiment. Amongst these were five in whom the thyroid gland
was larger than normal ; it was considered that these subjects might
respond more readily to goitrous influences than those in whom the

gland was perfectly normal.

7

The duration of the experiment was fifty-five days. It was
carried out concurrently with the preceding one. In no case could
the slightest increase in size be detected by any method of
examination. In the five cases, in whom the thyroid was normal at
the commencement of the experiment, there was no alteration
whatever. In the other five subjects, in whom at the commencement
the gland was somewhat larger than normal, the original and final

measurements were as follows :—

Original Measurement. Final Measurement.
De Bes 34 ae in ws 334
ibs. Rew 34 i a rien eg
Dy Rac 32 os sf ea gos
K. 35 oof ae, -.- 343
R. Pe eee re oe Sa

These results show that the tendency to alteration in size of the
thyroid was, in these five individuals, in the direction of
diminution and not of increase.

The results of the foregoing experiments may be summarized
as follows :—

1. Of twenty-three individuals who consumed the suspended
matter of goitre-producing waters, six showed an increase in size of
the gland, which persisted in a more or less well-marked manner up
to the end of the experiment. Three others showed a thyroid
hypertrophy of a transitory character.

2. Of twenty-three individuals who consumed the Jdozled
suspended matter of goitre-producing waters none showed the
slightest tendency to an increase in size of the thyroid gland.

These results are to be contrasted with those of my former
series, which were :—

1. That of thirteen individuals who consumed the untreated
residue of the goitre-producing waters of Kashrote, four developed
a noticeable swelling of the thyroid gland, while two others showed
an increase in size of the organ demonstrable by measurement and
evident to the touch.

2. That of eight individuals who consumed the doz/ed residue
of the goitre-producing waters of Kashrote, none developed any
swelling of the thyroid gland, and this although three were
individuals peculiarly likely to respond to goitrous influences.

8

The combined results of both series of experiments show : —

1. That of thirty-six individuals who consumed the untreated
suspended matter of a notoriously goitre-producing water, ten
developed a noticeable hypertrophy of the thyroid gland, while five
showed a swelling of the organ of a transitory character.

2. That of thirty-one individuals who consumed the same
suspended matter which had been previously boiled, none showed
any reaction in the direction of increase in size of the thyroid gland.

The results of these experiments justify the following
conclusions : —

1. There exists in suspension in the water of goitrous localities
some unknown agent which is capable of initiating an hypertrophy
of the thyroid gland.

2. This agent can be destroyed by boiling for ten minutes.

lI. THE EFFECT OF THE ADMINISTRATION OF THE
FILTERED GOITRE-PRODUCING WATERS TO MAN
UNDER EXPERIMENTAL CONDITIONS

Having demonstrated that there exists in suspension in
goitre-producing waters a substance which is capable of initiating
an hypertrophy of the thyroid gland, and that this substance is
readily destroyed by boiling, it became necessary to ascertain by
carefully conducted experimental observations whether filtration
deprived the water of its goitre-producing properties. For this
purpose seven individuals were selected who consumed the filtrate
of Kashrote water at the same time that the subjects of Experiment I
were consuming the suspended matter separated from it by filtration.
The same minute precautions were adopted in the case of these
men as in those of Experiment I. In four of these the thyroid
gland was perfectly normal on the 15th March, when the
experiment commenced ; the other three were the subjects of incipient
goitre. All seven drank only the filtered Kashrote water for fifty-five
days, with the following results:—The four normal individuals
showed no change whatever, while the three men in whom the
thyroid was enlarged showed a considerable reduction in size of
this organ. This result indicates that the process of filtration
renders water innocuous which was previously goitre-producing ; the

9

suspended matter removed from the water by this process having
actually produced a thyroid hypertrophy in five out of. ten
individuals who consumed it, while the subjects of the present
experiment were consuming the filtrate. Water so purified not only
does not cause a thyroid hypertrophy, but it exercises a curative
influence on incipient cases of goitre.

The beneficial influence of filtration of goitre-producing waters
is nowadays so well recognised, and has been demonstrated on such
a large scale in the case of many public water supplies that it
appears almost unnecessary to emphasize it. There are still,
however, many scientific men who adhere to the chemical cause of
goitre, and these are seeking in radio-active substances fresh support
for their view. My experiments make it clear that, for the Hindu
Kush region at least, where my researches have been carried out,
the noxious principle of goitre is found only in suspension in the
water and does not exist in solution; and, that if the cause of goitre
is a chemical one it is of such a nature as to be unable to pass
through pores of a Berkefeld filter and to be readily destroyed by
boiling.

The hypertrophy of the thyroid gland, which is capable of being
induced in the way described in the foregoing experiments, exhibits
the following characteristics : —

1. It makes its appearance usually between the tenth and the
fifteenth days of the experiment.

2. It shows a marked tendency to fluctuate in size.

3. It reaches its point of maximum size between the twenty-fifth
and thirtieth days of the experiment.

4. It may completely disappear under the conditions of the
experiment.

5. The hypertrophy of the gland is not great nor is it
progressive under the conditions of the experiment. The tendency
being towards a diminution in size from the thirtieth day onward.

6. It is accompanied, as a rule, with certain subjective
symptoms of throbbing in the neck, feelings of fullness and
discomfort.

The fluctuations of the thyroid gland observed in the individuals
under the conditions of my experiments, exhibit a very striking
resemblance to those of the endemic of goitre which are known to

pe)

occur in large communities. The endemic is subject to periods of
increase and decrease. It shows a marked tendency to increase in
an infected locality till a point of maximum intensity for that
locality is reached, after which the disease tends to decline. This
increase or decrease of the endemic in a given locality is rarely
uniformly progressive but exhibits a marked periodicity.

NOTES ON SECONDARY FACTORS IN THE PRODUCTION OF GOITRE

In the report of my first series of experiments on man, carried
out on the lines which I have here indicated, and which was
published in full in the ‘Quarterly Journal of Medicine’ (April,
1909), I made the following comments with reference to artificially
produced thyroid hypertrophy:—‘The conditions of these
‘experiments differed from those under which the inhabitants of
‘Gilgit live in certain important respects. The men were not
‘subjected to the debilitating influences of defective hygiene,
‘vitiated atmosphere, imperfect dietary, endemic disease and the
‘like. Nor did that potent source of infection among an agricultural
‘community, namely, contamination of the hands and food by the
‘soil of an infected locality come into operation in their case. It is
‘to the absence of such influences as these that I attribute the fact
‘that the experimentally produced goitres were neither large nor
YPIOPTEessive. <.° . .-: I am convinced, therefore, that there are
‘conditions provided by a residence in a goitrous locality apart from
‘the water supply which are important determining factors in the
“production of the disease, and that in the absence of these
‘secondary factors the organism, which is the real causal factor is
‘of feeble pathogenicity.’ My study of goitre during the past
eight years has led me to attach great importance to the following
factors as possessing a marked secondary influence on the
development of thyroid hypertrophy in endemic localities : —

A. Factors directly influencing the thyroid gland which render
it less able to counteract the action of the toxic agent of goitre
without undergoing hypertrophy :—

(a) Hereditary influences: Other things being equal, the
children of goitrous women appear to be more likely to
develop goitre than the children of normal women.

II

(6) The marked influence of age, sex, puberty, menstruation,
pregnancy, sexual activity on the functional activity of
the thyroid gland: the added strain of goitrous
influences at a time when the gland is already working
at high pressure very markedly favours the development
of a goitre.

(c) The influence of unhygienic conditions of life: defective
air space, on which the so-called ‘epidemics’ of the
disease are largely dependent; improper food or
defective food supply ; damp soil; the ‘ causes multiples ’
of the older French writers.

(2) The influences of certain infectious diseases on the thyroid
gland such as rheumatism, rheumatoid arthritis,
malaria, etc.

(e) The influence of emotional states.

B. Factors which favour infection :—

(a) The influence of occupation and habits of life, whereby
individuals are rendered more liable to infection from
the soil which is the natural habitat of the toxic agent
of the disease.

(6) The influence of temperature: goitre is more likely to
develop in temperate climates or at temperate seasons of
the year.

(c) Susceptibility : new-comers to a district are very prone to
contract the disease.

C. Factors favouring the action of the virus of the disease at
the time of its entry into the body :—

(a) An organically impure water.

(6) Much mineral matter in suspension in the water.

(c) Very hard waters, especially those containing much lime,
magnesium, or iron in solution.

These factors, I believe, act by inducing abnormal states of the
lining membrane of the gut, and thus favour the development or
action of the toxic agent of goitre. To them subsequent experience
may add others, but they are those which I have found to be of the
chief importance in the regions where my researches have been
carried out. These influences are, in short, those which observers

I2

from the remotest times have considered to be primarily causal in
the production of the disease. Their importance is undoubtedly
great, but secondary only to the true causal factor which remains
still to be discovered.

Iv. THE ACTION EXERCISED BY THE LACTIC FERMENTS
WHEN APPLIED TO THE TREATMENT OF GOITRE

I have previously drawn attention to the curative action of
intestinal antiseptics, notably thymol and beta-napthol, in cases
of goitre, and I have regarded the action of these drugs as strong,
though not conclusive evidence that the responsible agent in the
production of the disease has its habitat in the intestinal tract of
man. The action exercised by the lactic acid ferments when applied
to the treatment of goitre affords additional evidence in favour of
this view. In carrying out this line of treatment I have used the
fresh cultures in milk of the Baczllus bulgaricus. I have treated
up to the present time only eight cases in this way, but the results
have been so striking that it is necessary to record them in this
place. I have hitherto employed only milk as a medium for the
administration of this bacillus but, owing to the scarcity of milk in
this country, some other medium must in future be employed.
Twelve to twenty ounces of ‘soured milk’ were given to each patient
every morning before the first meal of the day for periods of one
month to six weeks. The cases were all of several months standing,
and during treatment there was no change whatever in the manner
of life of the patients. They were treated as external cases, and
carried out their work in the fields as usual. The results cannot be
attributed, therefore, to change of locality, habits of life or water
supply. Of these eight cases four were cured, two improved, and
two showed no appreciable difference after six weeks. In those
cases which were benefited by the treatment, it was observed that
the thyroid gland began to show evidence of diminution in size
about the tenth day of treatment, and, that the patients lost flesh.
This latter fact is of considerable interest, as it is observed to
occur also in the treatment of goitre by means of thymol,
beta-naphthol and iodine. Figs 10, 11 and 12 represent various
stages in the treatment of one of these cases. Fig. 11 shows the
case after fifteen days, and Fig. 12 after thirty days of treatment.

3

V. THE EFFECT ON DOGS OF THE ADMINISTRATION OF
EXTRACTS FROM THE FAECES OF GOITROUS
INDIVIDUALS

EXPERIMENT K.—Carried out from the 1oth December, 1900,
to 8th March, rgro.

Nine healthy puppies were confined in a pen for a period of
eighty-eight days and were fed during this’ time on watery extracts
from the faeces of goitrous individuals. All due precautions were
observed to prevent infection from other sources, such as the
provision of a pure water supply, etc. Three control animals of a
like age were confined in a neighbouring pen for the same length
of tine. The results were entirely negative. Post-mortem
examination of the thyroid gland in these animals revealed no
deviation from normal in the direction of hypertrophy. The
thyroid gland of the nine puppies, to which extracts of the faeces
were given, varied in weight from one-twelve-hundredth to
one-twenty-four-hundredth part of the body weight. In the case
of the control animals the weight of the thyroid varied from
one-thousandth to one-fifteen-hundredth part of the body weight.

Vir RESULTS OF..THE RESEARCH

1. There exists in suspension in waters which are known to be
goitre-producing an agent which is capable of initiating an
hypertrophy of the thyroid gland.

2. This agent is destroyed by boiling, and is removed from
the water by filtration.

3. This agent is, therefore, either a living organism or a
chemical substance the noxious properties of which are destroyed
by heat.

4. The incubation period of experimentally produced goitre is
usually about ten to fifteen days.

5. Goitre can be cured by the administration of intestinal
antiseptics. The lactic ferments exercise a curative action when
applied to the treatment of incipient goitres.

6. It is very probable that the agent which is responsible for
the production of goitre is a living organism parasitic in the human
intestine.

7. The disease cannot be communicated to dogs by means of
watery extracts from the faeces of goitrous individuals.

These results confirm in detail those which I communicated to
the Royal Society on 26th November, 1908.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig.

Fig,

Fig.

Fig.

Fig.

14
EXPLANATION OF PLATES I anp II

1.— G.M..,’ referred to in Experiment I. Shows appearance
of neck at the time the experiment was commenced.
Measurement, 333 centimetres.

2.—The same subject. Photograph taken on thirtieth day
of the experiment. Measurement, 35 centimetres.

3.—‘S.A.,’ referred to in Experiment I. Shows appearance
of neck at the time the experiment was commenced.
Measurement, 33 centimetres.

4.—The same subject. Photograph taken on the thirtieth day
of the experiment. Measurement, 35 centimetres.

5.—The same subject. Photograph taken on the thirtieth day
of the experiment. Shows subject’s method of fastening
his shirt-band which buttoned comfortably prior to the
commencement of the experiment.

6.— A.M.,’ referred to in Experiment I. Shows appearance
of the neck at the time the experiment was commenced.
Measurement, 34 centimetres.

7.—The same subject on the thirty-sixth day of the
experiment. Measurement, 354 centimetres. The
enlargement of the isthmus and of the right lobe of the
gland is well seen.

8.—A.D.,’ referred to in Experiment I. Shows the
appearance of the subject’s neck prior to the commence-
ment of the experiment. Measurement, 34 centimetres.

g.—The same subject on the thirtieth day of the experiment.
The increase in size is slight but evident. Measurement,
354 centimetres.

10.—Boy, aged twelve years, the subject of a goitre said to
be of several months standing.

11.—The same case after fifteen days’ ‘soured milk’
treatment.

12.—The same case after thirty days’ ‘soured milk’ treatment.

PEATE

PLATE 1]

11 12

15

NON-ULCERATING ORIENTAL SORE :
THE CULTURAL CHARACTERISTICS OF
THE PARASITE AS COMPARED WITH
A NEW SIMILAR PARASITE IN
Pei AESINA FULLO“(THUMB), (A
PENTATOMID BUG

BY

CAPTAIN R. MARKHAM CARTER, I.M.S5.
(Received for publication 15 February, 1911)

In tropical and sub-tropical countries several peculiar types of
skin lesion occur endemically and often limited in distribution to
certain districts. These skin lesions have a very varied
nomenclature but are loosely grouped under the name Oriental
sore. In November, 1909, a brief differentiation between three
types of Oriental sore was first made, and for the purposes of this
communication, which is confined to experimental work on the
parasites of the non-ulcerating type, as compared with a new and a
similar parasite in Erthesina fullo, the three types of Oriental sore
will be termed (1) the non-ulcerating Oriental sore, (2) the superficial
flat Oriental ulcer, (3) the deep-seated Oriental boil. It is of
interest to note that Thomson and Balfour* confirm the presence
of the first type in Egypt, and wholly agree with me as to the
possibility of there being different varieties of Leishmania hitherto
undifferentiated.

Recent experience has shown that the non-ulcerating type of
Oriental sore is in India and neighbouring countries a not uncommon
affection. In a mixed population of over 2,000 patients from all
parts of Northern, Western, Eastern and Central India, Burma and
Assam, who sought anti-rabic treatment at the Pasteur Institute of
India, seven cases of non-ulcerating Oriental sore were seen in

* Jour. R.A.M.C., Jan., 1910,

16

sixteen months. Neither of the two other types occurred, though all
three were sought for. From the characteristic painless indolent
nature of this cutaneous lesion, it can, however, readily be
understood how easily it is overlooked unless seen in an advanced
condition, or in a site such as the face or hand under frequent
observation. It is thus probable other early cases in this number
of patients escaped notice.
The history of these seven cases is as follows :—

CasE I.—Boy, Singh, aged 19, native from Patiala State. Patient
presented a large swelling on the right cheek, similating a gumboil. In the centre
of this swelling was a slightly raised area, masuring 4 inch by ? inch, pale straw
in colour, and covered with fine papery scales. The edge of this raised area was
slightly indurated and could be differentiated by the touch from the soft resilient
raised patch.

The upper right eyelid presented a small circular raised nodule, reddish in
colour, about 4 inch in diameter. The left forearm presented four small raised
nodules, one exactly like that on the eyelid, the other three resembling the area on
the face in all details. There was the scar of an old ulcer on the back of the left
forearm, said by patient to have resulted from an injury. No other person in his
family shows or has ever shown similar cutaneous lesions.

History of the lesions.—The spot on the cheek first appeared fourteen months
previously. It started as a minute itching red spot, which in a few weeks became
anaesthetic to the touch and gradually extended to its present size. Six months
later patient noted a similar spot appearing on the front of his right forearm. The
other lesions on the forearm and eye developed subsequently.

Patient is in the habit of working in the open air, inspecting fields, etc., at
his village. He often sleeps in the open during the day. He was shown specimens
of ticks and biting flies to see if he could recognise them. He recognised Tabanids,
Hippoboscidae and Haematapota as common biting flies of his district attacking
animals, Patient does not remember being bitten by either ticks or flies, but states
he has seen Tabanids bite persons as well as animals. The bite has been described to
him as not being painful, but bleeding freely after a few seconds’ smarting.

He described other flies in his district that bite, especially very small species
in the neighbourhood of hill streams. These probably are Simulium and
mosquitos.

Smears were taken from each spot, and also of peripheral blood from the right
arm and ear. The blood clotted with unusual rapidity.

In all slides from the spots the mononuclear cells were found distended with
Leishmania-like parasites. There were many free forms, those in the mononuclear
cells were mainly oval and vacuolated. The small torpedo-form described in Delhi
boil was comparatively rare.

In the films of peripheral blood, malarial parasites alone were seen.

Repeated punctures healed up invariably within a few days. The patient’s
spleen and liver were slightly enlarged.

Case II.—Miss C., European, Medical Missionary from Gujrat, aged 24.
Presented a small raised area of pale straw colour cn the forehead. This area was
smooth and hairless over the greater part of its surface, the periphery was covered
with fine paper-like scales of epithelium. The margin was sharply defined by a
fine reddish indurated line of inflammation, which faded for about 4 inch into the
normal surrounding skin. The affected area measured 4 inch by 3 inch. The
lesion was completely anaesthetic. The red margin was sensitive. Miss C. names
the affection ‘ Monghyr Phora,’ this being the name given to this lesion in her
district.

17

The history of the case is as follows :—Miss C. first noted a small, itching, red
pimple, like the bite of an insect, on her forehead thirteen months previously. This
shortly became anaesthetic, but gradually increased. When the area was 4 inch
in diameter it was painted with pure carbolic. This treatment did not arrest the
progress of the lesion. A second minute pimple of the same type as the first
appeared on the point of the elbow some months later. Patient states she has had
no fever for eight years. She presents no enlargement of liver or spleen. She
has, however, noticed that since the appearance of the spot on the forehead she has
been feeling ill.

Examination of blood films from punctures of each spot showed the
mononuclears distended with parasites as in the previous case. This blood clotted
very rapidly. The peripheral blood showed no parasites of any kind.

Miss C., when asked if she recognised three or more types of Oriental sore,
stated three were recognised in her district : (1) ‘ Monghyr Phora,’ above described,
a non-ulcerating form; (2) a large superficial ulcer, common on the hands, wrists,
ankles, and feet, whose floor was composed of exuberant granulations covered with
pus and débris—this type is known locally as ‘ Chambal’; (3) a deep crater with
raised sides, the former filled with and the latter undermined by foul pus and
epithelial débris, this is known as ‘ Delhi boil.’

Popular belief is that the cause of the first type is the bite of the sand-fly or
mosquito.

Case III.—Mr. E., European, aged 45, from Scinde. Patient presents multiple
infection with non-ulcerating Oriental sore. The first appeared at Gumbat, outside
Kohat, as a minute itching pimple on the forehead. This has slowly increased
for fourteen months, is of pale straw colour, and raised above the surface of the
adjacent skin, from which it is separated by a red thickened edge. The affected
area is under 4 an inch in its greatest diameter.

A second spot appeared on the back of the forearm eight months ago, this is
similar to the first, but encroaches cn to an old healed scar, which seems to have
limited its spread on that side. There are seventeen other scars on this forearm,
patient states they are different from ulcerating ‘ Scinde sore.’

A third similar area was noted on the front of the left wrist. It was larger but
similar to the previous two described, covered with fine papery scales, and
completely anaesthetic. Patient first had a small ulcerating ‘ Scinde sore,’ 24 years
previously on the forehead, this healed with difficulty and left a small, smooth,
white, depressed scar. Four similar ‘ Scinde sores ’ developed later on the forehead,
and fourteen sores on and around the right elbow joint. The scars were most
distinct, many of them had a brown tinge round. their edges. The raised lesions
were examined for parasites and a condition found as in the previous cases. The
blood clotted rapidly. There was but little enlargement of the spleen. Patient had
a malarial history, but no parasites were found in the peripheral blood. He
describes three forms of sore in Scinde: (1) the flat indolent and often multiple
ulcer, known as ‘ Scinde sore,’ which is the commonest met with; (2) the deep-seated
affection like a deep boil or carbuncle; (3) the non-ulcerating form, comparatively
rare. Patient asked, as he had now had both the first and third varieties, if he was
liable to contract the second.

Patient states the sore on the forehead appeared at the beginning of the hot
weather. He attributes these to the bite of either mosquitos or sand-flies, which are
commoner than other pests outside Kohat. None of his servants or friends were
suffering from the same disease.

Case I1V.—Ephraim, son of W., native Christian from Gujrat, aged 7 years
2 months. Patient presents a small raised pale brown area the size of a three-penny
bit, 4 inch from the lower edge of the right nostril. The skin in the centre is
smooth, and is seen under a lens to be devoid of fine hairs. The edges are covered
with fine papery scales, the margin of the affected area is dark and slightly more
indurated than the adjoining healthy tissue.

18

This spot first appeared seventeen months previously at the commencement of
the hot weather, as a small itching point in the skin which gradually increased in
size. Though anaesthetic at the time of examination, patient’s parents state the
child complains of intense itching in the affected area at intervals of many days.
The child suffers from occasional fever every fifteen to twenty days. There were
no malarial parasites in the blood. The liver and spleen were normal. On looking
at the area with a high-power hand-lens, the appearance of the affected skin was
as if it were distended with fine saccules of serous fluid throughout the whole
thickness of the skin. By this serous sacculation the affected area was raised 3 of
an inch above the surrounding normal tissues. No enlarged glands were noted
in the neck.

Films were frequently made from the spot, by puncturing the area in the centre,
also in the indurated margin. The blood clotted rapidly. The wounds rapidly
healed. There were myriads of free parasites and the mononuclear cells were
distended as in the previous cases.

One other person in the house was affected with a similar lesion.

Case V.—Minnie, daughter of W., native Christian, Gujrat, aged 1 year
8 months. Patient presented what looked exactly like herpetic affection of the
mucous surface of the upper lip. However, where the upper edge of the spot met
the skin of the upper lip the characteristic slightly indurated red margin was seen.
The child looked very ill. The patient’s mother states the spot first started one
year previously as a small itching spot, which gradually grew.

History of constant fever attacks.—Patient has looked ill for months. Spleen and
liver normal. Blood taken from the affected area clots rapidly and presents a similar
condition to Case IV. These two last cases have lived in the same house since the
birth of Case V.

Case VI.—Abdul Rahman, a Pathan boy from Kohat, aged 10 years. Patient
presented a straw-coloured, hairless, raised area on the tip of the nose, which had
spread symmetrically on each side towards the nostrils. The edges of the affected
area was covered with papery epithelial scales. The fine thickened margin of the
affection seen in previous cases was well marked. The history of the case was
confirmed by the political naib tahsildar, who brought the patient to the Institute
The child’s attendant was well acquainted with the progress of the affection as he
was a friend of the patient’s parents.

Fourteen months previously the sore commenced as a _ small itching
khaki-coloured spot. It spread for some three weeks fairly rapidly, then remained
practically in the same condition as when it first came under examination.

The nose was slightly enlarged, the affected area was anaesthetic. No enlarged
glands were found. The spleen and liver were of normal size. There was no
history of fever. None of the child’s relations were infected.

On puncturing the area and making films, the same conditions as in previous
cases were noted. The blood clotted rapidly. There were no parasites of any kind
in the peripheral blood. Both the patient and his guardian state that three kinds of
Oriental sore are recognised in the Kohat district. The first is called ‘ Spumai,’ »
a form that never ulcerates, and is extremely common. The second a flat ulcer,
known locally by the name ‘ Aurungzebe phora,’ and by Englishmen as ‘ Frontier
sore. The third is a deep-seated boil, with a deep-pointed core, this is
comparatively rare, and is called locally ‘ Naroo.” The native treatment for the
non-ulcerating type is to apply a wafer of flour soaked in hot oil to the affected
site and its immediate vicinity as hot as the patient can bear it. The native
opinion around Kohat is that the lesion follows on the bite of the sand-fly, and
appears most commonly at the commencement of the hot weather.

Case VII.~Major C., British officer serving on the Aden Boundary Commission.
A small typical non-ulcerating Oriental sore developed on the front of the wrist and
lasted for eight months, when it was excised and the area painted with pure carbolic
acid. The sore first developed when the Commission encamped at Sanawi at the

19

foot of the Jehaf plateau in South Arabia. Two Sepoys, one other British officer and
a native servant developed similar sores at this camping site. The water supply of
the camp was obtained from two wells situated in a belt of tamarisk, where the
camels were kept during the breeding season. Films made from these cases showed
swarms of typical parasites. The blood clotted rapidly, and though repeatedly
punctured there never was any tendency on the part of the sores to break down.

Case VIII.— Gunner K, 4th Battery, Mian Meer. Patient presents multiple
infection with what seems to be the flat ulcerating type of Oriental sore. There are
eight small flat ulcers on and around the left knee, sixteen similar sores on the left
leg, front of left foot and round the left ankle joint. Seven small ulcers occur
round right knee on right foot and leg, one larger flat ulcer above the right buttock.
There are twenty-five old white scars on left leg and foot, twenty-two scars on right
leg and foot. One small ulcer presented itself on the inner side of the left eyebrow.
Patient has only been stationed at Mian Meer. There was no history of syphilis.
The first ulcer appeared eleven months previously. Examinations of all the ulcers
showed bacterial invasion alone. The peripheral blood showed benign tertian
parasites but no other protozoa. The blood clotted slightly more rapidly than
normal blood, but much less rapidly than in the previous seven cases. None of the
patient’s comrades are or have lately been similarly affected.

The point of interest in the first set of cases as compared

with the last are: —

1. Non-ulcerating character.

2. Increased coagulability of blood.

3. High constant infection of mononuclear cells.

4. Rare infection of polynuclears.

5. Long history, and appearance of other similar lesions at
long intervals.

6. Possible infection from another case in close daily contact.

7. Primary itching followed by anaesthesia.

8. Lesion presents constantly a central smooth surface, papery
epithelial scales at periphery, margin indurated and
visible to the naked eye.

g. Liver and spleen unaffected.

10. Possible exacerbations at irregular periods.

11. General malaise.

12. Occurs at all ages and in Europeans as well as natives.

13. First area affected usually on exposed surface.

The method of staining adopted for all smears and films from
the tissues of the patient or culture tubes was as follows:—To
10 c.c. distilled water add 12 drops of ripened Giemsa stain, shake,
and allow to act on films for twenty minutes. Then rinse each film
with tap water and stain in a watery solution of eosin I in 50,000,
until the film, which was dark purple, has changed to a rosy violet,

20

and the erythrocytes are seen under a low power to be of a rosy
pink colour.

The films should be fixed in methyl alcohol for five minutes and
blotted dry before the stain is applied.

The best method of making films from culture tubes is described
in my paper, ‘ British Medical Journal,’ Sept. 11, 1909, page 650.
In a differential leucocyte count made from the affected areas as
compared with the peripheral and normal blood, the result in all
cases showing protozoal infection presented a marked increase in
the polymorphonuclears. Difficulty was experienced in making
evenly spread films, as the blood drawn from the lesions clotted in
a few seconds. No pain is felt whilst boring into the lesion with a
sterile glass pipette until the point has passed through the anaesthetic
layer into the deeper tissues, about a quarter of an inch from the
surface. As a rule mononuclear cells are found distended with oval
parasites; polymorphonuclear cells rarely included any parasites.
Many free and dividing forms were seen amongst the cells. These
results are very similar to those seen by Thomson and Balfour in
Egypt.

The following work on the developmental forms of the
parasite of non-ulcerating Oriental sore is based on the material
taken from a series of cases in India during the last eighteen
months. Flagellated forms were first obtained in November,
1908.

In order to obtain development of the parasites of non-ulcerating
Oriental sore the method advocated in September, 1909, is the
most certain. To four units of clear non-activated human serum
add four units of non-activated red blood cells and mix freely in
a bulbed pipette with three units of a mixture of sterilised citrate
10 per cent. and salt 0°75 per cent. When the mixture is complete
add four units of a similar citrate solution which has been heavily
infected with organisms expressed from a puncture of the infected
area. By passing a fine glass tube under the skin surface in all
directions from one point of puncture, a free discharge of
straw-coloured fluid and blood is easily obtained on pressure, and
contamination is reduced to its minimum point. The fluid
expressed must be collected and discharged into the citrate as soon
as possible, as it clots rapidly. It is necessary to aerate the

21

contents of the culture each day by drawing air up the stem of the
pipette and shaking the mixture in the bulb. The cultures should
be kept at 22°C. Under such conditions flagellates appear in
forty-eight hours, living symbiotically with masses of cocci and
bacteria. These flagellated forms increase rapidly up to 120 hours,
and then degeneration processes set in.

In seventy-two hours flagellated organisms of two types are
frequently found singly and in pairs. One is of monadine form
and stains blue, the other is oval or circular, and stains rosy pink.
In ninety-six hours large clusters of rosy pink bodies are found,
some flagellated, others not. Amongst such clusters a smaller
number of blue staining monadine flagellates are usually seen.

The process of development in culture is as follows:—The
minute parasites packed within the mononuclear cells liberate
themselves, and rapidly multiply, dividing by simple fission.
Each daughter cell grows to the size of the parent cell and divides
into two or more grand-daughter parasites. After a series of such
divisions, differentiation takes place as revealed by staining films
from cultures, certain of the parasites now stain a fine rosy tint.
These rose-staining parasites possess but little nucleus or chromatin,
they slowly enlarge and a flagellum is extruded from the
extra-nuclear centrosome. The other type of parasite develops into
an oval pyriform body, which increases in size and divides by
fission several times. A_ flagellum with three plications is
extruded from the extra-nuclear centrosome, and the flagellated
daughter cell breaks away from a tangle of pale rose-staining
material to which these parasites are frequently found attached.
The flagellum is usually the same length as the body of the
monadine parasites. The zooglea mass of rose-staining material
above mentioned seems to have been extruded by the parasites as
a protective measure to enable them to fix themselves to one small
area whilst development and division by fission takes place. It is
no uncommon thing to find groups of many hundreds of parasites
in all stages of development, thus differing from the parasite of
Delhi boil worked at by Row in 1908 and 1909 in non-acidulated
human serum. Surrounding such groups masses of bacilli and
cocci are seen growing symbiotically. Groups of eight or ten blue
monadine flagellates have been seen surrounded by mixed colonies

22

of germs living in perfect harmony, thus differentiating their
specific nature from the allied parasite that gives rise to Kala Azar.
Further differences, morphological and cultural, between the three
parasites will be dealt with later.

A curious feature in cultures of non-ulcerating Oriental sore,
first noted by me in October, 1909, is the constant occurrence of
enormous clusters of what seem at first sight to be giant cocci.
These bodies stain purple, and have often a reddish margin or film
round them. They vary from forms the same size as an
erythrocyte to smaller forms, altogether like cocci, diplococci, etc.
This suspicious and interesting point has since been confirmed
by Thomson and Balfour in their work on _ non-ulcerating
Oriental sore in Egypt. In the description of material from
an affected area on the neck, they note in addition to the
parasites found free and in mononuclear cells groups of what
seem to be large cocci, also a number of pale _ blue
homogeneous structureless masses—a condition which is seen to
occur also in cultures of the parasite from the intestinal tract of
Erthesina fullo, to be described later. In material from affected
areas on the thigh similar coccoidal bodies were found. These
observers describe these blue coccoidal bodies as four to six times
the size of the small cocci present. They stain feebly in their
centres, often present unstained areas, occur in clumps or pairs,
and may resemble huge gonococci.

In the light of recent experiments I am of the belief that the
life-history of the parasite of non-ulcerating Oriental sore is as
follows :—

The cockle-shaped form found in the tissues and mononuclear
cells represent the form of the parasite which multiplies in the cells
of the host by simple fission. In their earliest form they are seen
as an exceedingly minute protoplasmal ring, containing a dot-like
nucleus. Such forms are occasionally seen amongst the more
maturely developed forms.

After a series of divisions by simple fission in cultures, what
would seem to be sexual elements are formed, which stain
differently. These seem to pair with interchange of elements.
From this point the cycle becomes obscure, and light alone is thrown
by observations on the life cycle of a similar parasite to be

23

subsequently described. If dot-like forms are released from the
female cell, as recently seen in Trypanosoma gambiense, on
examination of infected salivary glands in the intermediate host,
the tsetse, these elemental forms might well be the minute bodies
occasionally seen in infected mononuclears.

With a view to throwing light on these points the subsequent
series of experiments were performed.

Before describing these, a few remarks on the insect host
are necessary.

The order of the Rhynchota is divided into three main
sub-orders: the Heteroptera, Homoptera, and Phytophthires.

The sub-order Heteroptera is divided into two series. The
Gymnocerata having conspicuous antennae and the Crypotocera
having their antennae more or less concealed.

The Pentatominae is one of the most extensive of the nineteen
sub-families of the Gymnocerata, and includes the largest number
of common species. E7rthesina fullo and Halys dentatus are two
speckled drab-coloured species commonly found in India, and
supposed by competent authorities, such as Maxwell Lefroy to be
predacious habitually or occasionally. This insect is widely
distributed throughout India, Burma, Assam, Java, Japan,
Formosa, Hainan, China and Ceylon. On hatching from the egg,
there are four nymphal instars, lasting roughly about a month
before the adult stage is reached.

Mature specimens of E7rthesina fullo are common in _ the
Himalayas throughout the year. Larvae and nymphs are most
frequently seen in the spring. The mature insect is readily
attracted by light at night and enters houses freely. The younger
forms can only be taken on the bark of trees, and have not been
noticed to frequent human habitations. In connection with
Rhynchota of similar habits, it is as well to recall Donovan’s
observations in Madras, where he noted Conorhinus rubrofasciatus
a member of the Acanthaspidinae is a common blood-sucking insect
of local distribution found at night, whose nymphs frequent
corners and crevices in houses. This insect, though not
commensurate in its range with the occurrence of Kala Azar, is of
extreme interest, as both nymphs and adults suck human blood if
opportunity is afforded. Other Rhynchota with similar habits are

24
C. infestans, fed by Darwin on his blood, and C. sanguisiga,
found in Arizona, in which latter case the site of puncture from
which the insect has sucked human blood becomes painful,
inflammation and even pus formation ensuing.

The point of this seeming discursion will be seen later.
Forty-three adult specimens of Erthesina fullo were dissected, and
the contents of their intestinal tract examined. In forty-one
insects the crop contained large numbers of a crithidial organism
freely motile. In one the infection was scanty, and one insect was
found negative.

A series of nymphs and adult specimens of Erthesina fullo were
dissected, and it was found that infection of the intestinal tract
was common in all stages of the insect’s life history. No eggs were
examined. A few specimens of Halys dentatus and another
species, aS yet undetermined, were dissected and found negative,
an interesting point showing the selective preference shown by the
parasite for one species of insect. The further selective preference
shown by the parasite of Evthesina fullo for human blood in
culture as compared with blood of other animals is highly
suggestive, and opens a large field for research work to those
interested in discovering the definitive hosts that transmit the
parasites of Kala Azar and the forms of Oriental sore to man.

Adult specimens of E7thesina fullo fed readily on my blood,
citrated, when it was presented to them. A series of insects were
fed thus twice a week through the winter and thrived on the diet.

Stained films prepared from the gut contents, showed all stages
of the parasite, from the small cockle-shaped form simulating
Leishmania to pyriform and flagellated bodies of two varieties as
seen in non-ulcerating Oriental sore. The parasites were found
living symbiotically with myriads of cocci and bacteria in the crop
and intestine.

First series of culture experiments :—The crop of an infected
insect, having been dissected out, was placed in a drop of citrate
solution, I per cent. with 0°75 per cent. salt, and kept in a moist
chamber for twenty-four hours. When the contents were examined
the same conditions were noted as occurred in material from freshly
dissected insects. There was, however, a marked increase in the
number of the flagellate forms.

25
Ten drops of citrate solution were infected with the contents of

the crop of two infected insects, and a series of cultures were made
with bulbed pipettes as before.

A. A mixture of human blood citrate solution and infected
citrate in equal units.

B. Ditto.
C. Human serum and infected citrate, equal units.

A similar series of culture tubes were made up, in which the
blood from the rat, guinea-pig, rabbit, fowl and lizard (Agama
tuberculata) was put up with equal units of citrate solution and
infected citrate.

A similar series of serum cultures from these latter were also
put up, and controls in each case made without infected citrate.
The result of these experiments is rather striking, showing the
selective preference of this parasite for human blood. In a culture
of this parasite in citrated human blood the whole culture was
found seething with active flagellates in twenty-four hours. This
rapid increase was still more marked in forty-eight hours. Large
groups of flagellates and parasites in all stages were common.
After 192 hours a few live flagellating parasites were seen, most
of them were motionless. In fresh preparations put up under a
vaseline ringed cover-slip, the same process of multiplication was
watched daily. The flagellated parasites were found to arrange
themselves in large clusters around air bubbles with their flagella
attached to the under surface of the bubble. Death, apparently
due to want of air, was seen to occur in this confined area, usually
in 168 hours.

The formation of pairs of monadine flagellate and circular or
oval bodies was noted frequently, a condition checked by other
workers in the laboratory.

In human serum the parasites vanished in forty-eight hours,
a few cyst-like bodies with thick capsules alone were seen.

In blood and serum cultures from the common grey rat, no
increase in the number of the parasites took place. They became
motionless in four hours and had vanished in ninety-six hours. In
blood and serum cultures from the white rat the parasites were
found still motile after twenty-four hours, but were all motionless,

26

and the slide showed no increase in the parasites in forty-eight
hours. In cultures made from the blood and serum of the
guinea-pig, fowl and lizard there was no increase, a few parasites
only were found dead and motionless after twenty-four hours.
In lizard and fowl’s blood and serum the parasites became
motionless in four hours.

In the blood of the rabbit many single flagellates were seen
actively motile up to ninety-six hours. There were no groups
formed by multiplication of the parasites. After 120 hours the
parasites became sluggish and finally motionless. Stained films
showed no evidence of multiplication at any time.

Several interesting points were noted both in the fresh and in
stained preparations from the culture tubes.

In cultures of the parasite in human blood it was frequently
seen that at about the end of twenty-four hours pairs of parasites
of different form were found constantly in apposition. The one a
monadine flagellated body, the other a_ small parasite
either boat-shaped, oval, or round, which was apposed to the
monadine form in the neighbourhood of the nucleus in the case of
the oval form, and often near the commencement of the flagellum in
the case of the early boat shaped type. The character of the two
forms, as shown in the plates, will be seen to be of the same nature
as seen in the parasites of non-ulcerating Oriental sore during the
stage of numerical increase in culture tubes. Stained preparations
showed the monadine form stained bluish, the cyst-like body in
connection with it rosy pink and with a flagellum often attached.
In the case of the minute boat-shaped form this usually stained a
bright blue in the early stage, and contains the same structural
elements as the early form of Leishmania. Increase in the number
of the parasites continued up to 144 hours in citrated cultures of
human blood. After 196 hours but few motile flagellates were
found, the majority of the parasites were motionless, and many
showed commencing disintegration.

The monadine flagellate type had a characteristic movement.
The anterior part of the parasite moved to and fro with the
flagellum. The posterior one-third rarely is distorted. The
flagellum would give five or six flicks then the whole parasite would
vibrate, next the body of the parasite rapidly curves in § forms

27

for a few seconds, and becomes quiescent for a short period. The
tip of the flagellum is applied by the parasite to the edge of the
red cell frequently as if deriving nutriment from its contents.

The flagellated oval or round form of the parasite streams
slowly along the field of the microscope propelled by the flagellum.

A detailed description of the parasite when stained takes up
space, and is_ best elucidated by accurate drawings, the
morphological details of the various stages of this parasite are
shown in the accompanying plates, all drawn with the large Abbé
camera lucida and Bernhard’s drawing table.

A point of interest, and which may throw light on the rosy
zooglea formation seen in culture groups of the parasites of Kala
Azar and the varieties of Oriental sore, is the formation of what I
propose to term ‘anchor cords,’ first noted in the monadine
flagellate form of this parasite.

On putting up this parasite with citrated guinea-pig’s blood,
as above, and examining fresh-ringed cover-slip preparations after
four hours a curious condition was seen for the first time. A group
of four monadine flagellates were noticed to leave a certain
refractile glass-like spot on the slide, and with the aid of their
flagella explore the various channels that lay between adyacent
groups of red corpuscles. It was noticed they invariably returned
to the same spot after their excursions, often almost beyond the
limit of the field of the microscope. When the light from the
condenser was markedly reduced it was now noted that the
posterior end of each parasite was connected by a fine thread
to a small spot in a minute patch of sticky material adhering to
the slide, which material was faintly ground-glass in appearance.
These ‘anchor cords’ were extremely fine and highly elastic, and
enabled the free flagellated parasite to wander far from the central
spot, but yet return to its starting point from any angle with
absolute certainty. There was no coiling up of these threads as
the parasites returned to their common central point in the zooglea
mass, they contracted and expanded with the requirements of the
parasite, but seemed to have an equal length as regards the
extreme limit of distance to which the parasites are capable of
wandering. Such an apparatus can have only one or both objects
in view: exploration for food material or the opposite sex.

28

It is probable this morphological detail occurs at this stage in
the life-history of other allied flagellates and has hitherto escaped
notice. These ‘anchor cords’ are extremely difficult to focus, but
once located were shown to and detected by two other workers in
the laboratory.

A series of adult Ervthesina fullo were placed in a sterilised
glass box and fed only on distilled water in a pledget of sterile
wool for three winter months. The air in the box was changed
once a week. This shows the vitality of the insect and its parasite.
On dissection, the crop of each insect was found distended to about
the size of a pea, with clear greenish fluid.

Examination of this fluid revealed numerous clumps of pyriform
forms and free flagellates. Earlier forms were seen in large
numbers.

A similar series of experiments with a series of bloods as before
gave the same results.

On separating a series of insects fed only on water for three
months, an insect was noted to defaecate a large quantity of dark,
watery, greenish fluid. On examination of this fluid it was found
to contain large numbers of parasites in all stages, the flagellate
forms were, however, all motionless.

On making citrated blood cultures of the fluid with human,
rabbit, fowl, guinea-pig, and white rat’s blood, and preparing
vaseline-ringed cover-slip preparations, it was noted the flagellates
previously motionless and seemingly dead had all become freely
active in four hours.

In the case of blood cultures from the rabbit, fowl, and
guinea-pig with this material, the flagellates again were found
motionless in twenty-four hours, and disintegrating. In the blood
of the white rat flagellating parasites were seen after twenty-four
hours, but dead in forty-eight hours.

In human blood, however, it is interesting to note there was an
enormous increase in the numbers of the parasites by the end of
forty-eight hours. Many pairs of monadine flagellates and
cyst-like bodies were seen. Parasites in all stages of development
were found increasing in numbers up to 144 hours; from this point
they grew more sluggish in their movements and became motionless
and degenerated.

29

From a primary culture of the parasite in human blood which
showed flagellates after ninety-six hours, a sub-culture was made
into fresh citrated human blood.

This sub-culture showed freely moving flagellates in 120 hours,
none could be found after 168 hours.

That mature insects void the parasite in all its stages indicates
the method of infection of others of the same species. It is not
uncommon to find several of these insects huddled together in one
spot. The fact that motionless and seemingly dead flagellar forms
capable of revivifying in a suitable medium and multiplying in it
are voided, shows that infection of other insects need not necessarily
be confined to the infection by cyst forms, a commonly accepted
belief amongst some workers at this branch of protozoology.

In addition to the stages in the life-history of the parasite
mentioned above, we note that in the ileum, and below the crop,
many cyst-like bodies occur with granular contents. These, if a
phase in the life cycle of the parasite, and not another gut
organism, would seem to be the stage previous to the liberation of
the earliest form of the parasite.

To review the points of similarity between the parasite causing
non-ulcerating Oriental sore and that infesting the intestinal tract
of Erthesina fullo.

They both multiply and develop pyriform forms, monadine and
circular or oval flagellated forms, in a culture of human blood
acidulated with sodium citrate. Neither parasite will develop in an
alkaline medium. They both live and develop symbiotically with
masses of cocci and bacteria.

Bodies like giant cocci and bluish homogeneous bodies are found
in cultures of both parasites.

Pairs of dissimilarly shaped and staining flagellated parasites
are seen in both cases, whilst in the parasite of Evthesina fullo
cyst-like bodies have been found suggestive of the formation of
multiple early forms from a fertilised female.

The crithidial parasite of Evthesina fullo, when examined
in the living condition, affords many interesting points likely to
throw light on the life cycle of allied parasites. When a drop of
human blood-culture fluid has been kept under a vaseline-ringed
cover-slip, for eighteen hours, and a few small bubbles of air

30

similarly enclosed amongst the medium, the fully formed monadine
flagellates are found often to have arranged themselves in clusters
around the edge of the air space, with the tips of their flagella
pointing towards its centre. The parasites do not leave the bubble,
but remain vigorously swaying to and fro, as if having found air
they fear to leave it. In early fresh-infected cultures of human
blood one of the most striking and frequent features is the linking
up of a small motile boat-shaped body to the fully-formed
crithidial monadine flagellate. The smaller body is found
frequently apposed to the body of the former at the root of the
flagellum. It may, however, be apposed opposite the nucleus or
even on the flagellum itself. Once the small body has fixed itself
to the larger parasite it seems to become securely stuck to the site,
and accompanies the flagellate monadine in its peregrinations
without being dislodged. Such pairs are commonest seen in the
first period of rapid multiplication of the parasites.

The monadine flagellate presents, as a rule, pointed ends, the
anterior flagellated end tapers finely, and in many cases passes a
marked way up the flagellum. Occasionally the monadine form
presents a globose posterior end, looking like a pouch filled with
numerous brightly refractile granules.

The monadine form presents in the fresh condition an easily
seen granular nucleus, a clear centrosome with indications of a
surrounding vacuole. Small golden granules are commonly seen
at either end of the body of the parasite, but usually occur in
greatest number in the posterior portion.

When the monadine forms die, one often sees in fresh
specimens a clear vacuole-like body in the centre of the parasite.
Occasionally one sees evidence of a kind of prolongation of the
flagellum into the body of the parasite, giving it a bladed
screw-like appearance. Other parasites after death are globose in
their centres, this globular area is clear as a rule, whilst collections
of golden granules are seen at the posterior end and round a dark
pigmented area, anterior to the nucleus, probably the site of the
extra-nuclear centrosome.

In the case of the monadine form found in apposition with
cyst-like bodies at the level of, or in the neighbourhood of the
nucleus of the former, a detailed description can be given, as it is

31

based on a large series of observations by myself and other workers
in the laboratory.

The smaller rounded cyst-like body may, or may not, present
a flagellum arising from a clear staining spot, the extra-nuclear
centrosome. The body of the parasite in favourable cases is seen
to have (1) a fine, clearly defined, smooth capsule; (2) a
comparatively large area of scattered granular material; (3) the
nucleus a large dark spot is seen in one portion of the cell. Round
this nucleus are often arranged a ring of fine dark dots.

A reniform ground-glass area, looking like a vacuole, is
occasionally seen separated from the cuticle of the cell by a series
of dark dots, seemingly equidistant from each other. These dots
vary in number and in position, they may be found in a loose clump
at one side of the cell.

The monadine form may or may not present a certain degree of
globular enlargement at the point of contact between the two
bodies. Where the small cell lies apposed at the level of the
nucleus this globular enlargement has been most frequently seen.
The nucleus of the monadine form has a ground-glass appearance,
and its centre is usually situated at the junction of the anterior and
middle thirds of the body of the parasite. The flagellum is usually
about the same length as the body of the parasite, and presents two
to four wide undulations. The root of the flagellum passes down
the sinuous tapering anterior point of the parasite to end in the
immediate vicinity of the extra-nuclear centrosome. When this
latter is seen clearly, it is usually recognised as a brilliant clear
oval spot, with a dark edge to it, the whole surrounded by a fine
band of ground-glass-like material.

Between the extra-nuclear centrosome and the filamentous
anterior extremity an area thickly sown with fine granules is seen.
The posterior third of the parasite may show two definite dark
refractive dots or a varying number of granules. Occasionally a
cyst-like body, non-flagellated, showing a reniform vacuole and a
large number of granules, as it were boiling within it, has been seen.
Further experimental work on the later stages of the mature
parasites is necessary, and these cyst bodies are recorded without
comment.

32
EXPLANATION OF PLATES

PLATE III

I.—Shows pairs of flagellated organisms from a culture tube of
non-ulcerating Oriental sore. These stain differently;
the blue monadine form, the rosy pink oval form.

These organisms are in relation with a group of large
coccoidal bodies, whose nuclei stain dark-bluish purple,
and are often surrounded by a fine zone of cytoplasm,
staining pink.

Film made after seventy-two hours’ culture at 22°C.

II.—A cluster of parasites in all stages of development in a
ninety-six hours’ culture of non-ulcerating Oriental sore
kept at 20° C.

The parasites are seen living symbiotically with
bacteria and cocci. A large clump of parasites showing
early differentiation into the two forms which stain
differently and present flagella seen at the edge of the
clump.

Two rosy-staining zooglea masses are shown.

IlI.—Pairs of bodies seen in a culture of non-ulcerating Oriental
sore seventy-two hours old.

IV.—A group of rosy bodies, amongst which a few blue-staining
monadine forms are seen. Note the group of coccoidal
bodies, also the curious apposition of a flagellated rosy
body to that of a blue monadine flagellate at the level
of the nucleus.

From a culture of non-ulcerating Oriental sore
ninety-six hours old.

V.—Blue monadine flagellates, dividing forms, also others in
conjunction with rosy bodies.

Note a globose enlargement in a blue monadine form
opposite the point of apposition of a flagellated rosy
body.

Drawn from a culture of non-ulcerating Oriental
sore ninety-six hours old.

PLATE III.

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VI.—A group of organisms drawn from a culture of parasites from
Erthesina fullo after seventy-two hours.

Note the parasites are seen in all stages of
development, large blue coccoidal masses are seen
attached to two rosy zooglea masses. The morphological
details of the bluish monadine form are chiefly
crithidial in type. The rosy bodies similate those found
in non-ulcerating Oriental sore. The parasites are seen
living symbiotically with bacteria.

34
PLATE IV.

Series of drawings of living specimens of the gut parasite from
Erthesina fullo in blood cultures. I-XII and No. XIX drawn with
1/12 objective No. 2 eye-piece, XIII-XVIII and No. XX drawn
with 1/12 objective and No. 4 eye-piece.

I.—Oval body showing nucleus, nucleolus and vacuole.

II and III.—Monadine parasites (4) with small oval or boat-shaped
bodies, (a) apposed at the root of the flagellum.

IV and V.—Flagellated large oval parasites.

VI.—Monadine flagellate (0), and small oval body (a). Note
granular posterior and anterior ends of the former.

VII and VIII.—Flagellate parasite with the posterior ends globose
and filled with granules.

IX and X.—A monadine flagellate dividing by fission from the
anterior end of the parasite.

XI.—Monadine flagellate with globose granular centre.

XII.—Edge of an air bubble in a cover-glass preparation showing
the arrangement of an aggregate of monadine
flagellates, with their flagellae towards the centre of the
lower surface of the bubble.

XIII.—A pair of parasites, the one (6) a monadine flagellate, the
other (a) a cyst-like body unflagellated apposed at the
level of the nucleus of the monadine parasite. The
monadine parasite (4) presents a ground-glass-like
nucleus about its centre. The posterior end of the
parasite presents two dark dots. The anterior portion
of the parasite presents fine granules and a small, clear,
dark-edged oval area surrounded by a fine vacole.
Occasionally the root of the flagellum can be traced
ending in the neighbourhood of the vacuolar area.

The oval parasite (2) presents a fine capsule. To
one side lies a large dark dot surrounded by a ring of
fine dots. These adjoin a reniform ground-glass-like
area, seemingly like a faint vacuole. This latter is
sepirated from the capsule of the parasite by a line of
equidistant dots of equal size.

PLATE

39

XIV, XV, XVI and XVII.—Similar pairs of parasites, the oval
parasite flagellated or not.

XVIII.—Cyst-like bodies packed with small golden granules,
which seemed to boil within the capsule.

XIX.—Four monadine flagellates with acicular posterior
extremities, each parasite attached by a fine elastic cord
to a spot in a mass of zooglea material.

XX.—A parasite showing the posterior end of the parasite contains
clear fine granules.

A and B show two positions of the parasite leaving the zooglea
clear mass with the flagellated anterior extremity
forwards.

6.—The fine elastic posterior ‘ anchor cord.’

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INFANTILE LEISHMANIASIS
(MARDA TAL BICCIA) IN MALTA

BY

A; GRITTEN,-M. Ds; DiPAe, D.T.M: (LIiv.)
(Received for publication 23 January, IgII)

There exists in these Islands a morbid condition characterised
by great enlargement of the spleen and profound anaemia. It is
met with almost exclusively in very young children, and is nearly
always fatal.

Although, with the exception of splenic leukaemia, all the
anaemias with chronic swelling of the spleen are clinically little
differentiated, there always has been a feeling among local
practitioners that they were dealing with a special pathological
entity, which being in its clinical manifestations very much like
some of the better known disorders of the blood and haematopoietic
organs, they could not very well, in the absence of some special
element of diagnosis, dissociate from the latter.

Etiologically its connection with syphilis, tubercle, rickets, or
amyloid degeneration is not apparent; as to malaria, it is not
endemic in these Islands. The disease is variously certified at
death as leucocythaemia, splenic leukaemia, splenic anaemia,
pseudo-leukaemia, splenitis, splenopathy; but in the Maltese
language it is referred to by the professional and the layman alike
under one name, ‘ Marda tal biccia.’

The disease begins very insidiously with spells of fever of a slow
type, at a period of the child’s life when slight ailments are very
frequent and not made much of. If the initial pyrexia tends to
establish itself without any obvious explanation such as dentition or
gastro-intestinal troubles, Mediterranean fever is apt to be suspected,
especially as on percussion the spleen is already found somewhat
enlarged. More often, the initial attacks of fever do not attract
attention until there arrives a time when the child, having lost its

38

usual brightness and desire for food, becomes pale and begins to lose
flesh. By this time the spleen can be felt as a distinct tumour in
the left hypochondrium, and the little patient is shown to a doctor.
From this stage the malady has a protracted course of from six to
eighteen or twenty months.

The following is a short clinical picture of the disease when fully
developed :—The skin is waxy white or sallow, according as the
subject is fair or dark; the lips and mucous membranes are blanched ;
the eyes are full of sadness and look abnormally large in the
emaciated little face; all the muscles are flabby and more or less
atrophic, the distended abdomen contrasting with the wasted thorax,
its fulness more pronounced on the left. The outlines of the splenic
tumour may sometimes be easily made out by inspection. On
examination the spleen is found generally to extend down to the
level of the umbilicus, firm but not hard, freely movable, only
slightly tender on pressure or not at all, its margins rounded but
well defined, its notches well pronounced. In growing downwards
as a rule it keeps to the left of the navel, reaching very often down
to the iliac crest; but in some cases it fills the pelvis, and, crossing
the linea alba, occupies the right inferior quadrant, where in an
extreme case I have found it in close apposition to the anterior
border of the enlarged liver. The occurrence of the caput medusae
is very common. The liver is also enlarged, but to a less degree,
not more than one or two fingers’ breadth below the costal margin.
In the cases observed the presence of fluid in the abdominal cavity
could not be detected. The lymphatic glands accessible to
examination are not sensibly enlarged, but when the wasting is very
pronounced they can easily be felt and seen. Transitory oedema of
the feet, hands and eyelids is common. The appetite is very
indifferent, but sometimes there is a great craving for food; it is
rarely perverted: in some cases the patients pick and chew bits of
plaster or little stones. Gastro-intestinal troubles are the rule,
manifested by intercurrent attacks of very fetid diarrhoea. A
symptom met with sooner or later consists of a solitary or repeated
attack of dysenteriform diarrhoea with tenesmi, slimy motions
containing blood and mucus or mucus only. The frequent passage
of loose stools is not infrequently accompanied by temporary
shrinking of the spleen. In one case this was observed to such a

39

degree and the improvement of the other symptoms was so marked
and continued that the mother firmly believed the abdominal
tumour—the spleen—had been passed with the motions. Curiously
enough the case ended in complete recovery; the patient, a boy of
six, when seen by me was in perfect health. Evidently, he also had
had cancrum oris, as the upper middle incisors were missing and the
gums were badly scarred. Intercurrent attacks of bronchitis are by
no means rare. Epistaxis is a common occurrence, so is bleeding
of the gums, and the appearance of one or more crops of purpura
all over the trunk and face. In one case a few vibices were
observed. A frequent terminal complication is cancrum oris. The
mortification may assume formidable proportions in a few days,
or it may evolve less acutely, death supervening in a month or six
weeks. It sets in very stealthily, almost without pain, the increased
flow of saliva at first being ascribed to irritation of the mouth due
to dental evolution. The process in the cases observed started from
the gums in connection with the upper incisors, lower or upper
premolars. The gangrene is often very extensive and exceedingly
repulsive to the eye and nose, and the deformity is generally such
that no plastic operation could ever remedy. The blood in
advanced cases is quite watery; it separates quickly into clot and
plasma. Prognosis is very bad. Nearly all practitioners, however,
quote from experience one or two instances of the disease ending in
recovery ; but some maintain that all recoveries are cases of mistaken
diagnosis. It is wonderful how some patients can go on living;
on the other hand death occurs when least expected. Bronchial
complications are very frequent towards the end.

The disease, as outlined, was found to have reached a more or
less advanced stage in the twenty-one cases, to which the following
notes refer : —

1. Girl, 4 years, seen in May, 1gog. Ill since October, 1908, after whooping
cough. Intercurrent waves of fever of a remittent type, profuse perspiration, no
appetite, anaemia, muscular atrophy, transitory oedema of both legs, spleen reaches
down to iliac crest, liver also enlarged, purpura, dysenteric diarrhoea with great
loss of blood, bleeding from gums, noma, fall of upper incisors. Peripheral blood
examined : two Leishman-Donovan bodies in a large mononuclear, well-marked
large mononuclear increase. Died about one month after. No post mortem or
puncture of spleen after death allowed.

2. Girl, 44 years, seen in July, 1909. Very scanty notes taken at the time.
Died in January, 1910, after an illness of about fourteen months. Two weeks before
death a swelling of the left cheek and a very foul condition of the mouth foreboded

40

the very common final complication, noma. The disease had started with spells of
fever with very high temperature of a remittent type, then anaemia, great pallor of
the integuments, enlargement of the spleen, intermittent attacks of diarrhoea,
oedema of the extremities followed. Liver moderately enlarged, lymphatic glands
not affected. No other cases in the same family. No dogs kept. Smears and
sections from spleen post mortem: smears were literally studded with Leishman-
Donovan bodies, but the parasites were not so abundant in the sections.

3. Girl, 34 years, seen in October, 1909. Had measles in April, 1908. About
four months ago had fever for twenty days, no high temperatures, no regular type.
She gradually became anaemic and lost flesh. Now her skin is of an earthy
pallor, marked muscular atrophy, no oedema, no haemorrhages, the appetite very
poor, is at times abnormal, has diarrhoea every now and then, no blood with
stools. Spleen is enlarged down to two fingers’ breadth below the navel. Anterior
border of liver is two fingers’ breadth below costal border on mammillary line
Glands not enlarged. No noma. Died about three months after.

4. Boy, 25 months, seen in October, 1909. Ill since six months. Mother did
not notice any fever at first; two months after he had a spell of dysenteric
diarrhoea with loss of blood. Now he is very pale and has lost flesh, very fretful,
glands not enlarged, no oedema, fever of an irregular type. Spleen reaches down
to three fingers’ breadth below navel, no marked increase of liver. Mother stated
that she had had the same disease when a child. Case has not been seen again.

5. Boy, 6 years, born in Malta of English parents, seen in October, 1909.
Ill since one year. Spleen began to increase in size about five months ago.
Extreme anaemia and wasting, spleen enormous, spells of fever every now and then,
appetite good, no purpura, no bleeding from gums, no blood with stools, one or
two vibices on the back. Later on had several purpuric eruptions, bleeding from
gums, profuse diarrhoea. The doctor attending noticed an almost complete
retraction of the spleen a few days before the end and the diarrhoea ceased, but
there was no amelioration of the other symptoms. The child died of exhaustion in
April, 1gto.

6. Girl, 3 years, seen in November, 1909. Ill since one year. Moderate
anaemia, no marked wasting, no oedema, has a temperature every now and then,
appetite good. Spleen reaches to just below navel. Has had purpura and
dysenteric attacks but no bronchial phenomena. Glands not enlarged. Blood
smears from ear: no parasites. Child lost sight of. Doctor attending stated to
have observed a great improvement following a course of injections of methyl
arsenate of iron.

7. Girl, 6 years, seen in December, 1909. III since one year, after sustaining
a fractured clavicle. Earthy colour, extremely anaemic; very extensive gangrene of
gums, both lips, nose and cheeks. Great emaciation. Spleen, but for the enlarged
liver which reaches to about four fingers’ breadth below costal margin, occupies
the whole abdomen; has dysenteric diarrhoea; appetite fairly good. Died four
days after. No examination of the blood or spleen puncture allowed. A sister
died from the same disease when two years old: had noma followed by same
. extensive gangrene. An elder brother, who is stated also to have had the disease
when six years old, is now quite well. An aunt and a cousin on the mother’s side
are supposed to have died of the same complaint.

8. Boy, 3 years, seen in January, 1910. II] since eighteen months, spells of
fever with profuse perspiration, intercurrent attacks of diarrhoea and bronchitis,
purpura, epistaxis, bleeding from gums, extreme pallor and emaciation. Face has
a very old and sad look. The spleen free, firm, notchy, easily movable, not painful,
reaches down to the inguinal fold ; liver has grown to four fingers’ breadth below the
costal margin. Died suddenly in February. Two dogs had been in the house for

a long time. A fragment of spleen obtained post mortem: Leishman-Donovan
bodies in smears and sections.

41

g. Girl, 5 years, seen in January, 1910. Ill since November, 1909. Had
whooping cough a year ago. The disease began with spells of fever and anaemia,
then swelling of the spleen; this organ now reaches to inguinal fold; liver is little
enlarged. She is very pale and very sad, has diarrhoea, no blood with stools, no
epistaxis, no bleeding from gums, no purpura, feet are oedematous. A cousin on
mother’s side died from the same complaint when eighteen months old. Seen again
in April, no change; administration of 77. semegae suggested. Seen again in
October : very marked improvement, the spleen has receded to one finger’s breadth
below the costal margin, the child has recovered her gaiety, has a healthy colour
and good appetite. One dog in house.

10. Boy, 34 years, seen in January, 1910. Ill since seven months, after a
fright, as stated. Spells of fever, loss of appetite, great pallor and emaciation,
oedema of feet, hands and eyelids, great sadness, dysenteric diarrhoea and
bronchial catarrh. The splenic tumour fills the left inferior and part of the right
inferior quadrant ; the liver reaches down to a finger’s breadth below costal margin.
Seen again in February, the spleen maintains the same curved configuration but
does not reach quite down to the ilium, the liver is also smaller, the diarrhoea
persists, general condition worse. Peripheral blood examined, no parasites. Died in
November, 1g10. A brother died of the same disease in June, 1907, when two years
old, after an illness of fourteen months. No dogs.

11. Boy, 3 years. He is one of six, of which the eldest is 14 years old and the
youngest 15 months. No other children have had the disease. III since fifteen
months. It was only after three months of irregular fever that the enlarged spleen
began to attract attention. Iron preparations were prescribed. After three months’
treatment the splenic tumour was so reduced in size that the mother believed him
cured. He then contracted whooping-cough and the spleen started growing again.
About the time this patient sickened two other children living near were suffering
from splenic anaemia, both developed cancrum oris and died. A dog was owned
by these people. I saw the child in March, 1o10, three days before death: great
pallor and emaciation, oedema of feet and eyelids, spleen reaches down to one
finger’s breadth from iliac crest, liver moderately enlarged, diarrhoea, a black
stool occasionally (melaena?). Cancrum oris started opposite right upper
premolars, now mortification of right cheek, exposure of buccal cavity; no
epistaxis, no purpura. Post mortem: Cancrum oris, extensive destruction of right
cheek and gums, ioss of teeth. Lungs: right, caseous lobular pneumonia; left,
emphysema. Heart: all cavities dilated. Liver: enlarged, consistency increased.
Spleen : weight 8} ounces, about three times normal size, rounded margins, many
notches, very firm, capsule thickened and adherent; the cut surface greyish towards
the middle, brownish-red at periphery, malpighian corpuscles prominent.
Mesenteric glands enlarged, not caseous, the other glands normal in size and
appearance, bone marrow body of femur reddish and swollen. Smears and sections
of spleen and liver show a fair number of Leishman Donovan bodies. Smears from
mesenteric glands, a few parasites present. Smears from bone marrow, owing to
defective fixation, could not be stained successfully.

12. Girl, 4 years, seen in March, 1910. Has had fever and diarrhoea since
three months, moderate emaciation, skin and mucous membranes anaemic, loss of
appetite, the child is listless and sad. Diarrhoea every now and then, with tenesmi
and passage of mucus, but no blood. Splenic tumour rather narrow, it does not
reach below navel, easily movable, not painful; liver cannot be felt on palpation,
lymphatic glands normal in size. Seen again in December. Abdomen greatly
distended as the splenic tumour has grown down to the iliac crest and, to the right,
under the linea alba, the liver is two and a half fingers’ breadth below the costal
margin, cervical glands are larger than normal, eyelids are oedematous,
respiration is much hindered, hollow cough, fever, no haemorrhages.

December 3rd, 1910.—Peripheral blood examined: no parasites. Relative
leucocytic values : Large mononuclear, 36:2; small mononuclear, 29°4; transitional,
7°0; polynuclear, 26-2; eosinophile, 1°2.

42

13. Girl, 3 years. One of a large family, but no other member ever had the
disease. A child next door died of splenic anaemia in 1902. Patient has been ill
since December, 1909. In January, 1g10, had a slight attack of diarrhoea with
tenesmi; after three or four weeks of fever, attended with profuse perspiration,
mother noticed the splenic tumour just below costal margin. Seen by me in March :
no marked emaciation, moderate anaemia, no oedema, no purpura, appetite fair,
no great depression. Splenic tumour reaches down to three fingers’ breadth below
costal margin, liver is just palpable, no diarrhoea. Splenic puncture with an
ordinary hypodermic needle, usual antiseptic precautions: Leishman-Donovan
bodies present in a fairly large number. Treatment with senega preparations
suggested. Marked improvement during the next two months; the case, however,
ended fatally in September.

14. Boy, 21 months. Ill since August, 1g09. Two cousins on mother’s side
of about the same age died from same disease after a year’s illness. In August,
1909, after a fright, the boy started having a temperature at irregular intervals,
with perspiration; several attacks of dysenteric diarrhoea, slight epistaxis and
crops of purpura followed. Seen in March, 1910: great pallor of skin and mucous
membranes, loss of flesh, profuse salivation, initial mortification of gums at the
base of left premolars and slight bleeding, oedema of feet and eyelids. Spleen
enlarged down to iliac crest, moderately hard, freely movable, not painful, very
marked notches, liver just palpable. Died in April.

15. Boy, 19 months, seen in March, 1910. No history of splenic anaemia in
the family—a large one. Ill since 44 months; spells of fever with perspiration.
No diarrhoea, no bronchial catarrh, no epistaxis. Appetite good but perverted, is
always picking and chewing stones. On examination : moderate anaemia, no great
loss of flesh, gums normal, splenic tumour reaches down to iliac crest and to the
right, 14 inches beyond linea alba, liver can be felt two fingers’ breadth below
costal border. Splenic puncture : Leishman-Donovan bodies present in all smears
in moderate numbers. Treatment: tinctura senegae in large doses. Seen again
in April: extreme pallor, slight bleeding from gums, purpura. Died the same
month.

16. Boy, 18 months, seen in April, 1910. There is a history of short spells of
fever before mother noticed that spleen was enlarged, five months ago. Since then
has had diarrhoea off and on, slight bleeding from gums, a few spots of purpura,
no oedema. Now skin and mucous membranes anaemic, loss of flesh, appetite fairly
good, spleen reaches down to iliac crest and laterally almost to umbilicus, caput
medusae, liver one finger’s breadth below costal margin, lymphatic glands not
enlarged. Splenic puncture: no parasites met with. Died in August.

17. Boy, 2 years. Weakly child from birth. Seen in April. About two
months before had enteritis with tenesmi, passage of mucus but no blood, no
haemorrhages. Cancrum oris started three weeks before I saw him, when a small
slough formed in the gums over the upper incisors. Mother never noticed any
enlargement of spleen. Now great pallor and emaciation, diarrhoea, prolapsus ani,
mortification of upper lips, nose, cheeks and lower eyelids. Spleen and liver can
hardly be felt on palpation, but abdomen is very distended. Died 2oth April, 1910.
Post mortem, partial : spleen exceeds costal border by about two fingers’ breadth.
Spleen smears swarming with Leishman-Donovan bodies. About a year ago they
had a small dog in the house. Eldest sister died five years ago; two other boys and
a baby, of whom the former are older than patient, all alive. Eldest sister was ill
for one year, and presented the following symptoms: progressive anaemia and
emaciation, fever, attacks of diarrhoea with passage of mucus and prolapsus ani, but
no enlargement of spleen was noticed by mother. Then gums in connection with
lower left molars underwent a process of mortification which extended to cheek,
sloughing through. At that time they also kept a dog different from one mentioned
above,

43

18. Girl, 25 years. Seen in May, 1g1o. III since five months: Anaemia,
emaciation, loss of appetite, diarrhoea with passage of mucus and tenesmi. Now
great dejection, bronchitis, no oedema, splenic tumour reaches to about four fingers’
breadth below costal margin, liver enlarged but to a less extent, lymphatic glands
normal. Died August, 1910.

19. Boy, 15 months. Not seen during life. Post mortem, twenty-four hours
after death, 2nd June, 1910 : extreme anaemia, mucous membranes bleached, no great
emaciation, spots of purpura, oedema of lower limbs, liver very large especially
left lobe, splenic tumour reaches toabout two fingers’ breadth from iliac crest, and
is pushed to the left by the enlarged liver, cancrum oris with loss of upper incisors,
mortification of gums, upper lip, left cheek, and nose up to lower eyelids on both
sides, lymphatic glands normal. Spleen: 5 inches by 3 inches, weight 6 ounces,
perisplenitis, patches of infarction, free edges rounded, deep notches, firm but not
hard. Liver: 7 inches by 4} inches, weight 144 ounces, uniform yellowish white
colour, on section very anaemic, dry, mottled appearance. Abdominal cavity
contains a small amount of clear yellowish fluid. Mesenteric glands colourless,
normal size. Lungs very anaemic, otherwise normal. Heart flaccid and anaemic,
pericardium contains a moderate quantity of clear transparent fluid. Smears and
sections from spleen, liver and kidneys contain Leishman-Donovan bodies, numerous
in the spleen, very few in the kidneys.

20. Girl, 2 years. II] since April, 1910. At first fever of slow type lasting
two weeks. In September the child, who had acquired a sickly hue and lost flesh,
started again having a temperature, and by the middle of November the spleen was
so enlarged as to be easily palpable. Had no diarrhoea, appetite maintained, no
petechiae, no epistaxis, slight bleeding from gums, no oedema. She is the fifth
child in a family of six, all in good health; but a cousin on the mother’s side died
of the disease. No dogs kept. Seen on the 25th of November: very pale and
emaciated, no oedema, no purpura, has a hollow cough. Spleen comes down to the
level of navel, liver is not palpable. Abscess the size of a large walnut just behind
angle of right mandible, gums normal, diarrhoea with tenesmi and passage of
blood-tinged mucus. Spleen puncture : Leishman-Donovan bodies in large amount,
mostly free, some in large mononuclears, others in groups of 8 to 11.

21. Girl, 25 years. Il] since three months (?). Seen in December, 1910. Great
anaemia and emaciation, rise of temperature at irregular intervals, diarrhoea with
tenesmi but no blood, cancrum oris starting over first left upper premolar, cheeks
swollen hard and tender, has had no haemorrhages, no oedema, accessible
lymphatic glands normal, appetite maintained. Spleen freely movable and painless,
occupies upper and lower left quadrant, the liver reaches down two and a half
fingers’ breadth below costal margin. P. is the youngest of five, of which two
died when quite small, eldest two are living and in good health. A dog kept up
to ten months ago. Splenic punctures : Leishman-Donovan bodies present in fairly
large numbers.

For the observation of these cases I am indebted to the kindness
of medical colleagues practising in different parts of the Island,
especially Dr. Cannataci, Dr. Wirth, and the staff of the Central
Hospital. The symptomatology, the age of the patients, the almost
constantly fatal termination, all point to a common morbid
condition. It is likewise justifiable to infer that the majority of
deaths catalogued under the different names referred to before,

more especially if belonging to a certain age period, are instances
of one and the same disease.

44

The question now arises whether all the deaths due to this
disease are caused by Leishmania infection. Up to the time of
writing* Leishmania infantum has been found in nine out of ten
cases by examination of the spleen and other organs, and in one out
of three cases in which films of peripheral blood were stained. These
facts do not as yet justify generalisation, the more so as no case of
infantile leishmaniasis should be counted as such unless the clinical
diagnosis be supported by the demonstration of the specific
protozoa; but they go very far to show that the bulk of deaths
under five certified as due to leucocythaemia, splenic leukaemia,
pseudo-leukaemia and splenitis are cases of anaemia infantum a
letshmania (G. Pianese, 1905), or infantile kala-azar (C. Nicolle,
1908).

The conditions I have found associated with the disease will
now be reviewed. The disease is not notifiable, hence all
considerations regarding its incidence are based on the deaths
imputable to it. Having regard to its fatality, the deaths must fall
very little short of the number of attacks.

Locality. During the ten years, 1899-1908, 686 deaths under
five were registered in Malta and 58 in Gozo. In order to form an
idea of the prevalence of the disease in the various localities the
population under five for Malta and Gozo and for the different
populated centres in Malta, as estimated in the last census, 1901,
have been divided into the number of deaths for the said decennium
and the results multiplied by 100. The figures obtained may be
taken to represent a sort of endemic index expressing with some
degree of approximation the intensity of the disease in the different

* The following are some notes kindly given me by Captain W. L. Baker,
R.A.M.C., of a case under his care :—

Male child, aged 1g months. First seen end of June, 1910. Spleen then
enlarged to umbilicus.

Blood Count ... R.C. 4,000,000 per c.cm.
W.C. 4,400 os
Hgb. Index 25 per cent.

Spleen became more enlarged and child developed diarrhoea with passage of
blood, small haemorrhages also occurred in skin of limbs and trunk. Anaemia
increased, and in the middle of August the count was :—

R.C. 2,050,000 per c.cm.
W.C. 3,000 5
Hgb. Index 20 per cent.

Before death, which occurred early in September, spleen retracted two fingers’
breadth above umbilicus. Leishmania sp. found post-mortem in spleen in enormous
numbers, and in lesser numbers in liver.

places.

45

Populations under five, deaths and indices have been
tabulated as follows :—

TABLE I
Persons under 5. Deaths,
Locality Census 1901 1899-1908 Endemic index
|
FB 3 aa eae es aet OE 8! se
Malta ... 19,684 686 34
Gozo . 2,256 58 2°5
Malta—
Valletta 25295 40 1-7
Floriana 559 4 o'7
Senglea 914 fe) be)
Cospicua 1,475 43 2.9
Vittoriosa ... 693 14 2:0
Calcara 128 17 132
Zabbas : 720 24 a-3
Tarxien and Paola 576 40 6:9
Zeitun and Marsascirocco 908 55 6:0
Asciak 217 20 9:2
Luca 335 13 38
Gudia 117 6 51
Chircop 82 9 I0'9
Micabiba 158 12 7°5
Safi ... 53 4 7°5
Zurrico 452 38 8-4
Crendi : ne 192 17 8:8
Misida and Pieta ... 539 21 378
Sliema and St. Julians 1,415 39 2-7
Hamrun 1,394 40 27
Birchircara ... 1,057 49 46
Curmi 1,193 22 18
Zebbug 624 20 372
Siggieui... 395 32 8-1
Balzan Lia and Attard 464 16 3°4
Naxaro 414 6 I°4
Gargur 170 I O'5
Musta Fe 6 635 29 4°5
Imgiar and St. Paul’s Bay 117 5 4:2
Melleha_... 8 372 2 O'5
Rabato and Dingli 1,007 36 3°5

Reference to Table 1 will show that the disease prevails in Malta

more than in Gozo, and that the rural population, on the whole, is
more heavily affected than the urban, the east of the Island more
than the west, with an intermediate zone exhibiting intermediate
intensity. Endemicity is lowest in Gargur, Melleha, Floriana and
Senglea; Naxaro, Valletta and Curmi come next; whilst Calcara
represents the highest. Zeitun, Tarxien, Asciak, Gudia, Safi,
Micabiba, Chircop, Crendi and Zurrico, a group of villages to the

46

east of a line passing along the greater axis of the Island, and at
no great distance one from the other, appear to suffer heavily, their
indices varying from 6 to 10°9. One fact stands out: the low
endemicity in the towns and the comparatively moderate
endemicity in the suburban areas. To what extent this difference
is attributable to certain conditions found to be more closely
connected with our rural populations it would be rash to say at
present. But the disease is such that less personal and domestic
cleanliness, worse housing conditions, more frequent excremental
pollution of soil and water, closer and more indiscriminate contact
with domestic animals may very well help to spread.

Analysis of deaths, besides showing the prevalence of the
disease to vary in the different populated centres, furnishes data for
stating that the influence of locality is still more selective.
Endemicity, in fact, is often found to be restricted to, or more
intense in, some neighbourhoods or streets in preference to others.
To a certain extent this may be accidental. But given the very
protracted course of the disease, when the deaths do not happen
to be separated by a lapse of several years, inference is justifiable
that the specific virus has been conveyed from one house to the
other by some common carrier. Allowing for changes of residence,
which are not frequent in the villages, bringing together persons that
were infected in different, and parting those that were infected in,
the same streets, I believe the following graphs to be of interest.
The broad lines represent roadways between blocks of buildings,
the dots stand for houses from which deaths were registered, the
figures in italics indicate the number of the houses, the others the
year in which death occurred.

Age and sex. The total deaths under 5 for the period 1899-1908
were made up of 392 males and 352 females. Of 41 deaths at ages
over 5, 16 were between 5 and Io years of age.

wn

1904

47

1902

* ®
26 6
19OI 1908
16
© 1907
4

1899 1899
J J

T5
I9O0I

48

9
1907
98
RO Se ees
8 8 8
ssf) 23 15

1904 1906 1903

49

10

1905

.
: e
be r16
3 190
e 3 1903 903
I9OI
II =e e
1907 code
1906 % -
; co)
. G @
6 ‘
i ae 1904
1905 904
2 1907
1907 Be
17 A
®
14
15
CHURCH
) .
9 IZ

1900

1900

50

In the succeeding table deaths have been grouped according to
sex and age. (Table 2.)

Deaths under I appear to be equally distributed; but there is
a distinct predominance of males over females at age group I to 2,
which is responsible for nearly all the excess observed in the total
males over females. Again, age-groups I to 2 contribute almost
half the total deaths, 328, while the age-groups immediately
preceding and following these account between them for 252 deaths.
Owing to the protracted course of the disease, deaths recorded at
ages below six months cannot be counted as caused by the disease
unless it be assumed that this may prove fatal in a comparatively
short time. As yet no such instances have been met with by me.
Ponos, however, a disease endemic in the Greek islands of Spetsae
and Hydra identical with ‘marda tal biccia,’ both as to its
symptomatology and age of persons attacked, may prove fatal in
one or two months.

Social circumstances are not specially restrictive of the disease,
as cases do occur in families of well-to-do people, where the
usual conditions associated with poverty are absent; its prevalence,
however, appears to be more extensive among the children of the
lower classes. Children born of English or Italian parents are not
immune.

Recurrence of the disease in the same family and amongst
velatives. Investigation has shown instances of brothers and sisters
or first cousins dying of the disease to be not infrequent. Besides
the cases that have come under my observation, I have been able
to trace several others. These do not represent all that could be
collected; but inquiry over a period reaching sometimes ten or
twelve years back is for obvious reasons not easy, the more so when
one has to overcome a not unnatural reticence founded on the belief
that a sort of taint attaches to the disease. The view maintained
by some practitioners that ‘marda tal biccia’ is a_ hereditary
complaint is based on its aptitude to recur in two or more members
of the same family. This standpoint is untenable both as
regards direct transmission or transmission of proclivity if
the following facts be duly considered, viz.: the special age
incidence, the almost inexorably fatal termination of the disease,
the healthiness of parents whose children are attacked, and of the

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52

brothers and sisters of the children attacked who very often are
members of a large family.

Instances of recurrence in the same family and amongst cousins
are here appended, showing sex, age at death, year and month in
which they died. (Table 3.)

In Groups 1 to 12 and 18 the patient died before his or her
sister or brother were born, in some cases several months intervening
between the two events. The disease, therefore, cannot have been
transmitted by direct contact. As it happens, belief in the
communicability of ‘marda tal biccia’ is so rooted in the mind
of the people that all articles of clothing and bedding used by the
little patients are destroyed. Transmission by fomites is thus
hindered to a considerable extent. Hence the existence of an
intermediate parasite host becomes extremely probable. The sphere
of action of an animal host would extend to members of different
families in so far as their connections are more intimate and
frequent. Generally speaking this is true of persons related by
marriage and of their children. Instances of the disease among
first cousins can thus be accounted for. 1

The specific cause of ‘marda tal biccia’ is a protozoon of the
genus Lezshmania. The parasites are morphologically identical
with Leishmania donovani. Described first by G. Pianese, in 1905,
in the splenic tissue from some cases of infantile splenic anaemia,
they were observed in three cases of infantile splenomegaly in Tunis
by C. Nicolle and E. Cassuto, in 1908, of which the former
succeeded in cultivating the parasite and named it Lezshmania
infantum. Since then many similar observations have been made
by Gabbi, Basile, Jemma, and Feletti in Southern Italy and Sicily;
Sluka in Vienna; the writer in Malta* ; Alvarez in Lisbon. Infantile
leishmaniasis is found to have a daily widening endemicity. In
common with other observers, the writer has found the Leishman
bodies in the spleen, liver, kidneys and mesenteric glands, and once
in films of peripheral blood. As the morphology and staining
reactions of the parasite are well known, any mention here would
be superfluous. Splenic puncture zuztva vitam was performed in

* Kala Azar Infantile 4 Malte. Note préliminaire. Archives de 1’Institut
Pasteur de Tunis. II. 1g10.

53

TaBLE 3—BRoTHERS AND SISTERS

Io.

Il.

13.

14.

15.

17.

18.

19.

20.

Sex, and age at death

oP Ps-2. male fo:

M.P., 6/12, female

. R.C., 1 8/12, female

A.C., 1 1/12, male

- E.P., 1 9/12, female

F.P., 10/12, female

eye sIVi-. 3) anale

N.M., 1 4/12, male

2 C-C.,,3, female

A.C., 2, male

. C.V., 14, female

A.V., 1 3/12, female

a .CG. 1, sale

N.G., 1, female

. A.P., 24, male

C.P., 14, male

. A.X., 1 8/12, male

E.X., 14, male

N.A., 24, male
N.A., 2 4/12, male

M.C., 3, female
R.C., 2 1/12, female

Eez.1Ds, 25;male

S.D., 1, female

G.A., 2 3/12, male
C.A., 1 2/12, male
R.A., 13, female

P.C., 2 2/12, male
S.C., 4, female
M.C., 9/12, female

P.M., 34, male
C.M., 24, female ...

. A.M., 3, male

C.M., 1 10/12, male

M.F., 14, female .
G.F., 1, male

A.C., 1 7/12, female
R.C., 1 4/12, female

C.B., 1 8/12, female
G.B., 3, female

S.S., 3, male
E.S., 2, female

Year and month of death

...| Died March, 1904 ...

», November, 1904

»» May, 1904
35 July, 1905

»» September, 1903
»» January, 1907

» March, 1903 ...
March, 1906 ...

» April, 1903
5, August, 1907...

» August, gor...
», November, 1907

», October, 1902
» February, 1905

March, 1902 ...
>> April, 1904

55 January, 1903
3» December, 1908

3, December, 1899
»» May, 1904

5» June, 1900
3, November, 1902

», October, 1906
» May, 1908

5, June, 1g00

” July, IgoI
» October, 1903

»» May, 1904
», November, 1907
>» April, 1908

“- October, 1902
»» April, 1904

» July, 1907
», December, 1907

55 October, 1gor
»» February, 1go2

» March, 1904 ...
jypeearch, 1908)...

5, September, 1899
5) June, 1902

5, November, 1904
»» May, 1905

A]
ean

Residence at time
of death

Same house

Different houses

Same house

Same street,
different number

Same house

21.

n
N

54

TaBLe 3—continued.—First Cousins

Sex and age at death

S.B., 1 8/12, male
S.B., 5, male

. C.M., 14, female ...

G.M.M., 2, male ..

2?

Year and month of death

.| Died February, 1908

September, 1908

January, 1902
July, 1907

«if }
aly

Residence at time
of death

Different houses

23. C.F., 1 10/12, female 3» October, 1908 ”
(Cousin to Na. 4)
24. A.C., 64, male », May, 1903 ...| | Same street,
N.C., 1.7/12, male » February, 1903 ...|) different number
A.C., 1 4/12, male 3, December, 1903 ...| Different street
25. A.G., 3, male : » February, 1908 .| Different houses
(Cousin to No. 5)
26. C.V.,10/12, female »» May, 1905 “F
(Cousin to No. 6)
five cases* : no untoward results were observed. The examination of

the contents of blisters raised by vessication resorted to in two
clinically typical cases of the disease proved negative. In several
cases material for examination was available twenty-four hours or
more after death, but the appearance of the Leishman bodies was
still characteristic, only they were a little smaller than those
obtained during life and their cytoplasm stained badly or not at
all. The best specimens are obtained by splenic puncture zutra
vitam. The free parasites in the same film vary somewhat in size
and shape, elongated and round forms are met with side by side;
some have typical chromatin masses, in others the blepharoplast is
punctiform, others, again, show the nucleus only. Forms are also
met with containing two large chromatin masses, or nuclei, with
or without a blepharoplast.

The writer found 7 out of 53 stray dogs examined post mortem
in April and May, 1910, infected with Lezshmania, sp.; 11 dogs seen
Some dogs were heavily infected, others
less so, the parasites being always more numerous in the spleen than
The bone marrow was not examined. Almost all the

in September were free.

in the liver.

* In March, 1911, another. case—a boy, 2 years—was diagnosed by splenic
puncture.

55

infected dogs were small mongrels, some were mangy and
extremely emaciated, one had chronic sores on the ischia and
suppuration of the conjunctivae. A few ticks, of which some were
gravid females off an infected dog, were dissected and examined
with negative result.

Only in a few instances dogs have been found associated with
human leishmaniasis, on the whole less frequently than expected.
Until it be known how the virus is eliminated from the body of a
naturally infected dog, and whether it may be withdrawn from its
blood by blood-sucking insects, the results of my enquiries in this
direction cannot minimise the importance of this animal as a
probable factor in the transmission of the disease. If the excreta
of an infected dog are able to carry infection or represent the means
by which the parasite is dispersed about in order to undergo some as
yet unknown developmental cycle, the presence of a diseased dog
in a given street or neighbourhood is sufficient to explain the
endemicity of ‘marda tal biccia’ in such street or neighbourhood.

In the light of this hypothesis the special incidence of the
disease at certain ages below five, and its preference for the children
of the lower classes are easy to explain.

By far the largest number of deaths occur between the 12th and
24th month of life, the next heaviest mortality is registered at ages
between 24 and 30 months, the next again between the 6th and 12th
months. It is not inconsistent with the variable duration of the
disease for infection to occur when the child, having manifested
more or less precociously a certain desire or ability to use its
limbs, is put down to crawl. As long as the child is unable to do
so the chances of infection appear to be very small. The families
of the poorer classes, as a rule, live in the ground-floor, in the
front room by preference, where they get more light and air; more
often than not they have no other accommodation. The children
crawl about through the doorway to the street. This ground they
hold in common with the dog, that trots from door to door at its
leisure. If the dog is a reservoir of the virus, its habits and the
habits of children are so fitted that infection by ingestion or
through skin abrasions is bound to occur.

The scope of this paper is to put on record that ‘ marda tal biccia’
and anaemia infantum a leishmania, or infantile kala azar, are

56

one and the same disease, and to contribute to the study of some of
the conditions associated with it.

As a specific parasitic complaint ‘marda tal biccia’ becomes
ipso facto preventable.

The ease with which dogs contract experimental leishmaniosis,
and the presence of infected dogs wherever infantile leishmaniosis
has been shown to exist, make it extremely probable that the dog is
a very important factor in the propagation and continued existence
of the disease. The exact way of transmission is occupying the
attention of several observers. Whether the dog be the only
channel of infection, with or without the mediation of insects, or
whether the disease be also contracted by one human being from
another without the intervention of a lower animal, it is hoped that
the epidemiology of the disease may be soon cleared up so that
prophylactic measures may be applied on sound scientific lines.

57

I—A RESEARCH INTO THE PRODUC-
TION, LIFE AND DEATH OF.CRESCENTS
PN MARIGNANYT “TERTIAN’MALCARTA,
IN TREATED AND UNTREATED CASES,
BY, AN~ ENUMERATIVE METHOD

BY

DAVID THOMSON, M.B., CH.B. (EDIN.), D.P.H. (CAMB.)

(Received for publication 23 February, 1911)

PREFATORY NOTE.

This research has been carried on in the Tropical Ward of the
Royal Southern Hospital, Liverpool, under the direction of Major
Ronald Ross, C.B., F.R.S., and is a continuation of the research
described in a former paper (Ross and Thomson [1910]). The
funds were supplied by the Advisory Committee of the Colonial
Office. The work has been facilitated by a new instrument, which
enables one to estimate the number of parasites, leucocytes, etc., in
a given volume of blood by a method based on Ross’s ‘ Thick Film
Process’ [1903]. A following paper will describe this instrument
and the method of its use.

INTRODUCTION

Knowledge regarding ‘Crescents,’ or the sexual forms of the
malignant tertian malarial parasite (Plasmodium falciparum), is of
considerable importance owing to the fact that mosquitos are
infected by them, and thereby transmit the disease from man to
man. As is well known, there are three distinct stages in the life
history of the malarial parasite, namely (1) the stage of asexual
parasites (fever forms); (2) the stage of sexual parasites or gametes ;
and (3) the stage of the parasite in mosquitos.

All these stages are essential for the spread of malaria, so that
by dealing successfully with any one stage the disease can no
longer be propagated, and must therefore dwindle and die.

58

It is, however, the second stage (sexual stage) of the parasite
that I wish to consider. Less is known concerning it than of the
first and second periods. No one can demonstrate how the sexual
forms are produced, nor how long they live; and no effective
method of killing them has been found. Research regarding this
obscure stage is therefore necessary, and of great importance.
When we know how to destroy the sexual malarial parasites, or how
to prevent their production, we will have another powerful weapon
whereby we can exterminate the disease. In this article I shall call
the sexual parasites ‘crescents,’ as I have dealt only with cases of
P. falciparum. The accompanying table has been compiled from
the cases studied by the enumerative method used in this research.
Some of the results have already been mentioned in the previous
paper referred to above.

THE PRODUCTION OF CRESCENTS

From the figures given regarding the forty-two cases of
P. falciparum studied, it is clearly noticeable that the production
of crescents is extremely irregular. Certain paroxysms of fever
result in a numerous brood of crescents even up to 7,000 per c.mm.
of the patient’s blood. Other paroxysms produce very few or none
at all. Thirty-one, or 74 per cent. of the cases, showed crescents at
some time during the period of examination. Eleven, or 20 per
cent., showed no crescents at any time while under observation.

A. HOw ARE CRESCENTS PRODUCED AND WHERE ARE THEY
DEVELOPED? All malarial experts seem agreed that the crescents
are developed from the ordinary asexual spores or merozoits of the
parasite.

Mannaberg [1894] stated his belief that they were produced
from the conjugation of two asexual parasites within a red
corpuscle. If this is so, then the more numerous these asexual
forms are, the greater is the likelihood of two or more finding their
way into a red cell, that is according to the theory of probability.
In Case 13 where there were 300,000 asexual parasites per c.mm. of
blood, many of the red cells contained more than one parasite. In
Case 18 asexual parasites were few and difficult to find (1,860 per
c.mm.), and no doubly infected corpuscles could be detected; yet

59

in the former case no crescents were produced, whereas in the latter,
286 crescents per c.mm. of blood appeared. Again, as shown in
Table A, the cases with very numerous asexual parasites produced
on the average fewer crescents than cases with much less numerous
asexual parasites. These facts would appear to bring strong
evidence against Mannaberg’s hypothesis. The following quotation
is from Stephens and Christophers [1908] :—‘ The sexual cycle, it
has been thought, commences in the blood when the conditions are
unfavourable for the continuance of the asexual cycle, and, in fact,
has been taken as a sign that the patient has already developed
immunity against the fever-producing young parasites (spores).
Thus it is well known that in malignant tertian the sexual forms,
gametes or crescents, first appear a week to ten days after the first
febrile attack. If this view be true, then it follows that the gametes
develop from forms already present in the system, viz., the asexual
forms, and possibly the divergence into sexual forms takes place
from the youngest form of the parasite, i.e., the spore. But it is
possible that the divergence takes place at a stage previous to the
youngest form of the parasite, i.e., at a stage immediately
preceding the entry of the sporozoits into the blood, so that we have
from the first indifferent and sexual forms present, involving indeed
the existence of three kinds of sporozoits. Sexual development has
been supposed to proceed mainly in the internal organs, e.g., the
bone marrow, but it is being gradually recognised that young forms
of gametes are also found in the circulation.’

This research would appear to support the idea that the
crescents are developed from the asexual spores when a certain
amount of immunity has developed, but it seems to me that they do
not come from special asexual spores, but that they arise merely
owing to a transformation of an ordinary asexual spore into a sexual
parasite. I will give evidence to show that the development of
immunity is necessary for their production, and I fail to see why, if
they do develop from special spores they cannot be produced at any
time independently of immunity. There seems to be little doubt
also that they develop chiefly in the internal organs, and when
completely developed they appear suddenly in the peripheral blood ;
for, although small undersized crescents are sometimes seen, yet
their occurrence in the peripheral blood is rare. I have never seen

60

in the peripheral blood anything which could be considered as an
intermediate stage between an asexual spore and a true crescent.

B. WHEN ARE CRESCENTS PRODUCED? The accumulated
evidence of the crescent cases studied seems to show that a period of
ten days elapses between the appearance in the peripheral blood of
the asexual spores, and the crescents which are produced from these
spores. As a general rule the crescents appear in the peripheral
blood on the fifth day after the attack of fever, and increase in
number for four or five more days, so that they are most numerous
on about the tenth day after the height of the fever. Those
crescents appearing on the fifth day after the paroxysm correspond
to asexual parasites existing in the blood five days previous to this
paroxysm. Asexual parasites can exist in the blood in numbers as
great as 2,000 and rarely 10,000 per c.mm. without producing any
temperature reaction. They gradually increase in number by
sporulation till they are numerous enough to cause a paroxysm of
fever. The numbers may then fall spontaneously or by quinine
treatment so that only one single paroxysm results. It is by the
study of such single paroxysms that the time required for the
appearance in the peripheral blood of the corresponding crescents
can be best determined. In such cases the graphs representing the
numbers of asexual parasites and crescents show a striking
similarity, the points on the crescent graph occurring on the tenth
day after the corresponding points on the graph of the asexual
parasites. It is very difficult to demonstrate this with mathe-
matical accuracy, and very frequent examinations of the blood
require to be made.

The chart of Case 20 shows the correspondence fairly well. A
careful study of all the charts leads one to the conclusion that the
corresponding points on the crescent curve occur about ten days
after those of the asexual curve. This conclusion is strengthened
by the chart of Case 38. In this case the numbers of leucocytes,
crescents and asexual parasites were estimated several times daily
for twenty-three days.

This case is extremely interesting, as the patient had nine
successive daily paroxysms of fever. Four paroxysms on the four
days before admission, and five paroxysms on the next five days.
Corresponding to these nine fever paroxysms or sporulations we

61

have nine outbursts of crescents into the peripheral circulation, each
occurring on the tenth day after the corresponding fever paroxysm.
The asexual parasites were rapidly destroyed by quinine after the
ninth paroxysm of fever, and a corresponding diminution in the
production of crescents is evident ten days later. It is stated in
the above quotation from Stephens and Christophers that many
have observed the appearance of crescents on the eighth to the tenth
day after the first paroxysm of fever, but this delay is attributed
to the development of immunity, whereas it is due to the fact that
crescents take that time to develop from the asexual spores before
they appear in the peripheral circulation. The following
diagrammatic charts A and B represent the correspondence between
asexual parasites and the crescents developed therefrom, where
only one blood count is made per day.

DIAGRAM A. (Single fever paroxysm.)

PREWEN PALLET TTT | 22|

oe 3

ra Wits

<9 dine 36
2S

1

3

= hy See

CUTE

ee ie PV Pe ee

C. WHY ARE CRESCENTS PRODUCED IN SOME CASES AND NOT IN
OTHERS ? Crescents would seem to be developed from the asexual
spores, due to a development of immunity towards the latter.
When the asexual spores find that their environment is becoming
unsuitable, they undergo a transformation into a sexual generation
and thereby save themselves from destruction. In this new state
they remain passive, waiting for their transference into a more
suitable host. Schaudinn and other observers have stated that

Te mera ture.

<7 Ca. am. of

Nawbers A

Temperature.

Bloed.

62

they have seen sexual gametes undergoing a change back into
asexual spores by parthenogenesis. Although this has never been
observed by workers in the Liverpool School of Tropical Medicine,
yet in the light of the above theory it would seem quite possible
that such a retransformation might take place, especially in cases
where the acquired immunity had become less or disappeared.
This phenomenon must, however, be very rare, as it would otherwise
have been noticed more often.

DIAGRAM B. (Showing tertian paroxysms with corresponding outbursts of crescents ten
days later.)

Pays [1 [2] 3/4 | slo [y|s |o [oly v2 [ra |e [rs]e [> [8-9 [20] [2e]2s] 2n]es] 26/27 [25 |

ARIA TTI

APM ar Te Cy Y Min

The following evidence would seem to supply seven points in
support of the statement that crescents are formed after the develop-
ment of partial immunity.

(1) The relationship between the number of asexual parasites
and the number of crescents produced. Taking very acute cases,
I find that out of eight paroxysms of fever caused by numbers of
asexual parasites over 50,000 per c.mm. of blood, only three, or
37°5 per cent. resulted in crescent production. A total of 724,000
asexual parasites per c.mm. produced a total of 1,354 crescents per
c.mm., giving a ratio of 535 asexual parasites to one crescent.

Eleven subacute paroxysms had asexual parasites varying from
20,000 to 50,000 per c.mm. of blood; of these, seven, or 63°6 per
cent., resulted in crescent formation. A total of 318,700 asexual

63

parasites per c.mm. produced a total of 3,952 crescents per c.mm.
of blood, giving a ratio of 81 asexual parasites to one crescent.

Twenty-six mild chronic cases had asexual parasites, varying
from 1,100 to 20,000 per c.mm. of blood. Eighteen of these, or
69°25 per cent., resulted in crescent production. A total of 172,360
asexual parasites per c.mm. produced a total of 3,343 crescents per
c.mm. of blood, giving a ratio of 52 asexual parasites to one
crescent.

The mild, chronic, and probably partially immune cases had
therefore a crescent-producing power fully ten times greater than
the very acute cases.

(2) The duration of the disease in relation to the production
of crescents. Sixteen paroxysms occurred during the first thirty
days of the disease. Of these 43°7 per cent. produced crescents. The
total average number of crescents was 1601 per c.mm. of blood.

Twenty paroxysms occurred between the thirtieth and the
sixtieth day of the disease. Of these 65 per cent. produced
crescents, the total average number of crescents being 551 per c.mm.

Sixteen paroxysms occurred after the sixtieth day of the disease.
Twelve, or 75 per cent. of these produced crescents, and the total
average number of crescents was 650 per c.mm. of blood.

Thus it would appear that crescents are more likely to be
produced in cases of long standing, where a certain amount of
immunity has had time to develop.

(3) Crescent production in cases which have had previous
attacks of fever one or more years previously. Sixteen cases had
had previous attacks of fever. Of these, 87 per cent. developed
crescents during the period of observation. There were twenty-six
cases which had no history of previous attacks; of these, only 46 per
cent. produced crescents. It is reasonable to suppose that these
cases, which had a history of previous attacks, were more immune
than the primary cases.

(4) The relationship between crescent production and the age
of the patient. From the table it can be seen that 50 per cent. of the
cases up to twenty years of age (average eighteen years) produced
crescents, the average number being 130 per c.mm. of blood.

64

Cases between twenty and thirty years of age (average twenty-
sIX years) gave an average of 526 crescents per c.mm., 74 per cent.
of the paroxysms resulting in crescents.

Cases between thirty and sixty-eight years of age (average forty-
five years) gave an average of 1,018 crescents, 71 per cent. of the
paroxysms producing crescents.

There is apparently an increase in crescent-producing power in
older patients. This might be attributed to a greater power in
adults of developing immunity, as compared with the young
growing patients. Many of the older patients, however, had been
in the tropics for a long time, and had had previous attacks of
fever. The young patients had not been long in malarial districts,
and in most cases it was their first attack of malaria. The greater
crescent-producing power in the older patients may therefore have
been due to immunity developed from previous attacks.

(5) The relationship between crescent production and the
percentage of the patient’s haemoglobin. Nineteen paroxysms of
fever, occurring chiefly in different cases where the haemoglobin
during the next ten days was 75 per cent. and under, produced only
an average of twenty-two crescents per c.mm. of blood. Of these
paroxysms, 52 per cent. produced crescents.

Twenty-seven paroxysms, where the haemoglobin during the
next ten days was over 75 per cent., produced an average of 428
crescents per c.mm., and nineteen, or 70 per cent., of these
paroxysms produced crescents.

It would seem therefore that a low percentage of haemoglobin
is not so favourable for crescent production as a fairly high
percentage. This again might be explained by the supposition
that immunity to the asexual parasites is more likely to be
successfully developed in cases where the blood standard is fairly
healthy.

(6) Crescent production in relation to the size of the spleen.
Twenty-six cases had palpable spleens. Of these, fifteen, or
75 per cent., produced crescents. In twenty-three cases the spleen
could not be palpated. Twelve, or 52 per cent. of these produced
crescents. ;

65

According to Ross [1910] the number of asexual parasites
tends to vary inversely as the degree of splenomegaly, that is, the
parasites tend to die out in persons with very large spleens. Again,
N. F. Surveyor [1910] states that malignant malaria is more fatal
in cases where the spleen is not enlarged, and less fatal in those
with splenomegaly. These statements would seem to indicate that
immunity to the disease increases pari passu with the size of the
spleen; hence the increased crescent production where the spleen is
enlarged.

(7) Crescent production in relation to the number of leucocytes.
The average number of leucocytes in the ten-day periods following
paroxysms which produced no crescents was 7,284 per c.mm. of
blood (56 per cent. mononuclears). After paroxysms producing up
to 100 crescents per c.mm., the average number of leucocytes during
the same period was 7,411 per c.mm. (52 per cent. mononuclears).
While after paroxysms producing over 100 crescents per c.mm., the
average number of leucocytes was 8,924 per c.mm. (53 per cent.
total mononuclears).

Again in thirteen cases, which produced no crescents at any
time, the average number of leucocytes throughout was 8,646 per
c.mm. (total mononuclears 55°9 per cent.), as compared with an
average of 10,970 per c.mm. (total mononuclears 489 per cent.), in
sixteen cases with numerous crescents throughout the period of
examination.

It would appear therefore, that greater numbers of crescents are
produced in cases where the leucocytes are numerous.

The leucocytes in malaria increase markedly in number,
simultaneously with the quiescence of the disease. About one week
after the last paroxysm of fever, the leucopenia (characteristic as a
rule of the febrile period in malaria) disappears, and a leucocytosis
takes its place, provided the fever does not return. Thus a high
leucocyte count is characteristic of quiescent malaria and in post-
. malarial conditions, and would appear to be coincident with
periods of immunity. This increase in the number of peripheral
blood leucocytes occurs after the fever abates, whether quinine has
been given or not. Where quinine is given, it occurs earlier and
remains permanent, because during the treatment no true relapse

E

66

can occur. The leucocytes decrease in number previous to the onset
of arelapse. The chart of Case 20 shows the fall in the number of
leucocytes with the onset of a relapse, and later an increase in
crescent production when the leucocyte count again becomes high.
These facts would tend to show that a high leucocyte count and
immunity are co-existent, the latter explaining the increase of
crescents.

(8) Crescent production in velation to the month of infection.
We have not sufficient cases on record to make any reliable
deductions. We can only state, that from West Africa, cases
infected in all months of the year except June produced crescents,
and again cases infected in every month, except May and July,
showed no production of crescents. From this evidence there seems
no reason to suspect that crescent production depends upon the
month of infection.

D. THE EFFECT OF QUININE ON CRESCENT PRODUCTION*. It
would appear that quinine in doses of ten grains three times daily,
given just before and during the paroxysm of fever, diminishes the
subsequent formation of crescents. The cases showing the greatest
numbers of crescents had little or no quinine for several days
previous to the producing paroxysm, and little or no quinine during
that paroxysm. Case 23 seems to show the good effects of quinine
in this respect. In this case the first paroxysm of fever produced
852 crescents per c.mm. of blood, resulting from 50,000 asexual
spores per c.mm. Twenty grains of quinine were given during this
paroxysm, but none was given for several days before or after it.
The next relapse where no quinine was given till the day after the
paroxysm produced 468 crescents per c.mm., while the next
paroxysm of this relapse produced 344 crescents per c.mm. from
54,000 fever forms per c.mm. During this last paroxysm and
afterwards thirty grains of quinine was given daily. A subsequent
relapse treated similarly with quinine gave a production of only
four crescents per c.mm. from fever forms amounting to 16,000 per .
c.mm. of blood. This case and others would seem to indicate that
if quinine is given in doses of ten grains three times daily during

* The various salts of quinine used were kindly supplied by Messrs. Burroughs,
Wellcome & Co.

67

the fever paroxysm and afterwards, it helps much to prevent the
formation of crescents. The destructive action of quinine in these
doses on the asexual spores is so powerful and rapid that one is
surprised at the subsequent appearance of even a few crescents. If
quinine is withheld till one or two days after the fever paroxysm
and then given in the above daily doses, the crescents may still
appear in large numbers—vzde Cases 20, 22, etc. This shows that
when once the crescents have commenced to develop, the quinine
does not then prevent them from reaching maturity and appearing
in the peripheral blood on the tenth day or thereabout. I would
therefore conclude that quinine in large daily doses, given during
and after the fever paroxysm, diminishes the crescent-producing
power of that paroxysm, not by acting on the crescents themselves,
but indirectly by destroying the asexual spores from which the
crescents are produced. However, if quinine be given in smaller
doses, say five grains daily, or ten to twenty grains irregularly,
then instead of the crescent production becoming less, there is
evidence to show that it may become even more prolific. Thus in
Case 18, crescents became much more numerous after quinine was
administered in daily doses of five grains. This case with such
treatment showed a very high crescent-producing power, the ratio
of asexual spores to crescents being as eight is to one approximately.
It is quite reasonable to suppose that in such cases, quinine given
in small doses destroys only some of the asexual spores, but enables
the host to keep the disease under control, and to develop some
resistance or immunity. In the consequence more crescents develop
from the remaining parasites. Case 18 and others showed the
presence of asexual parasites for many days during the administra-
tion of quinine in doses of five grains daily.

Quinine given continuously in daily doses of twenty to thirty
grains, has never failed in our cases to reduce the crescents to
numbers less than one per c.mm. of blood in a period not exceeding
three weeks, vide chart of Case 49. This reduction of crescents by
quinine has also been noted by Darling [1910].

E. THE EFFECT OF METHYLENE BLUE ON CRESCENTS. It would
appear from the careful investigation of six cases treated alone
with methylene blue, that this drug in doses of twelve grains daily,
given by mouth (pill form), though not so potent in destroying

68

the asexual parasites, is yet more potent than quinine in preventing
crescent formation. It would seem also to have some direct
destructive effect on the crescents. It is good treatment, therefore,
to give methylene blue along with quinine, especially where one
cannot give large doses of the latter due to the idiosyncrasy of the
patient.

THE DURATION OF LIFE, AND THE DEATH OF
CRESCENTS

(a) There is evidence to show that the duration of life of a
crescent cannot be more than twenty days. During the first ten
days of this period they are developing somewhere, but not in the
peripheral blood. They then appear in the peripheral blood, some-
times small in size. The majority of them must perish in the
peripheral blood in a very few days. This must be so, for if they
lived for say four days or longer, then the summit of the crescent
curve would not be a sharp point as it always is, especially when
the numbers are great.

(6) The appearance of the graph, representing the life and
death of crescents in the peripheral blood. ‘The crescent curve has
a definite formation. Where the number of crescents is estimated
only once daily, it assumes more or less the appearance shown in the
diagram C.

DIAGRAM C. (Crescent curve, numbers estimated once daily.)

This is the usual type of crescent curve obtained after a single
isolated paroxysm of fever, where the number of crescents is
estimated once daily. When no quinine is given, a single isolated
paroxysm is rare, and the crescent curve will as a rule be quite

Crceseents ~

/

Pree Cu. ree. of flood

Nawmober o

69

different, as in Cases 1, 14, 16, 18, 23, 24, etc. In these cases the
number of crescents remains high for some days, the graph
resembling a kind of plateau containing several sharp peaks, as in
diagram D.

D1iAGRAM D. (Crescent curve with plateau formation.)

: 6

°
‘\
* \

The explanation of this plateau is quite simple, for if no
quinine is given the asexual parasites remain alive, even though
there is no fever to indicate their presence, and keep on producing
new crescents. The source of crescents is not cut off, so that the
supply is replenished by new broods of crescents, appearing every
day, or on alternate days, or irregularly, according as the fever or
asexual sporulation occurs every day, on alternate days, or
irregularly. The sharp peaks on the plateau of the curve show
that although the crescents are dying rapidly, yet their numbers are
replenished by fresh broods coming into the circulation. As
pointed out by Ross and Thomson [1910], these peaks on the
crescent curve often show a tertian tendency.

In ne case is there a plateau formed when quinine has been given
in doses of thirty grains daily ten days previous to the height of
the crescent curve. If a plateau has formed and quinine is then
administered in large doses, its effect will not be manifested for
about ten days, because although it very quickly reduces the source
of supply, yet those crescents which commenced to develop during
the previous ten days are not affected, but continue to appear in the
peripheral blood replenishing the loss. Thus quinine, as is clearly
seen in Case 23, takes about ten days to destroy the plateau
formation. Hence, though quinine makes the crescents disappear
from the peripheral blood more quickly than they would otherwise

Number of Cresecents

Act Gy wm. of Clood

70

do without its administration, yet this effect is not due to any direct
destructive action on the crescents themselves. It is due indirectly
to the destruction of the asexual spores from which the crescents
are developed. The length of time that crescents will remain in
the peripheral blood therefore depends upon the persistence of the
asexual parasites. If immunity develops so strongly that the
asexual parasites almost disappear, or if they are destroyed by
quinine, then the crescents also will disappear in due course.

In cases where immunity remains, but is only sufficient to keep
the number of asexual parasites in check, crescents may continue
almost indefinitely. Crescents have been observed to continue in
the peripheral blood for eight weeks, Surveyor [1910]. Case 18,
where the asexual source of supply was not destroyed by quinine
till late, had crescents for forty-four days, and probably longer
than this, as they were present when the case first came under our
observation. Sufficient has been said to point out the fallacies
regarding the duration of life of crescents. I must, however, once
more refer to the chart of Case 38. Here the number of crescents
per c.mm. of blood was estimated several times daily. The
crescent graph obtained shows the great importance of making
numerous observations, for had the numbers been estimated only
once a day, the daily variation in the number of crescents would not
have been noticed. Here we have a pure quotidian case of fever,
resulting in quotidian outbursts of crescents into the peripheral
circulation, each crescent outburst corresponding to a sporulation
of the asexual parasites occurring ten days before. It is noticeable
that the quinine in doses of thirty grains daily did not appreciably
diminish the numbers of crescents till the tenth day after its
administration. The number of crescents then diminished, rapidly
at first and afterwards more slowly, for nine more days. This
would seem to indicate that the quinine quickly destroyed the
majority of the parasites of the asexual source, the remainder dying
more slowly. The curve also clearly shows that the crescents die
very quickly in the peripheral blood stream; a very marked fall
occurs each day, but this fall is compensated for by a fresh brood
each day. It is clear that but for this compensation the crescents
would only remain in the peripheral blood for a very few days.
Again it will be observed that although asexual parasites could no

71

longer be detected in the blood after the third day of quinine, yet
crescents continued to be present till the eighteenth day of quinine
treatment. Thus those crescents found after thirteen days of
quinine administration had either a life of five days in the peripheral
blood, or else they were new crescents produced from surviving
asexual spores, so few in number that they could not be detected.
When quinine has been given in the above doses for a few days
asexual parasites can no longer be found, but the fact that relapses
occur (even when there are no crescents), shows that they were still
present. They may exist in numbers below the detectable limit,
or (?) as resisting forms in the internal organs, and it is possible
the crescents found more than thirteen days after the administration
of quinine come from these.

In other cases, where the number of crescents was estimated
several times daily, the graph obtained was much more irregular
than in Case 38. In these, daily irregular variations took place,
vide chart of case 49. This is easily explained, for in many cases
of malignant tertian, sporulation is extremely irregular. It is very
seldom that one gets so well a defined quotidian sporulation as in
Case 38. In cases of malignant tertian therefore with irregular
temperature indicating irregular sporulation, one would expect the
crescent graph, as estimated from several counts daily, to be
irregular also. The majority of our cases have shown this
irregularity. The following diagrammatic chart will indicate the
idea more clearly.

DIAGRAM E. (Represents a pure tertian fever or sporulation.)

Pays |e [2 [3 [els fe [> [5s [of ef] nf | | a] sr [ae| = [eden ze

date HH

Nuwbers Av Crim.

i eal . Nile VE ALAAA IT

72

This chart represents the true relationship between crescents and
the asexual spores in a pure case of malignant tertian fever. A case
with an irregular fever, indicating irregular sporulations, would
give an irregular crescent graph. These points, however, still
require to be worked out more thoroughly.

There is, however, one very constant law regarding the crescent
graph, viz., that the curve representing the diminution or death rate
of the crescents is always a parabolic line (vide curve shown in
Diagram C). This shape of curve arises from the fact that they die
off by a constant fraction, say one-half to one-fifth of their daily
number. One might give two explanations of this law regarding
their death rate.

(1) That it is a case of the survival of the fit, a certain
proportion dying day by day according to their varying powers of
resistance.

(2) That it depends upon the law of probability regarding their
contact with leucocytes, by which they are ingested. The mono-
nuclear leucocytes, especially the large forms, undoubtedly ingest
crescents, either when alive or after their death, for in cases where
only numerous crescents are present in the blood, many pigmented
mononuclear leucocytes are to be found. It is quite reasonable to
suppose that there may be some truth in these two hypotheses, but I
think the true explanation is to be found by studying the curve of the
asexual forms from which the cresents arise. It is clearly seen from
the charts of Cases 17, 38, and others, that the curve representing the
death rate of the asexual parasites 1s also a parabolic line, as shown
diagrammatically in Chart A. The curve is the same, whether they
die off spontaneously or under the influence of quinine. Now, when
the producers of crescents show this death rate curve, and if the
crescents produced have approximately all the same duration of life,
then necessarily the curve of their death rate which will occur ten
days later will assume the same form. Hence the peculiar form
of the crescent curve depends probably in every respect upon the
form of the curve of the asexual parasites. The form of the asexual
parasite curve most probably depends in its turn upon (1) The law
of the survival of the fit; (2) The law of probability of leucocyte
contact and ingestion. The large mononuclears undoubtedly ingest
the asexual parasites, especially the spores. When the spores are

73

numerous many will be ingested, but as they become fewer the
chance of contact and ingestion by these leucocytes will become less.

CONCLUSIONS REGARDING PROPHYLAXIS

From the above research one might give the following deductions
regarding the prevention of malaria : —

(a) It is a bad practice to give quinine in small doses of five
grains daily, or irregularly, even though the doses be larger, for
such treatment tends to increase the power of crescent formation.

(6) All cases of malaria should be treated early and con-
tinuously with doses of quinine of about twenty to thirty grains
daily, as such treatment during and after the fever diminishes the
subsequent formation of crescents. Continuous treatment with the
above doses has never failed, as stated above, to reduce the number
of crescents to less than one per c.mm. of blood in a period not
exceeding three weeks. That is to say, it renders infective cases of
malaria non-infective to mosquitos in a period not exceeding three
weeks.

SUMMARY

1. Crescents are produced from the ordinary asexual spores of
P. falciparum, due to a development of immunity towards the
latter.

2. They develop somewhere in the internal organs and then
appear suddenly in the peripheral blood.

3. The period required for their development is about ten days.

4. Crescents do not generally live more than a few days in the
peripheral blood.

5. Crescents may be present in the peripheral blood during
periods as long as eight weeks, not because the individual crescents
survive for that time, but because their numbers are constantly
replenished from surviving asexual forms.

6. Fresh broods of crescents come into the circulating blood
daily, or every other day, or irregularly, according as the asexual
sporulations occurring ten days before were quotidian, tertian, or
irregular.

74

7. Quinine has no direct destructive action on crescents, either
during their development or afterwards, but it destroys the asexual
source of supply.

8. Quinine reduces the crescents to numbers less than one per
c.mm. of blood within three weeks, provided it be given in daily
doses of twenty to thirty grains.

g. Quinine in small doses tends to increase crescent pro-
duction (?) by favouring the development of immunity to the
asexual parasites.

10. Methylene blue in doses of twelve grains daily reduces the
number of crescents, and would seem to have some _ direct
destructive action upon them.

LITERATURE

DARLING (1910), /é¢d. Vol. IV, No. 2.

MANNABERG (1894), ‘The Parasites of Malaria Fever,’ Marchiafava and Bignami,
and Mannaberg, New Sydenham Society, 1894, p. 289.

Ross (1903), ‘The Thick-Film Process for the Detection of Organisms in the Blood,’
Thompson Yates and Johnston Laboratories’ Reports, Vol. V, part I, 1903.

Ross (1910) ‘Prevention of Malaria,’ p. 129. A. Celli, p. 406.

Ross and THOMSON (1910), ‘Some Enumerative Studies on Malarial Fever,’ Annals
Trop. Med. and Parasit., Vol. IV, p. 267.

STEPHENS and CHRISTOPHERS (1908), ‘ The Practical Study of Malaria and other
Blood Parasites,’ p. 52.

SURVEYOR (1910), ‘Some Observations on Malaria in relation to Splenic Enlargement
and the Treatment of the Crescentic Stage,’ Zézd., Vol. IV, No. 3.

75

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83

I.—THE LEUCOCYTES IN MALARIAL

FEVER: A METHOD OF DIAGNOSING

Pees A ena AFTER TY ps
APPARENTLY CURED

BY

mv it) THOMSON, NMEG., CHB. (EDIN.), D.P.H: (CAMB,)

(Receztved for publication 8 February, 1911)

PREFATORY NOTE.

This research was carried on in the Tropical Ward of the
Royal Southern Hospital, Liverpool, under the direction of
Major R. Ross, C.B., F.R.S. The new facts obtained are the
result of numerous daily observations extending over periods of
several weeks. The laboriousness of the work was much
diminished by the use of a new blood-counting pipette devised by
the author. The method is based on ‘The Thick Film Process’
[Ross, 1903]. The funds for the research have been supplied by
the Advisory Committee of the Colonial Office.

I. THE LEUCOCYTES DURING ACTIVE MALARIA *

During active malaria the number of leucocytes is, as a general
rule, less than normal, but the numbers vary with the rise and fall
of temperature. When the fever is in full force, that is during
the rigor and high temperature, then there is a coincident
diminution of the total leucocytes in the peripheral blood. In this
diminution the polymorphonuclear as well as mononuclear
leucocytes take part. If we take 7,000 per c.mm. as the normal
leucocyte count, and the normal percentage of polymorphonuclears
and total mononuclears as 65 per cent. and 35 per cent. respectively,
then normally the polymorphonuclear leucocytes should number

* In this article I refer to charts in Paper I (‘The Production, Life and Death
of Crescents,’ etc.).

84

about 4,550 per c.mm. of blood and the total mononuclears about
2,450 per c.mm. of blood. During a malarial temperature, however,
the polymorphs are frequently less than 2,000 per c.mm., and the
total mononuclears are also frequntly below this number. In some
cases the total leucocyte count during the pyrexia may be as low as
2,000 per c.mm. of blood. In other exceptional cases, however,
I have seen a marked leucocytosis during a high malarial
temperature. This occurred in two cases of comatose malaria. It
is rare, however, and I think it may be taken as a general rule that
there is a leucopenia in malaria when the temperature is above
normal. In other words, during the actual sporulation of the
malarial parasites there is a diminution of the total leucocytes in
the peripheral blood. This rule holds good with the benign tertian,
malignant tertian and quartan parasites. Some observers have
described a transient polymorphonuclear leucocytosis lasting only
for one half-hour just at the commencement of a rigor. I have so
far been unable to confirm this. After the sporulation is over and
the temperature has fallen, it will be found that the total number
of leucocytes has increased even up to normal numbers or more,
so that in the intervals after the temperatures the leucopenia
disappears, and may occasionally even have its place taken by a
leucocytosis; but on the advent of the next sporulation and
temperature, the numbers again fall. Thus with the rise and
fall of temperature in malaria we have a corresponding fall and
rise in the total number of leucocytes. The diminution of total
leucocytes corresponds exactly with the rise of temperature, and
the rise of total leucocytes corresponds exactly with the fall of
temperature. See Charts N.B. Case 9, J.W. Case 30, and Billet’s
Chart [1908]. We may therefore enunciate a general law as
follows :—

LAW I. During active malaria the number of total leucocytes
in the peripheral blood is below normal, and varies more or less
inversely with the temperature.

When one analyses the different varieties of leucocytes, it is
found that the mononuclears, and especially the large mono-
nuclears, play most part in the variation enunciated in this law.
In this research, however, I have considered only the total poly-
morphonuclear leucocytes and the total mononuclears. So many

85

transitional forms of leucocytes occur between the recognised
varieties that a considerable personal factor comes into play, and
in dealing with all varieties one increases the complications very
much. Thus if we consider only the two chief classes of
leucocytes with regard to Law I, it may be stated that the total
polymorphonuclear leucocytes vary only slightly with the
temperature in active malaria, but that the total mononuclears vary
very much and inversely with the temperature. In other words,
the sporulations of the malarial parasite cause a greater disturbance
or reaction among the mononuclear than among the polymorpho-
nuclear leucocytes. When the spores of the malarial parasite
escape free into the blood plasma there occurs a mononuclear
leucopenia, followed later by a mononuclear excess. This is
exactly the reverse of what happens when a bacterial culture is
injected intravenously into an animal. Here the result is a poly-
morphonuclear leucopenia followed by a polymorphonuclear excess
(F. W. Andrewes [1910]).

In malarial fever the mononuclear leucocytes, especially the
large variety, are undoubtedly the soldiers for defence. They
ingest the spores of the malarial parasite in large numbers. Some
hours after a malarial paroxysm or sporulation the large mono-
nuclear leucocytes are found in great numbers filled with vacuoles
and pigment, due to the active ingestion of parasites. If it were
not for this phagocytic power of this class of leucocyte, it is likely
that the parasites would become more and more numerous after
each paroxysm, and the patient would soon succumb. When the
mononuclears gain the upper hand and exist in large numbers the
fever disappears spontaneously for a time. This is noticeable in
Chart J.W., Case 30, where the patient had no relapse, though no
quinine was given for fourteen days. This natural mononuclear
defence is greatly assisted by the administration of quinine. After
a dose of twenty to thirty grains of quinine the fever frequently
disappears quite suddenly. Corresponding to this sudden
disappearance of fever there is a fall in the number of parasites
and a large increase of mononuclear leucocytes. This phenomenon
occurs in many of the cases studied. The mononuclear increase
continues so long as the fever does not return. It would therefore
appear that an increase in the total mononuclear leucocytes is

86

coincident with periods of resistance or immunity to the disease.
Previous to a relapse the number of leucocytes invariably falls
(vide Charts W.M. [P. vivax], Case 17, R.B., and Case 20, F.B.).
From these facts we have therefore the basis of another law, though
this law is really only a different expression of Law I.

LAW II. Jn malarial fever the curve representing the percentage
of total mononuclear leucocytes is the exact inverse of the
temperature curve: in other words, if one makes frequent daily
differential counts of the leucocytes in malarial fever, on charting
the varying percentage of total mononuclears, one finds the curve
obtained is the exact inverse of the temperature curve. See
Charts M.G. (P. vivax), and G.J., Case 34 (P. falczparum).

When the temperature in malaria is rising, the total mono-
nuclear percentage is falling, and vice versa the fall of temperature
is exactly simultaneous with a rise in the total mononuclear
percentage. At the height of his fever, J.W., Case 30, had only
39°4 per cent. of total mononuclear leucocytes. He received twenty
grains of quinine. Next day he had no fever, and the total
mononuclears were 80 per cent. This rise is chiefly due to the
large mononucleated variety. In Case 8, P.D., after the first dose
of quinine the fever soon ceased, and the large mononuclears rose
from 19 per cent. to 65 per cent. in one day. I have observed
occasionally a total mononuclear percentage as high as go per cent.
I have not met with any exceptions so far to Law II. It would
seem that a high mononuclear percentage is incompatible with a
true malarial temperature.

From the above law it would seem reasonable, as a therapeutic
measure in malaria, to attempt to increase the leucocytes, especially
those of the mononuclear type. Recently it was discovered that an
injection of an extract of leucocytes, causes a marked increase of
leucocytes in the peripheral blood in a few hours (Ross and
Thomson [1911], Alexander [1911]). A case of malaria was
treated with this substance (Lambert [1909]), and it was found that
the injection prevented the rigors from’ occurring. It is highly
probable that this phenomenon was due to the increase in
leucocytes following these injections. The extracts made and used
by Alexander consist chiefly of the polymorph variety of leucocytes,
and produce, after injection, chiefly a polymorph excess. We would

87

expect to obtain better results from injections of a mononuclear
leucocytic extract, but unfortunately so far, it is very difficult to
obtain such a substance. Before leaving the question of
therapeutics, I would refer to Charts J.M. (leprosy), D.T. (normal
person), and J.L., Case 53 (P. vivax). From these it will be seen
that injections of pilocarpine in doses of one-tenth of a grain
produce quite a definite excess of the mononucleated variety of
leucocytes in the peripheral blood, (vwzde researches by Waldstein
[1895]). In Chart J.L., Case 53, it will be noticed that during the
injections of pilocarpine in the above doses the mononuclear
percentage as well as the total leucocytes maintained a high level,
and that the fever disappeared without other treatment. As this,
however, frequently occurs naturally without any treatment when
patients are kept in bed in comfortable circumstances, one cannot
come to definite conclusions until further work has been done in
this line.

II. THE BEHAVIOUR OF THE LEUCOCYTES IN QUIESCENT
OR LATENT MALARIA, AND IN MALARIA APPARENTLY
CURED BY QUININE OR OTHER TREATMENT

Laws I and II deal with the leucocytes where the malarial
parasites are numerous and easily found and where true paroxysms
of fever occur. When, however, we observe further the leucocytes
during the latency or apparent convalescence and cure of the
disease, we find some remarkable differences. During active
malaria, as already stated, the leucocytes, on the whole below
normal numbers, show periodic variations in number, due chiefly
to fluctuations of the mononuclear variety. In convalescent or
apparently cured malaria, however, where the temperature remains
more or less below normal, we have a similar periodic variation;
but this time the variations are due chiefly to fluctuations in the
polymorphonuclear variety, and the leucocytes on the average are
greatly in excess of the normal number (vzde Chart C.H., Case 38).
This change from a fluctuation in the number of mononuclears to a
greater fluctuation in the number of polymorphonuclears seems to
take place invariably. <A certain time elapses after the active
malaria before this new leucocytic phenomenon begins. If the
malaria has been severe, or if the patient is much debilitated, it 1s

88

not noticeable for perhaps ten days or longer, while in other cases,
usually less severe, it may occur in a few days to about a week.
It. also occurs whether the patient has recovered as a result of
quinine treatment or not, and is therefore not due to quinine treat-
ment (v7de Chart W.M. (P. vevar)). Again the fluctuation is very
markedly periodic, occurring daily or on alternate days or
irregularly. Thus if the fever was quotidian, tertian or irregular
there is evidence to show that these fluctuations in total leucocytes
(chiefly due to polymorphs) are also quotidian, tertian or irregular.
This would seem to indicate that there is some malarial periodic
virus lingering in the system in spite of vigorous quinine treatment
of thirty grains daily, and where no fever parasites can be detected
on microscopic examination of the blood. If the total mononuclear
percentage is charted as before; it is found that the percentage is
low when the total leucocytes are high, and the curve representing
the total mononuclears does not vary nearly so much as the curve
of the total polymorphonuclears (vzde Chart 38, C.H.). Law II,
however, still holds good, the mononuclear percentage being as a
rule lowest when the temperature is highest, although the
temperature may at no time rise above normal. It would thus
appear that this transient periodic increase of leucocytes, chiefly due
to polymorphonuclears, is due possibly to minute numbers of asexual
parasites sporulating at regular intervals. If this is so, then it
would appear that the sudden liberation into the blood of large
doses of the malarial virus causes a leucopenia, while small doses
cause a leucocytosis. This periodic leucocytosis occurs at the time
of the day at which the patient previously had a rigor and fever.
Another possible explanation is that it is due to the periodic
outburst of sexual forms or gametes into the peripheral circulation.
(See accompanying paper on ‘Crescents.’) Against this explana-
tion, it is found to occur when no gametes have been produced, and
also long after they have disappeared owing to quinine treatment.
In Case C.H. 38, it almost seems as if the daily outburst of
crescents caused a daily polymorphonuclear increase. It would
seem, however, that the polymorphonuclear leucocytes seldom or
never ingest crescents. In cases with very numerous crescents
and absence of asexual parasites many pigmented mononuclear
leucocytes are found, but only very occasionally does one find a

89

pigmented polymorphonuclear leucocyte. It would therefore
appear that the mononuclear leucocytes ingest not only the asexual
parasites, but also the sexual forms or gametes. | Whether the
gametes can be ingested alive or only after their death is a matter
for speculation. It is interesting to note that the change from a
mononuclear into a polymorphonuclear swing often takes place
about the time that crescents first appear in the peripheral
circulation, that is a week to ten days after the last paroxysm of
fever. In the preceding article on ‘Crescents,’ pages 65 and 66,
I have pointed out that an increase of leucocytes seems to be
coincident with increase of immunity and consequent crescent
production, and that in quiescent malaria the leucocytes are in
greater numbers than normal. These periods of quiescence often
continue for ten or more days, though no quinine be given, and it
would almost seem that the relapses tend to occur when the poly-
morphonuclear leucocytes begin to play more part in the increase
of leucocytes. This, however, is only speculation. Beyond these
suggestions, I am unfortunately unable to give any explanation
of this most interesting phenomenon, which I will enunciate as

Law ITI.

LAW III. Ix convalescent or apparently cured malaria, transient
periodic leucocytoses occur in the peripheral blood, and these
leucocyte fluctuations arise chiefly from a polynuclear variation.

In these periodic fluctuations the leucocytes often reach numbers
as great as 40,000 to 50,000 per c.mm. of blood, and the mono-
nuclear percentage may rarely fall as low as 20 per cent. of the
total leucocytes. On one occasion I observed a leucocytosis of
125,000 per c.mm. Two hours later the number had fallen to
22,000 per c.mm., and eight hours later to 6,000 per c.mm. (Chart
Eek (GP. falc.):)s

III. A METHOD OF DIAGNOSING MALARIA NOT ONLY DURING
THE ACTIVE STAGE BUT ALSO IN LATENT CASES,
AND IN CASES APPARENTLY CURED BY
CONTINUED QUININE TREATMENT

This new method of diagnosis depends on the constancy of
Laws Il and III. As already stated, there is a periodic fluctuation
in the percentage of total mononuclear leucocytes in malaria. In
active malaria this is due mainly to a variation in the number of

go

mononuclear leucocytes. This periodic fluctuating mononuclear
percentage still continues in latent malaria and in cases thoroughly
treated with quinine, but in these latter it 1s due mainly to a
variation in the number of polymorphonuclear leucocytes. To
apply this diagnostic test one must take frequent smears of the
suspected person’s blood; one smear every four to six hours is
sufficient. These should be taken during a period of two to three
days. Make differential counts estimating the total mononuclear
percentage in each smear and plot the curve representing these
percentages. If there is a more or less periodic variation in the
percentage amounting to over 20 per cent., then the patient has, or
has had, malarial fever. In active malaria the total mononuclear
percentage usually varies somewhere between 20 per cent. and
80 per cent., the periodic swing having usually an amplitude over
20 per cent. and often as high as 40 per cent. and more. In latent
and apparently cured malaria the mononuclear swing occurs at a
lower level somewhere between 20 per cent. and 60 per cent. The
amplitude as before is usually about 20 per cent. or over.

If the swing is irregular, then it denotes that the patient has,
or has had, malarial fever of an irregular type. If the swing 1s
distinctly quotidian or tertian, it denotes that the patient has, or
had, quotidian or tertian paroxysms of fever. On examining the
blood of a normal person in this way, I find that the swing of the
total mononuclear percentage does not amount as a rule to more
than 10 per cent., if one counts from 150 to 200 leucocytes. This
10 per cent. swing is quite irregular, and is due mainly to the error
arising from the estimation of an insufficient number of leucocytes.
Slight changes do, of course, take place in normal persons after
exercise, meals, etc., but Chart D.T. (normal), shows clearly that
these natural variations are much smaller than the marked
oscillations which take place in malaria cases (vide Chart D.O.,
Case 52 (P. vivax), and others). Again in certain septic diseases
one may have a swing in the total mononuclear percentage, but in
such cases the percentage falls to a very low limit, as low as
8 per cent. to IO per cent., and will seldom rise as high as 30 per
cent., whereas in malaria the highest point of the mononuclear
percentage will seldom be lower than about 45 per cent. to 50 per
cent. Thus I am led to bélieve that a large periodic variation in
the total mononuclear percentage amounting to 20 per cent., and

gi

where the upper limit reaches over 45 per cent., is pathognomonic of
malaria, and is furthermore so delicate a test that the presence of
the disease or its dregs can be detected long after continued quinine
administration. Moreover, by careful estimation of the percentage
in this way one can also state whether the previous active fever was
quotidian, tertian, or irregular in nature. It is also of great value to
estimate several times daily the total leucocyte count; because, as
already stated above, remarkable periodic variations in number
take place, especially in quiescent or latent cases. Most of the
cases show a quotidian rise of leucocytes (vide Charts C.H.,
Case 38, T.H. (P. falc.), etc.). The height of the leucocyte rise
corresponds exactly with the lowest point of the total mononuclear
percentage. This shows that this rise of leucocytes is not a
digestion leucocytosis. The increase in the number of leucocytes
after a meal is principally a lymphocyte increase, also the variation
in number is too great to be accounted for by the natural increase
after a meal. In normal persons the leucocytes seldom reach
15,000 per c.mm. after a meal. When they reach numbers over
15,000 per c.mm. it denotes some pathological condition (Beattie
and Dickson [1908]). In malaria the height of the periodic rise is
often as great as 30,000 per c.mm. of blood, and more rarely
reaches a number as high as 40,000 to 50,000 per c.mm. Chart
D.T. (normal person) shows that the natural daily variation seldom
reaches 14,000 to 15,000 per c.mm. of blood.

One naturally wonders how long this abnormal leucocytic
phenomenon continues after the last active attack of malarial fever.
I am unable so far to answer this question, but can give some
remarkable instances which show that it may continue for months,
and possibly even for years.

(a) Case 38, C.H., shows the phenomenon with unabated force
after three weeks’ continuous administration of quinine 30 grains
daily. Case D.O., 52, shows the same after one month of
continuous treatment with quinine twenty grains daily and
methylene blue twelve grains daily.

(6) Case J.M. (leprosy) showed the same phenomenon. This
was noticed while examining the blood to see if there was any
leucocyte change taking place which might be characteristic of
leprosy. To my surprise the mononuclear percentage curve
indicated a previous quotidian malaria. On enquiring into the

Q2

past history of this case, it was found that he had suffered from
malaria four years previously. He had had no recurrence of fever
since then, but stated that he remembered having a slight shivering
attack five months previous to this blood examination. So far as
I am aware, no one has ever described a mononuclear leucocyte
excess as a characteristic of leprosy, so that one is led to the
conclusion that the high mononuclear percentage with large
variation was due to an infection of malaria long since apparently
cured.

In the above method of diagnosis it is important to remember
the already well-known fact that there is an excess of large mono-
nuclear leucocytes in malaria. Stephens and Christophers [1908]
consider a value of this variety of leucocyte above 15 per cent. as
diagnostic of malaria, and there is no doubt but that these large
mononuclears play most part in the total mononuclear fluctuation
occurring in active malaria. They also occur in numbers above
normal in apparently cured cases of malaria. Thus a diagnosis
of malaria might be made from one single differential
leucocyte count where the total mononuclear percentage is high,
say above 50 per cent., and especially where the large mononucleated
variety is in excess. Normally the large mononuclears vary from
I per cent. to 3 per cent. of the total leucocytes (Beattie and
Dickson [1909]).

The two following cases show that malaria may be diagnosed
from one single differential count even months after an apparent
cure :—

(a) Case I, W.M. (mixed infection), left hospital in March,
1910, after a thorough course of quinine and methylene blue treat-
ment. On his discharge no parasites could be detected in his
blood. He continued to take quinine in doses of five to ten grains
daily until July, 1910, when he returned to hospital to report
himself. During this time he had not left England. Examination
of his blood revealed no parasites, but showed a total mononuclear
percentage of 67 per cent. His blood was therefore still abnormal
more than five months after his last attack of fever. The patient
was at this time quite well.

(b) Case A.L.B. was treated in hospital during June and July,
1910, for malignant tertian malaria followed by blackwater fever.
He returned to hospital six months later in February, 1911, to

93

report himself. He stated that he had been quite well for several
months. His blood nevertheless showed that the total mononuciear
leucocytes amounted to 62 per cent. No parasites could be
detected.

Though malaria may therefore be diagnosed, or at least
suspected long after its apparent cure, from one differential count,
yet one count is not sufficient as a rule in these cases. The blood
might happen to be taken at the lowest limit of the mononuclear
swing, and might reveal a total mononuclear percentage of, say,
only 20 per cent. Thus one estimation only might give a negative
result, whereas several differential counts taken six hourly for two
to three days would give a positive result. I] think sufficient has
been said to show that malaria, or at least its dregs, remain in
the system for a long time after an apparent cure. It would
almost seem certain that the parasites remain in the host for very
long periods in spite of vigorous quinine treatment, and that they
continue to develop and sporulate, giving rise to the periodic
leucocyte variations described above. The case of P. T. Manson
is well known, in which a relapse occurred nine months after the
original infection followed by three months of quinine treatment,
and where re-infection was impossible.

Before concluding, I should state that the leucocytic phenomena
in blackwater fever show the malarial origin of that disease. In
blackwater fever, as in malaria, the mononuclear percentage would
seem also to vary inversely with the temperature.

SUMMARY

(1) During active malaria the number of leucocytes in the
peripheral blood is decreased. During quiescent malaria, and in
cases apparently cured by treatment, the leucocytes in the peripheral
blood are much increased.

(2) During the rigor and temperature in malaria, the mono-
nuclear leucocyte percentage (more especially that of the large
mononucleated variety) is low. With the fall of temperature,
however, the mononuclear percentage rises very high, sometimes
even to go per cent. of the total leucocytes. This fluctuation in the
percentage of total mononuclear leucocytes occurs also long after

94

continuous quinine treatment, and is observed for months and even
years (?) after the last attack of fever.

(3) In these apparently cured cases of malaria, the mononuclear
percentage is lowest at the time of the day at which the rigor and
fever occurred during the previous active malaria; and, moreover,
at this time there also occurs a very marked leucocytosis, which
continues only for a few hours. The leucocytes often reach
numbers as great as 30,000 to 50,000 per c.mm. of blood. On one
occasion they were as numerous as 125,000 per c.mm. Two hours
later they had fallen to 22,000 per c.mm., and in eight more hours
there were only 6,000 per c.mm. This case showed a regular daily
periodic variation in the number of leucocytes, averaging from about
6,000 per c.mm. to 50,000 per c._mm. The height of the rise
always occurred about noon. This was the time at which the
rigor and fever, which was quotidian, was wont to occur
previously. This post-malarial leucocyte phenomenon occurred
always without exception in the forty cases examined, and would
therefore seem to be an infallible sign of previous malaria, as, so
far, it has not been observed in any other disease.

(4) It would appear that large numbers of malarial parasites
on sporulating cause a leucopenia, while a very small number on
sporulating cause a leucocytosis.

REFERENCES

ALEXANDER (1911), ‘The Use of Leucocytic Extract in Infective Processes.’
Brit. Med. Journ., Feb. 18.

ALEXANDER, Nauss, and WILLIAMS (1911), ‘The Use of Leucocytic Extracts
in Infective Processes.’ Liverpool Med. Chir. Journ., Jan.

ANDREWES (1910), Croonian Lectures, Lancet, July.

BEATTIE and DICKSON (1909), Special Pathology, p. 88.

BILLET, A. (1901), ‘ De la Formule Hémo-leucocytaire,’ Bulletin Médical de l’Algérie.
LAMBERT (1909), Amer. Journ. Med. Sciences, Vol. CXXXVII, p. 506.

Ross (1903), ‘ The Thick Film Process for the Detection of Organisms in the
Blood.’ Thompson Yates and Johnston Laboratories’ Report, Vol. V,
Part I.

Ross and THomson (1911), ‘A case of Sleeping Sickness Studied by Precise
Enumerative Methods: Further Observations,’ Proc. Royal Soc., B,
Vol. LXXXIII,

STEPHENS and CHRISTOPHERS (1908), Practical Study of Malaria and other Blood
Parasites, p. 221.

WaLDSTEIN (1895), Berliner klinische Wochenschrift, April 29 and May 6.

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97

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103

NOTE. UPON YELLOW, FEVER. IN, THE
BLACK RACE AND ITS BEARING UPON
fiat, QUESTION OF THE ENDEMICITY
OF YELLOW FEVER IN WEST AFRICA

BY

SIR RUBBERY BOYCE, ERS; 328,
(Receztved for publication 25 February, 1911)

The question of the susceptibility of the various races of
mankind to yellow fever is one which has always attracted
considerable attention, and one about which there still exists very
great misunderstanding.

The medical authorities on yellow fever in the 18th and 19th
centuries held that no races were absolutely immune, with the
possible exception of the Chinese. They held strongly, however,
to the view that the disease was very rare amongst the native
inhabitants of tropical countries, whilst very common on the other
hand amongst new arrivals. In other words, the older authorities
stated that yeilow fever was essentially an acclimatising fever.

Inasmuch as the Latin races were the first to colonise, they were
the first to suffer from yellow fever. Thus we have very complete
records of continual epidemics amongst the Spaniards, Portuguese
and French. So frequent, in fact, were these epidemics that the
Latin races were considered more susceptible than the other races.

When, however, the northern races began to colonise, yellow
fever proved itself equally virulent amongst them as the records of
Dutch, Danish, English, Norwegian and Swedish colonisation and
immigration amply testify.

It thus came about that yellow fever was a measure of
commercial and maritime expansion, and of labour movements in
the various industrial centres in the tropical world.

A period then arrived when, as in the 18th century, medical
men observed that the offspring of whites who were born in yellow
fever districts later escaped the disease during epidemics of yellow

104

fever. Then it also became apparent that the size of the epidemics
was strictly proportional to the number of new arrivals.

This observation brought out into still greater prominence the
fact that the permanent inhabitants of tropical towns did not die
in the same proportion as the new comers. These facts were
observed over and over again in New Orleans, in the West Indies,
in Central and in South America, the most careful tables for
comparison being furnished by Rio de Janeiro.

It was therefore concluded that the permanent inhabitants,
whether Creoles as in the Southern States and in the West Indies,
or Indian-Spanish as in Central and South America, were to a large
degree immune, but that they lost this apparent immunity if they
went to reside in Europe or a cold climate for a long period.
The question why they were immune, however, only attracted scant
attention, and at most, shrewd surmises were attempted like those
of the great Faget of New Orleans. It was not, in fact, until the
mosquito doctrine was firmly established that scientific attention
was given to this exceedingly interesting fact.

In the slave trade period, when black labour was introduced into
the West Indies, Central and South America, and into the Southern
States of America a new series of facts became patent. Sometimes
the blacks died in very large numbers from yellow fever, as in the
Philadelphia epidemic, but in most cases where we are in
possession of ‘reliable figures like those furnished by Chassaignac,
Roche, Lazard, Brady and numerous others, the proportion of
deaths amongst the slaves or their descendants was relatively small.
It was conclusively shown during the 1905 New Orleans epidemic of
yellow fever that the blacks could get yellow fever. Thus Lazard
states that in that eprdemic there were 452 fatal cases amongst the
whites and six amongst the negroes, whilst Chassaignac observed
that’the blacks were liable to the disease equally with the whites,
but had it in a particularly mild form. Chassaignac’s figures are
as follows :—

In one series Of the observations the mortality amongst ninety
white cases was 20 per cent., and amongst 950 coloured cases 1°2 per
cent. In another series of 500 cases amongst the whites there was
a mortality of fifty-one, and amongst 200 coloured a mortality
of one. La Roche states that the mortality from yellow fever in

105

Jamaica amongst the troops was 102 per thousand amongst the
white soldiers, and eight per thousand amongst the blacks. Blair
states that of the 1,790 black men imported into Demarara none
died of yellow fever during the 1852 epidemic of that disease.
The fact was therefore abundantly proved that whilst yellow fever
did occur in the blacks, it nevertheless did not assume the same
severe type as in the whites; fatal cases did, however, occur from
time to time amongst the blacks, and in some epidemics there was
a comparatively high sickness rate. It became evident that,
therefore, the black possessed no natural race immunity, and that it
was only a question as amongst the whites of ‘ acclimatisation,’ that
is, of coming from a district or country where yellow fever was
rare into a city where yellow fever happened to be endemic.

The new comer, whether black or white, was liable to the
disease. In this connection it is interesting to note that Coolies
and Chinamen are also liable to yellow fever.

All these are facts which go to prove that the various races of
mankind are susceptible to yellow fever, and that there is no
absolute racial immunity.

The question has now proceeded a stage further owing to the
increased attention paid to yellow fever in West Africa.

I have examined very closely the recorded outbreaks of yellow
fever in West Africa, and it soon became abundantly clear that
the so-called classical type of yellow fever was comparatively rare
amongst the native races. In the various recorded epidemics the
medical authorities of the time drew attention to the disproportion
of the death-rate amongst blacks and whites. This fact was all
the more remarkable as the natives far outnumbered the whites,
and lived in notoriously over-crowded and insanitary conditions,
and, as we now know, in an atmosphere crowded with the Stegomyia.

Why, therefore, if there was yellow fever on the coast of
Africa, as was abundantly shown by the very numerous outbreaks
amongst the whites, did no large epidemics occur amongst the
black natives, and depopulate the West Coast? This is a very
pertinent question and requires a very definite answer.

The West African blacks can get yellow fever, of this we have
absolute proof, notably in the epidemics of yellow fever on the
Coast in 1910. As far back as the epidemic of 1884 in Freetown,

106

a case of yellow fever was recorded in a native, and two cases
amongst the black soldiers of the West Indian Regiment. In 1910,
however, we have recent and positive evidence from the clinical
histories and post mortem examinations. At Freetown, for
example, one fatal case was recorded in a West Indian native
soldier in July of last year, and also a fatal case in a native of
Freetown. At Sekondi, in 1910, two cases amongst black men are
recorded during the outbreak. In the two places there was a total
of seventeen cases recorded amongst the white and five amongst
the black residents. In October, 1910, according to Sorel, a small
outbreak occurred at Grand Bassam, and three cases were recorded
amongst natives. Therefore, it is beyond dispute that yellow
fever can occur in its severe and fatal forms amongst the West
African black races. This, then, corroborates the opinions of the
older clinical observers that the black races were not absolutely
immune. A new light, however, is thrown upon the problem by
the 1909 epidemic of yellow fever in Barbados, which I was called
upon to investigate. In this epidemic, yellow fever proved more
fatal amongst the blacks than the whites. Out of a total of
eighty-six cases, fifty-four occurred amongst the black inhabitants.

The blacks of Barbados are the descendants of the original
imported African slaves; clearly, therefore, there was no hereditary
racial immunity.

But why should the same race in West Africa appear to be
immune? The answer to this question is the solution of the
question of the presence of yellow fever in Africa. The Barbadian
black lived in recent years under favourable conditions. The
Stegomyia, there is every reason to believe, was greatly reduced in
numbers by the introduction of a pipe-borne water supply laid on to
the houses or to stand-pipes along the roads; puddles of water are
not met with owing to the very porous nature of the soil, and the
yards had been kept fairly free from odd water containers. There
is practically no bush in the towns and villages, and the island is
very much wind-swept. All these are factors which would tend to
the diminution of the Stegomyia. The last recorded epidemic of
yellow fever prior to the 1909 outbreak occurred in 1881, that is
twenty-seven years previously. Therefore, it is reasonable to
assume that yellow fever was not endemic on the island, and that

107

all those natives born since the 1881 epidemic were absolutely
non-immunes. It is not surprising, therefore, that they became
infected in those districts where the Stegomyia was present in
sufficient numbers, and when the virus had been introduced into
the island from without. The reverse is the case in West Africa.

The evidence, therefore, is conclusive—
1. That the negro can contract and die from yellow fever.

2. That he has, as a rule, yellow fever of a much milder type
than that met with amongst whites who have recently arrived in
a tropical country.

Naturally it follows that it is reasonable to ask: Does yellow
fever occur amongst the natives of West Africa in a mild form,
difficult of recognition; just as we know it did amongst the Creoles
of the West Indies and the indigenous inhabitants of New Orleans,
Cuba, Rio, Vera Cruz, Para, etc.? In my opinion this is the only
reasonable hypothesis which the facts will support and, moreover,
it 1s one which has become formally adopted by those who have
specially studied yellow fever, notably Marchoux and Simond,
Otto and Neumann, Durham, and the American Cuban
Commissions.

It is notorious that in places like Rio, Parad, and other endemic
centres in the past, yellow fever was regarded as a disease of
the foreigner or new-comer and not of the native; only the
foreigners acquired the severe black vomit and died, the permanent
inhabitants escaped. From the time of Faget, of New Orleans, up
to the present date observers have, however, come to the conclusion
that yellow fever does occur in the native children, and that it can
occur more than once amongst adult natives. In other words, the
natives suffer in early childhood and may suffer from subsequent
attacks. We now can understand why fatal or severe yellow fever
is rare amongst the native population—the natives are partially
immunised. They contain the virus, nevertheless, in their blood,
and can infect the Stegomyia. If, on the other hand, these same
natives are removed in childhood from a yellow fever endemic area
and protected, they become rapidly non-immunes, as shown above
in the case of Barbados, and as has been proved many times
amongst the Creoles and Indian-Spanish races.

108

The evidence is that yellow fever, like most other infectious
diseases, does not confer permanent immunity. Also, that just as
in other infectious diseases, mild ambulatory forms of the disease
are probably far more common than is usually supposed. The
outbreaks of yellow fever in West Africa last year, IQIO,
corroborates this view in a remarkable manner. From May to
October there were five outbreaks of yellow fever, viz., at Freetown, .
Sekondi, Axim, Saw Mills, and Grand Bassam. In the case
of four, at least, of these outbreaks no connection could be traced
between them, they appeared to originate de novo. But more
significant still, the first to suffer from the disease and to show the
medical authorities that yellow fever was present were the Syrians,
small traders who, with ¢hezr families, live in the midst of the
natives in the most Stegomyia-haunted parts of the towns. The
1910 outbreaks showed that those who lived outside the native
towns remained absolutely yellow fever free. In my opinion,
therefore, the evidence is overwhelming that yellow fever is endemic
in West Africa, and that the reservoirs are the natives of West
Africa. How far the natives of all coast towns in West Africa are
reservoirs of the virus, I am not prepared to state, as we require
more evidence, but the facts warrant us in stating that in many
places, yellow fever is endemic amongst the native inhabitants in a
particularly mild form, very much as malaria occurs amongst
them. Unfortunately, we have so far no blood or animal test which
will prove the presence of the virus, and have only to rely upon a
severe Case occurring in a non-immune to prove the existence of the
disease. In West Africa the non-immune who serves as the test
appears to be the Syrian, who happens to live most in contact with
the native. From these facts it follows that the great practical
lessons to be learnt are that segregation of the non-immunes and
Stegomyia destruction are the absolute remedies against yellow
fever, also that the answer to the question propounded in the
beginning of this paper, viz., why have not the native races in the
large towns been decimated or completely wiped out? is that they
are completely immunised by mild attacks of yellow fever from
childhood. It must also be borne in mind, however, that a
considerable proportion of the infantile mortality in the native
races may be due to mild yellow fever as well as malaria.

109

‘nal

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Blow. Fever 1890 | 1891 1892 | 1893 1894 | 1895 | 1896 | 1897 | 1898 | 1899 | 1900 {1901 |1902 | 1903
m Rio.

Diagram to illustrate the great difference between the cases of Yellow Fever amongst new arrivals, i.e. foreigners, and the
native residents, i.e. the Brazilians in Rio. (Otto and Neumann.)

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Diagram to show the chief outbreaks of Yellow Fever in British West Africa from 1825-1910.
“From the year 1884 the cases amongst the natives have been recorded. Note the
marked difference between the whites and blacks respectively. Compare it with the
Rio table.

=. 4 “al

Ii!

ON” THE “AMOEBAE “PARASITIC IN
THE HUMAN INTESTINE, WITH
REMARKS ON THE LIFE-CYCLE OF
ENTAMOEBA COLI IN CULTURES

BY

H. B. FANT HAM, D:Sc: Lonpb., (BrA., CANTAB., A.R.C.S.

(Received for publication 21 March, 1911)

INTRODUCTION

The study of the parasitic Amoebae, although of the greatest
importance, is one of considerable difficulty. Many investigators, in

all parts of the world, have engaged in this study during the last

twenty years with the most conflicting results. Consequently,
to-day, the utmost confusion prevails as to the pathogenicity,
morphology and culturab:lity of the parasitic Amoebae of the
human digestive tract, and at present nearly a dozen species are
recorded from the human intestine alone. Further, nearly as many
more species have been recorded from other organs of man.

Recently, while studying cultures of Extamoeba colt, as well as
stools from patients being treated for dysentery at the Royal
Southern Hospital, Liverpool, I have had occasion to examine the
scattered literature on the subject. Before recording the _pre-
liminary results of my own studies it will be convenient to set forth
a short critical review of the recent work on the parasitic intestinal
Amoebae of man, as it seems to me that there has been a tendency
to give undue prominence to Schaudinn’s researches.

My work has been done in the Liverpool School of Tropical
Medicine, under a grant from the Tropical Diseases Research Fund.

112
THE PARASITIC AMOEBAE OF THE INTESTINE OF MAN

Without adding to the complexity of the subject by a discussion
of the history of the association of parasitic amoebae with human
dysentery, we may at once give a list, with brief diagnoses, of the
species of Amoeba recorded from the human intestine up to the
end of 1910. We adopt the generic name Entamoeba of
Casagrandi and Barbagallo (1895), and divide the parasites into
those which are said to be pathogenic and those which are said to
be non-pathogenic, beginning with the latter, thus : —

(A) NON-PATHOGENIC FORMS.

1. Entamoeba coli (Losch, 1875). Diameter, 12 to 25 m, but
variable.

No distinct ectoplasm apparent except at the beginning of
pseudopodia formation. Endoplasm granular, filling up the body
space when the organism is at rest. Nucleus large, sub-central,
spherical, vesicular, containing much chromatin. Nucleus visible in
the fresh state. Motility rather feeble.

Multiplication by binary fission or by schizogony, with
formation of eight merozoites.

Encystment total and endogenous. Cysts 28 m in diameter.
Sporogony, after nuclear reduction and autogamy, with formation
of eight amoebulae.

This parasite lives in the lumen of the large intestine, on the
contents thereof; it is incapable of penetrating the mucosa. It may
occur in the stools of healthy persons. It is usually considered to
be non-pathogenic. Its possible pathogenicity is not above
suspicion according to the researches of Billet (1907) and others.
It can be cultivated in association with certain bacteria.

2. E. tropicalis (Lesage, 1908). This parasite is said to be
non-pathogenic, and to occur in the intestine of man in the tropics.
Though exhibiting a general resemblance to &. colz, and having a
nucleus charged with chromatin, it is said to have a clearly
distinguishable ectoplasm and to form small cysts (6 to Io p» in
diameter). The small size of the cyst is due to the amoeba having
previously divided. Further the nucleus of the cyst is said to break

113

up into a variable number of daughter nuclei, so that from three
to thirteen amoebulae may occur inside a cyst. Several varieties
of this species are said to exist by Lesage. It is culturable in
symbiosis with bacteria.

3. £. hominis (Walker, 1908). Diameter, 6 to 15 » when at
rest. Ectoplasm apparent only in the pseudopodia, endoplasm
granular, nucleus circular. A single contractile vacuole present.
Encystment total. Cysts small (4°6 to 7:7 #). Sporulation frequent,
spores spheroidal, measuring 0°3 to o'8 p.

Culturable with bacteria, but with difficulty. Original strain,
now lost, from an autopsy in Boston City Hospital.

This species would appear to be closely allied to &. tropicalis.

(B) PATHOGENIC FORMS.

4. Entamoeba histolytica (Schaudinn, 1903), also described by
Jiirgens in 1902.

Diameter, 25 to 30 mw.* Ectoplasm clearly defined. Stout
pseudopodia entirely composed of ectoplasm and capable of
burrowing into the mucosa and sub-mucosa of the intestine.
Nucleus variable in form, excentric and often lateral, poor in
chromatin. Nucleus usually invisible in the fresh state. This
parasite often ingests red blood corpuscles.

Multiplication by binary fission or by budding. Reproduction
by exogenous encystment, giving rise peripherally to minute spores
about 3 sm in diameter. The spores become encysted, and,
according to Lesage, contain three nuclei.

It is stated that series cultures of this parasite, in association
with bacteria, cannot be obtained, at any rate to retain their
pathogenicity. Lesage (1907), however, claims to have cultured the
parasite in leucocytic exudation from the peritoneum of infected
guinea-pigs. The parasite has been found in cases of liver abscess
and dysentery in Egypt, China and Japan.

5. Entamoeba sp., cultivated by Noc (1909), from cysts derived
from liver abscess, from dysenteric stools and from the water
supply of Saigon, Cochin China. Noc cultivated this amoeba in
association with bacteria. It is, apparently, pathogenic, closely

* Hartmann (1909) gives a smaller size, 15-204.
H

114

allied to E. histolytica, perhaps showing more marked znxéernal
budding (schizogony) than £. histolytica (judging by Noc’s figures).
It exhibits polymorphism, and may be a separate species, but is
unnamed.

6. EE. tetragena (Viereck, 1907), synonym £. africana
(Hartmann).

Recorded from dysenteric cases in various parts of Africa,
Brazil and India.

Diameter, 20 to 30 #, according to Viereck.

Although the trophozoite of this amoeba bears a general
resemblance to that of Z. colz, yet it is said by Hartmann to possess
a distinct ectoplasm which is only clearly visible when a
pseudopodium is protruded. However, its granular endoplasm may

contain ingested red blood corpuscles. There is a large round
nucleus visible in the fresh state. Chromidial masses occur in the
cytoplasm.

Multiplication proceeds by binary fission.

Sexual reproduction by endogenous encystment, which is
preceded by nuclear division into two, reduction and then
autogamy. The cysts contain four nuclei.

E. tetragena is pathogenic to man and to kittens, but the
dysentery resulting is said to be more benign than that resulting
from E. histolytica, and liver abscess is said to be rare.

E. tetragena is not culturable.

Hartmann (1909) stated that EZ. Azstolytica is a rare amoeba,
and that in nearly all cases of amoebic dysentery the £. ¢etragena
of Viereck is found.

Personally I have met with a case of chronic dysentery under
treatment in Liverpool (probably infected in Nigeria), the parasite
obtained from the stools being £. ¢tetvagena.

7. . phagocytotdes (Gauducheau, 1908).

This parasite was discovered in a case of dysentery at Hanoi,
Indo-China. The amoeba is very small, 2 to 15 uw in diameter. It
is active, and possesses a well-developed ectoplasm. It ingests
bacteria and red blood corpuscles, while peculiar spirilla-like bodies
are found in its cytoplasm.

It multiplies by binary and multiple fission.

=~

115

Young cultural forms of this amoeba inoculated intravenously
into a dog produced dysentery.

8. E. minuta (Elmassian, 1909). Found in association with
E. coli, in a case of chronic dysentery in Paraguay.

It resembles E. ¢etvagena, but is smaller, rarely exceeding 14 mu
in diameter. No differentiation between ectoplasm and endoplasm.
Nucleus invisible in fresh preparations, and when stained is richer
in chromatin than that of Z. coli.

Multiplication by schizogony into four merozoites.

Encystment total and endogenous, giving rise to cysts containing
four nuclei, after nuclear reduction and autogamy.

g. . nipponica (Koidzumi, 1909). Found in the motions of
Japanese suffering from dysentery or from diarrhoea, in the former
case in company with £. histolytica. It is also said to occur in
healthy persons.

Diameter, 15 to 30 p.

Clear distinction between ectoplasm and endoplasm. Pseudo-
podia are not spinose, but are lobopodia. Endoplasm vacuolated,
and phagocytic for red blood corpuscles. The nucleus is well
defined, and can be seen in the fresh condition; it is rich in
chromatin, resembling that of &. colz and LE. tetragena.

Multiplication by binary fission and by schizogony into six or
eight merozoites.

Encystment total, and accompanied by formation of chromidia.
The complete stages of sporogony have not been followed.

Experiments are necessary to determine the pathogenicity and
culturability of this amoeba.

10. £. undulans (Castellani, 1905). Found, in company with
other Protozoa, in the faeces of persons suffering from diarrhoea in
Ceylon.

Diameter, 12 to 30 mw. There is an undulating membrane present,
and long straight pseudopodia which appear rapidly, but only one
pseudopodium is protruded at a time. Cytoplasm not differentiated
into ectoplasm and endoplasm.

Obviously, further knowledge of this parasite is needed.

116

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117

SOME CRITICAL REMARKS ON THE VARIOUS
INTESTINAL AMOEBAE OF MAN

It is now usually recognised, since the experimental researches of
Schaudinn (1903) and others, that amoebae of two kinds may occur
in the human digestive tract, namely, pathogenic ones and others
which are non-pathogenic. To the latter class belong Entamoeba
coli and E. tropicalis. In the former class must be placed Entamoeba
histolytica and E. tetragena. Further, the non-pathogenic forms
are culturable with symbiotic bacteria, while the pathogenic ones are
not so culturable, or doubtfully so.

Musgrave and Clegg (1904) were the pioneers of successful
modern cultural methods as applied) to amoebae. These
distinguished workers, however, suggest that ‘all amoebas [in the
intestine] are, or may become pathogenic,’ and state that ‘amoebas
cultivated from various sources, including the dysenteric intestine,
the Manila water-supply, lettuce, etc., have proved pathogenic
under certain conditions, which reverses the view held of some of
those formerly considered harmless.’

The discrepancy between Musgrave and Clegg’s results and those
of Schaudinn is usually ascribed to impurity of cultures, due to the
presence, unnoticed, of the small cysts of EF. Azstolytica in the
cultures of the Philippine observers. Lesage (1908) considers that
Musgrave and Clegg cultured chiefly E. tropicalis, while Werner
(1908) thinks that they had Amoeba limax in their cultures, and he
similarly criticises Walker. But we must still carefully consider
Musgrave and Clegg’s results, especially when we remember that
the possible pathogenicity of Z. colz is not above suspicion (cf.
Billet, 1907), and that-quite recently (November, 1909) Elmassian
asks how we are to interpret the occurrence of EF. histolytica in
non-dysenteric natives in Asia. Obviously, many further researches
are needed.

Regarding the large number of species of parasitic amoebae
recorded from the human intestine, I think that few of the so-called
species are really good ones. With respect to plurality of species,
we must carefully consider the phenomenon of folymorphism, a
phenomenon markedly exhibited by amoebae. Many of the species
are apparently only separated by slight morphological differences,

118

such as in the distinctness or otherwise of the ectoplasm from the
endoplasm, in the structure of the nucleus, or even in size. Such
differentiation is unsatisfactory, as must be evident to any
investigator who has worked for any length of time on one species
of organism, who has fixed and stained it by various methods from
day to day, and compared the results obtained. Morphological
variation, then, must not be overlooked when separating species.
Both Musgrave and Clegg (1906) and Noc (1909) have recorded
the occurrence of variability in morphological characters of
amoebae in cultures started from a single, isolated cyst.

Certain observers, again, have cultivated species of amoebae, and
then omitted to test the pathogenicity or otherwise of the cultural
forms by experiments on animals. Further, the life-cycles of
cultural amoebae must be carefully examined and compared with
the natural forms, a point which appears to have been largely over-
looked.

It seems to me that the methods of reproduction supply the
most valid grounds on which to base species differences, taken in
conjunction with possible pathogenicity. We then have three
fairly well recognised species of amoebae parasitic in the human
intestine, namely :—

(1) Entamoeba coli, with its varieties E. tropicalis and
possibly £. hominis. These are apparently non-
pathogenic. The encystment is total and endogenous.

(2) EB. histolytica, the pathogenic agent in certain cases of
dysentery and liver abscess recorded from Egypt and
China, and perhaps from Europe. The encystment is
not total but exogenous, and minute spores are produced.
The organism has been best studied by Schaudinn (1903),
Craig (1908) and Hartmann (1909).

(3) E. tetragena, the pathogenic agent of dysentery in cases
recorded from various parts of Africa, Brazil and India.
The encystment is total and endogenous. The organism
has been studied by Viereck (1907) and Hartmann (1908).

Probably E. minuta is merely a variety of E. tetragena, while
E. nipponica seems to belong, as a variety, either to E. colz or to

11g

E. tetragena. The position of E. phagocytoides is unsatisfactory
until its sporogony has been investigated.

Gauducheau (1909) states that at a later stage of the culture the
organisms (E. phagocytoides) are difficult to keep alive, and then
are only about I m in diameter. Brown (1910) considers that
Gauducheau’s organism clearly shows affinities with Z. histolytica,
like Noc’s entamoeba.

Morphologically &. colz and E. tetragena are somewhat alike
and form endogenous cysts, though the daughter forms within the
cyst are eight and four respectively. It is interesting to note that
Viereck (1907) first thought that &. ¢etragena was a variety of
E. colz. Our knowledge of £. tetragena is not yet quite complete.

PRELIMINARY NOTE ON THE LIFE-HISTORY OF ENTAMOEBA
COLI AS SEEN IN CULTURES

Two separate cultures of Entamoeba coli have been examined.
They were derived from dysenteric cases from Manila, and have
been maintained on Musgrave and Clegg’s medium (at 20° to 25° C.)
by sub-inoculations for some three years. I have much pleasure in
thanking Dr. Stephens for the material.

The life-cycle of the parasite in cultures has been studied, a
point which does not appear to have been fully recorded in previous
literature.

The results, not yet complete, may be summarised as follows.
On Musgrave and Clegg’s medium the amoebae leave their cysts
after rupturing them, and some discarded empty cysts may be
found in preparations. Sometimes, however, the encysted parasite
first appears to swell up or grow, the cyst wall gradually becoming
thinner meanwhile until it appears ultimately to be absorbed.
Small vacuoles may occur in the cyst. The amoeba when first free
usually contains one or two small vacuoles which after combining
slightly enlarge and travel to the periphery. This vacuole aids in
the protrusion of the first pseudopodium. The pseudopodia are
composed chiefly of ectoplasm, though endoplasm flows in to some
extent later. The greater part of the body of the amoeba is
composed of granular endoplasm, and some of the larger granules
may stain metachromatically with methylene blue zv/7a vitam. The

120

amoeba now feeds, grows and moves about in a restricted area
which is often approximately circular. The nucleus of the amoeba
is round and vesicular with a central karyosome, and is clearly
visible in life. There is a clear area in the endoplasm around the
nucleus. The parasite at this stage may be called a trophozoite, in
preference to the term ‘vegetative’ stage which is so often used.
The amoeba divides by binary fission with nuclear promitosis,
in which the karyosome plays an important part. The parasite also
divides, occasionally, by schizogony, forming eight merozoites.

On the culture-media which I have been using, the amoebae
begin to encyst in about four days, the cyst wall of each being
formed by differentiation at the periphery of the now rounded
amoeba. The encystment is total. The cyst at first contains a
centrally-placed nucleus, with a karyosome. Inside some of the
cysts division occurs, and eight daughter forms are produced. The
cytological details of sporogony are now being studied, and will
be published later. When all the amoebae in a culture have
encysted and remained in that condition for some time a new
culture must be prepared. The cultures with which I have been
working are renewed about every fortnight or three weeks.

I have tried the action on Extamoeba coli of some of Dr. H. C.
Ross’s ‘ auxetics ’"—substances capable of inducing division in living
cells. I wish to thank Drs. H. C. Ross and J. W. Cropper for
providing me with some of these substances. Many of these
auxetics occur naturally in the body, and my attention has been
especially directed to some which are found in the intestines, such
as tyrosin, leucin and skatol.

These substances are best used in a jelly with agar, sodium
chloride and alkali (sodium bicarbonate), forming a slightly
alkaline culture medium. When such a medium, containing about
o°2 per cent. of tyrosin, is inoculated with cysts of &. colz obtained
from a culture on Musgrave and Clegg’s medium, the period of the
life-cycle is shortened, and the amoebae on the culture reproduce
for several generations. I have a culture which has already gone
through five generations. Somewhat similar results occur on
a culture-medium containing a similar quantity of leucin.
Unfortunately the ‘growth’ does not increase much, for there is

E21

apparently rapid death of some of the amoebae, probably resulting
from insufficient food-supply in the media.

One interesting and novel result on tyrosin-containing media,
compared with cultures on Musgrave and Clegg’s medium, is that
a complete life-cycle of E. colz is passed through in about three
days (at 20° to 25° C.), when all the amoebae of a given generation
have encysted. Then a large number of the cysts produce eight
daughter forms inside them, and the amoebulae come out of the
cysts and start a new generation on the same medium. I have seen
these phenomena continued through five generations, whereas on
Musgrave and Clegg’s medium only one generation of amoebae is
usually produced, and few of the cysts give rise to eight daughter
forms. Further, binary fission of amoebae occurs on a tyrosin-
containing medium more frequently than on a Musgrave and Clegg
medium. The process of binary fission involves a primitive mitosis
(or promitosis) of the nucleus, caps of chromatin derived from the
karyosome being formed at the ends of the rudimentary spindle.
Stages of schizogony have also been seen.

Skatol added to a preparation containing free &. coli rapidly
induces encystment. This is of interest since skatol occurs
naturally in the hinder part of the digestive tract and in the faeces.

Auxetics such as supra-renal extract and metaphenylenediamine
also apparently induce division. The binary fission in such cases is
of the nature of unequal promitosis, probably due to the rapidity
with which it is induced.

These researches are being continued, and I hope to publish a
more detailed and illustrated account later, in which the cultural
forms will be compared with the amoebae in their natural habitat.

122

REFERENCES

The following list is confined principally to papers published
during the last ten years. Only a few of the older papers are
included, but further references to these may be obtained at the
end of some of the memoirs listed.

BENSEN, W. (1908), ‘Die Darmprotozoen des Menschen.’ Arch. f. Schiffs- u.
Trop.-Hygiene, Bd. XII, pp. 661-676.

BILLET, A. (1907), ‘Sur un cas de dysenterie ‘‘ nostras’’? 4 Amibes.’ C. R. Soc.
Biol, LXIl, p. 1232:

Brown, W. C. (1910), ‘ Amoebic or Tropical Dysentery, its Complications and
Treatment.’ 271 pp. London, Bale & Danielsson.

CASAGRANDI, O. e BARBAGALLO, P. (1897), ‘ Entamoeba hominis s. Amoeba coli
(Losch).? Ann. d’Igiene speriment., VII, pp. 103-166, 1 pl.

CASTELLANI, A. (1905), ‘ Observations on some Protozoa found in Human Faeces.’
Centralbl. f. Bakteriol. u Parasitenk., Abt. 1, Orig., XX XVIII, pp. 66-69.

CouNcILMAN, W. T. AND LAFLEUR, H. A. (1891), ‘ Amoebic Dysentery.’ Johns
Hopkins Hosp. Reports, II, pp. 395-548, 7 pls.

CratG, C. F. (1908), ‘ Studies upon the Amebae in the Intestine of Man.’ Journ.
Infect. Diseases, V, pp.324-377, 2 plates.

ELMASSIAN, M. (1909), ‘ Sur une nouvelle espéce amibienne chez |’ homme, Entamoeba
minuta, n. sp.’ Centralbl. f. Bakteriol., Abt. 1, Orig., LII, pp. 335-351, 2
plates.

GasUDUCHEAU, A. (1908), ‘ Culture d’une amibe dysenterique’ (Z. phagocytotdes),
C. R. Soc. Biol., LXIV, p. 493.

GAUDUCHEAU, A. (1909), ‘ Sur une culture amibienne,’ Bull. Soc. Path. Exotique, II,
PP- 247; 370, 568.
HARTMANN, M. (1908), ‘ Eine neue Dysenterieamébe, Entamoeba tetragena (Viereck),

syn. Entamoeba africana (Hartmann),’ Arch. f. Schiffs- u. Trop.-Hygiene, Bd.
XII, Beiheft 5, pp. 117-127.

HARTMANN, M. (1909), ‘ Untersuchungen tiber parasitischen Amében. I. Entamoeba
histolytica Schaudinn.’ Arch. f. Protistenkunde, XVIII, pp. 207-220, 1 plate.

JURGENS (1902), ‘ Zur Kenntnis der Darmamében der Amében-enteritis.’ Veroff.
a.d. Gebiete d. Militar-Sanitatswesens, XX, p. 110.

KorpzuMt1, M. (1909), ‘ On a new parasitic Amoeba, Entamoeba nipponica, found in
the intestine of Japanese,’ Centralbl. f. Bakteriol., Abt. 1, Orig., LI, pp.
650-653.

LesaGE, A. (1905), ‘ Culture de 1’Amibe de la Dysenterie des pays chauds,’ Ann.
Inst. Pasteur, XIX, pp. 9-16, 2 plates.

LesaGE, A. (1907), ‘Culture du parasite de l’amibiase humaine (Dysenterie
amibienne),’ C. R. Soc. Biol., LXII, pp. 1157-1159.

123

LEsAGE, A. (1907), ‘ L’amibiase chez le chat (Dysenterie amibienne),’ C. R. Soc.
Biol., LXII, pp. 1191-1193.

LEsaGE, A. (1908), ‘ Note sur les entamibes dans la dysenterie amibienne des pays
chauds,’ Bull. Soc. Path. Exotique, I, pp. 104-11.

Loscu, F. (1875), ‘ Massenhafte Entwickelung von Amoeben ein Dickdarm,’
Virchow’s Archives, LXV, pp. 196-211.
MusGrave, W. E. and Criecc, M. T. (1904), ‘ Amebas: their Cultivation and

Etiologic Significance,’ Dept. of Interior, Bureau of Govt. Labs., Biol. Lab.,
Manila, No. 18.

MuscraVE, W. E., and CLEGG, M. T. (1906), ‘ The Cultivation and Pathogenesis of
Amoebae,’ Philippine Journ. of Sc., I, pp. go9-g5o.

Noc, F. (1909), ‘ Sur la Dysenterie amibienne en Cochinchine,’ Ann. Inst. Pasteur,
XXIII, pp. 177-204, 4 plates.

SCHAUDINN, F. (1903), ‘ Untersuchungen uber die Fortpflanzung einiger Rhizopoden,’
Arb. a. d. Kaiserl. Gesundheitsamte, XIX, Heft 3, pp. 547-576 (see p. 563).

VIERECK, H. (1907), ‘ Studien tiber die in den Tropen erworbene Dysenterie,’ Arch.
f. Schiffs- u. Trop.-Hygiene, Bd. XI, Beiheft 1, pp. 1-41, 3 plates.

VINCENT, H. (1909), ‘ Note sur la latence prolongée de l’Amibe dysenterique dans
Bull. Soc. Path. Exotique,

eh

Vintestin humain. Les “‘ porteurs d’amibes.
II, pp. 78-80.

WaLKER, E. L. (1908), ‘ The parasitic Amebae of the Intestinal Tract of Man and
other animals,’ Journ. Med. Research, XVII, pp. 379-459, 4 plates.

WERNER, H. (1908), ‘ Studien tiber pathogene Amoben,’ Arch. f. Schiffs- u. Trop.-
Hygiene, Bd. XII, Beiheft 11, 18 pp., 6 plates.

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125

SOME FURTHER OBSERVATIONS ON
THE TSETSE-FLY, DESCRIBED IN THESE
ANNALS AS GLOSSINA GROSSA, Etc.

BY
R NEWStfe AD: O1Sc.; A.L.S.,. &c.

(Recewved for publication 29 March, 1911)

I have recently ascertained that Bigot’s type of Glossina grossa
is preserved in the British Museum (Natural History) at South
Kensington, and that it is morphologically distinct from the tsetse-
fly which I described under this name in December, 1910.

Had I known at the time that the type was available, I should
have taken steps to have verified my conclusions, and thereby have
avoided the confusion in the synonomy of this insect. Now that I
have had an opportunity of examining Bigot’s type, I have no
hesitation in stating that my examples are specifically distinct, and
the name xzgrofusca which I suggested must now be adopted, the
synonomy of which is appended below :—

Glossina nigrofusca, Newstead. Annals of Tropical Medicine
and Parasitology, Vol. IV, 3, pp. 370, 373, 1910.

Glossina grossa, Newstead (vec Bigot). Annals of Tropical
Medicine and Parasitology, Vol. IV, 3, p. 373, 1910.

The distinguishing characters of this insect are that the terminal
segment of the antenna is much more strongly recurved at the tip
than in G. grossa; it is also clothed with much longer hairs; the
thoracic markings are more sharply and clearly defined, and the
general colour of the insect is darker than in any other species of the
fusca group.

Glossina palpalis, var. wellmani, Austen.

Mr. E. E. Austen has very kindly afforded me the opportunity
of examining a para-type male of this variety of G. palpalis, and I
find that the morphological characters of the armature are
specifically the same as in G. palpalis. The example in question
bears the following data: —‘ Katumbela River, Benguella, Angola,
November, 1904, Dr. F. C. Wellman. 1906. 139.’

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127

ON THE CORRELATION BETWEEN
TRYPANOSOMES, LEUCOCYTES,
COAGULATION TIME, HAEMOGLOBIN
AND SPECIFIC GRAVITY OF BLOOD

BY
VISHNG We RORKE, MIR: C.P?, DTM,

JOHNSTON COLONIAL FELLOW, THE UNIVERSITY OF LIVERPOOL
(Received for publication 4 April, 1911)

During daily observations of the blood of animals, viz., guinea-
pigs and rats infected with 7. gambiense and T. rhodesiense, it was
observed that the coagulation rate of blood and the rate of haemolysis
by a dehaemoglobinising fluid on thick films varied from time
to time.

Wright’s method was used in determining the coagulation time of
blood at the temperature of half blood heat. In taking blood,
squeezing of the tissues was carefully avoided. Leucocytes and
parasites were counted by Ross and Thomson’s* quarter millimetre
pipette method on thick films, using an Ehrlich’s eye-piece. They
were counted on the same film and in the same field in specimens
stained by Romanowsky’s method. Many parasite counts were
made by Dr. H. B. Fantham, to whom my thanks are due. The
haemoglobin percentage value was determined by Sahli’s haemo-
globinometer.

The specific gravity was determined by Hammerschlag’s benzol-
chloroform method, a modification of Roy’s. In the case of infected
animals, the specific gravity was always below 1,060. In the case of
control animals I found the specific gravity was rarely below 1,055,
and as the instrument was not graduated above 1,060, readings above
this were expressed as ‘little above,’ ‘well above,’ or ‘far above’
the 1,060 mark. The observations were made every twenty-four
hours, practically at the same hour of the day, three hours after

* Annals of Tropical Medicine and Parasitology, Vol. IV, p. 268 (1910).

128

feeding the animals, viz., guinea-pigs and rats infected with
T. gambiense and T. rhodesiense.

I am indebted to Walter Stott, Esq., Honorary Statistician to
the Liverpool School of Tropical Medicine, for calculating the
following correlations. This. and the application of correlation
method to other observations, is one of the first attempts at applying
precise mathematical method to clinical investigations. From 173
observations on specific gravity, haemoglobin and parasites, the
following results were obtained :—

(1) Correlation between the number of parasites and the amount
of haemoglobin—
7 = O'1517 + 1902, error greater than 7.
Result = No correlation shewn.

(2) Correlation between the number of parasites and specific
gravity of blood—
7 = 0'2530 + 1899, error only slightly less than 7.
Result = No correlation shewn.

(3) Correlation between the amount of haemoglobin and specific
gravity—
7 = 0'9950 + O'0049.

Result = Strong correlation.

Tas ie showing the mean specific gravity value and amount of haemoglobin in different animals
infected with trypanosomes.

Mean specific Mean | No. of
Animals | gravity of blood | haemoglobin observations Remarks
value :
Guinea-pig 1. T. gambtense ... 1039°8 65°99 % | 26
ze Zs 5 a 1048-7 82-9 % 32
” 3- » oe 104373 761 % | 39
ds 1. T. rbodesiense... 1050 833% | 19
* Zs a aoe 1046-25 856% | 8
. 3. %» vee 1047°65 79°7 % | 19
29 4. %» oe 1O51*2 962% | 4
Rat 1. T. rbodesiense... 1050 87:38 % 7
” 2. ” ooo 1047°65 77°5 % 2
3- sf ae 1048-7 81-2 % | 4
+ 4- ‘> asd 1044-1 729 % 12
5 ce + ain 1060 110 % | I On the day of
inoculation
Total 12 Total n73

129
I. PARASITES AND LEUCOCYTES

From 224 observations on twenty-seven animals no correlation
was found between the number of parasites and leucocytes per mm.#
in animals infected with 7. gambiense and T. rhodesiense. The
mean value of leucocytes per mm.* in infected guinea-pigs and rats
was found to be 11,700 and 33,800 respectively.

From twenty-four observations on two control animals the mean
leucocyte value per mm.* was found to be 9,344 in a normal guinea-
pig weighing 800 grammes and 18,863 ina rat weighing 215 grammes.

The normal value of leucocytes per mm.* in guinea-pigs and rats
varies according to the age and weight. Leucocytes appear to be
abundant—(a) during the incubation period ; (4) when parasites have
temporarily disappeared ; (c) towards the end of infection. Leuco-
cytic values in infected animals may vary for the following
reasons :—(i) Owing to osmotic disturbances between the tissue fluids
and lymph and blood, dilution and concentration of the blood
plasma giving rise respectively to apparent leucopenia or leuco-
cytosis. *(11) It is probable where the infection is lymphatic that this
may give rise to what may be called ‘ passive lymphocytosis’; for
when the leucocytes appeared to be abundant, the majority of them
were lymphocytes. But whether this was an ‘active’ or ‘ passive’
lymphocytosis remains to be seen.

II. PARASITES AND THE COAGULATION TIME OF BLOOD

From 118 observations on twenty-three infected animals no
correlation was found between the number of parasites and the
coagulation time of blood, The mean value of coagulation time in
infected guinea-pigs and rats was found to be three minutes eleven
seconds and five minutes one second respectively.

From twenty-four observations on two control animals the mean
coagulation time was found to be four minutes fourteen seconds in
guinea-pigs, and three minutes thirty-six seconds in rats.

III. PARASITES AND HAEMOGLOBIN

From 291 observations in thirty-five infected animals no
correlation was found between the number of parasites and the

* Lazarus Barlow. Experimental or General Pathology, 1904, p. 154.

130

percentage of haemoglobin. The mean value of Hgb. in infected
guinea-pigs and rats was found to be 81 per cent. and 80 per cent.
respectively.

From fifty-one observations in eleven control animals it was
found that the haemoglobin percentage was rarely below 95 per
cent. In the majority of observations it was between 100 per cent.
and 120 per cent.

In animals infected with T. gambiense and T. rhodesiense there
is a fall in the haemoglobin percentage, but it is not of a marked
degree, and further it is irregular.

IV. PARASITES AND THE SPECIFIC GRAVITY OF BLOOD

The correlation figures of 173 observations on twelve infected
animals have been given earlier.

From forty-two observations on sixteen control animals it was
found that there is a fall in the specific gravity of the blood in
animals infected with 7. cambzense and T. rhodesiense. .

In the majority of the control observations it was found that the
specific gravity value was always ‘a little above’ or ‘ far above’ 1060
mark. It was rarely as low as 1050.

It would be natural to ascribe the cause of the fall in specific
gravity to the number of trypanosomes in blood. However, this
does not appear to be the case. There is no correlation whatsoever
between the number of parasites and the specific gravity values.
Consequently one has to look for a cause which is intimately related
to the trypanosomes. Such a cause may be the toxins* of trypano-
somes. The liberation of these products is under the influence of
diverse circumstances, unknown at present. Hence there is no direct
clinical method of demonstrating a possible correlation between
toxins and specific gravity.

If the liberation of toxin is irregular it would account for the
fall in specific gravity, which is irregular also.

As the density of the blood depends on the intra- cogeuactien
haemoglobin and the blood plasma, and as there is no evidence at

* Used in the sense of product or products of metabolism, disintegration of
trypanosomes or the liberation of their toxin.

131

present of haemolysis zz vivo in trypanosomiasis, consequently the
fall is probably due to dilution of the blood plasma.*

There exists, however, a strong relation between the specific
gravity value and haemoglobin percentage under normal conditions. t
So strong is this ratio that the specific gravity value can be estimated
approximately by determining the haemoglobin _ percentage.
Consequently one might infer the fall in specific gravity is due to
the fall of haemoglobin. No doubt a strong correlation exists
between them in experimental trypanosomiasis. But from
Hammerschlag’s tablet on specific gravity and Hgb. percentage, one
sees that there is a greater fall in specific gravity value than could
be accounted for by the haemoglobin percentage. | Unless, then,
dilution of blood plasma occurs, an explanation is very difficult.

Finally we may note that oedema is a characteristic of human
trypanosomiasis. This oedema is not due to those causes which
produce oedema in cardiac, renal, pernicious anaemia and cachectic
conditions, such lesions are not pathognomonic of human trypano-
some infections. The oedema in the latter is fairly early in its
onset. In experimental observations it was noticed that the specific
gravity value fell from 1,040 to 1,030 when the animals were in the
incubation period and when they were showing few trypanosomes
(1 to 90 per mm.?).

We may conclude then : —

(1) That the number of trypanosomes in the peripheral blood is
not responsible for the fall in specific gravity value.
(2) That the probable cause of the fall is a product, toxic in

nature.

(3) That this toxic product damages the osmotic membranes and
increases their permeability to the tissue fluids, whereby the blood
plasma gets diluted.

(4) That the dilute nature of the toxic blood plasma facilitates
the onset of oedema in human trypanosomiasis.

* For a full discussion of this subject, see Lazarus Barlow, Experimental
Pathology, 1904, pp. 189-225 and 692-709.

+ Cabot, Clinical Examination of Blood, 1897, p. 31.

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Volume V August, i911 No. 2

ANNALS

OF

TROPICAL MEDICINE AND
PARASITOLOGY

ISSUED BY

| fae, LIVERPOOL SCHOOL OF TROPICAL MEDICINE

Editor

Prorgssor Sir RONALD ROSS, Major I.M.S. (Rer.), D.P.H., F.R.C.S.,
Sey LL.D; FE. RiSg Kic.B:

_ In Collaboration with

J. W. W. STEPHENS, M.D., Canras., D.P.H.
R. NEWSTEAD, M.Sc., A.L.S., F.E.S., Flon. F.R.H.S.
J. L. TODD, B.A., M.D., C.M. McGill, D.Sc., Liv.
H. WOLFERSTAN THOMAS, M.D., C.M. McGill.

ANTON BREINL, M.U.Dr.

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ed ‘ fy L 4i a -

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an :

C. Tinling & Co., Ltd. .
Printers to the University Press of Liverpool —
53 Victoria Street

= me gsie flicg By po Rey —
MAD LM 2AMORE Atos
- 3M Se Pe

IN MEMORIAM

Professor Sir RUBERT BOYCE, F.R.S.

We deeply regret to record the death of Sir Rubert Boyce, Dean
of the Liverpool School of Tropical Medicine, and one of the editors
of these Annals. His death, from cerebral haemorrhage, occurred
on Friday, June 16th, 1911, at the early age of forty-eight. In
1900 he suffered from a severe attack of hemiplegia, from which it
was hardly expected he would recover. Not only did he regain
comparative health, but continued to work with the same ardent
energy so characteristic of him, and even after a minor attack some
weeks before the last fatal one, his indomitable spirit would not be
checked. His manifold activities in Liverpool, especially in helping
to found the University, are well known to all in this city, and
his spirit of progress found wide scope for its play when Mr.
Chamberlain, at the Colonial Office, first expounded in practical
form the idea of tropical schools for the instruction of medical
officers in the Tropics, and for the study of tropical diseases. Sir
Rubert at once threw himself with unbounded confidence into the
new movement, and, together with Sir Alfred Jones, established
the Liverpool School of Tropical Medicine in 1898. It does not
become us here to point out what part this School has played in the
history of tropical medicine. Sir Rubert was not content with
merely starting the movement in Liverpool, but with unremitting
interest and zeal devoted the remainder of his short life to tropical
medicine. His foresight was remarkable; and to quote only one
instance, it was to his eager persistence and untiring effort that we
owe the two professorial chairs of Tropical Medicine and
Entomology associated with this School. Latterly, the subject of
yellow fever more particularly claimed his attention, and in 1905,
at the invitation of the American Government, he visited New
Orleans to study the epidemic there, and he also visited British
Honduras. In 1907 he again set out for the West Indies for the
same purpose, and finally, in 1910, to West Africa. The results

134

of his observations have been published in several official reports ;
but in order that the public might be interested and instructed in
sanitation, he published three works—‘ Mosquito or Man,’ 1909;
‘Health Progress and Administration in the West Indies,’ 1910;
‘Yellow Fever and its Prevention,’ 1911—in popular form, which
had an immediate and great success.

We need not discuss here his views on yellow fever in West
Africa. Whether they be finally accepted or no, certain it is that
he focussed attention on the subject in a way which had never been
done before, and even before his death practical action was being
taken by the Home Government based on his recommendations.
There are many in the Tropics who will mourn the loss to the

Empire; there are many who will miss his most cheery optimism ;

there are many who will never know what they have owed to him.
For ourselves we must express our deep grief at the loss of a chief
of amazing energy, of magnetic inspiration, and beyond everything,
one who was a devoted friend. Vale.

135

NOTE “ON “TROPICAL “DISEASES IN
SOUTHERN ITALY

BY

PROFESSOR UMBERTO GABBI, OF ROME.

(Received for publication 26 March, 1911)

For several years clinicians of Sicilian Universities (Gabbi,
Giuffré, Jemma) have called the attention of practising physicians
and of the Government to certain diseases, as yet unknown, or
very little known, which belong to the great chapter of tropical
pathology. The first, which Gabbi, Giuffre and their pupils made
known, was Medzterranean fever, which, designated in Palermo by
Federici as ‘febbre miliare,’ and by Tomaselli in Catania, as
‘febbre continua sudorale epidemica,’ was _ bacteriologically
determined by my researches and by those of my pupils, and
confirmed, principally, by the subsequent ones of Trambusti,
Pollaci and Pulvirenti.

In Naples it had, from 1872, in which year it was discovered,
twelve different designations; and only after my communication to
the Medical Congress in Rome, in 1906, where, in consequence of
a controversy between myself and Castellino, of Naples, aroused by
my decisive affirmation that the ‘ febricola,’ or ‘Naples fever,’ was
nothing else than Mediterranean fever, Arnaldo Cantami, junr.,
undertook bacteriological researches, by which it was proved by
Wright’s sero-diagnostic method, and by blood culture, that the
germ producing the infection was Micrococcus melitensis.

In 1901 Zando and Tiberti, in Florence, had isolated a
micro-organism, which they identified as that of Bruce; in 1904 it was
isolated by Professor Carbone in Pisa, from a patient coming from
Catania, and who died inthe medical clinic of Professor Queirolo;
in 1905, by Cippitelli in Rome. After 1906 the question was much
more seriously studied, and, from 1906 to 1gio it was found that
Mediterranean fever was widely diffused in Sicily and Southern
Italy, and is to be found also in Central Italy, especially in the
provinces of Leghorn, Lucca and Florence; and in Upper Italy

136

(Bologna, Padua, Milan). During the same period (1906-1909) I
have, with my pupils, made examinations of goats and have
succeeded in demonstrating that amongst us, in Sicily and in
Southern Italy, goats are infected, and not only those imported
from Malta, but both those of our own country, and also the crossed
ones. An approximate estimate, which my pupils are now making,
by means of the milk test, in Sicily and Southern Italy, proves the
high average of the infection (from 3 to 17 per cent.) in the goats
which furnish milk to these populations, as they make use, almost
exclusively, of goats’ milk.

The symptoms which Bruce’s septicaemia present among us are
not so grave as those in Malta, and neither so extraordinary as
those which our French colleagues describe in cases which, for two
years, they have discovered in various departments of Southern
France, and also in Paris.

Another disease, believed to be typically tropical, viz.,
Kala-azay was demonstrated five years ago among us by Pianese
of Naples. He announced in 1905 at the Congress of Pathology in
Rome, that he had found parasites in the spleen of children
suffering from Anaemia splenica pseudoleucaemica, discovered by
Professor Cardarelli in 1880, and afterwards designated by
Fede under the name of Anaemia splenica infettiva. Two varieties
were distinguished, one with fever and one without. Pianese found
Leishmania, sp., in the first, and, in a paper published more than
three years and a half afterwards, agreed with Nicolle’s
observations, considering it to be a species of Leishmania, and on
morphological grounds named it Leishmania infantum, proposing
to change also the name of the disease to Anaemia infantum a
Leishmania, Pianese. In 1907, during a course of clinical lectures
to practising physicians (of Sicily and Calabria), I stated the
existence of Kala-azar in Sicily; and, in 1908, four months after
Pianese’s publication, I communicated twelve cases of Kala-azar,
observed in Messina, Calabria and the Lipari Islands (two with
Leishmania in the spleen), and afterwards my publications
led to a full series of studies, which demonstrated how, in reality,
the febrile variety of Anaemia splenica infantum is nothing else
than Kala-azar. Contrary to Pianese’s statements I and my scholars
demonstrated : —

137

1. That even youths and adults can be affected by Kala-azar,
though very seldom.

2. That the disease appears at the beginning of Spring.

3. That it is greatly diffused in littoral towns of the South.

4. That it affected principally the lower classes.

Studies upon the agent of transmission of Leéshmania are being
actively pursued, and, besides the Czmex Jlectularius (Patton,
Rogers), and Conorrhinus rubrofasciatus (Donovan), Basile
implicates also Pulex serraticeps. At the same time that I discovered
Kala-azar in Sicily and Calabria, Basile found Leishmania sp. in
the dog.

A year afterwards, together with Dr. Lacava, we found cases
of Oriental sore in Bovalino (Calabria) and Bordonaro (Messina),
and others have observed them in various countries of Reggio
Calabria (Palozzi, Bova), and in Catania and Palermo. I had the
luck to observe the first example of multiple Oriental sore in a
woman of a district near Messina (Tremestier1).

Besides these, typically tropical, sub-tropical, and endemic
diseases, I found in Messina, in 1907, and described an epidemic
of Dengue fever (150 cases) imported by merchants of Tripoli
(Africa), and afterwards little epidemics were observed in
Francavilla (Messina) and in Bovalino (Calabria).

In 1910 I have clinically recognised the ¢hree days’ fever or
Pappataci fever in an epidemic which attacked more than 4,000
persons, and which diffused itself along the Ionic Coast of Calabria
on its eastern side. Examples of a summer fever, clinically corre-
sponding to the fever caused by Phlebotomus had been for some
years described by Italian military doctors, but the identity with
the three days’ fever was not yet declared by them before my
publications. I think this will prove to be a summer disease which
will regularly appear in Upper and Lower Italy; and, if the
physicians of Central Italy would pay attention to it they would
certainly discover it also.

I have observed and described undoubted cases of climatic
bubo; Dr. Lacava has recently found in Calabria cases of Ulcum
tropicum and of Myzasis ocularis.

The above-mentioned diseases are to be found likewise in North
Africa, and this community of diseases is explicable if we consider

138

that Arabs ruled for years in Sicily and Calabria; that commercial
intercourse was, and is, ever more active between Sicily and
Calabria and the Italian colonies of North Africa; that, moreover,
intermarriage has taken place between Italians and women of
African colonies. These data explain this area of common
pathology, and so much the more when we consider that the climate
and vegetation of North Africa and Sicily differ so very little,
permitting of the existence of similar parasites, and, again, the
habits of the classes which are the most affected by the described
diseases are very alike.

These studies, begun in the Medical Clinic of the University of
Messina, are now being continued, since the earthquake, in the
Medical Clinics of Professor Baccelli in Rome, with the aid of the
Government.

LITERATURE

‘ Studi intorno alle malattie tropicali della Calabria e Sicilia.’ Fasc. I, II, III.
‘ Malaria e malattie dei paesi caldi.’ Edited by Prof. U. Gabbi. Rome, 1911.

139

THE PAPATACI FLIES (PHLEBOTOMUS)
OF THE MALTESE ISLANDS’

BY

RAUNT Wome AD MISE yet. S?; “&:
(PLATES V—VII.)

(A report of the twenty-third Expedition of the Liverpool School of
Tropical Medicine.)

(Received for publication 15 May, 1911)

Acting under the instructions of the Liverpool School of
Tropical Medicine I proceeded to Malta on the 25th of June, 1910,
and stayed in the Island for a period of two months. The object
of this expedition was to investigate the problems connected with
the menace to health caused by the blood-sucking ‘ Papataci Flies’
of the genus Phlebotomus.t The greater part of my time was
devoted to searching for the breeding-places of these insects with
a view to devising practical prophylactic measures for the control
of the pest. Other phases relating chiefly to the bionomics of
Phlebotomus were also investigated; and attempts were made to
rear the insect from the egg.

On making a critica! examination of the material collected
during the first week of my visit, two distinct species (P. papatasii,
Scop., and P. perniciosus, n. sp.) were found to be almost equally
abundant; and examples of a third, though apparently rare, species
(P. minutus, Rond.) were subsequently taken. Since my return to
England, Captain P. J. Marett, R.A.M.C., has very generously
placed the whole of his collection of Maltese Papataci flies in my
hands for examination and report; and among the numerous
examples there were two specimens which have proved to be a
new and hitherto undescribed species (P. nigerrimus, n. sp.), SO
* Reprinted from the Bulletin of Entomological Research, Vol. il, pte 1, pps 47-78, 1911,

+ These insects are generally known to Englishmen as ‘Sand Flies.’

140

that altogether four distinct species of Phlebotomus are now known
to occur in the Maltese Islands.

These discoveries, though of much interest for the zoologist,
add considerably to the labours of those who are or may be engaged
in studying these insects more especially from a medical point
of view; as owing to the minute morphological differences which
exist between the females of these small midges the task of
separating the respective species, more especially the commoner ones,
is one which can be accomplished only after long and careful
microscopical examination and comparison.

Hitherto the only species recorded from Malta was the common
and widely distributed P. papatasiz; but judging from recent
experience, I have come to the conclusion that the almost equally
abundant P. perniczosus must have been seen, though not recognised,
by those who have been engaged in studying the bionomics of these
insects.

It is highly probable too, that examples of this species were
also used by those who conducted the transmission experiments, and
although one has no direct proof, it is possible that P. perniczosus,
like its near relative (P. papatasiz), may also act as a carrier of
Papataci fever.

THE SEARCH FOR BREEDING PLACES OF PHLEBOTOMUS

The results of my unremitting search for the breeding places of
these insects were that I secured two larvae from the crevices of the
loose rock in the ‘ caves’ or catacombs at Notabile near the centre of
Malta; thereby confirming the discoveries made by Captain Marett®*
a month or so previously. Had my searches been continued in the
same kind of habitat I have reason to believe that a few more larvae
would have been secured, but having trained the eye so as to
facilitate the finding of so minute an object the more readily on any
future occasion, I proceeded in other directions, and searched
innumerable places that were thought likely to form suitable
breeding grounds for these insects, unfortunately without
discovering either eggs, larvae or pupae; disappointment met me at

* Such numbers refer to the bibliography on page 181.

141

every turn and I am therefore unable to add anything that is new
or noteworthy regarding the breeding places of Phlebotomus
papatasii or any of the allied species.

In addition to the cave from which larvae were secured I also
inspected the places in which both larvae and pupae had been found
by Captain Marett; these were the cave at Gozo, the embankment
forming part of the Cottonera Lines, and the stone wall in Captain
Marett’s garden, which he had thoroughly explored and had also
kept under close and constant observation for a considerable time.
In all of these places the conditions were very similar, if not almost
identical.

In the caves the larvae occurred in the crevices and fissures
beneath the loose rock amongst the damp earth, etc., at some
distance from the surface, and I was informed that those which were
found in the stone wall, occurred low down near the foundations,
well within the centre, and attached chiefly to the under surface of
the stones; while those from the Cottonera embankment were found
at some considerable distance from the surface, where the stones
were damp’*.

The crevices between the loose rock in the caves were often found
partly filled with soil rich in organic remains. In the caves at
Notabile, in which the larvae were found, the soil had for the most
part been reconstituted by the burrowing larvae of various insects
and other allied animals. To such an extent had this been done in
some instances that quite 50 per cent. of the deposit consisted of
the rejectamenta of insects, woodlice (Oxzscus sp.), etc. Here and
there were found also large numbers of the empty pupae of
Stomoxys calcitrans and the pupae of other Muscid flies whose
larvae had matured in the stable refuse which had been stored in the
cave for agricultural purposes.

In all of these places the conditions were practically the same,
the three main factors being: (a) the presence of organic matter ;
(6) moisture, but not in excess ; and (c) the absence of light.

The principal places which were searched as being likely to
afford suitable breeding-grounds for Papataci flies were as follows:
The main sewers and ventilating shafts in various parts of the city
of Valetta; drains of various kinds, cesspools and latrines in many
places; cellars and prison cells in the Police Court; sewage works,

142

and the dark damp buildings used by the Customs as bonded
stores; refuse of all kinds, especially such as occurred in dark damp
places; the refuse ‘tips,’ and the roots of plants along the coast,
especially in localities which were known to be badly infested with
the flies; the decayed stems of the Prickly Pear (Opuntia sp.);
collections of stone and rock in shady places in gardens and
elsewhere; freshly excavated earth and rock; the empty shells of
molluscs (chiefly Helzx sp.) found in caves and other sheltered
situations ; refuse in caves which were used as stables for oxen and
other domesticated animals, and the faecal matter which was found
in those which had been used as latrines; the roots of trees,
ivy and flowering plants which were kept moistened by constant
supplies of water, also those growing in the rock fissures; the
accumulation of leaves in damp places, etc.; litter from rabbit-
hutches, consisting chiefly of faecal matter, especially at Casa
Leoni, where the adult flies were invariably found associated with
these animals.

Although one failed to discover either larvae or pupae in any
of these situations, it does not prove conclusively, in my opinion,
that these insects do not breed in some of them, especially as
Grassi? has found that in Italy the larvae of P. papatasz live in
dark damp spots amidst all kinds of refuse in underground places
such as cellars, and particularly on the sides of drains which are
kept moist by occasional splashes of dirty water.

Other investigators in Malta have met with results similar to my
own. Lieut.-Colonel C. Birt, R.A.M.C.?, who collected the most
varied materials, states that he did not succeed in detecting the ova
or larvae in any of the samples, ‘nor has the adult P. papatasz ever
hatched out from larvae which might have been hidden in the
materials.’ Captain Marett® has also made extensive search for
the larvae and pupae in similar places and in similar materials, and
has failed to find a single example of the insect in any of its stages.
In so far therefore as our present knowledge is concerned, the
only conclusion which can be drawn from the investigations in Malta
is that the chief breeding-places of the Papataci flies (P. papatasiz
and P. perniciosus) are the crevices between the loose rocks in caves,
stone walls, bastions and similar situations.

The task of finding such minute objects as either the larvae or

143

pupae of these flies is, however, very great; of the two, the larvae
are perhaps the more conspicuous, but these have the remarkable
habit of flicking themselves from off the surface of the stone or
other objects when exposed to light, and in this way numbers may
escape detection even under the most practised eye. The pupae are
the more difficult to detect, as, apart from their minute size, the
colour so exactly harmonises with the colour of the rock to which
they are attached that they are rendered almost invisible, and when
detected appear onlyas a naturally formed granular projection on
the surface of the stone. In every sense, therefore, they are highly
protective forms, and numbers must necessarily escape detection,
more especially when artificial light has to be employed in searching
for them. Bearing these facts in mind, large quantities of
detritus were collected from many and varied sources so that it
could be examined under more favourable conditions, but in no
single instance were these insects found in either of their
preliminary stages, though a lens of low magnification was almost
invariably employed in searching for them. Quantities of the
detritus were also kept in large vessels in the hope that adult flies
might be successfully reared from it; in this again complete failure
was the result. As to the detection of the ova in a state of nature
I believe this to be a practical impossibility, as when laid upon
dark substances they become absolutely invisible and can be
detected only by the aid of a microscope. Even when laid in
captivity in confined areas they are most difficult to detect, and
under the most favourable conditions can be seen only when laid
upon colourless or transparent surfaces such as white paper or the
surface of a glass tube.

HABITS AND OCCURRENCE OF THE ADULT FLIES

Though so evasive in their early stages, the adult flies may be
found almost everywhere throughout the Island in favourable
situations or localities. They outnumber the mosquitos, and the
females may be included among the most vicious of all the
blood-sucking Arthropods. They are distinctly ‘domestic’ in their
habits and may be considered among the most detestable of all
man’s ‘uninvited guests.’ It is a curious fact, however, that they

144

have their likes and dislikes both in regard to hosts and habitats.
I can fortunately place myself among the small numbers of those
who have proved immune to the bites of these blood-sucking pests ;
or at least I have never consciously experienced the effect of their
bites, any more than I have in the case of Pulex zrritans. And this
is all the more extraordinary because fresh comers to the Island,
especially children, generally suffer torture from the bites of these
insects, and many cases are admitted to the hospitals through the
infection which the Papataci flies are known to convey. To say the
least, they are an intolerable nuisance in every part of the world in
which they are known to occur. Man is evidently not the only
vertebrate which these insects attack, as examples were frequently
found which had filled themselves to repletion with the blood of
the domesticated rabbit; so that it is evident that they are not
entirely dependent upon man for food, and the probabilities are
that they subsist and flourish on any of the warm-blooded animals
when man is not available.

My experience with regard to the favoured haunts of these flies
is almost precisely the same as that of other investigators. In
certain parts of the island they were found to be abundant, while in
others, for some unaccountable reason, they occurred very sparingly,
though the conditions necessary for breeding purposes, especially
stone walls, abounded everywhere. In badly infested regions, too,
they favoured certain dwellings much more than others; of two
houses occupying the same aspect and surroundings, or a section
of the same block or street, one was often found to be infested while
the other was rarely visited. It was noted also that there was a
marked domiciliary distribution in many houses. Bedrooms on the
first floor, especially those occupying a position on the lee or
sheltered side of the house, were particularly favoured, while those
on the opposite side of the building were rarely visited; and rooms
at a greater elevation (second floor), which I had under close
observation for a considerable time, were only once found to contain
a single example.

The naval and military camps at Ghain-Tuffeiha afforded also
a remarkable instance of the local distribution of these flies, the
naval camp on one side of the plain being badly infested, while
the other and more extensive camp was said to be practically free

145

from the invasion of Phlebotomus. This remarkable localisation
was in all probability due to the fact that the naval camp was
bounded on one side by rocky ground and stone walls, affording
excellent breeding-grounds for the flies, while the military camp
was remote from such surroundings, and lying fully exposed in the
open plain.

At times also, when Papataci flies were literally swarming in
houses near the old bastion at Floriana, not a single individual was
discoverable in the city of Valetta, half a mile away. In this
instance also one may safely infer that the flies at Floriana were
breeding in close proximity, and it is highly probable that the
actual site was in the interstices between the masonry forming the
old fortifications, only a few yards distant from the dwellings.

The daylight retreats of these flies were often similar to those
in which they were found at night, providing always that there was
an absence of direct light. Thus in the dwelling-houses and
barracks, the flies were found at rest in the dark corners of the
rooms, under garments, behind pictures and in other similar places ;
but in nearly all cases they occurred in considerably smaller numbers
than at night, though there were one or two noted exceptions. In
one instance they could be found in considerable numbers in a
badly lighted bedroom at any time of the day, especially after a
still, damp night with a heavy sirocco. Odd examples were also
found in cellars and in the prison cells in the heart of Valetta; while
numbers could be found almost at any time in the small caves or
isolated catacombs at Notabile, and such retreats seemed to be one
of their favourite haunts during the day. In the early mornings,
shortly after daylight, examples of both sexes may frequently be
found inside the mosquito curtains, and after favourable nights
they sometimes get entrapped in large numbers by this means.
On the slightest disturbance the males may readily effect their
escape through the meshes of the net; but the females, which are
generally engorged with blood, are, under such conditions, much
more sluggish than at other times and may then be captured with
_ comparative ease, as they cannot escape through the net very
readily when the body is distended with food. In one or two
instances Papataci flies were dislodged from the interior of stone
walls by forcing tobacco smoke into the interstices; but one met

146

with such little success that this method was abandoned. Sections
of the lower portions of stone walls were also covered with chiffon
and carefully examined at intervals during the night, and although
the most favourable structures were selected for the purpose, and
areas 36 square feet in extent were most carefully covered, not a
single fly was entrapped by this method. This is all the more
strange seeing that Captain Marett has met with marked success
by adopting the plan even on a smaller scale. However this may
be, it is perfectly obvious that in the light of Captain Marett’s
experience stone walls, especially those from which the surface
‘pointing’ has fallen away in patches, leaving free access to the
interior, are the frequent and possibly the principal resorts of the
parent flies.

Atmospheric conditions have undoubtedly a marked effect upon
the flies. On still sirocco nights they take wing freely and occur
in dwellings in larger numbers under such conditions than at any
other time. On the other hand, when fresh cool breezes are blowing,
especially from the north-west, they are rarely seen; and it is
the testimony of everyone who has studied their habits that these
insects remain in their hidden and sheltered retreats and rarely
venture forth at such times. There is little wonder at this, as their
frail bodies and delicate wings are ill-suited for flight under such
conditions; moreover, it is a habit common to many members of
the same order; minute midges, in particular, are often seen to
swarm on still warm evenings, and rarely if ever assemble in
numbers under any other circumstances.

A general belief is held by the Maltese that certain kinds of
trees and shrubs (fig and loquat especially) form the principal
resorts of these insects, and many are also under the impression
that they breed either in the foliage or branches or in the fallen
and dead leayes which lie beneath them. There may of course be
a measure of truth in these theories; but we may at once dismiss
the statement that they breed in the trees. It is perfectly obvious,
however, that the presence of ornamental shrubs and fruit trees in
the walled-in gardens would afford them just the kind of shelter and
shade which they require, and would enable them in all probability
to travelthe more safely from their breeding-places to the house in
the immediate vicinity... It is just possible that rotting vegetation in

147

damp shady places, such as shrubberies, may form a breeding-place
also, but so far as our researches have extended up to the present
moment we have no evidence in support of this view. Considerable
attention was paid to searching such materials but with negative
results, as has already been stated. It is clearly evident moreover
that dry materials, whether in a state of decay or otherwise, do not
form a suitable breeding-place, especially dead leaves which may
accumulate on the surface of the ground beneath the trees; light
and dryness being both unsuitable conditions for the preliminary
stages of the Phlebotomus.

The characteristic attitude of Phlebotomus is portrayed on
Plates VI and VII. When at rest the wings slightly diverge and are
elevated at a considerable angle above the thorax and abdomen.
On the least disturbance the insects make short rapid flights,
almost invariably to the right or left, reminding one of the rapid
movements of a flea rather than those of a winged insect.
Occasionally, however, they will take long-continued flights, when
the course is more or less direct and distinctly midge-like. Their
movements on the wing can be followed with little difficulty in
daylight, but by artificial light it is almost impossible to do so for
more than a few seconds at a time.

Both sexes live but a short time in captivity, unless they are fed
upon human blood. Without this they will subsist on wet
blotting-paper or other damp materials, such as soil, fresh leaves,
etc. Under such conditions many examples survived for periods
varying from three to nine days though the majority died on the
third and fourth days, even although the females, in many
instances, had taken a meal of blood a few hours before they were
captured.

SEASONAL PREVALENCE

The adult insects were more or less prevalent during the whole
of my stay in the island (July, August, and the first week in
September). That the numbers fluctuated during this period has
already been mentioned, but this was apparently due, in a large
measure at least, to variations in temperature, humidity, and wind.
Relatively few Papataci flies occur before the middle of June, and

148

practically all observers of their habits informed me that they occur
most freely and are most troublesome during the hot, dry months of
the year. It is highly probable that successive broods are produced
during the summer months, but as the larval stage occupies
apparently a long period, the successive generations can be
produced only at extended intervals.

As to whether the larvae occur most frequently during the
summer remains to be seen. It is my impression, however, that
they may be found more abundantly in autumn and winter than at
any other season, and careful search should be made for them a
week or so after the adults have disappeared.

PROPHYLACTIC MEASURES

In consideration of the facts which have so far been brought to
light regarding the economy of P&lebotomus, it is clearly evident
that the task of suppressing these insects is an almost
insurmountable one. Had we to deal with insects as large and as
accessible as mosquitos, the adoption of prophylactic measures
would be comparatively easy, but owing to the extremely minute
size and almost flea-like habits of the adult insects, and the
enormous area over which the breeding-places may occur, we are
faced with a problem which is most difficult of solution.

As I was unable to devote any time to experimental work
bearing upon the control of these pests, the only course open to me
now is to suggest a few measures which may ameliorate the existing
conditions and lead to a reduction of the malady of which these
insects are transmitting agents. It seems to me, however, that the
only practical way of grappling with this question is to proceed
tentatively at first, and although I have discussed an extensive
field of operations which may be directed against these insects, I
would pin my faith rather to some of those measures which are
considered under the following headings. But in the first instance
it must be borne in mind that precautions against the bites of
blood-sucking insects, though feasible to intelligent and well-to-do
persons, are not as a rule employed by the mass of the people. Yet
any prophylactic measures which are calculated to diminish the
infection, even in a small degree, should be seriously and
persistently employed.

q

149

Repellents.—I had no opportunity of demonstrating the value
of these by experiment owing to my immunity from the bites of
these insects, but I was assured that several good formulae were in
general use, though proprietary preparations were rarely employed.
Judging by the testimony of those who had used such deterrents,
one of the best was that which was prescribed by Major Crawford,
R.A.M.C., and I am extremely indebted to him for giving me
permission to embody it in this report. It is composed of the
following ingredients : —

Ol. Anisi, one drachm.
Ol. Eucalypti, one drachm.
Ol. Terebenth, half a drachm.

Ung. Acid Borac, one ounce.

Spraying with repellents.—The least objectionable of these, and
at the same time one of the most effective, is formalin. The dark
portions and angles of sleeping apartments should be sprayed with
a I per cent. solution of this substance every day during the season
in which the flies are prevalent; a fine spraying apparatus is
necessary for its application, and an excessive amount must not be
applied. It is considered an excellent plan also to spray the
mosquito curtains regularly every day towards sunset; nets thus
treated are claimed to repel the attacks of these insects.

Fumigation.—There are several substances which are employed
as fumigants for the destruction of insects, but I fail to see the
practical utility of employing such means for the destruction of
Papataci flies in Malta or elsewhere.

Light.-—Daylight is a most important factor in driving away
these insects from man’s dwelling-places, and directly a flood of
light is admitted to a room in which Papataci flies may be present,
they immediately seek places of concealment behind garments or
draperies and pictures, or other furniture which may be suspended
from the walls or placed in dark corners. It is important, therefore,
that as much light should be admitted into the rooms as is possible,
and this can easily be done either in the early morning or evening,
or when the windows are lying in shadow.

Beds should be arranged in the best-lighted portions of the

150

room, and on no account should children’s cots be placed in
out-of-the-way corners in deep shadow. Decorative drapery in
such apartments should be abolished, and the walls rendered as
free from pictures and other furniture as possible.

Artificial light does not, unfortunately, act as a repellent; on
the contrary, it would appear to serve as an attraction for these
insects, as it is well known to do with other groups belonging to
widely different orders.

Artificial air movement.—In India, if not.also in other parts of
the tropics, it is a recognised fact that punkahs and fans will repel
the attacks of mosquitos if continuously and properly employed.
It seems to me, therefore, that if a similar method could be applied
in Malta, we should be able to dispense with almost every other
form of prophylaxis which is discussed in this report. As it has
been abundantly proved that Papataci flies do not take wing
when the slightest breezes are blowing, one may safely infer that
they would not face a strong current of air such as would be
produced by either fans or punkahs. It is unlikely that the latter
will ever be employed in Malta, but it is my firm belief that if
electric fans were fitted so as produce a current of air in the direction
of the window in sleeping apartments, that very few, if any, of the
flies would be able to pass through the open window into the room
beyond. I venture to recommend, therefore, that this method be
put to the test, and if found to give satisfactory results, that it be
employed in all cases where the cost of running such an apparatus is
not a serious consideration.

Traps.—lf a modified form of the biscuit-box trap, such as is
used for capturing mosquitos, were fixed high up in the dark
corners and angles of the rooms, I believe that numbers of Papataci
flies would be entrapped. The trap should be made in the form of
a corner-cupboard in miniature, and should measure about eighteen
inches in length; the basal portion should be left open, and the
interior should be lined with dark cloth or similar material. These
should be examined daily and the flies killed with ammonia fumes.

Nets.—The use of ordinary mosquito nets is of no avail against
the bites of these pests, as they readily pass through the meshes,
and attack persons just as freely as if nets were not used; but if
they could be rendered repulsive to the insects by spraying them

151

with formol or other repellents, as has been suggested, so much the
better; but experiments in this direction must be conducted before
we can say definitely that such a method would prove effectual.
Fine nets made of strong chiffon or other similar material would
undoubtedly prevent the approach of these flies, but the use of
such nets would render sleeping almost impossible in the hot
weather unless electric fans were used at the same time. If such
preventive measures as these could be employed to the complete
satisfaction and comfort of patients in hospitals, especially those
suffering from the Papataci fever, or to the community in general
we shall have succeeded in devising an excellent prophylactic
measure. If a net of this type is used, it should have a strip of
calico about two and a half feet in width stitched all round the
bettom, so that at least twelve inches of it extends above the
bedding, the remainder to be tucked in under the mattress. The
use of this is obvious; the strip above the bedding would prevent
the flies from biting any portion of the body which might be
brought into contact with it, and the lower portion of it would
stand the strain of ‘tucking-in,’ and consequently last for a very
much longer time than such flimsy material as chiffon.

Destruction of breeding-grounds.— As to the operations
necessary for reducing the number of breeding-places, it is perfectly
obvious that we can never expect to be able to deal with these in
any of the rural districts, owing to the fact that the fields and
roads extending over the whole of the country are bounded by
stone walls, and elsewhere there are fissured rocks, caves, and
other suitable places which afford just the right conditions
necessary for the breeding of Papataci flies. On the other hand,
we may reasonably hope to reduce them in the principal centres of
population, if persistent efforts are made to accomplish this, and
if financial considerations do not prohibit the employment of
such methods as are herein suggested. If it should be considered
advisable to carry out any section of this part of the propaganda,
one of the smallest and most isolated of the infested areas should
be chosen as an experimental ground, and an officer who is
thoroughly acquainted with the habits of the insects should be
appointed to, direct the operations. If loose rubble walls exist in
the immediate neighbourhood of the selected area, these should be

152

either demolished and the materials removed, or they should be
completely covered with a thick layer of cement.

If such a type of wall exists as has the jointings partly filled
with plaster (‘ pozzolani’), then all openings and fissures should be
carefully filled in with cement, so that no holes are left for the
ingress or egress of the flies, remembering always that a crevice
sufficiently large to admit a flea will also afford ample space for
the admission of the fly.

If it should be found necessary to replace the old walls with
new ones, it is imperative that these should be built of solid
masonry to a height of at least two feet above the level of the soil
on either side, as it is the lower portions of the walls that are,
according to Captain Marett’s experience, selected as breeding-
places; but it would be better, in my opinion, to make all new
walls of solid masonry from the foundation to the topmost course
or layer; and if the old walls could be substituted by any other
form of boundary, so much the better.

There are also other kinds of walls which may have to be dealt
with, and these are they which form the old bastions and other
extensive fortifications at Cottonera and elsewhere. In cases where
such structures are backed with rubble and finally protected with
loose rock, it would be a comparatively easy task to prevent the
egress of the flies through such loose material by breaking or
pulverising it, or by covering it with soil; but unfortunately the
question of pointing the Ashlar work forming the facings of the
bastions and curtains presents not only a serious financial difficulty,
but a task which could be accomplished only by a huge army of
men; and in consideration of these facts it seems to me that in the
present stage of our inquiry such a method of procedure would be
extremely unwise and irrational. For the time being, therefore, I
should strongly advise that in selecting the experimental area a site
should be chosen which is as remote from the old fortifications or
similar structures as is possible.

Though there is no evidence which will lead us to believe that
Papataci flies breed in the cellars and drains in Malta, at the same
time we must not lose sight of the fact that Grassi3, as has already
been stated, has found larvae of P. papatasiz in such places. It is
highly probable, therefore, that this species breeds in similar

153

habitats in Malta also; but it is impossible without more study to
make any definite statement on the point. Taking all the facts
into consideration, therefore, I consider that the only really
practical prophylactic measures which can at present be taken are
those which are considered as precautionary against the bites of
these insects. It is perfectly obvious, moreover, that any operations
which will not bring about an almost complete destruction of the
breeding-grounds are not likely to make an appreciable reduction
in the numbers of these insects.

SYNONOMY, AFFINITIES, AND MORPHOLOGY OF THE
GENUS PHLEBOTOMUS

Though the differential characters of this genus have been given
by several authors, and Grassi* has published an elaborate memoir
on the morphology and biology of Phlebotomus papatasi, I

consider that this report would be incomplete without giving some

~

details concerning the morphology of these insects; all the more so
because Grassi’s paper, in Italian, is now very difficult to obtain and
also a very costly publication, in fact the price (41 Ios.) for so
small a work, is practically prohibitive, and certainly not within
the reach of students in general.

I do not claim, however, to treat of this phase of the subject in
an exhaustive way, but rather to point out the salient characters of
these insects in a measure that may be helpful both to the medical
profession and to the zoologist.

The genus Phlebotomus was established by Rondani in 1840,
though the species for which it was founded had been placed by
various authorities in other genera, such as Szbzo (Scopoli, 1786),
Musca (Gmelin, 1788-1793), Cznzphes (Costa, 1840). But as
Rondani’s name is now generally accepted, one need not go into
further details regarding the nomenclature and synonyms of
Phlebotomus. The taxonomic position of this genus is with the
family PSYCHODIDAE, and it is included in the sub-family
PHLEBOTOMINAE. All the members of this family are small
Nemocerous insects characterised by the possession of relatively
large wings which are clothed with either scales or hairs; and one of
the most familiar representatives, and one also which is widely

154

distributed and nearly related to Phlebotomus, is the genus
Psychoda (sub-family PSYCHODINAE) the members of which are
known generally to Englishmen as ‘ Moth-flies’ or ‘Owl-midges.’
The short diagnosis which follows will serve at once to distinguish
Phlebotomus from any of the allied genera in the PHLEBOTOMINAE
and also from the midges belonging to the PSYCHODINAE.

Genus PHLEBOTOMUS, Rondani

Mouth formed for piercing and sucking; palpi of five segments;
antennae long, filiform and composed normally of sixteen segments ;
wings hairy, narrow, second longitudinal vein twice forked,
cross-veins placed near the basal fourth of the wing; body clothed
with hairs; sexual dimorphism distinct.

The larva (Pl. V, figs. 7-8) is characterised by its caterpillar-like
form (eruciform); by the presence of two pairs of long caudal
bristles, which may equal the length of the body; and by the
absence of true legs.

The pupa (Pl. V, fig. 12) 1s obtectate, and may be recognised by
the presence of the larval skin which invariably remains attached
to the last two segments of the abdomen. It should be borne in
mind, however, that the partial retention of the larval skin by the
pupa, is not peculiar to the genus Phlebotomus, as Speiser® has
shown that the larval skin of Helea (Forcipomyia) regulus, Winn.,
one of the members of the CHIRONOMIDAE, also remains attached
to the anal segments of the pupa. The larva of this genus does
not, however, possess the long caudal bristles which are so

characteristic of PZlebotomus, though in other ways it is not unlike
the latter.

EXTERNAL MORPHOLOGY

Head (figs. 1 and 9) somewhat elongated, but distinctly
narrowed at the nape, vertex clothed with long hairs; clypeus large
and also clothed with hairs on the upper surface. Eyes large and
intensely black.

Antennae (fig. 2) very long and slender, and in all of the
Maltese species consisting of sixteen segments; the first and second
segments forming the scape are short and stout, the second one

E55

being somewhat spheroid in shape; the third is much the longest and
of uniform width throughout ; the remaining segments are gradually
swollen proximally, especially the terminal ones; all are clothed
with hairs; those arising from the swollen portions being
much the longest and considerably longer than the individual
segment to which they are attached. In all of the Maltese species
there are also present on several of the segments, and in both sexes,
a patr of relatively large geniculated spines (fig. 2). These curious

--- ,
---- Se

So eee =o pa/ 7

pro.

Fic. 1. --Head of Phlebotomus papatasit; ant, antenna; ¢, eye; cl, clypeus;
pal, palpus; pro, proboscis.

appendages are rendered practically invisible when the segment to
which they are attached is mounted so that a dorso-ventral aspect
is presented under the lens of the microscope; and for this reason
apparently they have been hitherto overlooked by all the students
of this genus of insects. It is true that Grassi? (p. 12) has noted
that ‘here and there one can observe a short hair curved and
relatively thick’; but that he failed to recognise the true
character and arrangement of these spines is perfectly clear. Now
that they have been discovered it is highly probable that they will
be found to exist in the majority of species, if not in all, and may
I think be considered of generic importance. Annandale, in his
description of the genus Brvunettia, a new Psychodid discovered in
Southern India, refers to a similar character, but in this instance
the paired spines are somewhat S-shaped and relatively much
stouter than the corresponding spines in Phlebotomus. In the light
oi these discoveries, therefore, it is possible that similar spines may
be discovered in various other members of the same family, though

156

it is highly improbable that such structures will eventually be found
to exist in all of them.

Palpi (figs. 1 and 13).—These organs are generally said to be
composed of four segments, but there are undoubtedly five, and

Fic. 2.—Antenna of Phlebotomus papatastt.

this number may, I think, be considered common to all the members
of this genus. Annandale! has pointed out that ‘a minute basal

i537

joint can sometimes be distinguished in fresh specimens,’ but that
it is ‘often difficult to see and appears to be imperfectly separated
from the others.’ That the small basal segment is clearly
articulated to the second there can be no doubt, as it can be seen
quite distinctly when mounted so that it is not obscured by the
surrounding structures. All of the segments are clothed (in
P. papatasi at least) with variously formed scales, intermixed with
a few hairs. The scales on the first three segments are for the most
part very long and somewhat hair-like, those on the remaining
segments short and closely packed together. The fourth and fifth
segments, especially the latter, are distinctly but somewhat
irregularly annulated or ringed, a character which has also been
hitherto overlooked by former investigators. In life, when these
organs are at rest they are bent downwards and backwards at the
articulation of the third and fourth segments, so that the anterior
half of the palpus is folded back more or less upon the proximal
half; by this curious arrangement practically the whole of the
proboscis is covered or protected (fig. 1, fal. 1).

Proboscis (figs. 1 and 3).—Slightly shorter than the head,
inclusive of the clypeus; in form it is somewhat cylindrical and
slightly recurved distally. In the female it 1s composed of the
following parts: —The labium (fig. 3, 1b). This is much the largest
organ, and as far as one can judge by viewing it in optical section,
it almost completely embraces the labrum-epipharynx ; the proximal
half is sparsely clothed with lanceolate scales, and the first third is
markedly narrower than the rest; immediately in front of the dark
chitinous apodeme or sclerite is a curved row of long fine hairs;
the labella are scarcely broader than the widest portion in the
region of the apodeme, and are clothed with a number of fine and
rather long hairs. Zhe labrum-epipharynx (fig. 3, /b7) is relatively
narrow and the sides are parallel, but the apex is suddenly
attenuated the tip bluntly pointed, and the margins furnished
with a series of long spinose teeth set closely together
and numbering about twenty on either side; ventrally it is
deeply and broadly channelled but does not appear to possess
interlocking teeth or other structures. Zhe hypopharynx (fig. 3, hy)
is similar in width and general form to the labrum, _ but
tapers off much more gradually towards the end, and the marginal

158

spinose teeth are much shorter and placed so closely together as to
present a finely serrated edge; its upper surface is distinctly and
broadly concave or trough-like and the salivary duct which is small,
occupies a central position. The mandibles (fig. 3, md) are broad
and blade-like, and have the outer edges faintly serrated, the

Fic. 3.—Mouth-parts of Phlebotomus papatasit, 9; 1b, labium; /br, labrum-epipharynx ;
hy, hypopharynx; md, mandible; mx. maxilla.

The upper figure represents the labrum (2dr 1) and hypopharynx (hy 1); male, P.
papatasii, as seen in profile.
serrations being rather widely separated. When at rest they lie,
apparently, superimposed one over the other. The marillae
(fig. 3, mx) are much narrower than the mandibles, curved
transversely, and attached to a broad trough-shaped sclerite, not to

>_>"

B59)

a long slender stalk as Grassi has shown. One edge is provided
with five relatively large and widely separated teeth; the opposite
edge with smaller ones set closely together.

Thorax (fig. 8)—This consists largely of the meso-thoracic
division, the prothorax being represented by a very short extension
which can be seen more or less distinctly in examples which have
been macerated and mounted in Canada balsam. The scutellum
and post-scutellum are well developed and conspicuous in mounted
preparations.

Abdomen.—This is composed of ten segments, the last being
modified by the external genitalia. In the female the appendages
are simple, flattened, leaf-like structures, densely clothed with
hairs and arranged in two pairs (figs. 8 and 10). In the Maltese
species they are all so similar in structure as to afford no
diagnostic characters of importance. Annandale! states (p. 41) that
these organs ‘become distorted and shrivelled in dried specimens.’
These structures can, however, be restored by maceration in caustic
potash but the best results may be obtained by preserving the
specimens in alcohol.

External genitalia of the males.—These are large and complex
structures (figs. 14-18) and afford a ready means of determining
the sexes, moreover, their morphological characters are of great
importance as they present very marked specific differences
whereby the closely allied species may be readily distinguished.
These appendages are arranged in five pairs as follows :—Szferior
claspers (sc in all the figures). These are placed dorsally and are
larger than any of the other structures; they are composed of two
distinct segments, of which the terminal or distal one is the smaller
and is provided at the apex with large spines, which in some species
are curiously modified. They are generally densely hairy and
large scales may also be present; but both hairs and scales are
easily deciduous and the greater portion of them usually fall away
during the process of mounting for microscopical study. The
accompanying illustrations must therefore be considered as
representing these structures in a partly denuded condition.
Inferior claspers (tc). These ate unisegmented and much shorter
than the superior pair; they are ventrally placed and may or may
not have modified spines at the distal extremity. Swbmedian

160

lamellae These lie between the inferior claspers, and although
they are usually short, thin, leaf-like structures, in some instances
(P. minutus) they are very similar to the clasper both in form and
length. Intermediate appendages (za). These occupy a median
position and are often curiously modified ; they form a branch of the
superior clasper and are sometimes bi-lobed. Jlntromittent
organ (20). This is homologous with the ‘juxta’ in Glossina, and
is described as the penis by Grassi. It consists of a pair of long
slender and highly chitinised organs which le between the
intermediate appendages. These completely ensheath the two long
filamentous processes which form a continuation of the ejaculatory
duct leading from the penultimate segment of the abdomen. In
P. papatasi they have not been seen to extend beyond the
intromittent organ or penis, while in P. perniczosus (figs. 16, 17),
though lying apparently in a normal resting position, they project
beyond it to a distance equalling one-half the length of the sheath.

W2zxg.—This is densely hairy, and may at once be distinguished
from that of the mosquitos (CULICIDAE) by the entire absence of
scales, the double fork of the second longitudinal vein, and the
proximal position of the cross-veins. The hairy character 1s well
shown in the illustrations (Pl. VI, fig. 2, and Pl. VII, figs. 1, 2),
and when denuded (figs. 4-7), the venation can be seen with
little difficulty in properly prepared specimens. The costa is
the thickest of the veins. The sub-costa, in comparison with that of
the CULICIDAE, is very short, curves downward distally, and joins
the first longitudinal vein at or about one-fourth of the distance
between the base and tip of the wing. The first longitudinal is
sumple, and unites with the costa about one-third from the tip;
the second longitudinal is twice forked, and extends almost to the
base of the wing; the third longitudinal is simple, and
originates from the mid cross-vein; the fourth has origin at the
base of the wing and is forked near the middle; the fifth and sixth
are simple and united basally, the former curving upwards and
uniting with the fourth considerably in advance of the base of the
wing. The first cross-vein unites the costa with the sub-costa at a
point immediately opposite to the turned-down portion of the
latter, so that in effect they produce two cross-veins: the first

Fic. 4.—Wing-venation of Phlebotomus papatasi ; upper, 9 ; lower, d.

Fic. 7.—Wing-venation of Phlebotomus minutus.

162

extending from the first longitudinal vein to the sub-costa, the
second from the tip of the latter to the costa. The mid cross-vein
arises from the base of the third longitudinal and passes obliquely
to the fourth; while the supernumerary vein is placed immediately
above it, and passes obliquely to the second longitudinal.

Legs.—These are very long and slender and densely clothed
with scales, the majority of which are flat and closely resemble
those which are found in the CULICIDAE. The ungues are simple
in all of the Maltese species, and do not offer any differentia!
morphological characters.

INTERNAL MORPHOLOGY

The Alimentary Canal (fig. 8).

This structure differs from that of the mosquito in having a true
sucking stomach, and also in the possession of four malpighian
tubules instead of five. The general form and relative position of
these organs in the female are as follows :—

The buccal cavity lies at the base of the clypeus; it is dilated
distally, but almost immediately contracts and forms a slender
tube which leads to the pharynx.

Scute/lum Sucking
stomach

Mid- gut

Malpighian tubules

Fhorax

ti
Fic, 8.—Internal morphology of Phlebotomus.

The oesophagus divides at a point a little in advance of the
posterior margin of the head (nape), one tube leading to the sucking
stomach, or food-reservoir, the other to the digestive canal.

163

The sucking stomach.—This is a large, thin-walled pouch,
connected with the end of the oesophagus by means of a very
slender tube. It lies on the left side of the digestive canal, and
extends distally as far as the region of the fourth abdominal
segment.

The mid-gut or chyle stomach.—This is capable of great
distention, and when filled with fresh blood occupies a_ large
portion of the abdominal cavity; but when such food has been
partly comminuted it becomes much smaller, and can be easily seen
as a black, elongated pouch in the anterior portion of the body.

\ 7/4
\Y
N\ f NP Antenna
Clypeus
Eye
4 Salivary
duct
QOesophagus:
. Salivar
Mid- gut Bland

Fic. 9.—Head ot Phlebotomus, showing position of salivary glands,

Malpighian tubules.—There are two pairs of urinary organs,
each pair being united at their bases, where they form a single tube,
which is connected with the intestine immediately below the mid-gut.
They are of great length, extending forwards as far as the first
abdominal segment, where they are folded and doubled backwards
upon themselves, and aiso form loops in the mid-region of the
ventral portion of the abdominal cavity.

The salivary glands (fig. 9).—These consist of two broadly
dilated or lobe-like acinous glands, lying one upon either side of
the prothorax. The periphery of these glands presents an even or

164

smooth surface, and immediately within the exterior wall is a
series of rather large secretory cells. The ducts leading from the
acini unite near the mid-region of the head, forming a common duct,
which enters the buccal cavity close to the base of the clypeus.

The Sexual Organs of the Female.

The ovaries occupy a variable position in the different stages of
their development. In the early adult stages of the insect (fig. 8) they
are very small, and are seen to extend from just behind the origin
of the Malpighian tubules to the region of the penultimate segment
of the abdomen. When fully matured (fig. 10) they occupy
practically the whole of the abdominal cavity, extending forwards

Fic, 10.—A female Phlebotomus, showing fully matured ovaries.

as far as the second segment. They are bi-lateral, and each ovary
comprises 20-25 ova, representing a full complement of 40-50, so
that these insects cannot be considered very prolific. The tubular
oviducts unite at a point just before reaching the base of the inferior
claspers, where they form the common oviduct.

The spermathecae (fig. 11) lie in the median line in the region of
the oviducts. They consist of a single thin-walled, sub-spherical
sac, and are relatively very large; at their junction with the duct
they are strongly chitinised, and consist of usually ten transverse
and convex ridges, which are so constricted at the margins as to
present, in optical section, a distinct and well-marked crenulation.
The tubular ducts, which are long and straight, open into the
oviduct near to its termination, apparently.

Ovaries

Sebaceous
gland

Spermatheca
Oviduct

Inferior clasper

Superior clasper

Fic. 11.—Female generative organs of Phlebotomus.

Sexual Organs of the Male (fig. 12).

The external characters of the male armature or copulatory
apparatus have already been discussed (p. 159) and although the
internal sexual organs have been but briefly studied, from

Testis

Seminal vesicle

Ejaculatory duct

Ejaculatory
ducts

Penis Ejaculatory

duct

Eyaculatory ducts

Fic, 12,—Male generative organs of PA/ebotc mus,

166

preparations examined in optical section, yet a brief account of them
may not be without interest. These consist of the following : —

The testes.—These may present a somewhat variable outline,
though normally they are elongate-ovate ; they are distinctly paired
and widely separated, each possessing its own duct which enters
the seminal vesicle at its anterior lateral margin.

The seminal vesicle consists of a large pyriform sac, the
proximal portion of which gradually narrows and merges into the
short, tubular, ejaculatory duct.

The ejaculatory duct is connected with a singular morphological
structure which, together with the chitinous rods, presents a slender
and somewhat club-shaped though cylindrical process. The outer
wall of the swollen portion is formed’ of thin transparent chitin;
and occupying a central position is a piston-like rod which is
dilated at both extremities; the distal portion somewhat resembles
an inverted bulb, the opposite dilatation forming a more or less
spherical sac. The space between the central structures is seen to
contain delicate muscular fibres; and the ejaculatory duct leads into
the spherical cavity at the lower end of the piston-like rod. Grassi’s
interpretation of the mechanism of this structure is that it acts like
a little pump (fomfet¢a) and regulates the exit of the spermatozoa.
Beyond this structure the ejaculatory duct is protected by two
slender hair-like rods which are highly chitinised and form the
‘intromittent organ’ which has already been described (p. 160) as
extending into and in some cases considerably beyond the penis-
sheath or juxta.

OVIPOSITION OF PHLEBOTOMUS IN CAPTIVITY

The act of oviposition was observed on several occasions and was
not without interest, as the insect assumed a position which seemed
altogether unique and extraordinary. In the first instance, a female
with ripe ova was placed in a small glass-topped box, the bottom
of which was within focal distance of a lens magnifying eight
diameters. She was supplied with blotting paper which had been
soaked in clean water. On placing this in the box the insect
immediately alighted upon it, brought her proboscis into contact
with the paper, and after a few seconds appeared to be perfectly

Le

167

intoxicated and helpless. Unfortunately she struggled away and
was finally hidden beneath the paper so that further observations at
the time were impossible. After an interval of a few minutes she
reappeared, crawled up the side of the box, and one and a half
hours later seemed as active as when first captured. On the
following day, at 9.30 a.m., a fresh supply of wet blotting paper
was placed in her cage when in less than sixty seconds she alighted
upon it and assumed the same extraordinary attitude as on the
previous evening at 6 p.m., collapsing immediately and placing her
legs so that the middle and hind pair were crossed behind the
abdomen, the front pair remaining almost in a normal position.
The abdomen was then elevated and extended to the full and three
eggs were laid at short intervals. Each egg appeared under the lens
as a tiny translucent drop of fluid and was ejected with considerable
force to a distance equal to about three times that of the length of
the abdomen. This process lasted for about two minutes, and
afterwards the female crawled slowly away and up the side of the
box, appearing weak and fatigued. Here she remained almost
motionless for nearly three hours, gradually raising the whole of the
body until it assumed a normal resting attitude.

On removing the blotting paper which had been placed in the
cage the previous evening, seven additional eggs were found and
these were evidently laid the previous evening when the insect was
observed to go through the evolutions which have just been
described. At 12.30 a.m. the same day she repeated the process
when freshly moistened blotting paper was supplied. On this
occasion two eggs were laid and these were found attached together
side by side. At 5 p.m. two additional eggs were laid, the same
curious attitude being assumed as before, but although frequently
supplied with fresh wet blotting paper she did not produce any
more eggs, and at 10 p.m. she died. On making an examination of
the abdomen it was found to contain eight fully developed ova so
that it is quite evident that this female had laid eggs elsewhere and
previously to her capture.

The act of oviposition was seen on subsequent occasions, but in
two instances the females died after remaining in a collapsed
condition for periods of two and a half hours, and three hours and
three-quarters, respectively. Both examples had their abdomens

168

well filled with ripe ova and had apparently not laid any eggs
before they were captured.

SYNOPSIS OF MALTESE SPECIES OF PHLEBOTOMUS
A. Abdominal hairs recumbent.

(a) Integument black. Large species. Palpi with second
segment slightly longer than the third.
nigerrimus, n.Sp., p. 168.

(6) Integument ochreous. Small species. Palpi with second

segment one half the length of the third.
minutus, Rond., p. 169.

B. Abdominal hairs more or less erect.

(a) Legs in both sexes relatively short; average length of
hind leg, 3 mm. Terminal segment of superior clasper
of male scarcely half as long as the inferior clasper.

perniciosus, n.sp., p. 172.

(6) Legs in both sexes relatively long; average length of
hind leg, 4 mm. Terminal segment of superior clasper
of male slightly longer than the inferior clasper.

papatasi, Scop., p. 174.
PHLEBOTOMUS NIGERRIMUS, 1. sp.

FEMALE.—Colour. Head, thorax, and abdomen brownish
black; hairs bright ochreous buff, those on the thorax being slightly
paler and erect, those on the abdomen recumbent. Basal segment
of antennae dark brown. Palpi pale to dark brown, hairs similar
in colour to those on the body. Legs pale ochreous buff, with.
ochreous white, ot silvery white, refulgence. Wings ochreous buff
or dull golden in some lights.

Head. Proboscis long; eyes black, deeply emarginate in front.
Palpi and antennae very like those of P. papatasi. Legs very
long, femur of hind pair nearly as long as the abdomen; tibia one
and one-third times the length of the femur; tarsi longer than the
tibiae by about one-sixth, or nearly as long as the wing; ungues
simple. Wings (fig. 5) with the hind margin strongly arched; sixth
longitudinal vein short, terminating near the centre of the hind
margin, the length equal to the distance, in a straight line,
from its tip to the tip of the third longitudinal vein; the anterior

169

branch of the second longitudinal vein twice the length of the
distance between the two forks.

Length 2°50 mm.

The black or brownish-black colour of the integument of this
insect will serve as a ready means of distinguishing it from any of
its allies. It may also be separated from P. papataszi, to which it
is closely related in its morphological characters, by the shape of
the wing and the shorter sixth longitudinal vein. The only two
examples which were secured were taken by Captain P. J. Marett;
both are females, one of which bears the data: ‘Black species,
Gozo, 20, X, 10’; the other ‘P. papatasw, dark variety,
Heel, 10, FF.”

Captain Marett had evidently, therefore, noted the black or
dark colour of this insect in life; and when questioned regarding
this he was absolutely certain that the colour was not due to
post mortem changes. It is undoubtedly a rare insect in the
Maltese Islands, otherwise more specimens would have been secured.
We trust that Captain Marett will be able to obtain examples of
the males so that the characters of the armature may be examined

and described.

PHLEBOTOMUS MINUTUS, Rondani.

MALE.—Colour. Integument rather opaque, dull golden
ochreous. Antennae with black and ochreous hairs mixed.
Head with the. clypeal and occipital tufts of hairs pale
ochreous. Thorax with a median mane-like tuft, a lateral tuft
in front of the insertion of the wings and also a tuft on the
scutellum, all pale ochreous with a golden tinge, but with a few
intermingled black hairs. Abdomen densely clothed with
recumbent, dull, golden ochreous hairs; those covering the genital
organs intermingled with black hairs. Legs covered with scales
which appear smoky brown in some lights, silvery ochreous in
others. Hairs of the wing mixed black and ochreous, those of the
costa not darker than those on the surface of the wing.

Head. Proboscis relatively short; clypeus hairy. Andennae
with the third segment a little longer than the fourth, but not
nearly so long as the fourth and fifth together; the long verticillate
hairs extending to the apical segment. Pali (fig. 13) with the

170

second segment one-half the length of the third; the latter much the
stoutest and broadest; dorsally it appears incrassate towards the
base; fourth segment not quite so long as the third; fifth much the
longest.

Wings (fig. 7) very narrow, and bluntly lanceolate; divided into two
almost equal halves by the third longitudinal vein; the upper or
anterior branch of the second vein shorter than the distance between
the two forks.

Fic. 13.—Palpus of Phlebotomus minutus.

Legs. Hund pair a little more than three times the length of
the abdomen inclusive of the genitalia; tarsus a little longer than
the tibia.

External genitalia (figs. 14, 15) small; superior claspers
with four long spines: two apical and two subapical: inferior
claspers very slightly swollen in the middle; intermediate
appendage similar to that in P. fapatasi; intromittent organ nearly
three-fourths the length of the inferior claspers; genital filament
not protruding.

Length 1°5-1'65 mm.

FEMALE.—Colour. Wings with a distinct black costa and
fringe; wing-area also with numerous black hairs intermixed with
the ochreous ones. Legs with the femora ochreous beneath, darker
above; tibiae and tarsi blackish, with silvery grey scales. Thoracic
and abdominal hairs as in the male.

Antennae with the long hairs extending to the tip, the third to

171

the ninth segments, inclusive, with geniculated and paired spines.
Palpi as in the male.

Length 2 mm.

The distinguishing characters of this insect are its relatively
small size, especially in the male; the recumbent abdominal hairs;
the short third antennal segment; and the marked character of the
palpi. The male may be easily distinguished also by the form of
the external genitalia.

Fic. 14.—External genitalia of Phlebotomus minutus, 3; sc, superior claspers ;
ic, inferior claspers ; za, intermediate appendages ; ed, ejaculatory duct.

Fic. 15.—Superior clasper (sc) and intermediate appendages (7a) of Phlebotomus
minutus, more highly magnified,

The first two examples were captured by Major F. L. Dibblee,
Royal Marine Artillery, at his residence at Sliema, August 2oth,
1910; and two additional specimens were taken by myself, one at
Casa Leoni, in a rabbit hutch, August 31st; the other at Floriana,
August 27th.

In captivity PA. minutus is much more active than any of the
other Maltese species, and when confined to a small area was almost
incessantly moving from place to place. Apart from its flea-like
actions it also has the remarkable habit of whirling round and
round with great rapidity, so rapidly at times as to render itself

almost invisible.

172

PHLEBOTOMUS PERNICIOSUS, n. sp.

MALE.—C olour immediately after death. Eyes black. Thorax
with or without dull red-brown spots; when present they are
arranged in a triangle, and there is occasionally a similar spot on
the vertex of the head. Thorax and coxae pale, translucent
ochreous; abdomen similar, but sometimes pale smoky grey. Hairs
pallid. Wings faintly iridescent in strong light; pale drab in
subdued light; costal fringe generally very dark or blackish grey,
though examples with pale costal fringes are not uncommon. Legs
silvery grey, in a strong light presenting a distinct metallic lustre;
in certain lights also those segments which lie in shadow appear
almost black and show up in marked contrast to those which are so
placed that their surfaces refract the light. In some lights the
under surface of the legs appears distinctly and regularly speckled,
a character due evidently to the regular arrangement of the scales.

Head densely hairy, with generally two ill-defined tufts.
Clypeus with a large tuft of hairs, some of which are directed
forwards, others backwards towards the forehead.

Palpi with segments 2, 3 and 4 equal in length and collectively
a little longer than the 5th. Aztennae with the second segment
much longer than the two succeeding ones; the longest hairs on
segment 14 almost equal in length to those on the preceding segment.
Thorax densely hairy, usually with a tuft on the front portion and
another on the scutellum. Addomen densely hairy, the longest
hairs arising from the apical margin of the segments but no distinct
tufts are found as in P. papatasii. The arrangement of the hairs
is similar in both sexes, but blackish hairs are often intermixed with
the pale ochreous ones on various parts of the body in the darker
forms of this insect. Legs shorter than those of P. papatasiz.
Wings (fig. 6) with the posterior border much more strongly
arched than the anterior border; the anterior branch of second
longitudinal vein nearly as long as the stem between the cross vein
and the proximal fork.

External genitalia (figs. 16,17). Superior clasper with five very
long stout curved spines; two apical, one external and two internal,
placed a little in advance of the outer one; inferior clasper nearly
twice the length of the intermediate appendage and clothed to the
apex with very long and slender hairs; intermediate appendage

173

somewhat finger-shaped and hairy, proximal portion with a large
keel-like extension ventrally, the distal margin of which bears
several (5-6) hairs; apex of intromittent organ deeply divided or
forked, with occasionally a minute central tooth; exposed portion of
the genital filament about half the length of the intromittent organ.

FEMALE.—With the palpi, antennae and legs similar to those of
the male. Wazmugs very slightly larger and broader than those of the
male.

Length 1°9g-2°2 mm.

ic.
Fic. 16.--External genitalia of Phlebotomus perniciosus, 3 ; sc, superior claspers ;
ic, inferior claspers; za, intermediate appendages ; ed, ejaculatory duct.

Fic. 17,—Portion of genitalia of P. pernictosus, 3, more highly magnified ;
$c, superior claspeis ; ta, intermediate appendage ; /, penis; 70, intromittent organ.
This insect is widely distributed over the island of Malta, and
was extremely abundant during the month of August and the
beginning of September, though many examples were captured also
in July. It was most abundant at Floriana, near the old bastion by
the Grand Harbour, on the evenings of August the 26th and 27th,

174

when, between the hours of 8.30 p.m. and 9.30 p.m., thirty-nine
examples were captured as they came into a lighted room; of this
total twenty-eight were males and eleven females.

Two examples of P. minutus were found in association with this
species; but strange as it may seem, not a single example of
P. papatasii was either captured or seen on these occasions.

It was common also during the last week in August at Casa
Leoni, the residence of the Hon. E. C. Roupell, D.S.O., Acting
Lieutenant-Governor. In this place it was found most abundantly
in a large outhouse which was tenanted by a number of rabbits. In
the early mornings, shortly after 6 a.m., numbers of sand-flies were
found chiefly in the corners of the room, but many were also seen
sitting about the walls in various places, though chiefly at the
junction with the ceiling. Later in the day they were rarely seen
in these situations ; but examples could always be found in the dark
earthen pots which were used, and generally occupied by the rabbits
as retreats.

The male is easily distinguished from that of P. papatasz by
its generally smaller size, shorter legs, and much smaller genital
armature, which is little more than half the width of the abdomen.
The female may also be distinguished by its shorter legs, and
generally darker colour. After a few hours in captivity it also
becomes generally much less active than P. papataszz, though it has
the same hopping flight so characteristic of these insects.

Pupa (Pl. VI, fig. 4).—Abdomen distinctly and sharply curved
upwards so that a somewhat S-shaped outline is produced; thorax
gibbose ; abdominal segments each provided with a fair of very large
tubercles (Pl. VI, fig. 5), the tips of which are furnished with a pair
of broad flat appendages; integument thickly covered with
squamose spines (PI. VI, fig. 4).

The larval skin attached to the pupa does not present any
morphological differences from that of P. papatasz, as far as one
can gather from its shrivelled condition. It possesses the same
kind of caudal bristles and hairy body-spines.

PHLEBOTOMUS PAPATASII (Scopoli)

Bibio papatasiz, Scopoli. Deliciae faun. et flor. Insubriciae, I,

p. 55, Pl. XXII, fig. B. a. b. (1786).

£75

Cyniphes molestus, Costa, Storia dei lavori dell’Acad. Aspir.
Natural., Artic. Zool. (1840); id., Annali dell’Acad. Aspir.
Natural. I, p. 4 (1843).

Hermasson minutus, Loew (nec Rondani), Stettin. Ent. Zeit. V,
p. ts; PRA figs. 15 (1844).

Phlebotomus papatasii, Grassi, Mem. d. soc. Ital. d. Sci. (3)
XV, p. 353 (1907).

This insect has been described so frequently that it seems
unnecessary here to do more than add such particulars as have
hitherto been overlooked, or imperfectly dealt with. In the first
place it may be noteworthy to state that there are two distinct colour
varieties of this common and widely distributed insect : —

(1) A uniformly pale form, which may be considered typical ;

(2) A form which differs from the foregoing in having a dark

coloured fringe to the costa and hind margin of the wing;
herein described as the dark form.

FEMALE.—Typical pale form (immediately after death).—
Almost uniformly pale translucent ochreous, thorax with a long
dull red-brown median stripe, and a single spot of the same colour
on either side, near the front margin of the thorax. Hairs on all
parts of the body greyish, their arrangement similar to that of the
male. Wing relatively broad (fig. 4). Wing fringe not markedly
darker than the hairs on the disc of the wing.

MALE.—Tyfical pale form (ammediately after death).—Colour
similar to that of the female. Clypeus with a tuft of eight to ten
hairs; head with a loose tuft, some of the hairs curving forwards,
others backwards; tuft on nape of slightly longer ones, chiefly
curved forwards. Thorax densely clothed; the hairs arranged in
loose tufts. Wing much narrower than in the female (fig. 4).
Abdomen uniformly hairy, with small tufts on the dorsum arising
from the apical margin of each segment; superior claspers densely
hairy, with a few black hairs intermixed with the pale ones; these
hairs are easily deciduous, with the exception of a large tuft which
is more or less permanent in examples mounted in Canada balsam.

FEMALE.—Dark form.—General colour similar to that of the
pale form. Wing fringes distinctly smoky grey; some of the hairs
on the veins are also dark grey or smoky grey.

MALE.—Dark form.—Not observed.

176

This form is not uncommon; but is very much rarer than the
dark form of P. perniciosus. It does not differ structurally from
typical pale examples so that the following description of the palpi
and antennae applies to both varieties.

Palpi of five segments; I very short, slightly dilated distally ;
2 a little longer than the succeeding one; 3 decidedly broader than
the rest; 4 a little shorter than 3; 5 as long as or slightly longer
than 2; 1 to 3 hairy; 4 and 5 scaly and with a few fine hairs.
Antennae (fig. 2) of sixteen segments; I and 2 the stoutest,
the former with one side longer than the other, the latter bead-like;
3 much the longest, being equal in length to the last five segments
together; 4-13 each very slightly shorter than the preceding one
respectively; 14 to 16, inclusive, more strongly incrassate (swollen)
basally than the rest; all the segments with the exception of 1 and
2 densely clothed with hairs, the longest of which arise from the
incrassated portion of each segment, except on the terminal
segments which are furnished with hairs of equal length; 4 to 15,
inclusive, also furnished with a pair of stout spines (fig. 2), which
are suddenly elbowed or bent at right angles to their insertion so
that for nine-tenths of their length they lie practically parallel with
the surface of the segment to which they are attached.

The external genitalia of the male are much larger than those of
any of the other Maltese species; a character which may be readily
recognised in life, under a low magnification. The morphological
characters are shown in the accompanying illustration (fig. 18).
Length, 2°5-2°65 mm.

In captivity this insect is much more restless than P. perniciosus,
so much so that after a few hours one may readily distinguish the
two species by this alone, apart from the other characters; z.é., the
generally larger size, paler colour, and much longer legs of
P. papatasiz.

Ovum (Pl. V, figs. 1-5). When forcibly expelled from the
body a day or so before the cuticle has become opaque the interior
(oolemm) can be seen; and in such examples also the micropyle is
distinctly visible as a short ring-like extension at the anterior pole
of the egg. The oolemm at this stage is filled with globular
particles of fatty matter, suspended in a structureless matrix.
When first laid the egg is translucent white and covered with a thin

—*

PY7

coating of viscous matter by which it readily adheres to the surface
upon which it may fall; five hours after it has been laid it assumes
its normal form and colour, which may be described as follows : —
Form very elongate, dark brown, shining, with longitudinal black
wavy lines, which in certain lights give the periphery of the egg a
faintly rugose appearance; these black lines are slightly raised and
are joined by slender cross-lines so that a faint but rather coarse
reticulation is formed. The transverse lines are, however, very .
difficult to trace unless they are illuminated by a strong beam of

light.

Sc.

ON LLM OEE ‘
SS SASNSS SA
IC. \

sc.

eee =
—

Fic. 18.—External genitalia of Phlebotomus papatasti, 3; sc, superior claspers ;

ic, inferior claspers ; ed, ejaculatory duct ; , penis.

The incubation period lasts for about nine days; but unless kept
in a moistened atmosphere the eggs will not hatch.

LarvaA.—First instar (Pl. V, fig. 8). Cylindrical and distinctly
caterpillar-like in its general form; head black; body white or
ochreous white; caudal bristles, long, black. Head (fig. 19) very
broadly pyriform; frontal hairs two in number; simple; dorsally
there are three similar hairs on each side; one arising from the
mid-region of the mandibles, one near the base; and a slightly

178

longer one towards the centre of the head, near the margin; besides
these there are at least four hairy spines on each side, arranged as
shown in the illustration. Antennae (fig. 19, aut.) composed,

apparently of three segments, the first two being quite rudimentary
and ring-like; third segment broad, flat and ovate in outline, the

. ‘2°
Steen et

VENTRAL

PROFILE

Fic. 19.—Head of larva of Phlebotomus papatastt. ant, antenna; md, mandible;
mp, maxillary palpus ; Zp, labial plate.

anterior edge faintly emarginate and furnished with a centrally
placed hair. Mandibles (fig. 19, md.) large and provided with four
distinct but rather blunt teeth, of which the apical one is much the
largest. Labial plate (fig. 19, 7) somewhat triangular in outline
with four teeth on each side, the median ones being much the

179

largest; in its general form the labial plate resembles those found
in the larvae of the CULICIDAE. Articulations of the body clearly
defined ; each segment bears from four to five hairy spines on each
side, all of which are broadly dilated apically. Caudal bristles in
two pairs, one of which is much the longer, almost equalling the
length of the body, the other pair are extremely short.

Lasi instar (Pl. V, fig. 7). Form resembling that of the first
instar; colour pale ochreous white; head black; caudal bristles
black, arranged in two pairs, each pair being attached to a large
tuberculous process; the inner bristle is much the longer, almost
equalling one-half the length of the body of the larva; all of these
bristles, under a high magnification, present a number of extremely
fine, equidistant, and intensely black surface lines, the intervening
spaces being distinctly pale; it is highly probable therefore that
these bristles are finely striated, but as no sections were cut it is
impossible to determine their true structure by examining them in
optical section only. Thoracic and abdominal spines (Pl. V,
fig. 10) much longer and stouter than ‘those in the earlier stages;
apices narrowly dilated and transparent, the remaining portion
clothed with minute stiff hairs; these hairy spines are arranged in
more or less regular transverse rows, there being four or five on each
side of the median line. Head with several large spines
similar to those on the abdominal segments, but they are pointed
instead of being dilated at the apex; besides these hairy spines
there are also several rather long stout hairs, four of which are
frontal. Sucker feet similar to, but relatively larger than those in
the first instar.

Length 2-3'28 mm.

Pupa.—(P1. V, figs. 11, 12).—When empty, clear ochreous buff.
Eyes in life black. Abdomen curved upwards distally in varying
degrees, but not apparently so distinctly S-shaped as_ in
P. perniciosus; considerably wider in the thoracic region than at
the distal segments of the abdomen; integument clothed with minute
squamose spines (P1. V, fig. 15), which are most conspicuous on the
abdominal segments. Thorax with two tubercles on each side, the
anterior one bearing two or three long slender spines. Abdominal
segments each with one (possibly two) extremely minute tubercles
at the apex of which is a minute broad flat spine; those on the 7th

180

and 8th segments more conspicuous than the rest; but all of these
processes are so minute as to be easily overlooked. Wing-sheaths
pointed apically and extending subventrally as far as the base of
the 7th abdominal segment. Head distinctly elongated and
somewhat triangular in outline; in the empty pupa this often breaks
away in the process of mounting when the outline may be seen to
bear a striking resemblance to the head of an ox in miniature
(Pl. V, fig. 13). Antennal sheaths distinctly segmented, lying
curved behind the eyes and subsequently following the costa of
the wing-sheath. Palpal sheaths originating near the centre of
the frons, extending backwards and then curving suddenly forward
so that the apex rests against the antennal sheath and lies pointing
in the same direction. Legs extending slightly beyond the wing
sheaths .

ACKNOWLEDGMENTS

The Liverpool School of Tropical Medicine is much indebted
to the Tropical Diseases Research Fund (Colonial Office) for the
Grant of £100 towards defraying the cost of the expedition, and to
the representatives of the Moss Steam Ship Company for granting
a free passage by their boats. I wish also to express my thanks
for assistance rendered in the furtherance of the objects of the
Expedition by His Excellency the Governor and Commander-in-
Chief, General Sir Leslie Rundle, K.C.B., K.C.M.G., D.S.O.,
etc., who very kindly gave permission to visit all Government lands
and Institutions and also sanctioned the use of the Laboratory at
the Public Health Department; to the Honourable E. C. Roupell,
D.S.O., Acting Lieutenant Governor, for much valued assistance
and kind hospitality; to the officials of the Civil, Naval and
Military Departments; to the Hon. C. Caruana Scicluna, Chief
Government Medical Officer, and the members of his staff,
especially to Professor T. Zammitt, who furthered the work of the
expedition in every possible way; to the officers of the Royal Army
Medical Corps, Captain Babbington, Captain Lloyd-Jones, Captain
Steward, Captain Beaman, and especially to Captain Marett,
R.A.M.C., for placing the whole of his valuable material in my
hands for examination; to Major G. S. Crawford, R.A.M.C., I am

ISI

also specially indebted for valuable aid and for his kind
hospitality; to Major F. L. Dibblee, Royal Marine Artillery and
Mrs. Dibblee for collecting extensive series of material for
investigation; to Surgeon Lancelot Kilroy, H.M.S. Diana, for
examples of Simulium from Crete; to the Government Veterinary
Officer, Dr. A. M. Macfarlane, for a valuable collection of
Helminths and other intestinal parasites for demonstrative and
other purposes in the Laboratory and Museum of the Liverpool
School of Tropical Medicine.

REFERENCES

1. ANNANDALE, N. (1910). Records of the Indian Museum, Vol. V, Pt. 3, Nos. 13 and rq.

ied

Brrt, C. (1910), ‘ Phlebotomus Fever in Malta and Crete.’ Journal of the Royal Army
Medical Corps, Vol. XIV, pp. 236-258.

Grasst, B. (1907), ‘ Ricerche sui Flebotomi.’ Memorie della Societa italiana delle Scienze,
Ser. 3a, XIV, pp. 353-395-

we

4. Grasst, B. (1908), ‘ Intorno ad un nuovo Phlebotomo.’ Rend. Reale Accademia dei Lincei
Ser. 5a, XVII, pp. 618-682.

5. Howtert, F. M. (1909), ‘Indian Sandflies.’ Indian Medical Congress, Section III,
Pp- 239-242.

6. Maretr, P. J. (1910), ‘ Preliminary report on the investigation into the breeding places of
the sand-fly in Malta.’ Journal of the Royal Army Medical Corps, XV, 3, pp. 286-291.

7. Nevev-Lemaire, M. (1906), Bulletin de la Societé de France, p. 65.

8. Sperser, P. (1910), Zoologische Jahrbiicher, Supp. 12, Heft. 3, Taf. 22.

Fig.

PEALE. ¥.

Phlebotomus papatasii, Scop.

Eggs, approximately natural size.

Egg, a few hours before extrusion, showing micropyle.
Egg, freshly extruded.

Egg, a few hours after extrusion.

Egg, much enlarged, to show reticulated surface.
Larva, approximately natural size.

Sketch of adult larva, enlarged.

Larva; first instar, enlarged.

Stigma of larva with spine.

Hairy spine of larva.

Pupa, approximately natural size.

Pupa enlarged: /s, larval skin with anal bristles
attached.

Front view of the head of the pupa: e, eye;
ad, antenna; 7, palpus.

Thoracic tubercles of pupa.
Squamose body-spines of pupa.
One of the abdominal papillae of the pupa.

REAGEONE

R. Newstead, ad. nat. del. Bale & Danielsson, Ltd., 4th.

PHLEBOTOMUS PAPATASII

Fig.

i)

PEATE Vi

Phlebotomus papatasu, Scop.

Imagos, approximately natural size.
Male, enlarged; from life.

Phlebotomus perniciosus, Newst.
Pupa, approximately natural size.
Pupa, enlarged.
One of the abdominal tubercles of the pupa.

Squamose spines of the abdominal segments of the
pupa.

PLATE VI.

}
sh S
we P4
1.
7 PP i ars:
| », [vee 7
ae 4 ve 5
| ?
d ; 4
, ; ry
ax
[ » |
3
R. Newstead, ad. nat. del. Bale & Danielsson, Ltd., th.

PHLEBOTOMUS PAPATASI!I AND P. PERNICIOSUS.

186

PLATE. Vil

Fig. 1. Phlebotomus papatasit, Scop., female, enlarged; from
life.

bo

Phlebotomus perniciosus, Newst., female, enlarged; .
from life.

3. Phlebotomus perniciosus, approximately natural size.

[NoTE.—The above enlarged figures and that of the male shown on Plate VI
are all drawn to the same scale. ]

PLaTE VII.

js

+”

R. Newstead, ad. nat. del. Bale & Danielsson, Ltd., 4th.

PHLEBOTOMUS PAPATASII aND P. PERNICIOSUS.

187

THE EXPERIMENTAL TRANSMISSION
OF GOITRE FROM MAN TO ANIMALS

BY

ROBERT McCARRISON, M.D., M.R.C.P. (LOND.),

CAPTAIN, INDIAN MEDICAL SERVICE,
AGENCY SURGEON, GILGIT, KASHMERE

(Received for publication 20 April, 1911)

Since the year 1906 I have repeatedly endeavoured to transmit
goitre from man to animals by infecting the water supply of the
latter with the faeces of sufferers from goitre. The assumption that
goitre can be so conveyed follows upon the results of my former
researches. I had previously employed dogs, but obtained no
results of a positive character (1906-11). This was due largely
to the fact that dogs suffer little from goitre in this country, though
these animals are known to be frequently affected in other localities.
I believe that the comparative freedom of dogs from goitre in Gilgit
indicates that the infecting agent of this disease is less toxic there
than elsewhere. Other facts point in the same direction, but these
I hope to discuss in another place.

The possibility that an intermediate host was concerned in the
spread of goitre having occurred to my mind, I suspected earth-
worms of playing this réle. I, consequently, devised an experiment
which would not only again test the assumption that goitre could
be transmitted from man to animals by infected faeces, but would
at the same time determine the influence, 1f any, which earthworms
might possess in spreading the disease.

Female goats were selected for this dual experiment. The
goats were between the ages of one and two years and were not
pregnant. This latter point is of importance, as pregnancy, owing
to the increased activity of the thyroid gland during that state,
would vitiate the results observed on microscopical examination of
this organ. The goats were brought from a non-goitrous locality,
high up in the mountains, about forty miles distant from Gilgit.
Their thyroid glands were carefully examined and were found to

TSS

show no signs of hypertrophy. In the majority the gland on each
side could be felt only with the greatest difficulty, or could not be
distinguished from the surrounding tissues. Where the organ was
palpable it was found to be oval in shape and about the size of a
very small almond. The goats were divided into batches, which
will be referred to as: I, ‘Controls’ (three goats); II, ‘Batch X’
(six goats); III, ‘ Batch Y’ (seven goats). Each batch was confined
in a separate house, the door of which was of netted wire. The
animals were all fed on the leaves and young branches of trees, in
order to exclude as far as possible sources of contamination from
the soil. The experiment was designed so as to foul the drinking
water of ‘ Batches X and Y,’ while that of the ‘ Controls’ remained
pure. The apparatus which I employed for this purpose is

represented in the figure :—

aan SET TARAS Eo
SE SSIULCUMT NUTT Gt GG

189

It consisted of a covered drum, A, which was fitted with a tap; a
covered wooden box, B, having a perforated false bottom of wire
gauze under which was a wooden trough sloping towards an outlet
pipe f. This outlet pipe led into a flask, C, which was fitted
with a perforated cork so as to ensure freedom from contamination
by soil, dust, etc. The drum A was filled with a non-goitre
producing water which was previously boiled. A trickle of water
from the drum A was allowed to pass continuously into the box B.
This box was partially filled with soil taken from the most goitrous
village in Gilgit. The soil was sterilized by steam at 230° F. for
thirty minutes, and was then mixed with the faeces of sufferers
from incipient goitre. Fresh faeces were added to the mixture
several times a week during the course of the experiment. The
water from the drum A trickled into the box B, saturated the
mixture of sterilized earth and faeces, and, passing through the
perforated bottom of the box, found its way, by means of the
wooden trough and outlet pipe 8, into the corked flask C. This
was the only drinking water provided for the goats of ‘Batches
X and Y.’

A separate apparatus was used for each batch. In the case of
“Batch Y,’ however, 500 earthworms were introduced into the box B.
It will be seen that ‘Batch X’ were provided with drinking water
which was grossly contaminated with the faeces of sufferers from
goitre, while in the case of ‘ Batch Y’ the drinking water contained
also such additional matter as was derived from the excreta of the
earthworms. The water which found its way into the stoppered
bottle was in both cases foul smelling and of a dark grey colour.

The experiment was commenced on the 13th October, 1910, and
terminated on the 15th December, 1910. The thyroid glands of
the control goats showed no change during the sixty-four days of
the experiment. In the case of the goats of ‘Batches X and Y,’
however, it was observed that

(1) the animals lost in weight, due doubtless to
confinement in a small hut for sixty-four days;

(2) many of them suffered from diarrhoea ;

(3) 50 per cent. of the goats in each batch showed
enlargements of the thyroid gland, which was most marked
in the right side.

1gO

To facilitate examination of the necks of these animals the hair
was kept closely clipped, but as measurement and photography
cannot be employed in the case of animals as aids to diagnosis,
palpation alone had to be relied upon during the course of the
experiment. It was observed that the thyroid gland in two goats in
‘Batch X’ showed signs of enlargement as early as the thirteenth
day of the experiment. In all cases in which enlargement occurred,
a noticeable feature was the manner in which the size of the gland
fluctuated. At one examination it would be found that the organ
was little, if anything, larger than in the case of the controls, while
a day or two later it would appear to be not less than twice the size.
Goitre in man, whether artificially produced or naturally acquired,
shows the same tendency to fluctuate in size in its early stages (1909)
(1911, Febr.). The animals were killed on the 15th December by
a Goorkha, skilled in severing the head from the body at one stroke
of his kookrie (knife). The neck was dissected immediately and the
gland rapidly exposed. It was observed in several cases that the
size of the organ diminished considerably before it could be
removed from the body. The right and left lobes of the thyroid,
with their long and narrow isthmus, were rapidly dissected out and
weighed. The following table shows the results in the case of the
sixteen animals employed in the experiment : —

I.—‘ Controls,’.3 in number, which consumed only pure water :—

Proportionate weight

Weight of goat Weight of thyroid of thyroid to body
weight

1

I 60 I|bs 3:2 gms. Woo
r)
I

2 A395 2°05 49 10,439
ae
I

3 65» 32 oy

191

II.— Batch X,’ 6 in number, which consumed only faecally contaminated water :—

Proportionate weight
Weight of goat Weight of thyroid of thyroid to body
weight
I
I oe 484 lbs. 3°6 gms. 6,654
I
2 263 5, 2°93 95 45500
I
3 38 2300 8,000
west I
4 So5yes, 2Sy Grey? Toisea
I
5 223 5, 2°32 9) 4,800
I
6 “Cc 2GMyw +3 Te3i6is ass ote

I1I.— Batch Y,’ 7 in number, which consumed faecally contaminated water, as in the case of

‘Batch X,’ together with the excrementitious products of earthworms :—

Proportionate weight

Weight of goat Weight of thyroid of thyroid to body

weight
I

I a. 50} lbs. 4 gms. 7,000
?
I

2 41b 5; 43 ” “4,850
I

3 56 »” 3°77 ”) 73530
I

4 554 3 36 45 “3,700
I

5 494 ” 5 4 ” 45272
; I

6 oes 45 49 Ig ” ~ 11,700.
I

7 eee 39 oy id ei "10,000

It will be seen from the tables that the weight of the control
animals’ thyroid was in all cases about jeeaoth part of the body
weight. In ‘Batch X’ the thyroids of Nos. 4 and 6 showed no
deviation from the normal weight. In two, Nos. 1 and 3, the

192

weight of the thyroid was considerably more than normal, while
in the remaining two, Nos. 2 and 5, it was twice as much as that of
the controls. In‘ Batch Y’ Nos. 6 and 7 had thyroid glands of the
normal weight; in Nos. 1, 3 and 4 the weight of these organs was
considerably more than normal; while in Nos. 2 and 5 the thyroids
were more than twice the weight of those of the control animals.
Allowing for variations in size of the normal thyroid of from
seoth to yonth part of the body weight, it will be admitted
that about 50 per cent. of the animals in ‘Batches X and Y’
showed enlargements of the thyroid gland.

MICROSCOPICAL APPEARANCES OF THE GLANDS

Striking differences were observed in the microscopical appear-
ances of the thyroids in these animals. In the control animals the
vesicles were found to be small, round, compact, and lined with
cubical epithelium. The space between the vesicles was filled with
masses of round cells, and many vesicles were seen in the field of
the microscope. In the thyroids of those animals which showed the
greatest increase in size as determined by weight, the vesicles were
much larger, their walls markedly thinner, and their outline much
more irregular, while fewer appeared in the field of the microscope
than in the case of the normal gland. The epithelium lining the
vesicles was much flattened, and the intervesicular cellular tissue
was markedly less in amount than in the case of the normal gland.
Sections of the enlarged organs often showed fields in which
the colloid had fallen out during the process of staining; these
fields, when viewed as a whole, had a peculiar ‘netted-wire’
appearance, which differed very markedly from the compact
structure of the normal gland. Glands which showed an inter-
mediate degree of enlargement presented in some parts of the
section the appearances of normal tissue, while in others the dilated
vesicles, flattened epithelial lining and the scarcity of intervesicular
cellular tissue were characteristic of the more enlarged organs. In
short, every degree of variation was met with from the small,
round, compact vesicle of the normal organ to the cyst-like
dilatation of the vesicle of the hypertrophied gland. In none of the
enlarged organs was there any evidence of active cell proliferation
or of inflammatory change. There appeared to be little or no
alteration in the connective tissue stroma of the enlarged glands.

193

The increase in size of the hypertrophied organs was due wholly to
the distension of the vesicles with colloid material. It seems
evident that this distension must result in thinning and _ rupture
of the walls of the vesicles and in the formation of small, cystic
cavities in the gland; and that distension of existing vesicles, and
the formation of new vesicles from the intervesicular cell masses,
are the earlier stages in the development of parenchymatous goitre.
In some cases colloid was seen in the vessels of the gland (Photo-
micrograph V1).

This experiment proves that a hypertrophy of the thyroid
gland of goats can be induced by infecting the water supply with
the faeces of sufferers from goitre. It is, at present, impossible to
state whether this hypertrophy is due to the action of the infecting
agent of goitre, or only to the organic impurity of the water thus
contaminated.

Earthworms do not appear to be concerned in the spread of
goitre.

Goitre is essentially a disease of country localities, that is, of
localities of unprotected water supplies. In this country I have
found, from a study of the topography of a very large number of
villages, that the freedom, or the reverse, of any particular village
from this disease depends very largely on the extent to which the
drinking water is contaminated by urigating fields, which are
fertilized with human and animal excreta. The source of the
water supply and the geological formations from which it is derived
are, in comparison, of minor importance.

Further experiments on goats are at present in progress, with a
view to confirming the results detailed above. These experiments
have not long been in progress, but already five out of twelve goats
which are drinking faecally contaminated water show signs of
enlargement of the thyroid gland, while no changes have occurred
in the thyroids of twelve control animals. The final results of these
experiments and the microscopical appearances of the glands will
be detailed in a subsequent report.

REFERENCES

McCarrison, R., (1906), Lancet, Dec. 8.
s—, (1908), Proc. Roy. Soc,, B. LXXXI.
—_—_—_—.,—, (1909), Quart. Journ. Med., II, 7,

--—, (1911), Proc. Roy. Soc., Febr. 28, B. LXXXIII.
—_—_——_——.,—, (1911), Ann. Trop. Med. Parasit., Apr. 20, V, 1.

194

DESCRIPTION; OF PLATES

PLATE VIII

Fig. 1.—Photomicrograph of normal thyroid gland of goat.
x 100. Shows numerous rounded vesicles holding
colloid. The cubical epithelial lining and the cell
masses between the vesicles cannot be seen with this
magnification. Section taken from the thyroid gland of
control goat No. 2.

Fig. 2.--Photomicrograph of artificially produced parenchymatous
goitre in goat. x 100. Shows the marked dilatation
of the vesicles and the thinning of their walls. Section
taken from thyroid gland of goat No. 1, ‘Batch Y.’

PGA TE - Ve

do lhwnle Bw
TE 7) >
Str rege bevel yt an
Pe tes tw hil |
: wae ;

“a ' e
ih eiborw rast] ‘ 2)

a earth pl rh Fyn

yi) a bon .ilidiw abies
bork bey a1 apie)

+ ,
bie 4 ifodntl : if 7 73 |

19)

PEATE: LX

Fig. 3.—Photomicrograph of normal thyroid gland of goat.
x 100. Shows same appearances as in Photomicrograph
No. 1. Section taken from thyroid gland of goat No. 6,
Mbaich xX.’

Fig. 4.—Photomicrograph of artificially produced parenchymatous
goitre in goat. x 100. Shows marked dilatation of the
vesicles, thinning of the vesicle walls, and the ‘ netted-
wire’ appearance alluded to in the text. Section taken
from thyroid gland of goat No. 5, ‘Batch Y.’

LX

PEATE

l\

IG,

3 in
a
birt
v. 7
A
a .
Mass >

i
mr

yinepionsrey LbAwibara errs , -
| ~

j

f ez tt hy othe “i i
qloviteraqeass ayvone olait ao) 10° 7

simousiont nods? 2s Ageractelt
--

198

PLATE X

Fig. 5.—Photomicrograph of normal thyroid gland of goat.
x 100. The cubical epithelial lining can be seen in a
few of the vesicles. Figs. 1, 3 and 5 illustrate the
variations in the size of the vesicles which are met with
in the normal thyroid gland of the goat. Section taken
from thyroid gland of control goat No. 5.

Fig. 6.—Photomicrograph of artificially produced parenchymatous
goitre in goat. x 100. Shows colloid in vessel of
gland. Part of the field shows comparatively normal
appearances. Photograph was taken from the same
section as Fig 2.

The sections, from which the photomicrographs were taken, were
stained in aqueous solutions of magenta red, picric acid and indigo
carmine (Borrel’s stain).

t5)s)

REDUCING ACTION OF TRYPANOSOMES
ON HAEMOGLOBIN

BY

RALPH W. NAUSS
AND

WARRINGTON YORKE

From the Runcorn Research Laboratories of the Liverpool School
of Tropical Medicine.

(Received for publication 21 April, 1911)

Amongst the blood changes occurring in animals infected with
trypanosomes there is one which, so far as we can ascertain, has
escaped observation. Whilst performing experiments with the
object of investigating the cause of auto-agglutination of red blood
cells*—a phenomenon which is frequently to be observed in
cover-slip preparations of the fresh blood of infected animals—it
was observed by one of us (W.Y.) that the colour of the
erythrocytes in certain of the capillary tubes had changed from the
normal red tint to a deep purple.

Further examination showed that this alteration in colour only
occurred when the plasma, which had been added to the suspension
of washed erythrocytes in normal saline solution, contained a
considerable number of actively motile trypanosomes. The
appearance was most striking in the tests carried out at 37°C. It
was not so distinct in the tubes kept at 15° C., and was absent from
those which had been placed in the ice-chest.

The fresh blood of a large number of animals of different species
in various stages of infection was examined as regards its colour.

Technique.—A few drops of blood were allowed to fall from
a vein of the ear into a tube containing a small amount of citrated
saline solution. The red blood cells were then quickly thrown
down by centrifugalisation and their colour noted. As a rule the

* Yorke. ‘ Auto-agglutination of Red Blood Cells in Trypanosomiasis,’ Roy
Soc. Proc., 1910, Vol. LXXXIII, p. 238.

200

purple colour, when present, could easily be seen as the blood
escaped from the wounded vein.

Only comparatively rarely were the red blood cells found to
exhibit a markedly purple colour. It was occasionally seen in
infected rats when the parasites in the peripheral blood were
exceedingly numerous. As a rule, however, the blood of these
animals was found to present the normal red colour even when it
was swarming with trypanosomes.

Durham,* in a recent paper, has drawn attention to this fact.
He states that, in rats suffering from Nagana, the blood in an
advanced stage of the disease is sometimes of a dull purplish or
chocolate colour. Examination with the spectroscope showed the
bands of oxyhaemoglobin.

The blood of some of our infected rabbits exhibited this change
of colour in a marked degree during the later stages of the disease.
In the case of a rabbit infected with T. rkodesiense, the blood, as
it escaped from the incised vein, was of an exceedingly purple
colour.

The blood of this animal was subjected to various tests with a
view to obtaining information as to the cause of this appearance.

In the first place it was found that when the blood was
thoroughly shaken with air, or, when air was blown through it,
the dull purple tint gradually gave place to the bright red colour
of normal oxyhaemoglobin, until, finally, the appearance was
identical with that of normal blood. The conversion of the purple
colour into the red was not particularly rapid, and the blood
required to be intimately mixed with air before it was accomplished.
The fact that the red blood cells could be washed many times in
large volumes of normal salt solution and still retain a distinctly
purple colour illustrates how stable is the condition.

It will be noted that this observation is at variance with the
results obtained by Durham with the blood of rats infected with
T. brucez. Durham states that the dull coloured blood of these
animals may be shaken with air, or allowed to stand for a week or
more without developing the full red of normal oxyhaemoglobin.

Spectroscopic examination of a solution made by adding a few

* “Notes on Nagana and some Haematozoa observed during my travels,’
Parasitology, 1908, p. 227.

20I

drops of the blood of this rabbit to distilled water revealed the
ordinary bands of oxyhaemoglobin. No abnormal absorption bands
were seen. When, however, the blood was added to previously
boiled distilled water, avoiding as far as possible contact with
air, the spectroscopic appearances were very different. Well-marked
bands of oxyhaemoglobin were visible as before, but the space
between the bands was also considerably darkened and the zone of
absorption extended further towards the red. In other words, it
was obvious that instead of the spectrum of a pure solution of
oxyhaemoglobin we were dealing with that of a solution of
partially reduced haemoglobin.

The dull purple colour of the blood is due, therefore, to the
presence of a certain proportion of haemoglobin in the reduced
form.

Some years ago Ehrlich demonstrated the ability of living cells
to decolourise solutions of methylene blue.

Later it was shown by Klett,* Neisser and Wechsbergt and
others that living leucocytes, spermatozoa, pancreas and kidney
cells, and various micro-organisms all possessed this property.
Neisser and Wechsberg found that these cells and bacteria lost their
reducing capacity after they had been treated with toxic substances.

Ricketts} observed that emulsion of nervous tissue caused
reduction of a solution of methylene blue. Further investigation
indicated that intact cells were not essential for this reduction, and
that emulsions which had been kept in the ice-chest for a week still
reduced, although less vigorously than when fresh.

Experiments were performed by us with the object of
ascertaining whether actively motile trypanosomes had a reducing
action on solutions of methylene blue.

Solutions of various concentration were prepared by dissolving
the dye in water to which sufficient sodium chloride had been added
to make it isotonic. Small cells of a capacity of approximately
I c.c. and 10 mm. in depth were completely filled with a mixture

** Zur Kenntniss der reducirenden Eigenschaften der Bakterien,’ Zeit. ftir
Hygiene, 1900, S.137.

+ Ueber eine neue einfache Methode zur Beobachtung von schadigungen
lebender Zellen und Organismen (Bioskopie),’ Miinch. med. Woch., rgo0, S.126.

t ‘ Reduction of Methylene Blue by Nervous Tissue,’ Journal of Infectious
Diseases, 1904, Vol. I, p. 5go0.

202

consisting of equal portions of citrated plasma containing many
actively motile trypanosomes in suspension and the isotonic solution
of methylene blue. The cells were then sealed with cover-slips and
placed in the incubator at 37° C.

It was found that in the course of a few minutes the fluid in
the cells containing numerous trypanosomes and only weak solutions
of methylene blue (0°05 per cent., or less) had completely lost its
bluish tint and become colourless, indicating that complete
reduction of the dye had resulted. On the other hand, no
reduction was observed in the cells which contained the more
concentrated solutions of methylene blue. It was found that
solutions of the dye equal to o’5 per cent. speedily caused the
parasites to become motionless and die.

In view of the injurious effect of solutions of methylene blue
on the parasites we were obliged to seek another indicator, the
presence of which did not prevent the trypanosomes from carrying
out their physiological functions.

Ultimately, it was decided to use isotonic solutions of rabbit
haemoglobin. Solutions of this substance were found to possess
many advantages.

(1) It had no injurious action on the parasites.

(2) The strength of the solution could be easily measured,
and we were thus enabled to perform experiments of a
quantitative character.

(3) An obvious change of colour occurs when such solutions
are undergoing reduction.

(4) By the aid of the spectroscope it is possible to determine
when reduction is complete.

Technique.—Blood from a vein of the ear of a rabbit was
allowed to drop into citrated saline solution. The red blood cells
were then separated from the citrated plasma by centrifugalisation,
and subsequently washed several -times with physiological salt
solution. The washed erythrocytes were then laked by the
addition of distilled water, and after the lapse of a few minutes
sufficient sodium chloride was added to render the solution isotonic.
A light precipitate, consisting for the most part of red cell
stromata, appeared upon the addition of the salt and was thrown

203

down by means of the centrifuge and the clear solution of
haemoglobin withdrawn.

The haemoglobin content of the solution in terms of human wet
red cells was then determined by means of a haemoglobinometer
reading. *

The suspension of trypanosomes was obtained by adding four
volumes of the blood of an infected animal to one volume of a
solution containing I per cent. sodium citrate and og per cent.
sodium chloride. The red corpuscles having been thrown down by
the aid of the centrifuge, the citrated plasma containing the
trypanosomes in suspension was decanted off. The number of
parasites present was determined by suitably diluting a small
portion with sodium chloride solution and counting by means of a
Thoma Zeiss haemocytometer.

A spectroscope tube of known capacity and 10 mm. in height
was then completely filled with a mixture consisting of equal parts
of the haemoglobin solution of known strength and of the
suspension of trypanosomes containing a definite number of
parasites per cubic millimetre. The tube was then sealed with a
cover-slip and placed in the incubator at 37° C.

Control experiments were always made with the same solution
of haemoglobin and the plasma of a normal animal of the same
species diluted to a similar degree as the infected plasma.

After the expiration of only a few minutes a distinct change in
colour was observed in the tubes which contained the suspension of
trypanosomes. The bright red of the oxyhaemoglobin was being
replaced by the dull purple colour of reduced haemoglobin. This
process continued, until, finally, the colour became dark purple.

From time to time the tubes were examined spectroscopically by
means of a Zeiss comparison spectroscope and the absorption bands
compared with those of the standard solution of oxyhaemoglobin.

Since the contents of all the tubes gave absorption bands
practically identical in appearance at the beginning of the
experiment it was easily determined in which of the tubes changes

* Barratt and Yorke. ‘ An Investigation into the Mechanism of Production
of Blackwater,’ Annals of Tropical Medicine and Parasitology, 1tq09, Vol. III,

p. 14.

204

were occurring by comparison with the standard solution which was
kept in contact with the air at 15°C.

As the colour of the solutions became more and more purple the
oxyhaemoglobin bands became less well defined. The space
between the bands at D and E became darker, whilst the zone of
absorption embracing the D line appeared to extend further
towards the red. Finally, in place of the two distinct bands of
oxyhaemoglobin there was only the single band of reduced
haemoglobin.

As will be seen from Table 1 the rapidity and degree of
reduction varied, as a rule, directly with the number of parasites
present in the citrated plasma. There were, however, certain
well-marked exceptions. In Experiment 12, where the citrated
plasma was derived from a rat heavily infected with 7. eguzperdum,
there was scarcely any reduction of the haemoglobin solution, in
spite of the fact that the infected plasma solution contained
numerous trypanosomes (300,000 per c.mm.). When cover-slip
preparations of the blood of this rat were examined it was found
that the parasites were sluggishly motile and quickly clumped
together into large masses and became motionless. Other analogous
observations indicate that for appreciable reduction to occur it is
essential for the trypanosomes to be actively motile. Trypanosomes
killed by heating for a short time to 50° C. caused no reduction of
methylene blue or haemoglobin solutions.

Having determined that actively motile trypanosomes exert a
marked reducing action upon haemoglobin, we decided to continue
our investigations with a view to ascertaining so far as possible in
what manner the gaseous contents of the blood are altered by the
action of living trypanosomes.

With this object in view, an analysis was made of the gases
contained in a definite volume of defibrinated rabbit blood which
had been treated for one hour at 37°C. with a certain amount of
citrated plasma, containing numerous living trypanosomes. In the
control experiments a similar volume of the same defibrinated blood
was treated for a like time with an equal quantity of citrated
plasma from a normal animal of the same species as the animal
yielding the infected plasma. As a further control the gases present
in the defibrinated blood alone were determined.

205

Tasre 1. Reducing action of trypanosomes on haemoglobin.

Eauat Portions oF INFECTED ©

PLasMA AND HAEMOGLOBIN _ .
SOLUTION |
No. of Variety of Time in which
experi- trypanosomes _ complete reduction
ment Number of Strength of occurred

parasites per | haemoglobin
cubic millimetre] solution

I T. gambiense ae 10,000 O73, Complete in 1 hour
2 $ ahs 18,250 7s V6 _ Partial in 15 minutes
a ae ae 30,000 0°86 Y% _ Complete in 1 hour
% 4 - see 36,500 075 % Complete in 1o minutes
5 + ae 600,000 0-72 % a
Gel Lin b7zicete | 3s. dy 300 o-75) Nil in 1 hour
7 3 ace 503 32,000 nn OF ‘5
Ss ar ss aon 40,000 O75 5 Complete in 10 minutes
an Quay i. coast ... aa 3,000 Tom 5 Nil in 1 hour
10 8 tis at 12,000 074 % Complete in 15 minutes
ir | ZT. equiperdum sie 100,000 0-72 % a
fagt 2 or soe 300,000 1:00 % Nil in 1 hour
— 13 | I. equinum ... vee 100,000 AN, Complete in 20 minutes
14 5 5 at gb 170,000 34% Complete in 15 minutes

N.B.—In the control experiments where normal plasma was used instead of that of

infected animals no reduction occurred.

200

Analysis of gases obtained from defibrinated blood and from mixtures of this with plasma of normal

and infected animals after incubation at 37° C. for 1 hour in the absence of air.

COMPOSITION OF BLOOD

TABLE 2.
No. of | Amount
experi- of
ment | defibrin-
ated
blood of
normal
rabbit
1 A Zic.c
B EA
Cc ea
2, A 2° Ge
B Phy
ga 3 cc.
B rn
Cc ”
4 Feree
B 33
C ,
5 AlP igrerc
B »
Cc =
6 A Zc
B ”
ce

9

MIxtTurRE

ANALYSIS OF GASES OBTAINED FROM BLoop MIxTURE AFTER
HEaTING FOR I HOUR AT 37° C. IN THE ABSENCE OF AIR

Amount of citrated

plasma of normal or |

infected animal
consisting of 2 parts
plasma and 1 part
citrated saline
solution

1°5 c.c. from normal |

rat

5 c.c. from rat in-
fected with T.
equiperdum
taining 640,000
trypanosomes per
c.mm.

1°5 c.c. from normal
guinea-pig

1°5 c.c. from guinea-
pig infected with
T. brucei, contain-

con- |

ing 340,000 try-
panosomes per
c.mm,

1-5 c.c. from normal
guinea-pig

1°5 c.c. from guinea-
pig infected with
T. brucet_ contain-
ing 450,000 try-
panosomes per

| cmm.

1°5 c.c. from normal
guinea-pig

| pig infected with
T. brucei, contain-

panosomes
c.mm.

per

1°5 c.c. from normal

rat
| 1-5 c-c. from rat
| infected with

| T. brucei, contain-
ing 270,000 try-
panosomes
c.mm.

guinea-pig

I°§ c.c. from guinea-
pig infected with
TI. brucet, contain-
ing 800,000 try-
panosomes per
c.mm.

|
!
|
|

ing 580,000 try- |

per

1°5 c.c. from normal

1°5 c.c. from guinea-

Total CO oO | N
| o73z0c.c. | o40cc. | o28¢.c. | 005 c.c.
Too c.c. | o-60¢.c. | 0275 c.c. | OFUZGIC.C-
0-745 c.c. | 07645 C.c. | 0'025 C.c. 0'075 ¢.c.
|
0775 c.c. | 0390 C.C. | 0°33 C.C. | C1055 C.c. |
| O97 c.c. | O57 CC. | O2QC.c. | OII c.c.
0-76 c.c. | 0°67 ¢.c. | O-007¢.c. | 008 c.c.
o64c.c. | 034c.c. | o26c.c. | o04c.c.
Iri4c.c. | 076 c.c. | O-3iL5 CG. | 01075) C.-C:
0-65 c.c. | 0°58 c.c. | cooc.c. | O07 cc.
|
067 c.c. | 0305 c.c. | O31 c.c. | 0055 cc. |
O-gI c.c. | 0°535 cc. | 0-305 c.c. | =°97 C.c.
0-815 c.c. | 0°64 c.c. 0°05 C.c. O'125 C.c.
0-935 c.c. | o-62¢.c. | 0275 c.c. - or04 uc.
I-I1 ccc: | 0°755 c-c. | 0°255 c.c. | 0:07 c.c.
0-73,c.c. | 0°645 c.c. | 0°03 ¢.c. | 07055 c.c.
1°09 C.c O'715 c.c | 0-315 c.c. | O-OGc.c.
1°355 c.c. | org6c.c. | 032¢.c. | 0075 c.c.'
Vizec. | og5c.c. | o09c.c. | 0:08 cc.

| Deficiency Variation
| in amount | in amount
of oxygen of carbon

in Cas | dioxide in
compared C as com-
with B pared with
/ B
0°25 C.c. | O'045 C.c.
| o283cc. of cc.
we a
0°315 c.c. |—or18 c.c.
}
O255c.c. | O1O5 Cc.
0'235 c.c. |—OrII c.c.
/ |
/ |
ae —
|
0°23 ¢.c. |—Oro! c.c.

.
No. of
>. experi-
; ment
4
b

CoMPosITION oF BLoop

207

TABLE 2—continued.

Mixture

ANALysIs oF GASES OBTAINED FROM BLoop MIxrurE AFTER
HEATING FOR I HOUR AT 37° C. IN THE ABSENCE OF AIR

blood of
normal

rabbit

aicics

Al

Amount of citrated
plasma of normal or
infected animal
consisting of 2 parts |
plasma and 1 part
citrated saline

solution

1-5 c.c. from normal
guinea-pig

1°5 c.c. from guinea-
pig infected with
T. evansi, contain-
ing 350,000 try-
panosomes _ per
c.mm. |

1°5 c.c. from normal
guinea-pig

I°5 c.c. from guinea-
pig infected with
T. evansi, contain-

ing 45,000 try-
panosomes per
¢.mm.

oe

1°5 c.c. from normal
guinea-pig

I°5 c.c. from guinea-
pig infected with
JT. evans, contain-
ing 395,000 try-
panosomes per
c.mm.

1°5 c.c. from normal
rat.

Ig c.c. rat
infected with
T. dimorphon, coa-
taining 250,000
trypanosomes per
c.mm.

from

1°§ c.c. from normal
rat.

15 c.c. from rat
infected with
T. evanst, contain-
ing 250,000 try-
panosomes per
¢.mm.

Total

_

"205 C.C.

=251C-G-

al

-

‘II c.c.

-

2.55 C-2
+505 C.c.

_

_

hits) (estes

°325 C.C.
68 c.c.

~

=2316:G-

0°90 ¢.¢.
1°40 C.C.

LG ZG.cs

0°755 C.c.
| 07955 c.c.

| 1'025 c.c.

| 0°82 c.¢.
1-08 c.c.

| 1:085 c.c.

1°225 C.c.

1°14 €.c.

1015 C.c.

0°62 c.¢.

O71 C.C.

0°865 c.c.

0°355 C.c.

Deficiency
| in amount
| of oxygen

O N
0°385 c.c. | 0°065 c.c.
0°32 C.C. | O°105 C.c.

| oor c.c. 0°075 C.Cc.
0°375 c.c. | 0:06 c.c.
0°345 c.c. | 0:08 c.c.
|
0025 c.c. | 008 c.c.
|
o36c.c. | o10c.c
0°37 ¢.c. | 0°85 c.c
005 c.c. | 0°85 c.c
0°43 €.c. | 0°05 C.c.
O45 c.c. | 0709 Cc.
o22¢.c. | 085 cc.
|
0°38 c.c. | 0°05 C.C.
0°39 C.c. | O'095 ¢.C.
O08 c.c. | 01085 c.c. |

in C as
compared
with B

O31 c:c:

0°32 C.C.

Variation
in amount
of carbon
dioxide in
C as com-
pared with

B

9°06 c.c.

208

TABLE 3. Data given in Table 2 re-calculated in volumes per cent.

ANALYSIS OF GASES OBTAINED FROM BLoop
MIxtTurE AFTER HEATING IN THE ABSENCE OF AIR
FoR I Hour art 37° C.

| | Defi- :
| ciency | Var ae
No. of in | tion
experi- 100 c.c. of mixture consisting of 2 parts amount ne
ment | defibrinated blood of normal rabbit and | of aniount
1 part of citrated plasma from the following | Total CO, O N | oxygen of CO,
normal or infected animals in Cas | n Cas
com ee
pare
— with B
1 B) Normal rat : | 22°2 c.e:|-19-3:c:c.| GT 'cce, |2B'ec. | ms ‘
C| Rat infected with T. equiperdum. “The citrated. | 16-6 c.c.] 14-3 c.c.| o-6c.c. | 1°6 c.c. 5°5 &-See.| ies
plasma containing 640,000 trypanosomes per |
c.mm.

B | Normal guinea-pig s-s| 20°6(C.€-| 12°7 c.c.|' 6:4'c.c. | 2-Averc.
C| Guinea-pig infected with iT ‘brucei. The | 17-0 c.c. 15-0\C.€.| (O°F5 G-C-| T-Siesc.
citrated plasma containing 340,000 trypano- |

|

|

| 6°25 c.c.| 2-3 c.c.

somes per c.mm.

3 B| Normal guinea-pig : <a) 25°3.c.c.| 16:9 c.c.| 7-0 c.c. | 1-7 C.c. “0 ¢.0 eee
C}| Guinea-pig infected with T. race. The | 14°4c.c.| 12-9 c.c.! oro c.c. | 1°6 c.c. 1 OSes plate
citrated plasma containing 450,000 trypano- |
somes per c.mm.

|

4 B)| Normal guinea-pig ; ---| 20:2 €.c.| I1°9 c.c.| 6:8 ¢.c. | 1-6 c.c. | _. ae aoe
C| Guinea-pig infected with T. ‘brucei. The | 18-1 ¢.c.| 14:2 c.c.| 11 c.c. | 2-8 c.c. aes sie:
citrated plasma containing 580,000 trypano-
| somes per c.mm. |
5 B) Normal rat : ae ++) 247 C-C.| 16-9 c.c.| 5B c.c. | 7 CC. | og ip ie
C| Rat infected with J. brucei. The citrated | 16-2 c.c.| 14°3.c.c.| o-7 c.c. | 1°2 €.c. as e?
plasma containing 270,000 trypanosomes per
c.mm. |
] ee eee eee ee
6 B| Normal guinea-pig : vo-| BOT CC. 21-3 cc.) 71 c.c. | 17 6c. | Oo | oo ce
C| Guinea-pig infected with T. brucei. The | 24:9 c.c.| arre.c.| 20¢.c. | rec. | 97% -
citrated plasma containing 800,000 trypano-
| somes per c.mm.
7 B| Normal guinea-pig oat s++| 30°7 CC. 21-2 €.C.| 771 CC. | 236C. | 6 Oo ee
C| Guinea-pig infected with T. evanst. The | 24°7¢.c.| 22°6c.c.| 0-2 ¢.c. | 1°7 c.c. is a
citrated plasma containing 350,000 trypano-
somes per c.mm.
8 B/| Normal guinea-pig ae +-| 33°4 C.c.| 24-0'¢.c.| 7°7 c.c. | 1°8'C.c. “1 cle
C| Guinea-pig infected with T. evansi. The | 26-4c.c.| 24:1 c.c.| o6c.c. | r8cc. | 77 o a
citrated plasma containing 450,000 trypano-
somes per c.mm.
| ae a i. ee ee oe =
g B)\ Normal guinea-pig = nig +++) 3773 C.c.| 27:2 c.€.) B2ec. | gcc.) | ace
C | Guinea-pig infected with J. evansi. The: | 27-3’e:c:|’ 25*4 c.c.| 1-1 €:c) 1°9 cc. i a ie
| citrated plasma containing 395,000 trypano-
somes per c.mm.
——— Se eS | /
| |
10 B| Normal rat os EA ++] 31°I C.c.| I9°1 €.C./IO°0 c.c. | 270 C.c. .
C.| Rat infected with T. dimorphon. “The citrated a4 C.c. oe Cic:|"4:9/c:e. | 1-9)e.c. “Ste Se as
plasma containing 150,000 trypanosomes per
c.mm.
11 B| Normal rat Se a my: --| 24°6 C.c.| 13°8.c.c.| 8°7 c.c. | 21 C.c.
C| Rat infected with T. evansz. he citrated | 19:6 c.c. es (spre 18 CLCan| eI-O.c.c- BPS er
plasma containing 250,000 trypanosomes per |
c.mm. }

—<——————<$<———— ee

209

Taste 4. Analysis of gases obtained from defibrinated blood and from mixtures of this with plasma of

infected animals before and after incubation at 37° C. for 1 hour in the absence of air.

ComposITION oF BLoop ANALYSIS OF GASES OBTAINED FROM BLoop MixTurE BEFORE AND
Mixture AFTER HEATING FOR I HOUR AT 37°C. IN THE ABSENCE OF AIR
{ = —
Amount | Amount of citrated | Deficiency | Variation
No. of of P plasma of in amount | in amount
experl- defi- infected animal of oxygen | of carbon
ment | brinated | consisting of 2 parts Total Cor O N ai ae eieedeae
= blood of plasma and I part : | compared Cas com-
_. _ | normal citrated saline | with B_ | pared with
ok rabbit solution B
PPA! «2 cc. = o83.c.c.| o26c.c. | o-§2c.c. | 0-08 c.c. — —
B i 1°5 c.c. from guinea- | 1-16c.c. | o-61c.c. | o48c.c. | 0-07 c.c.
pig infected with
T. brucei contain-
ing 37,500 try-
panosomes per
c.mm. Before
incubation O13 c.c. | 0*04 C.c.
Cc A As B after incubation | 1-07 ¢c.c. | o-65c.c. | 0°35 ¢.c. | 0°07 c.c.
Al zc. == ogi c.c. | 043 ¢.c. | 039 c.c. | 0:08 c.c. -- _
of 1°5 c.c. from guinea- | 1°13 c¢.c. | 0-64¢.c. | 0°36c.c. | o-12 ¢.c.
pig infected with
T. evanst, contain-
ing 100,000 try- 0°33. c.c. | 0708 c.c.
panosomes per
c.mm. Before
incubation
(¢ s As B after incubation | o-82¢.c. | o72¢.c. | 0:03 ¢.c. | 0:07 c.c.
7A) 3.c.c. — 1'04.c.c. | o-42¢.c. | o-§2¢.c. | 0709 ¢.c. — ~-
B an 1°53 c.c. from guinea- | 1:27¢.c. | o-66c.c. } o'§2¢.c. | O09 C.Cc. |
pig infected with
T. brucei, contain-
ing 35,000 try-
panosomes per
c.mm. Before
incubation O'13.C.c. | 0°02 C.C.
Cc a As B after incubation | 1-17¢.c. | o-68c.c. | o39¢.c. | ooc.c.
Ane|) 3-C.C. — I24.c.c. | o-68e.c. | ©47¢.c. | ‘0:09 c.c. — —
B - 1°5 c.c. from guinea- | 1°27¢.c. | o-77c.c. | o-4ic.c. | 0:08 c.c.
pig infected with |
T. evansi, contain- |
ing 287,000 try-
panosomes per
¢.mm. Before
incubation 0°36.c.c. | 0-22 C.c.

c 5 As Bafterincubation tt1c.c. | ogge.c. | o05c.c. | 0°07 ¢.C. |

210

TABLE 5.

Data given in Table 4 re-calculated in volumes per cent.

|

ANALYsIS OF GASES OBTAINED FROM BLoop

MIXTURE BEFORE AND arTER HeatinG aT 37°C.

ror 1 Hour 1n AssENcE oF AIR

|
Defi- | Varia-
' | ciency tion
No. of Too c.c. of mixture consisting of 2 parts ins pl) jae
experi- defibrinated blood of normal rabbit and amount | amount
ment | 1 part of citrated plasma of infected guinea- of | of carbon
pig Total co, O N oxygen | dioxide
| in Cas | in C as
com- com-
pared | pared
| with B | with B
|
Guinea-pig infected with T. brucei. The |
citrated plasma contained 37,500 trypano-
somes per c.mm.
1 B| Before incubation 25°8 c.c. |13°6 G.C.., |10°7/€.C.s|\. 16". Chul) 220)C-Ca ec ancres
C | After incubation ... 23-Bie-c. |E4A\G.Col.7°8.C.Cen|) E-e)G-e-
Guinea-pig infected with TJ. evansi. The |
citrated plasma contained 100,000 trypano-
somes per c-mm.
2 B| Before incubation 2571, C.C |_1422'C.c-) B0'c.c. |, 2-71C.c. 7-4 CCan men meetee
C| After incubation ... 18°2€:G.|,16:01c.c.|| 077 €-c., || T-b'c.c-
Guinea-pig infected with T. brucei. The |
citrated plasma contained 35,000 trypano-
somes per c.mm.
3 B| Before incubation | 28:2 c.c.| I4°7 c.c.|11°6 c.c. | 2:0.c.c. | 2°9 C.c. | O-4'e.c
C| After incubation ... | 26-0 c.c.| I5*1 c.c.|7B-7 c.c. | 2-2 ¢.c. /
Guinea-pig infected with TJ. evansi. The
citrated plasma contained 287,000 trypano-
somes per c.mm.
4 B| Before incubation |. 28-2.€.C.|17°T'C.€.| 9°, C.C. >| ‘1:8, C. Ca 8-O C.C. | AsgmeRGe
C | After incubation ... 24°7 C.C.| 22°O C.-C.) 1°l C.G& |. "5 7Gse

AUT

A pparatus.—The apparatus employed for this purpose was that
illustrated by Figure I. The portion of the apparatus between
Stop-cocks 1 and 2 was first filled with mercury. Through the
two-way Cock 2 half a cubic centimetre of phosphoric acid (1 per
cent. solution) was introduced into the vacuum Bulbs B to
facilitate the removal of carbon dioxide. Cock 2 was now closed
and a vacuum created in Bulbs B. The mixture of defibrinated
blood and citrated plasma was then introduced by means of the
receiver into Bulb A which communicated with the outside through
Cock 1. Subsequently mercury was poured into the receiver and
allowed to flow into the bulb until the blood just reached Cock 1,
which was then turned so that the blood was now in contact with
mercury both above and below. After allowing the mixture to
react :in Bulb A at 37° C. for one hour, Cock 2 was cautiously opened
and the blood allowed to pass slowly into the vacuum bulbs.
Co@k 2 was then closed and the blood exhausted and the gases
collected in Tube 1.

After complete exhaustion of the blood the gas collected in
Tube 1 was measured and analysed by means of the apparatus
indicated by figure 2.

Technique.—About 12 c.c. of blood was withdrawn from the
external jugular vein of a normal rabbit and defibrinated by
shaking with a few glass beads in a bottle. The blood was then
filtered through gauze and placed in the ice-chest over night. The
following morning it was thoroughly stirred and, after determining
the percentage of haemoglobin present (in terms of human wet red
cells), three samples each of 3 c.c. withdrawn and allowed to stand
at room temperature, 10-12° C.

One of the three samples of defibrinated blood was then
introduced into the apparatus in the manner described, and after
warming to 37°C. for one hour in Bulb A was exhausted in the
vacuum Bulbs B and the gases obtained measured and analysed.

To the second sample of defibrinated blood was added a definite
volume of citrated plasma from an infected rat or guinea-pig
containing numerous trypanosomes in suspension. (The number of
trypanosomes per cubic millimetre in the citrated plasma was
estimated by means of a Thoma Zeiss haemocytometer.) The
mixture of blood and citrated plasma was then heated at 37° C. for

NO
—
nt

Vacuum BurasB

25
fo é — || TAREE way Cock T

“MERCURY JOINT Butp A FoR HEATING <I es

=
MIXTURE © DEFIBRINATED
BLloop & CITRATED eS / Qajigp 2 WATER BATH
PLASMA iN ABSENCE ©

AS

/ Two Way Gok ©,

§
;
ston O
Fig =
MERCURY
RESERVOIR
=
MERCURY TROUGH nad i
AND TuBE T Wr / ma é
FOR COLLECTING ah '
gases a i

an a

Fic. 1. Apparatus employed for incubating the suspension of trypanosomes and
defibrinated blood in the absence of air, and for the subsequent removal of the
gases contained in the blood mixture.

Fic. 2. Apparatus employed for measuring and analysing the gases.

213

one hour in Bulb A and the gases it contained subsequently
determined.

A like volume of citrated plasma (diluted to the same degree as
in the previous case) of a normal rat or guinea-pig was added to
the third sample, which was then treated in the same manner as
the other two cases.

The results obtained in these experiments are set forth in
Table 2. It will be observed that in every case the action of the
trypanosomes resulted in practically complete disappearance of
oxygen from the gases subsequently exhausted from the mixture of
defibrinated blood and infected plasma. A second point of interest
is that the amount of carbon dioxide was not increased in a degree
corresponding to the diminution of oxygen.

In Table 3 the results given in Table 2 are re-calculated in
volumes per cent.; or, in other words, the quantity of gas present
in 100 c.c. of the mixture—defibrinated blood plasma—estimated.

In a second series of experiments the procedure was similar,
except that instead of employing as controls the plasma of a normal
animal that of the infected animal itself was used. Here, however,
reaction was not permitted to proceed in Bulb A of the apparatus,
but the blood and plasma were passed straight on into the vacuum
bulbs and immediately exhausted.

As will be seen from Tables 4. and 5 the results obtained are
similar to those of the first series of experiments.

Although actively motile trypanosomes cause so considerable a
reduction of solutions of methylene blue and haemoglobin zz vz/ro,
yet the mere presence of numerous parasites in the blood of the
living organism is of itself insufficient to give rise to a purple
condition of the blood. The blood of rats swarming with parasites
is generally of the normal red colour. Under these circumstances
the oxygenation of the haemoglobin occurring in the lungs 1s
sufficient to counterbalance the reduction resulting from the action
of the parasites.

As we have already mentioned, the purple appearance is most
frequently to be observed in the blood of rabbits in a late stage of
the disease. In these animals trypanosomes are usually absent from
the peripheral blood or present in small numbers only. All animals
presenting the phenomenon had, however, marked involvement of

214

the respiratory passages and the breathing was stertorous and
laboured. The external nares was often almost completely
obliterated by an oedematous and infiltrated condition of the skin
and mucous membrane. Post mortem examination showed
extensive thickening of the mucous membrane of the nose and
pharynx, and sometimes even of the trachea and larger bronchi.
Sections of the affected tissues usually revealed the presence of
trypanosomes often in very considerable numbers.

SUMMARY

The blood of certain animals in the later stages of trypanosomal
infections is frequently of a dark purple colour. This appearance
results from deficient oxygenation of the haemoglobin.

Living trypanosomes cause marked reduction of solutions of
methylene blue and also of those of oxyhaemoglobin.

The incubation, in the absence of air, of living trypanosomes in
defibrinated blood of a normal animal causes considerable reduction
or—if the parasites be numerous—total disappearance of the
oxygen combined with the haemoglobin. A corresponding increase
in the amount of carbon dioxide has not been found.

The mere presence of numerous parasites in the peripheral
circulation is not, however, sufficient to account for the purple
colour of the blood, since the blood of rats and guinea-pigs
swarming with parasites is usually of the normal bright-red
appearance.

The purple colour is most marked in the blood of rabbits in the
later stages of the disease. Although the peripheral blood of these
animals does not contain large numbers of parasites, yet the
respiratory passages are found to be considerably involved. The
nasal mucous membrane and that of the trachea and bronchial tubes
is often extremely oedematous and infiltrated, and the respiration
of the animal is stertorous and laboured. On microscopical
examinations of sections of these tissues trypanosomes were often

found in large numbers in the oedematous mucous and sub-mucous
tissue.

215

THE ANTI-MALARIAL OPERATIONS
AT ISMAILIA

BY

j; W.. W.. STEPHENS;..M.D. (CANTAB.)

(Received for publication 26 April, 1911)

That the Anti-malarial operations at Ismailia following on the
report of Sir Ronald Ross (1903) have resulted in completely
freeing the town from Anophelines and hence from malaria is a
well-known fact, but I do not think it is equally well known what
exactly was done at Ismailia to secure this result. I have attempted
here to give as complete an account as possible; the only existing
account so far being the brochure in French of the Suez Canal
Company, of which I have made full use*t. I have thought it of
interest also to attempt to trace historically the beginnings of
malaria at Ismailia.

I. HISTORICAL

The history of malaria in the Isthmus of Suez and more
particularly at Ismailia is bound up with the history of the water
supply of the region. The problem of supplying water to the
workmen engaged in cutting the Suez Canal through the desert,
was always a pressing one in the early days of the construction.
The three sources of water that were at successive periods available
were, viz., (1) the wells, (2) the alimentary canal, and (3) the
freshwater canal which flows from Cairo to Ismailia.

THE WELLS

1859. April 25. The work of constructing the Suez Canal was
commenced.

1859-1860. Water was found at the following sites near
Ismailia, the centre of the Isthmus.

** While this article was in the press a paper by Bruce (191!) has appeared dealing with
the subject from rather a different standpoint,

+ I beg to tender here my grateful thanks to Dr, Pressat and Dr. Cambouliu of the
Suez Canal Company for much information kindly supplied,

216

1. El Ferdann. Water was found at a depth of 2°50 metres,
but it was salt.

2. Sabah ’Byar (Seven Wells) in Wadi Tumilat.

3. Bir Abu Balah. Wells existed here from Biblical nates
Six other pits dug gave only salt water.

4. Lake Timsah. Workmen were employed in cutting the
reeds at the foot of the Nefisha sand-dunes, and the tamarisks
bordering the lake, showing that the soil contained sufficient
moisture to support vegetation.

5. Fawar. Passable water found at 3°30 metres. This was
the site of an ancient pit.

6. Tusum (plateau). Abundant but slightly brackish water
found at a depth of 13 metres. Previous to finding water here, it
was conveyed by dromedary from Awebet Station, six hours’
distance from Timsah.

1860-1861. Water in the centre of the Isthmus (Lake Timsah)
was procured from wells sunk at the following places.

7. Bir Abu Balah. Water was found at a depth of 4°70
metres. This water was distributed by a water-wheel used for
irrigating the attempts at cultivation of barley, cotton, melons,
haricots, etc., over an area of 14,200 square metres. In
October 6,400 square metres were ready to be sown. It is
interesting to note, that at this time even, possibilities of pool
formation existed, as very probably was the case with uncontrolled
irrigation.

8. Zusum. Wells were dug, and near them a layer of clay,
1°20 metres thick, was found. A small garden, 30 by 20 metres,
was planted with barley, wheat, bersim, cauliflowers, onions,
haricots, sea-kale, etc.

Q. Serapeum. Water was found at a depth of about 17
metres.

10. Abu Souer, near Makfar, 15 miles from Seuil (El Guisr).
These wells in the Wadi Tumilat Valley furnished excellent water.

11. Nefisha. Brackish but drunk by animals.

12. Sabah’Byar. Three miles from Makfar, close to Nefisha,
gave sweet water. ‘Excellent water throughout the year.’
(Anonymous, 1857).

13. Abu Eroug. East of the Suez Canal, near Working-
camp I, brackish but drunk by animals.

90:35 30:30

30°45 30:40

30:5

E\ Ferdane
oFawar Wells

—w

gaara

om a
— STi

OS :6%

Map
showing distribution of
Weis in NeiGuBouRHoop oF ISMAILIA

ahsama _|
Tation 9}

Orbe

R40

30:30

30-45 3040 30:95

217
ALIMENTARY CANAL.

In order, however, to secure a more abundant supply of water
for the working camp at El Guisr, about six miles north of Timsah
(Ismailia), water was got from Lake Mahsama (average depth
2 metre), in Wadi Tumilat.

A water-course, 26,800 metres long, 0°30 metres broad at bottom
and I metre at water level, was constructed as far as Bir Abu
Balah, where it was collected in a masonry reservoir. From here
it was brought in pipes to Timsah (Ismailia) to a well and pumped
up to a 64 square metre sheet-iron tank; from here, again, it was
taken in earthenware pipes to El] Guisr, where a second pump and
reservoir was again used for distributing it as far as El Ferdann
(about eight miles from Ismailia). The workmen digging this water-
course got their water by camel from Nefisha. During a temporary
failure of this supply, water was carried by camels from the sites
(10-13) just mentioned.

FRESHWATER CANAL

1862. Reached Timsah (Ismailia), and after the 23rd January
was opened for navigation. It comprised (1) an old canal from
Zagazig to Qassasin, and (2) a portion from Qassasin to Ismailia,
7°70 metres wide.

Sluices were constructed at Nefisha to carry off the excess of
water into the lagoons bordering Lake Timsah. The difference
of level between the freshwater canal and the Suez Canal was
6°6 metres, necessitating the intervention of locks.

1862. April 27. The first stones of the foundation of
Ismailia (originally Timsah) were laid (though in 1862 two or three
chalets had been begun), streets were laid out, palms planted. The
population in September, 1862, was:—Europeans, 150; Arabs, 593.

(1863. Timsah called Ismailia.)

1865. A new canal in lieu of the old one built from Abassa to
QOassasin, 10 metres wide.

1866. July. The canal termed the Ismailia Canal.

(1869. 17 November. Suez Canal opened.)

1870. The portion of the canal from Abassa to Qassasin
enlarged to 13 metres.

218

1874. Portion from Qassasin to Ismailia enlarged from 7°70
metres to 13 metres (commenced).

1877. The new canal inaugurated, 15 April.

1896. Hydraulic rams used for distributing the water at
Ismailia, previously it was done by the pumping station (wszue des
eaux) at Ismailia.

CIRCLE CANAL

1863. July-August. A pumping station was established at the
extreme East of the town, to supply the stations between Ismailia
and Port Said. This was supplied by a branch of the sweet-water
canal, starting 720 metres above the upper lock at Ismailia. It
was 2,670 metres long, and followed the North of the town,
o’5 metre wide at bottom, depth 0°75 metre: It furnished water for
the cultivations established along part of its course and for a large
experimental garden around the pumping station.

1866. It was cleaned.

1877. It was considerably enlarged at the same time that the
Abassa-Ismailia Canal was replaced by one of larger section.

1880. It was filled up and dried.

These data are, I think, sufficient to show that breeding places
existed from the earliest times, and that Anophelines also existed
will be shown in the next section, hence I consider that the view
of E. H. Ross (1909), that malaria came to Ismailia in 1877 with
the enlarged freshwater canal, is incorrect.

II. ORIGIN OF MALARIA

1861. ‘Among the numerous workmen employed at the
construction of the freshwater canal, there occurred nine cases of
simple intermittent fever which yielded easily to quinine. They
had contracted the malady on the borders of Lake Mahsama, where
the fever showed itself each year, especially after the rise of the
Nile.’ Voisin Bey (1906).

I conclude from this that malaria was, even at this time,
endemic in the valley of the freshwater canal, and Anophelines
also must have existed in the region.

219

1865. ‘The town (Ismailia) was extensively watered by a
system of pipes which conducted the water into each dwelling and
permitted the establishment there of vegetable and flower gardens.
The sanitary condition was normal in January, but in February
ordinary illnesses began to take more grave forms, especially among
the new arrivals—Calabrians, Dalmatians, Bretons and Greeks.
Illnesses begun in Europe, and unknown in the works-stations on
the Isthmus, showed themselves. Pernicious fevers, fever of a
remittent type, etc., occurred. The mortality was 2°5 per cent.’
Voisin Bey (1906).

1866. ‘Among some work-stations, among others E] Guisr
(about three miles from Ismailia), cases of simple intermittent, and
some cases of pernicious fever occurred. The Isthmus had, up to
this time, been free from this malady, but this year the fevers had
been fairly frequent, and had assumed the paludic character
without being otherwise dangerous or obstinate. There was a kind
of general paludic influence.’ Voisin Bey (1906).

It seems clear from this that malaria was fairly common in the
Isthmus, and that it is inconsistent to say that ‘the Isthmus had, up
to this time, been free from this malady.’ We see thus early the
seeds sewn of the crop which was eventually gathered in Ismailia.

After this date we have no further records, but it is hardly
credible that no more malaria occurred until the outbreak in
Ismailia in 1877. We believe rather that malaria was always
endemic in the Lake Mahsama region, that malaria and Anophelines
spread from there. Probably Anophelines were always present in
the Nefisha lagoons as they were in 1908. (Three cases of malaria
in 1905, Bulletin (1909), p. 8.) No doubt also many cases were
European in origin.

1877. Epidemic. Pressat (1905), attributes the outbreak to
the fact that the enlargement of the freshwater canal to 13 metres
led to a superabundant water supply, which, filtering through the
sand, formed a subterranean layer which formed pools at every
suitable spot, that subsequently Anophelines were introduced by
boats along the sweet-water canal, or by rail. The increase in water
very likely led to more pools in this way, but pools and Anophelines
must have existed long before this, as the freshwater canal reached
Ismailia as early as 1862, and irrigation was proceeding in 1863,

220

and, moreover, the sub-soil water was always present, making itself
apparent especially at high Nile, and, as a matter of fact, as we
have just seen, malaria was recorded at Lake Mahsama in 1861,
and at Ismailia itself in 1865. The data now available are,
however, insufficient to explain, with certainty, why malaria,
which we believe always to have been endemic at Ismailia, became
epidemic in 1877 (300 cases). Nor, again, is the cause of the rise
in 1886 (2,500 cases) explained.

III. THE ANTI-LARVAL OPERATIONS

Ismailia is situated on the North bank of Lake Timsah (Lake
of Crocodiles). The lake received at rather high Niles fresh
water (reaching it through the Valley of Gessen, roughly at
right-angles to the Suez Canal, and through which region the present
freshwater canal flows) according to an observer (Anon., 1857), who
also states that he found the muddy sediment of the Nile in its
swamps. Before the cutting of the Suez Canal, the water in it was
intensely salt (owing to the underlying bed of salt); its depth was
only o'6 metre, but along its margin reeds grew in abundance and
tamarisks (Anon., 1857) also occurred, deriving their nourishment
(presumably) from the underlying sub-soil water.

Over a great part of the Isthmus, according to Roux (1901),
one finds in the sub-soil a layer of water scarcely brackish, which
is held in the sand by a thin layer of clay, and which flows about
at the level of the sea and that of the Suez Canal.

Boyce (1904), notes: ‘freshwater grasses, and other freshwater
plants, growing along the margin of the canal, and even in the
water of the canal itself, which is strongly salt. The explanation
given to me of this occurrence was that the sub-soil fresh water in
the bank of the canal afforded the necessary moisture for the roots.’

Ross (1903, p. 5), states that ‘the sub-soil water is very near
the surface and, as we are informed, fluctuates with the rise and
fall of the distant Nile. In some spots near Ismailia, where the
surface of the desert is much depressed, this sub-soil water produces
considerable lakes and ponds, but owing to the extreme salinity
of the sand most of these pools are brackish, their shores being
encrusted with salt, supporting but little vegetation. There are

221

several spots, however, where the water is nearly, if not quite fresh,
and here we observe a considerable amount of cultivation—grass
and vegetation. There are even places where the fresh, natural
waters produce shallow marshes of small extent; where small pools
form among reeds and grass. And these can be found not only
close to Ismailia but, as I was informed, in many parts of the desert,
and can be seen along the railway to Cairo. But it must be under-
stood that such areas are very small in extent when compared with
the large surface of perfectly arid sand which surrounds the town.’

BREEDING-PLACES OF ANOPHELINES AND ANTI-LARVAL
MEASURES ADOPTED

Ross (1903, p. 12) says that ‘whenever we examined the marshes
connected with the natural waters which exist around Ismailia, we
succeeded in finding numerous larvae of Anopheles and also of
Culex. The insects existed especially among the short grass and
other vegetation growing on soil covered with a very thin layer of
water. On one series of pools situated to the East of the town,
we found innumerable larvae both of Anopheles and Culex
existing in water which was so brackish as to contain nine grams of
salt per litre.’*

He sums up the breeding-places as follows :—

1. The small marshes in the midst of the cultivation to the
East of the town (? Abu Rahan).

2. The still smaller marsh close to the abattoir. [(0) on map.]

3. A few were observed in an artificial fountain in the middle
of the European station.

The following account is based on the data contained in
(1) Pressat, ‘Le Paludisme et les Moustiques’ quoted as (P.) and (2)
‘Suppression du Paludisme a Ismailia,’ Compagnie universelle du
Canal maritime de Suez, quoted as (S.), and on personal communi-
cations from Drs. Pressat and Cambouliu.

Pressat (p. 132) describes the Abu Rahan marshes as_ being
infested with Anophelines, and that work was carried on there in

* These Anophelines may have been Pyretophorus cleopatrae Wilcocks, an Egyptian
species which commonly breeds in saltish water, whereas Cellia pharonesis does not.
Whether ?. chaudoyet, an Algerian species, also exists in Egypt is, I think, doubtful.

222

‘clouds of Anophelines’ (p. 132). Elsewhere he describes the
breeding places as being ‘pools, drains, camel foot-prints in the
environs of the town’ (p. 120).

ABU RAHAN MARSH.*

This marsh is situated to the North-east of the town, less than
a mile (1,640 yards) away. It has an area of between nine and ten
acres (44,252 square yards) and has an average depth at the
portions marked A and B of four and a half feet and a maximum
of about eight feet.

Presence of Larvae.

1901. August. Many were found in the irrigating channels
that supplied the plantations of this marsh.

1902. September. Ross’s data probably apply to this marsh.
Destructive measures.

1880-1881 or 1885. This marsh had been partly filled up in
these years and planted with Casuarina, Eucalyptus and Palms.
It had also been drained by a channel leading to the lake,
but this drainage had been ineffective for a forest of reeds had
grown over the whole of the Northern area. Moreover, at the rise
of the Nile (in the Autumn) all the inequalities of the soil filled
with water and especially the irrigating channels along the bases of
the trees.

NoTE.—These preliminary efforts were made before the mosquito
as the malaria agent was known.

1903. (@) The main drain was deepened and _ cleaned
throughout so as to maintain a proper fall to the lake. The fall of
the drain was 0'0005 per metre.

(6) Two encircling drains and three cross drains were
constructed.

(c) Further, sluices were constructed so as to dam up the water,
and subsequently to flush out the channels if necessary, but this
has not been required.

(zd) The reeds were cut everywhere.

(e) The soil was carefully levelled by filling up all depressions
and all the irrigation channels.

* Ihave been unable to ascertain when this marsh first came into existence.

223

Workmen were assigned to keep the drains at the proper depth
and to keep their banks free from vegetation, and also to keep the
marsh absolutely free from reeds.

Result.

1903. August. No larvae could be found either in the
contributory channels or in the main drain. It must be noted that
all these channels and the main drain contained fish, viz., Mugil
cephalus, M. capito and M. seheti and also Tilapia gallilea (Arabic
Chaba’r). Whether these fish destroy larvae is unknown,
but 7. gallilea will at least destroy them rapidly when hungry in
an aquarium (P.).

THE SUBSIDIARY MARSHES.

(1) MarsH H.
To the East of Abu Rahan is of slight depth.

Presence of larvae.
Along the margins there were numerous Anopheline pools.

Destructive measures.
It was filled up (with sand).
(2) MarsH I.
To the North-West of Abu Rahan, has an area of 22,962 square
yards, equal to 4-5 acres, and a depth of about 2 feet 9 inches.
Presence of larvae.

No records.

Destructive measures.
It was filled up and united to the drains of Abu Rahan by a
subterranean channel.

(3) MARSHY GROUND ALONG LEMASSON AVENUE.

Pools formed during the rise of the Nile.

Presence of larvae.

No records.
Destructive measures.

1903. These had been filled up many years before, but they
were now re-covered with a sufficiently thick layer of sand.

224

SOUTHERN REGION.

Small depressions near the North bank of the lake, which
contained water at high Nile.
Presence of larvae.

A few.

Destructive measures.

They were filled up with sand.
WESTERN REGION.

From the outskirts of the town to Nefisha Station, a distance of
more than 5 kilometres (3 miles), exists a stretch of land lying
between the sweet-water canal and the lake. The level of the
sweet-water canal along this district is 6 metres (19 feet 8 inches)
above the mean level of the lake. This land, like the rest of the
desert when irrigated, is fertile and is let out to cultivators, but
it is absolutely necessary for cultivation purposes that the water
should not lie stagnant but be drained away. For this purpose the
cultivators had constructed a number of canals but at the time of

the anti-malaria campaign they were badly kept, full of weed and
almost stagnant. We may consider these in more detail.

WESTERN REGION (N).

At this point there existed numerous small puddles and marshy
areas covered with grass or bulrushes.

Presence of larvae.
Present, a dangerous focus (S. p. 15).

Destructive measures.
Filled in with sand.

WESTERN REGION (O, Q).

At the points O and Q hydraulic rams exist for the supply of
water to the town and hospital respectively. The drains in
connection with O led into the lake, while that of Q ran into Nefisha
lagoon, an arm of the lake. Both were in a state of bad repair,
the gradients were imperfect so that water remained stagnant in
them, the banks had broken down, and water-cress beds had been
established along their margins.

225

Presence of larvae.

Abundant, ‘a dangerous focus’ (S. p. 15). Ross also cites the
marsh existing close to the Abattoir (O) as containing larvae.
Destructive measures.

The drains were cleaned, deepened, the banks repaired, the
cress-beds done away with, all vegetation removed. The drain of
hydraulic ram O was divided into branches, the eastern branch
draining the neighbouring cultivated land.

CGERVATED AREAS-R, S, ‘I, U:
The drains in these areas were exceedingly numerous.

Presence of larvae.
Abundant, ‘a dangerous focus’ (S. p. I5).

Destructive measures.

Numerous old drains were replaced by one large drain
traversing a dune and discharging into Timsah. In other cases the
drains were repaired, deepened and the banks kept in good order.

Result.

The result of these works on the cultivated lands was not at
first satisfactory, for Anophelines still continued to breed in the
gutters of the low-lying marshy parts opening into the lagoon, where
the water was shallow and almost stagnant. Accordingly the plan
was devised of damming (weekly) the irrigation water that supplied
these areas. As the freshwater canal is about twenty feet above
the lake level there is produced, on opening the dams, a very rapid
and powerful stream, sufficient to sweep out all larvae. The result
of this procedure was completely satisfactory. (This method of
damming the water is now practised in all the irrigation channels.
The water is led into those areas that require irrigation at definite
intervals. It is then shut off, the result being that the water
supplied to the area in question has all sunk into the ground in
two or three days).

Result.

From the summer of 1903, no larvae found in the whole of the

protected area* (S. p. 20).

* It is not clear whether the areas O, P, Q, R, S, I, U, were at this time included in
in the term ‘ protected area’ for in a letter Dr. Cambouliu informed me that the areas O, P,
Q, R, S, T, U, were first drained in 1904-1905. In a subsequent letter, however, he states
that 1903 is the correct date,

226

MarRsH I.

This is situated to the West of Nefisha, is deep and contains
several species of fish.

Presence of larvae.
None (Dr. Cambouliu).

Destructive (?) measures.
1906. Drainage commenced. Now completed, I9gI!o.

MARSH Y., SOUTH OF NEFISHA LAGOON.

Larvae.
Along the shore at the entrance of the drains. The drains
contain numerous fish.

Measures.
1898. Levelled, planted and drained. 1903 (?), the pools
containing larvae, filled with sand.

OTHER EXTENSIVE MARSHES.

Exist along the course of the sweet-water canal. They derive
their water from the great drain, Bir Abu Balah, beyond Nefisha,
but their drainage would be extremely costly.

ADULT ANOPHELINES.

Very few data exist as to their numbers in Ismailia, but it is
stated (S. p. 11) that the whole town was invaded, and elsewhere
(p. 16) that they appeared each year precisely in the autumn, but
in regard to the first statement in a private communication Dr.
Pressat states: ‘certainement trés peu parmi les grands quantités
d’autres moustiques.’

In Abu Rahan marsh, adult mosquitos in large quantity

(Sap. 46):
OILING OF POOLS.

Besides treating the culicine breeding grounds the brigade also
oiled ‘toutes les mares, rigoles, flaques d’eau, les pas de chameaus,
des jardins et des alentours de la ville’ (P. p. 134).

This, Dr. Pressat informed me, refers to the small collections
of water in and around the town, and not to the marshes such as

Abu Rahan.

227
SUMMARY

As stated above, in August, 1903 (?1904, 1905), there were no
larvae in the whole of the protected area, hence zfso facto from this
time all fresh cases of malaria must have ceased, so that the
anti-larval measures were a complete success. Apart from the
drainage as a whole, I consider that the following three factors
played an important part in achieving this success, viz. :—(1)
Ismailia is in the desert; (2) the intermittent irrigation system with
a fall of 20 feet from the freshwater to the maritime canal; (3)
the presence of fish in all the drains.

COST OF THE OPERATIONS

(1) Inztzal expenses, £2,000. This was incurred in filling up
swamps and drainage (S. p. 25). Presumably this is up to 1906,
the following data extend to 1909.

(2) The non-recurrent expenses incurred for filling up the pools
and drainage of the swamps and arable lands situate in the
neighbourhood of the town, and to which the Egyptian Government
has contributed a part, to-day (1909) reach a total of about 100,000
francs (£4000). The area of the improved land represents about
400 hectares (roughly 1,000 acres), so that the cost was 44 an acre
(Bulletin 1909).

(1) Permanent expenses. In keeping drains in good repair
cutting reeds and flushing drains, £312 per annum. This does not
include the sum spent in anti-Culicine work, which we have not
considered here (S. p. 25). To this would also have to be added
the expense of oiling small collections of water—possible
Anopheline breeding places.

(2) The permanent expenses for disinfection, the hunt for
mosquitos and larvae, and the maintenance of the improved lands
have remained at about 18,000 francs (4720) per annum since
1903 (Bulletin 1909). This, no doubt, includes expenses of
Culicine destruction, and accounts for the discrepancy between the
two statements.

(3) The following table is added for the sake of completeness,
though it does not appear that the expenses can be fairly put solely

228

to the account of anti-malarial measures. In fact, here, as in other
figures, no details are given, and in this case it is impossible to say
what proportion must be ascribed to each of the three headings.

Wages paid to fever patients whilst not working. Curative
medicines distributed to old malaria patients. General prophylactic
measures*.

TOTAL OF EXPENSES

1903 38,209 francs £1,528 approximately
1904 25,9860 ,, 1,039 y
1905 175420. 9, 696 5
1906 1639063.) 255 678 5
1907 T8421 i295 625 rr
1908 16,306, .;; 672 5

IV. THE QUININE PROPHYLAXIS

1902. February. This was instituted at this time, ‘@ tout le
personnel de la Compagnie qui en a retiré un grand bénéfice’
(P. p. 111), but I understand privately from Dr. Pressat that it was
optional in the case of employés (about 2,000) and obligatory in
the case of workmen (about 7,000). The latter, for example, if
sick, lost a part of their salary, otherwise paid to them, unless they
had taken their quinine in the fixed dose, in the presence of the
overseers (S. p. 9). The Company, in fact, were in a position to
ensure their orders being carried out.

Prophylactic dose.

This was given in the form of two pills of 14 grains each for
three consecutive days, then a week’s interval in the first half of
the year, and a three day interval in the latter half of the year
(the ‘fever season’) (S. p. 10).

* Ross (Bulletin 1909, p. 4) states ‘I am informed that a considerable part of these
expenses have been and are incurred not only for malaria prevention but also for agriculture
and other purposes.’ This is another illustration of the difficulty of finding out what
exactly the published data connote.

229

Result.

As an example, it is stated (P. p. 106) that ‘ces nombreuses
équipes (centaines des ouvriers)’ receiving daily, in this case,
3 grains of quinine, ‘travaillérent plusieurs mois,’ in Abu Rahan
marsh, ‘dévoreés par les Anopheles et pas un des ouvriers ne prit
la fiévre’; while, ‘a 14 méme époque une compagnie particuliére qui
exécutait dans la méme région des travaux de dragage sur le canal
Abassieh, et qui ne prenait pas les mémes précautions vit ses équipes
litteralement fondre sur les atteintes de la malaria’ (microscopically
confirmed). Here it is not stated that these latter workmen were
devoured by Anophelines, so that the difference might be attributed
to their absence. If we are to assume that the first set of men were
infected in the marsh then the mosquitos must have been infected
there by gamete-carriers among the men, so that we get a marsh full
of ‘infected’ mosquitos. If such a condition existed it is an
interesting and certainly an exceptional one, but it is, I think, more
probable that these workmen, like the other gang, were infected in
their houses, the difference between the two cases being the quinine
prophylaxis.

It should be stated, however, that, according to Pressat (p. 132),
Abu Rahan was formerly so unhealthy that one could not undertake
the least work without redoubling the malaria. Elsewhere
(P. p. 130) it is stated that circulars were issued. The employés
and workmen were informed that there would be a daily gratuitous
distribution of quinine. They were asked at the same time to
suppress water in their ‘bassins d’arrosage’ and to suppress all
stagnant water (P. p. 130).

Result.
This was appreciable, the number of malaria cases being :—
1901 s ae ae — 1990
1902 as se a ae 1551

We have no data, however, as to what proportion of these cases
were employés for whom the prophylaxis was optional.

Similarly in the dispensary we find a peculiar but more marked
fall, the figures being : —

1900... 2,591 1903, 1.206
FOOT” 3 476 1904 ..._ I (first six months)
B02... 85

From a private communication from Dr. Pressat I learn that
in 1901 he began systematic blood examinations of the dispensary

230

patients, nearly all natives, so that the figure 476 may be accepted
as accurate for 1901. We have then, in 1902, the great fall to 85.
This fall is, I think, certainly due to the quinine prophylaxis of
1902 (for it was in December, 1902, that it was first decided
systematically to undertake anti-larval measures after Major Ross’s
visit in September, 1902), for as we have seen the prophylaxis was
compulsory on the labourers. (This fall must have had, too, some
effect on the hospital statistics, for presumably some of these cases
would become hospital patients.) To what the extraordinary fall
of 2,591 in 1900 to 476 in Igo! is to be attributed is not clear. For
although diagnosis made by the ‘sisters’ of the dispensary may not
be very accurate, yet if these 2,591 cases of ‘fever’ were not
malaria, we are at a loss to what to ascribe them, for, as Ross
states, ‘without it (malaria) Ismailia would be a settlement almost
free from infectious diseases.’ It should be added that quinine at
this time was given by the ‘sisters’ to those who came to ask for it,
but to what extent it was given, what effect it had it is impossible
to say, unless one accepts these figures as affording the answer.

1903. The quinine prophylaxis was continued and in the
pre-epidemic months the supervision was more strict. Circulars were
issued explaining how to take it. It was distributed freely in the
office, workshops and at the dispensary.

The following figures (P. p. 138), indicate to what extent the
compulsory prophylaxis was carried out :—

QUININE PROPHYLAXIS.

1902 (Qmonths) ... 9,900 francs se 4396
1903 me -.~ 14,700 3 ie 5Q1'2
1904 ae ree © lee DP oa oy. 568°56

The following data differ again from these, but are official.

INTERNATIONAL CO-OPERATIVE PHARMACY AT ISMAILIA.
SALE OF QUININE.*

1902... 16,905 fr.10 ... £656'20 approximately.
eae. «15,075 IT. 2Or* asx 627°12 =.
meee... 11,024 in, 17. 464'90 58
1905... 6,482 fr. 4GoRr:. 259'28 +
1000 -*...-, ‘6,080 fr: OGL... 267°56 e
ieo7)...: .. 9,821 fr. Go %:. 352°84 iy
Peer 2... i, O27 tt Ady ay 237°08 3

** Bulletin 1904, p. 9.

231

It is impossible then to estimate definitely the result of the
quinine prophylaxis owing (1) to the incompleteness of the data,
and (2) to the fact that in 1903 it was complicated by the anti-larval
measures. We have, however, quoted some results that must be
attributed to it. Whether malaria would have been stamped out
at Ismailia by the quinine prophylaxis it is impossible to say.
Had not anti-larval measures been adopted in 1903 then we
would have had a quinine prophylactic experiment carried out under
ideal conditions, and the result would have been of great scientific
interest.

If, however, the Areas O, P, QO, R, S, T, U, were not drained till
1904-1905* it is probable that the decrease zz malaria in 1903 was
due in considerable part to the quinine prophylaxis, though the same
result would have been arrived at without the taking of a grain of
quinine.

NoTE.—-The map is adapted from that published by the Suez
‘Canal Company.

LITERATURE

Anonymous (1857), ‘Isthmus Suez Ship Canal, etc.’ London, John Weale.

— (1906), ‘Suppression du Paludisme a Ismailia.” Compagnie universelle du canal maritime

de Suez, Paris.
Bulletin (1g09) of the Liv. School of Trop. Med., No. 1. Liverpool.

Boyce, Sir Ruserr (1904), ‘The Antimalarial measures at Ismailia.’ Liv. School of Trop. Med.

Memoir XII, p. 5.

Bruce, Sir Davin (1911), ‘ Report on the present conditions of Ismailia as regards malarial fever.’

Journ. of the Roy. Army Med. Corps, p. 402.
Pressat, ANpRE (1905), ‘ Le Paludisme et les moustiques (Prophylaxie).’ Paris, Masson et Cie.
Ross, E. H. (1909), ‘ The Prevention of fever on the Suez Canal.’ Cairo.

Ross, Ronatp (1903), ‘ Report on Malaria at Ismailia and Suez.’ Liverpool School of Trop. Med.
Memoir IX.

Roux, J. Cuartes (1go1), ‘ L’Isthme et de Canal de Suez.’

Vorstn Bey (1904), ‘Le Canal de Suez, Paris,’ 6 vols., 1904-1906.

* Vide note antea.

y 7

ie

ne
z

Dat & ~
ile .

SI SCO'WS&YZSS!!SS

f = LA FIEVRE PALUDEENNE
A ISMAILIA

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14.000°

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11000
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; 1876 a 1905
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Note sur la Suppression du Patudisme a Isr a. C

233

ON SOME NEW SPECIES OF AFRICAN
MOSQUITOS (CULICIDAE)

BY

Rae Wote Ab, MSc., A.L.S., ETC.,
AND

BENE -CARTER,

ASSISTANT ENTOMOLOGIST, LIVERPOOL SCHOOL OF TROPICAL MEDICINE
(Recewed for publication 29 May, 1911)

Three of the Anopheline mosquitos described herein formed part
of the collections made by our colleague, Dr. Allan Kinghorn,
and Mr. R. E. Montgomery during their expedition to Zambesi in
1908, on behalf of the Liverpool School of Tropical Medicine.
In addition to these there were also present in the collection
examples of Myzomyia funesta, Myzorhynchus mauritianus, and of
the Culicines A porvoculex punctipes, Mansonia major, M. uniforms,
and a species of Chrysoconops, which may prove to be new.

Neocellia ( ?) christyi was presented to the School by Dr. Christy
some years ago. It is a very striking species, but we cannot from
the single specimen be quite certain as to whether it is correctly
placed in the genus Neocellza.

Cellia sqguamosa var. arnoldi has already been referred to by
Stephens and Christophers (1908), as Cellza arnoldi, and the
characteristics of the egg and larva are now given below.

Dr. A. S. Donaldson made an extensive collection of mosquitos
while stationed at Broomassie, Ashanti, during the years 1907-1909.
It has been our intention to publish a list of his captures, which
would add considerably to our knowledge of the mosquitos of this
region, but we have thought it desirable to defer this for a future
publication. Two new species were found among those he

234

collected; Cellza czncta, described in a previous number of these
Annals (1910), and Reedomyza stmulans described below. We take
this opportunity of expressing, on behalf of the School, our best
thanks for the valuable material which he was pleased to present

to us.

Pyretophorus distinctus, n. sp.

Under pocket lens x 16.

Head.—White in front, yellowish in the centre and darker
behind; palpi black, with four whitish bands, the apex white;
proboscis black, with the labella pale yellowish brown.

Thorax.—Slaty grey in the middle with a dark median
longitudinal line, yellowish-brown laterally; scutellum rather paler
in colour than the thorax; pleurae greyish brown.

A bdomen.—Dark brown, with pale golden brown hairs.

Legs.—Femora and tibiae uniformly brown, rather paler
beneath ; fore legs with distinct but very narrow white apical bands
on the metatarsus and first tarsal segments; middle and hind legs
with minute pale areas at the articulations.

Wzings.—Costa mostly black, with two pale spots extending on
to the first longitudinal vein; fringe with pale areas at the apices
of the veins.

Microscopical characters.

Head.—Integument dark grey to black, densely covered with
upright forked scales, those in front being pure white, those in the
median area yellow, and those behind black; projecting from the
centre of the anterior portion of the head is a tuft of long white
scales and at the margins of the eyes laterally, from four to five
dark bristles. Antennae grey, the basal segment pale yellow; the
second, third and fourth segments with a few white scales.
Clypeus black. Palpi of four segments clothed with very dark
brownish grey scales and with an apical band to each segment, the
band at the apex of the first being very small and yellowish, the
others white. Prvobosczs black, with the labella pale.

Thorax.—With a median longitudinal groove and with two
pronounced lateral ridges. Between these ridges the integument is
of a slaty grey colour, and on either side of them, dark reddish
brown. Anterior area of thorax with a mass of long, thin white

235
scales, some of which project over the nape, remaining portion
clothed with golden narrow curved scales. Scutellum denuded,
grey in the centre, pale yellowish brown laterally. Metanotum
dark brown, pleurae rather paler brown. Hal¢eres with very pale
stems and dark apices.

Abdomen.—Dark brown, with pale golden brown hairs.

Legs.—Brown, femora pale ventrally; first pair of legs with
two distinct apical bands on the metatarsus and first tarsal
segment, remaining segments of fore legs and also those of the
middle and hind legs with pale articulations.

Wings.—With the veins clothed with rather long, thin,
black and yellow lanceolate scales; ¢he costa deep black, with two
pale areas, one apical and the other on the distal half just above
the base of the first sub-marginal cell; sub-costa black. First
longitudinal vein pale at the base, with five black spots, the apical
and two basal ones being the smallest. The second long vein
mostly pale, there being two small dark spots immediately before
and after the supernumerary cross vein respectively; the upper
branch of the cell with a small basal and a large apical spot, the
lower branch similar. Third longitudinal vein with a small apical
spot and two basal ones almost directly under those on the second
long vein. Fourth vein for the greater part pale scaled but with
a fairly large, dark area just after the base of the fork; both
branches of the cell with two dark patches of scales. Fifth vein
with two dark scaled areas, the larger near the centre and extending
a considerable distance beyond the junction of the branch, the
smaller situated near the apex of the vein, the branch with three
spots, one apical and two basal. Sixth vein with three dark
patches. Fringe with nine pale areas, situated at the apices of the
veins and branches. First cell considerably longer than the second,
posterior cross vein about twice its own length distant from the
mid vein.

Length—4-5 mm.

Habitat.—Luapula river, below Chingola’s village, N. E.
Rhodesia, 17/9/07. (Dr. A. Kinghorn.)

This Anopheline may easily be distinguished by the dense black
costa, which is strikingly characteristic, and also unique among the
members of this genus.

236

Pyretophorus distinctus var. melanocosta, n. vat.

This differs from P. adistznctus in having the whole of the costa
black, with the exception of the small pale apical spot. There are
also other differences, the chief being that the small spot on the
apex of the basal segment of the palp is not present; on the first
long vein also there are six dark areas as compared with. five in
P. distinctus. The position of the posterior cross vein also differs,
it being about its own length distant from the mid cross vein.

Habitat—Luapula river below Chingola’s village, N. E
Rhodesia, 17/9/’07.. (Dr. A. Kinghorn.)

Cellia pseudosquamosa, Nn. sp.

Under lens x 16.

Head, white in front, black posteriorly; palpi, dark with three
white bands and a small basal spot; on the dorsal surface is a
white line running from the apex to the basal spot.

Thorax, dark with two large black ocelli, one on either side of
the median line and situated on the anterior portion at a distance
of about one-third the length of the thorax; clothed with
numerous white scales.

Abdomen, dark brown with dark lateral tufts; last two seg-
ments white.

Legs, dark brown, the femora and tibiae mottled, metatarsi and
tarsi unbanded. .

Wings, dark, very densely scaled; costa with two small and
three larger white spots.

Microscopical characters.

Head, dark, very thickly covered with upright forked scales,
those at the base black, the rest pure white. A tuft of long white
scales projects forwards between the eyes and several dark bristles
also extend outwards from the anterior and lateral portions of the
head. Axtennae, dark, the basal segments with a few white scales.
Clypeus, dark. Palfz, with dense brown scales, and white apical
bands. to the segments, the basal band very small; the upper
surface has a thick line of white scales along almost the whole of its
length, which gives the two apical segments the appearance of
being entirely white. Proboscis, black.

Thorax—-\ntegument dark greenish brown; with two large
black ocelli, covered with rather small white spindle shaped scales.

237

Prothoracic lobes, with outstanding black and _ white © scales.
Scutellum, dark with white spindle shaped scales. Pleurac, paler
than thorax with three white longitudinal lines. Metanotum, dark.
Halteres, with pale yellowish stems and dark apices. Addomen,
dark brown, densely clothed with long irregular scales, those on the
last two segments being white. There are also a few scattered
white ones on the 6th, 7th, and also the basal segments; the dark
apical lateral tufts are not present on the 7th, 8th and oth segments.
Venter, dark with numerous scattered white scales.

Legs, dark brown, unbanded. Femora and tibiae dark, mottled
with white, pale ventrally; the basal half of the metatarsus also
shows traces of mottling.

Wings with large black and white lanceolate scales, the greater
portion of the wing being dark. Costa black with five white spots,
the two smaller basal ones not extending on to the first longitudinal
vein, the third being represented on this vein by a few white scales.
The first longitudinal has, besides those already mentioned, a small
white spot almost under the centre of the second black costal area,
and another immediately before the posterior extremity of the third
dark costal area; for a short distance at the base the vein is pale.
Stem and upper branch of the second vein black, lower branch
with two white scaled patches; third longitudinal vein mostly dark
with several white scales intermingled with the black ones in the
central portion. Fourth vein black; branches of cell with two pale
spots and white scales intermingled with the remaining dark
portion. Fifth vein with two pale areas on the upper branch and
one just before the fork on the lower branch; stem with a white
spot towards the apex, base white. Sixth vein with three pale and
three dark scaled areas. Wing fringe composed of dark scales but
with a few pale scales at the apices of the first longitudinal vein,
and the upper branch of the second long vein.

Length.—5'5 mm.

This description was drawn up from a single perfect female,
taken by Dr. Allan Kinghorn in North Eastern Rhodesia (Chin-
yanta’s village, Luombwa river), and evidently is closely allied to
Cellia squamosa Theob. From this, however, it can at once be

separated by the unbanded tarsal segments and the somewhat
different wing markings.

238
Cellia squamosa, var. arnoldi. (Newstead and Carter.)
Cellia arnoldi (Stephens and Christophers, 1908).

The only marked difference between typical examples of the
imagines of Cellia sguamosa and the var. arnold: is that the latter
has no trace of the white pleural lines; in all other respects the
two insects are, so far as we can trace, identical There is, how-
ever, a marked difference between the larvae of these insects. In
the first place that of C. sgwamosa, according to Hill and Haydon
has no branched hair on the antenna, indeed, this structure is
apparently absent in many of the known African species of this
genus. It would seem therefore that as the var. arnoldi has a well
developed branched hair, that it may eventually be raised again to
specific rank. There are also other differences, especially in the
form and situation of the palmate hairs.

Ova (fig. 1), somewhat peculiar in form, the anterior extremity
being considerably broader than the posterior. It is reddish brown
in colour and about 0’5 mm. in length.

Larva (figs. 2, 3, 4).—Antenna with a distinct branched hair on
the shaft; terminal hair missing.

Frontal hairs apparently the same as in Cellia sguamosa, Theob.

Palmate hairs (fig. 4) rudimentary on the first and second
abdominal segments, fully developed on the third to the seventh
inclusive. In C. sguamosa the hairs are all well developed on the
abdomen, and there is also a rudimentary one on the thorax; the
shape and number of the leaflets to each hair also differs from the
latter species. In the var. arnoldi the filament is very short, and
there is a larger number of leaflets.

Neocellta ? christyi, n. sp.

Under pocket lens x 16.

Head.—Dark behind, creamy white in front; palpi dark brown
with two white apical bands and a narrower creamy median basal
band; proboscis black with the labella pale.

Thorax and scutellum black with creamy scales; metanotum

black.
Abdomen dark with distinct pale spots laterally.

239

Legs brown, femora and tibiae pale beneath with apical banding
to the tarsal segments.

Wings large and broad, very clearly spotted costa with five
black spots, the second and third the largest, the basal one small
and not extending on to the first longitudinal vein. Fringe with
eight pale spots.

Microscopical characters.

Head black, clothed with creamy upright forked scales in front,
dark ones behind, with a tuft of long irregular pale scales
extending distally between the eyes, and with several short golden
bristles projecting over the eyes laterally and anteriorly. Antennae
dark with pale scales on the first few segments. Palfz with dark
brown scales and with pale apical bands, the posterior pair small
and creamy, the distal pair white. Pvodosczs dark.

Thorax.—Integument very dark grey, almost black, with deep
cream coloured scales, which approximate more to the narrow curved
than to those of the spindle-shaped type; anterior portion with
long thin creamy-white scales projecting forward over the nape.
Scutellum dark, somewhat paler laterally with scales similar to
those on the thorax, and with numerous short golden bristles;
metanotum black. AHaléeves testaceous with slightly darker apices,
clothed with small flat scales of a dull golden brown colour.

Abdomen.—Integument almost black, with white, basal, lateral
areas to the middle segments. Covered with long irregular golden
brown scales and hairs; the basal segment with a median tuft of
long golden bristles. Venter dark with a few white flat scales.
Legs brown, the femora and tibiae covered with pale scales laterally
and ventrally. Tarsi of the first and third pair of legs dark with
fairly broad apical creamy bands, the last tarsal segment dark;
mid-legs missing.

Wengs much broader than is usual in Anopheline mosquitos, the
hind margin being markedly arched, and the veins are somewhat
thinly scaled. The costa with five dark areas, the third being much
the largest, and all, with the exception of the small basal one,
spreading on to the first long vein; the first and fourth evenly, the
second interrupted by a few white scales in the centre, and the
third by a small pale area towards the proximal end. The third
and fourth spread evenly on to the sub-costa. Second vein with

240

a large dark patch at the base, upper branch all black except at
the apex, lower with three dark spots; junction of the branches
pale scaled. Third vein with three patches of black scales, two
basal and one apical. Fourth longitudinal with two large dark
areas, each branch with two spots. Fifth vein with three spots
almost equidistant, the branch also with three, one at the apex, one
immediately in front of the posterior cross-vein and the other at
the base of the fork. Sixth vein with two dark areas. Wing fringe
with eight pale areas, the first at the apex of the first long vein, the
next at the apex of the lower branch of the second long vein, and
the others at the apices of the veins.

Length 7 mm.

A single female of this curious AzoPphelzne was taken in Uganda
by Dr. C. Christy; according to scale structure it appears to belong
to the genus Neocellia, Theobald, but in its general appearance it
is strikingly distinct, and is also widely separated geographically,
the members of the latter genus, as defined by Theobald, occurring
only in India.

Reedomyia simulans, n. sp.

Under pocket lens x 10.

Head black, with a brilliant white patch in front and a large
pale area on each side. Pvoboscis black, with a small indistinct
yellowish median band. Palgi about the same length as the
proboscis with three bands, the two basal ones being little more than
spots.

Thorax reddish brown, with two brilliant white shoulder-spots
and two smaller ones towards the centre, one on each side of the
middle line. Scutellum white; pleurae with five white patches.

Abdomen with basal and lateral spots ; apical segment all white.

Legs.—Femora dark brown above, pale ventrally, each with an
apical white spot and a small indistinct one a short distance before
the apex; tibiae and metatarsi dark brown with apical bands; tarsi
of the fore and mid-legs unbanded, the last two joints being pale
dusky brown; those of the hind legs with broad apical bands, the
last joint being entirely white.

Microscopical characters.

Head (fig. 5) with a triangular patch of silvery white and

rather broadly curved scales in front, this patch of scales is

241

connected by a narrow median line of somewhat narrower curved
scales, the latter finally expanding laterally at the base of the
head. The remaining portion of the head is clothed with flat
scales, the larger sub-median areas being composed of black ones,
which, however, gradually merge into lateral basal patches of pure
white ones; those scales bordering on the white areas are dark at the
base and of a brownish yellow colour towards the apex; these pale
areas are followed again with small patches of dark scales.
Several upright forked black scales are present over the greater
part of the head. Two long dark bristles project between the eyes
and also several shorter ones over the lateral margins. Axtennae
testaceous with whorls of long dark hairs. Palpz (fig. 6) composed
of four segments, slightly longer than the proboscis, and with three
small pale bands; the band at the base of the apical segment is the
most conspicuous and is composed of white scales, the others at
the bases of the second and third are very small and yellowish.
Proboscis black, with a pale yellow and somewhat indistinct band ;
labella yellowish.

Thorax reddish brown, covered with narrow curved scales, those
on the anterior lateral area forming large white spots; almost
immediately under these, towards the centre of the thorax, are two
minute patches of similar scales. Prothoracic lobes clothed with
white narrow curved scales similar to those forming the spots on the
thorax; scutellum with white flat scales. Pleuvae slightly paler in
colour than the thorax, with five patches of white flat scales, one
of which is situated immediately in front of the root of the wing
and another of somewhat dusky scales immediately behind.

Abdomen dark brown with basal dusky white bands expanding
laterally into large spots; the apical segment all white. Venter pale
dusky brown, the last three segments with basal patches of white
scales.

Legs.—Femora dark brown merging into black towards the apex,
pale ventrally, and with two white spots, one being apical the other
situated on the distal half; tibiae and metatarsi black with broad
white apical bands. Tarsi of the fore and middle pairs dark,
unbanded, the last two segments brownish yellow; the hind tarsi
with broad apical bands on the first, second and third segments,
the fourth being all white.

242

Wexgs with a white scaled spot at the extreme base of the costa;
first fork cell longer and narrower than the second, their bases
almost level; stem of the first sub-marginal cell rather more than
half the length of the cell, that of the second posterior almost as
long as the cell; posterior cross vein rather more than its own
length distant from the mid-vein.

Length 35 mm.

Habitat.—Broomassie, Ashanti, W. Africa (Dr. A. S.
Donaldson).

This pretty little mosquito comes nearer to R. albopunctata,
Theob. than the other members of this genus, but differs from the
latter in the thoracic ornamentation and the leg banding.

LITERATURE

STEPHENS, J. W. W., anp Curistopuers, S. R. (1908), Practical Study of Malaria, p. 175.

Newsteab, R., aND Carter, H. F. (1gto), Annals of Trop. Med. and Parasit., Vol. IV, No. 3,
p- 381.

Hirt, E., anp Haypon, L. G. (1907), ‘ A contribution to the study of the characteristics of larvae
of species of Anophelina in South Africa.’ Annals of Natal Government Museum, Vol. I,

Part 2, p. II.

244

EXPLANATION OF PLATE* 2X2.

Fig. 1.—Ova of Cellia squamosa, var arnoldz.

Fig. 2.—Larval head of Cellia squamosa, var. arnoldz.

Fig. 2a.—Labial plate of same larva enlarged.

Fig. 3.—Lateral comb of larva, Cellza syuamosa, var. arnoldz.

Fig. 4.—Palmate hair from third abdominal segment of larva,
Cellia squamosa, var. arnoldz.

Fig. 5.—Cephalic scaling of Reedomyzia simulans, n. sp.

Fig. 6.—Head and palpi of Reedomyia simulans, n. sp. CO.

Al

PILATE

» Carter, del,

H, F

245

THE DIAGNOSIS AND DISTRIBUTION

OF HUMAN TRYPANOSOMIASIS IN THE

COLONY AND PROTECTORATE OF THE
GAMBIA

First Report of the Expedition of the Liverpool School of Tropical
Medicine to the Gambia, 1911.

BY
JOHN. L.7 FODD, M.D., > 5B} WOLBACH, M:D:*
ASSOCIATE PROFESSOR OF PARASITOLOGY, ASSISTANT PROFESSOR OF BACTERIOLOGY,
MCGILL UNIVERSITY, MONTREAL HARVARD UNIVERSITY MEDICAL SCHOOL, BOSTON

(Received for publication 12 June, 1911)

CONTENTS
1. PREFACE 5a Sar Bat Fs Sac os i soe ad ates 3 246
2. INTRODUCTION 248
3. PROCEDURE ... 255
4. TECHNIQUE ... ae oe aie S00 oe see sas re 505 oad 257
(a) Fresh cover-slip preparations ... a nee 58: oe 500 ae 257
(b) Thin blood smears 257
(c) Thick blood films as 257
(d) Centrifugalisation of the bodat 2 sO $e “et mr 258
(e) Auto-agglutination dcr ae ae Se <a ae Pi aS 258
(f) Gland palpation ah ts and a Bat ee Ss ae 258
(g) Gland puncture ... ae ae ae aan soc ae des ce 259
5. FinpINcs.
(a) Fresh cover-slip preparations bs! 3 AG SEs au os 59
(b) Thin blood smears 5 ‘i jet Sie a et “it its 260
(c) Thick blood films ase 2 sel mat S58 sp? sea sh 260
) Centrifugalisation of the plod Ae a5 tis ae ote ws 260
(e) Auto-agglutination 1 Be = one 50 aes és Ess 260
(f) Gland palpation and puncture sie iat HO ss Sr te 261
(g) Pulse and temperature ... apr ae ae ie 595 vay a 273
6. Tue Mernops or DiaGNosinc ‘TryPanosomiasis DiscusseD AND COMPARED Ne 27
7. Tur Practica ApPLicaTION OF GLAND PALPATION AND PUNCTURE ... ie ne 280
8. Native Torerancr or TRYPANOSOMIASIS A wt sBs sere avy a 282
g. R&cOMMENDATIONS ate oe ve sae ess er +e sas ai 283
10. CONCLUSIONS as as wes “en a9 Ka ah sit in sew 286

*Sheldon Fellow in Tropical Medicine, Harvard University.

246

I. PREFACE

The main objects of an expedition sent to the Colony of the
Gambia, in 1911, by the Liverpool School of Tropical Medicine,
were to make additional observations on the efficiency of gland
puncture in the diagnosis of human trypanosomiasis, and to
determine the incidence of that disease in the territory visited by
the expedition. The present paper reports on this part of our
work; observations on other points will be reported on in later
papers. That a few cases of human trypanosomiasis may usually
be found in the Gambia is shown by the discovery there, in
IQOI-02, of Trypanosoma gambiense in two Europeans and in six
natives!. Since then the parasite has been found in one other
European and in # several natives. In a report made for the Under
Secretary of State, in 1910, Dr. Hopkinson states that there are
about six cases of trypanosomiasis among the 1,500 new cases whom
he sees yearly; he suggests that about 1 per cent. of the patients
coming to the hospital at Bathurst are cases of trypanosomiasis‘.
The figures published in the report of the Senior Medical Officer,
Dr. Hood, for 1909, would make it appear that the number of
cases is rather less than this; for there were no cases of
trypanosomiasis recorded among almost 8,000 patients treated
during that year at the Bathurst Hospital, and only one among
1,117 treated at the McCarthy Island Hospital. In 1908, however,
there was one death from trypanosomiasis among 593 patients
admitted to the Bathurst Hospital.

All of these observations prove that human trypanosomiasis has
been endemic in the Gambia for some years; the Gambia,
consequently, offered an excellent field for testing the efficiency of
gland palpation and puncture in the diagnosis of human
trypanosomiasis. The Gambia furnished an exceptionally good
opportunity for a test, since it has been suggested that gland
palpation is most likely to prove untrustworthy in those localities
where human trypanosomiasis exists in an endemic, rather than in
an epidemic form. It has often been suggested that palpation,
controlled by gland puncture, could not be usefully employed in
detecting cases of trypanosomiasis, because many natives who live
in areas where sleeping sickness occurs sporadically, have enlarged

247

glands without obvious cause, and that all of them could not
possibly be infected with trypanosomes. Indeed, when human
trypanosomiasis was first described in the Gambia!, it was
considered inadvisable to lay much stress on the diagnostic
importance of the enlarged lymphatic glands existing in the cases
in whom trypanosomes were found, since no trypanosomes were
found in the blood of other, apparently healthy, natives whose
glands were also enlarged. Eventually, experience in the Congo
Free State led to the publication of reports in which it was
concluded that, in areas where human trypanosomiasis exists, all
negroes with enlarged glands must be considered to be cases of
trypanosomiasis until the contrary is proved, and, although it was
recognised that all cases of the disease are not detected by gland
palpation and puncture, it was urged that this fact should be used
as a basis for measures designed to control the spread of sleeping
sickness. Many papers have since appeared on the same subject.
Some authors conclude that gland palpation and puncture form an
efficient diagnostic method, others do not. Those who decry it
have usually expected too much from it. Most of the papers which
had appeared up to August, 1908, have been reviewed and
discussed? by one of us. Those which have appeared since then
have been abstracted in the Bulletins of the Sleeping Sickness
Bureau. These Bulletins also contain full considerations of the
literature dealing with the measures employed in the diagnosis of
human trypanosomiasis, and the Director of the Sleeping Sickness
Bureau has published reviews of them®: 12. Broden and Rodhain*®,
Thiroux and d’Anfreville’, the German Sleeping Sickness
Commission in Togoland’, the Sleeping Sickness Commission in
French Congo’, the German Sleeping Sickness Commission in East
Africa’®, Kinghorn and Montgomery!!, Davey, Stannus, Park and
Barclay!*, Horn, Kinghorn!5: 16,21, Sanderson!8, May!®, Drew”,
have all written papers on this subject. All these authors have
employed gland palpation and puncture, and have found them of
value; there is, however, considerable variation in the opinions
they express concerning the exactness of the results obtained by
these methods.

References are given to the abstracts in the Bulletins rather than
to the original papers, because the former are much more easily

ioe)

24

obtained by most persons. It would be unprofitable to enter upon
a second consideration of these papers; especially since one would
necessarily traverse, almost exactly, the ground covered by a
previous review*. More recently Kinghorn and Montgomery have
given an excellent review of the subject!.

The figures obtained by our work in the Gambia speak for
themselves; the results there entirely coincide with those obtained in
the Congo. Consequently, the conclusions reached in the Congo are
left unaltered. It seems strange that our results should differ so
widely from those of other authors. Koch stated in East Africa
that ‘50 per cent. of trypanosome carriers could be detected by
a single examination of the blood’; the statement would have been
impossible if the carriers examined had been the early cases
occurring in the Gambia and in the Congo Free State.

Thanks are due by us for favours and assistance received during
this expedition from the Elder Dempster Steamship Company,
from the Governor, the officials and merchants in the Colony of the
Gambia, and from M. Legrand and M. Lanzerac in French
territory.

Il. INTRODUCTION

Because human trypanosomiasis is endemic in the Gambia, that
territory offered an excellent opportunity for testing the efficiency
of gland palpation and puncture in the diagnosis of that disease.
The expedition sent out for the purpose determined to examine as
many natives as possible in all parts of the colony, in order to
avoid any errors which might be produced by local causes.

The expedition reached Bathurst, the capital of the Colony of
the Gambia, on the 4th of February, 1911. Five days were spent
in making the necessary arrangements, and work was commenced
on the 1oth of February. Ninety days were spent in travelling
through the Protectorate and five in examining Bathurst and its
neighbourhood. In all, the expedition spent one hundred days in
the Gambia. During that time it travelled about 550 miles,
and it palpated the necks of 12,298 -natives drawn from
ninety-five towns and villages. Trypanosomes were found in
seventy-nine persons. If to these be added twenty-one persons with
much enlarged glands, whom it was impossible to puncture and who

249

were almost certainly infected, a total of one hundred is obtained ;
consequently, at least, o°8 per cent. of the whole population of the
Gambia are probably infected with trypanosomes. From _ the
observations made by previous expeditions to the Gambia, and
from the reports made by resident medical officers* it was already
known that Glosstna palpalis was very common everywhere along
the Gambia and its tributaries, and that Glossina morsitans also
occurred there. During this expedition Glossina palpalis was seen
in varying numbers, wherever the neighbourhood of the river, or of
a stream, was remained in for any length of time. Several areas,
on both sides of the river were passed through in which swarms of
Glossina morsitans occurred, and it was seen in very many places,
far from any water, in smaller numbers. No reason was observed
for the irregularity in their distribution. Tabanids and sand-flies
were very common.

On the accompanying map the towns visited are underlined, and
the route followed by the expedition is indicated by an unbroken
line; so far as it was possible, examples of every type of country,
included in the 5,000 square miles of the Colony and of the
Protectorate of the Gambia, were visited.

The expedition was undertaken during the dry season because
it is almost impossible, for Europeans at least, to travel in the
Gambia during the rains. The dry season there lasts from about
November to June.

During the height of the dry season there is very little water in
the country. The swamps are dry and the river becomes little more
than an arm of the sea in which fresh water lies; at this time of the
year the tides are felt at Fatta Tenda, about 240 miles inland
by the river from Bathurst. There is, consequently, very little fall
to the river, and the country through which it runs is very flat.
During the rains the river is swollen so that it sometimes passes the
banks and its current becomes so rapid that sailing boats require
weeks to make journeys which can be made in days during the dry
season. At this time of the year the swamps and creeks are all
flooded so that it becomes almost impossible to travel by any of the
roads running near the river.

At its mouth, and for some eighty miles up stream, the river
and the creeks tributary to it are bordered by dense fringes of

250

mangrove trees. The fringe of mangroves varies in width from ten
to fifty or more yards. Beyond the mangroves there are often
extensive grass-covered, swampy plains; such plains are very
characteristic of the Gambian Protectorate, and they occur on both
sides of the river all the way from the sea to the end of British
territory, some 200 miles inland. From the swamps the land rises
eradually to the level plain, of which the greater part of British
territory consists. This plain is composed of sandy soil and, where
it is uncultivated, is usually covered by forest composed of scrubby
trees and bamboo. Near water-courses, or on low-lying ground,
there are a few heavily forested areas. The plain varies
considerably in width, but practically all of it is included in British
territory, which is merely a strip of land, ten kilometres in width,
on both sides of the winding river. It is evident that the Gambia
has not always occupied its present bed, and that the level country
on either side of the river is part of the wide valley which it has
eroded. The valley is limited more or less abruptly by higher
ground. Sometimes it is limited by high escarpments of red,
volcanic iron-stone or by cliffs going down many feet to the river;
sometimes the rise is gradual te a plateau only a few feet above
the river, which is covered by scrubby forest and interrupted by
out-croppings of the constantly recurring, red, volcanic rock.

A few Niuminkas living at Bathurst are fishermen by profession.
Some of the Mandingoes and Jolloffs living along the sea-coast or
in villages situated near the banks of the river, in the lower part
of its course, own canoes and often catch fish. With these
exceptions the whole of the population of the Gambia 1s agricultural
or pastoral, and no tribe gains any considerable part of its food
from the river. Almost every village, however, looks forward to
scooping, with hand-nets, a few fish from the ponds left in the
dried-up swamps at the end of the dry season.

The population of the Gambia consists mainly of Mandingoes,
Jolloffs and Jolahs; these tribes are mentioned in the order of their
importance. There is also a considerable number of Foulahs. The
customs of Mandingoes, Jolloffs and Jolahs are very similar; they
are all agricultural peoples who build their villages near their fields,
and grow ground-nuts, millet of several varieties, rice, beans,
Indian corn, gourds, pumpkins, and medicinal herbs of various

251

sorts, as well as cotton. The chief difference between them is that
the Jolahs are more primitive than the other tribes. Unlike them,
they are not Mahomedans; they drink palm wine and live in
primitive hamlets, not in villages. They are very independent and
much more difficult to control than their more civilised neighbours ;
if it were advisable to do so it might be difficult to persuade them
to adopt measures designed to prevent the extension of sleeping
sickness among them. With the exception of a few insignificant
Mandingoe villages almost all of the native towns in the Gambia
are built at some distance—half a mile or more—from the banks of
the river. Indeed, one Mandingoe chief said that his people know
that ‘it is not healthy to build a town near too much water.’

The Foulahs are pastoral people. They are most numerous far
up the river; but they occur throughout the Colony. They move,
with their cattle, from grazing-ground to grazing-ground. In the
wet season they leave the river for the interior, where their cattle
are not exposed to fly-bites; in the dry season they return to the
river for the sake of the grass and water in the swamps along
its banks. Consequently, the Foulahs build no towns but, at the
most, only temporary collections of huts.

All the tribes in the Gambia are very prosperous. Their cattle
have increased enormously in number and their land is fertile.
They raise good crops of millet and rice for their own use, and
large amounts of ground-nuts which are sold to traders for export
to Europe. With the money obtained from the sale of ground-nuts
the natives are able to buy all the European articles, such as cloth
and powder, which they require. Because of the favourable
conditions for obtaining money by the sale of ground-nuts, large
numbers of young men yearly come to British territory in order to
make farms and raise crops of ground-nuts. They come from
French territory, from all directions; some of them come from
places distant eight, and even more, weeks’ travel.

Eleven cases of clinical ‘sleeping sickness’ were seen during
the expedition. Although the disease has existed in the Gambia
for some years, and although single cases have been seen in every
part of the colony, it has never become epidemic; neither do cases
ever seem to have been very common. In 1902? the blood of 1,043
natives was examined by cover-slip preparations; six of them,

252

0°5 per cent., were found to be infected with trypanosomes. During
this expedition the blood of 362 persons, selected by gland
palpation, was examined in exactly the same way; trypanosomes
were found in seven, 1°9 per cent. After examining three cover-slip
preparations the parasites were found in the blood of an eighth
case, in whose gland-juice they had been seen previously. From
these figures it seems possible that human trypanosomiasis in the
Gambia may be tending to increase. It is interesting that three
cases were found in Lammin among 100 persons examined, in 1902;
in IQI11 three cases were found there among thirty-five persons
whose blood was examined.

The natives of all the tribes know the disease well (the
Mandingoes call it Aanta bero,* the Jolloffs, Nelouan, and the
Foulahs, Dotngol or Danu). Nevertheless, answers to our questions
concerning the presence of sleeping sickness were sometimes given
which seemed to be almost wilful in their strangeness. For
example, the chief at Essau, where 5°4 per cent. of the population
had trypanosomiasis, professed to be able to remember only one
case of sleeping sickness among the people of his village.

Every headman was questioned; but none gave any hint of a
tradition that sleeping sickness had ever been more prevalent than
it is at present, and none knew when the disease first came to the
Gambia, though they all agreed that it had been in the country for
two or three generations. Natives told Dr. Hopkinson that,
formerly, no towns were built near Nianija Bolon (creek) on the
north bank because persons living there were in danger of catching
sleeping sickness. It is probable that the situation of the native
towns, among fields at some distance from the river, and the
agricultural habits of the natives—which make it unnecessary for
the men to go frequently to the river—have had some effect in
preventing the spread of the disease.

M. Legrand, the Administrator of the French Territory to the
South of the Gambia, wrote that he has been travelling through the
district of Fulladu for two years, and that all the natives living
in that district are well acquainted with sleeping sickness. He, with
Dr. Dufougére, estimated that about 0°15 per cent. of the natives

* This term really means ‘ neck stones’ and refers to the enlarged glands; Sima
jankers refers directly to the disease.

253

there were infected with trypanosomiasis. Most of the cases were
young men who had gone to British territory along the Gambia to
farm during the rainy season; but tsetses do exist in Fulladu about
the marshes, so that cases of trypanosomiasis do occur there among
natives who have never been to the Gambia. M. Lanzarec, the
French resident in the territory to the North of the Gambia, had
also noticed that sleeping sickness occurred most frequently among
those natives who had farmed in British territory. It is certain that
the men who cultivate millet and ground-nuts in the Gambia do
run some danger of being bitten by tsetse-flies. Natives often
stated that they were bitten while they were at work by flies, which
they called Solo-fing jolo or Kongjolo. Both Glossina palpalis ana
G. morsitans are probably included under these names; we saw no
other small species of tsetse-flies in the Gambia. It was frequently
said that the flies were most numerous in the wet season and that
—this we saw—Glossina palpalis often followed persons who had
come from the water-side into towns situated half a mile or more
from the river bank. It 1s, however, the women who are most
exposed to tsetse-flies. They alone cultivate rice. The rice-fields
are always placed in the swamps, and they often lie within a
hundred yards or so of the river, consequently, those who work in
them, as the natives freely admit, must be frequently bitten by
tsetses.

A French trader, who passed the rainy season of 1909 at Jamekunda, near
Sallikeni, said that this town has many rice-fields which are situated near an
arm of the river. As is usual, they are worked by the women, with the result
that many women have died of sleeping sickness, and one man lost five wives from
that disease in two years.

Young children are usually carried wrapped in a shawl on their
mothers’ backs. They are consequently little exposed to infection.
Boys are sent on errands, and run about everywhere; while girls
help their mothers in household work, in drawing water and in
farming ; so both boys and girls are exposed to the bites of flies; the
boys are, perhaps, more exposed than the girls. As has been
pointed out, the women are much more exposed than are the men,
because they cultivate rice. In maturity and in old age both men
and women, provided they have children or slaves to maintain
them, do little work afield, and remain very largely within the
villages. A consideration of the usual occupations of the natives
in the Gambia would lead to the conclusion that in childhood males

254

are more likely to be bitten by tsetse-flies than are females; but
that in adult life, where rice is grown, females are more likely to
be bitten than men and, moreover, that females are likely to be
bitten more often than any other class of the population. It would
consequently seem as though the women were more liable to become
infected by trypanosomes than are men. An inspection of Table I]
is interesting in this connection. It may be noted here that because
of their habits the pastoral Foulahs are little exposed to infection ;
few of them have much enlarged glands and no case of
trypanosomiasis was found among them.

In the Gambia, as in many other places along the West Coast of
Africa, the natives of all conditions and tribes realise that the
occurrence of enlarged glands is one of the earliest signs of
‘sleeping sickness.’ Some of them, at least, also recognise that
change of character, unstable emotions—easily excited tears or
laughter—and irritability are often early signs. Dr. Hopkinson has
been told by natives that an early sign is delay of the eyelids in
following the eyes when an affected person looks down. Many
natives realise that frenzy and mania are often late symptoms of
the disease which may exist before somnolence appears. One
headman had noticed that persons who scratched much often had
sleeping sickness.

Many tribes along the West Coast of Africa practice gland
excision as a preventative of sleeping sickness; the Jollofts,
Mandingoes and, apparently to a less extent, the Jolahs and
Foulahs all do so. Sometimes glands are undoubtedly removed
with a knife and a bit of wire. Often none can have been reached?
through the incision which is very frequently made high up, on the
ramus of the jaw, in a situation from which it would be almost
impossible to remove a gland; in these instances it seems almost
certain that no glands were removed. In other cases incisions have
been made in favourable places beneath the jaw, in the anterior or
posterior triangles of the neck and in the axillae; in these instances
it seems very probable that glands were excised. There are some
men who profess to remove glands by plasters, which ‘ draw’ them
out. Something of the same sort is done by natives in Nigeria and
on the Ivory Coast.

The natives, even those who are most educated, believe in the

255

advisability of gland excision, and believe, more or less firmly, that
the removal of glands will prevent the development of sleeping
sickness. They usually say, however, that excision is only of value
in the early stages, and that it is useless to remove glands when the
sickness has ‘ gone into the body.’

Very many of the natives living in the Gambia have had glands
removed. The fact that 150 out of 220 consecutive persons chosen
for gland puncture had had glands excised will give some idea of
the extent of the practice. In some districts excision is more
general than in others; the Jolahs seem to practise it least of all.
Usually, the operation is done but once on each individual; not
infrequently, persons are met with who have, as they say, had their
‘bumps pulled’ on several occasions. Some of these have
trypanosomiasis.

One woman, aged 24, in whom trypanosomes were found, had had glands
removed when she was about 13 years old. Since then, on three occasions, glands
were said to have been removed from the sub-maxillary, posterior cervical and
axillary groups.

One case of clinical trypanosomiasis had had glands excised on five occasions
during as many years.

Several persons in whom trypanosomes were not found had had
their ‘bumps pulled’ three times; usually the operation is only
done once, in childhood. The natives all know that people above
middle age rarely have enlarged glands and, consequently, that
they rarely have sleeping sickness (see Table I).

Ill. PROCEDURE

It is estimated that there are over 200,000 negroes in the
Gambia. In order that those whom we examined might represent
a fair sample of the whole population, persons were examined in
every part of the Colony and Protectorate. As many persons as
was possible were examined in each village visited. The natives
were not called together for us to see them, but we went from house
to house and entered the huts in order to make certain that cases
were not being concealed. As the posterior cervical glands of each
native were palpated, the age, sex, and degree or absence of
enlargement of the glands was dictated to a clerk who noted them:
these notes were kept for a series of 9,069 persons. Notes of only
those with puncturable glands were made for the remainder of the
12,298 persons palpated.

256

So soon as palpation of a village was finished, all of those who
had puncturable glands were told to go to the camp of the
expedition to be examined. One of the objects of the expedition
was to compare the efficiency of the various methods of diagnosing
trypanosomiasis; so, in a series of 283 persons, observations on
the following points were made for each native examined: personal
history, pulse, temperature, size of spleen, whether the glands had
been excised, presence of enlargement in all the groups of superficial
lymphatic glands, the presence of scurf, tartar, skin disease, or of
any cause which might produce glandular enlargement, and the
presence of trypanosomes in gland juice or blood. The blood was
searched for trypanosomes by the examination of fresh cover-slip
preparations, of thick films and of smears. At first it was intended
to also centrifugalise the blood by either our own method or by
that first employed by those who worked in Uganda®. Both of
these methods are tedious, and it soon became evident that it would
be necessary to abandon all hope of using them if a serious number
of gland punctures was to be done in the time at our disposal. In
the remainder of the 350 persons whose glands were punctured, the
full examination was made on only those in whom trypanosomes
were found; for the others the results of the examination of blood
and gland juice alone were recorded.

The names and descriptions of all those who were punctured
have been given to the Semor Medical Officer and to the
Commissioner of the district in which they were seen. It 1s hoped
to keep track of them, and in this way to gain information on at
least two very interesting points, namely: ‘Do all untreated cases
of human trypanosomiasis in natives end fatally?’ and ‘ Are any
of the persons with moderately enlarged glands, and in whom no
trypanosomes could be found, really cases of trypanosomiasis ?’
With the exception of the young men, natives in the Gambia travel
but little, because they are prosperous and contented. They are
well under control and they are usually very amenable to European
rule. It is, consequently, probable that it will be possible to keep
track of these persons. It is very important that they should not
be lost sight of ; the probability that they could, and would, be kept
under observation was one of the reasons which determined the
sending of this expedition to the Colony of the Gambia.

257

IV. TECHNIQUE

In order to establish a just basis for comparing the various
methods of diagnosing trypanosomiasis, a definite routine of
examination was established.

All the observations on each case used in making our comparison
were made at the same time. The blood preparations were made
with blood drawn from a single finger. The gland punctured was
usually the most convenient one in either of the posterior cervical
sroups. If glands from any other group were punctured the fact
has been noted. It has been strongly insisted that only perfect
specimens of gland juice should be examined, for a negative
examination of an imperfect preparation of gland juice has
absolutely no significance?. Consequently, only examinations of
good preparations of gland juice and of blood have been recorded.
As a rule only one preparation was made by each method of
examination, if more were examined the fact has been noted. A
microscope used in searching for trypanosomes should always be
fitted with a mechanical stage so that it becomes possible to make
certain of missing no part of the preparation examined.

A. Fresh cover-slip preparations.

The simplest method of finding trypanosomes in a patient 1s
to place a drop of his blood between a slide and a cover-slip and
to search in it for the living parasites with a microscope. The
trypanosomes are detected by their movement, so the preparation
must not be too thick lest the movements of the parasites should be
obscured by the blood cells. As usual, our fresh preparations were
made with cover-slips, three-quarters of an inch square, and they
were examined with a Zeiss, D. objective and a No. 4* eye-piece.
B. Thin blood smears.

In making a thin smear a drop of blood, half as large again as
that used in making a cover-slip preparation, is placed at one end
of a slide. Then, with a needle, placed transversely across the
drop, the blood is smeared along the slide in a thin layer. Our
smears were allowed to dry and they were then fixed in absolute
alcohol and stained by Giemsa’s method.

C. Thick blood films.

Four or five drops of blood as large as those used in making

thin smears, are placed on a glass slide over a circular area about

258

one centimetre in diameter. The slides are then usually fixed by
heat, de-haemoglobinised in distilled water and stained by some
modification of Romanowsky’s method. The value of the method
depends upon the well-known manoeuvre of removing the
haemoglobin; haemoglobin stains densely so that, were it present, it
would be impossible to see parasites lying among the red blood
cells.

D. Centrifugalisation of the blood.

Only a few specimens of blood were centrifugalised. We are
quite aware of the advantages offered by the method, but the length
of time required for employing it, as well as the large amount of
blood, 10 c.cm., required for the most usually employed method of
centrifugalising, makes it almost impossible for it to be used in the
routine examination of a large number of persons. We regret that
those in whom trypanosomes were found by other methods were not
examined by this one; but lack of time and the fear of frightening
natives by taking the necessary blood from them prevented us from
doing so.

E. Auto-agglutination of the red blood cells.

It has been noted frequently, in fresh cover-slip preparations,

that the red blood cells of persons suffering from trypanosomiasis

very often run together to form shapeless masses. The term of
auto-agglutination has been applied to this phenomenon.

F. Gland palpation.

The classification employed for grouping the persons palpated
according to the absence of glandular enlargement or according to
the degree of it, if it were present, was that proposed in the Congo?.
Those with enlarged posterior cervical glands are grouped in the
following way, according to the degree of enlargement present. As
“+? are classified persons with posterior triangles which contain
(a) one gland which is estimated to measure at least 1°5 by 0°75 cm.,
or (6) three or more smaller glands, the largest measuring perhaps
I by o5 cm. As‘ + —’ are classified enlargements, less than this,
but greater than those classified as ‘+ — —.’ As ‘+ — —”’ are
classified groups containing only one or two glands measuring
0°5 by 0°25 cm., or (4) many tiny shot-like glands which are only
just palpable.

259
G. Gland puncture.

No change was made in the technique developed in the Congo
Free State?. Ordinary hypodermic syringes were used. The gland
was fixed with the left hand while the needle of the syringe was
passed into it, with a sharp thrust, by the right. The plunger was
withdrawn by an assistant while the point of the needle was gently
moved about within the gland. The plunger was released and the
syringe withdrawn. The tiny droplet of fluid, which was usually
all that was obtained, was then blown out by a single thrust of the
plunger, and examined immediately in the same way as a fresh
preparation of blood was examined. The necessity of avoiding the
dangers mentioned in former papers? was more than ever evident.
The preparations must be thin, and care must be taken to use clean
slides and cover-slips and to see that the morsel of skin punched
out by the needle is not contained in the specimen. If the
preparation contains water from the syringe, or air bubbles, it 1s not
a good one, and the chance of finding living trypanosomes in it 1s
lessened. Trypanosomes are also less likely to be seen in pus-like
preparations, in which the cells are dull, than in preparations filled
with clear, brightly refractive cells. Those who attempt to examine
a person suspected of trypanosomiasis by gland puncture must not
record a negative examination until they have examined, in perfect
preparations, all of the material obtained by the successful
aspiration of a gland. Although there is usually very little blood in
a preparation of gland juice, the presence of even a considerable
number of red cells seems to make no difference to the likelihood of
trypanosomes being found.

V. FINDINGS

A. Fresh cover-slip preparations.

The blood of 362 persons was examined by _ cover-slip
preparations; trypanosomes were found in eight of them. A
series of 340 persons were examined by gland puncture and by
cover-slip preparation; gland puncture detected sixty-six cases of
trypanosomiasis, including all of six instances in which simul-
taneous examination of cover-slip preparations was successful in
finding trypanosomes. It was necessary to examine three cover-slip

260

preparations from one of these cases before the parasites, already
seen in gland juice, could be found in the blood.

B. Thin blood smears.

Thin blood smears were examined from 316 _ persons.
Trypanosomes were found in ten of them; the parasites had also
been seen in three of these cases in cover-slip preparations made at
the same time as the smears. They were also found by the
examination of cover-slip preparations in five persons in whom the
examination of thin smears had failed to find them. Gland
puncture found forty-eight cases of trypanosomiasis among these
316 persons.

C. Thick blood films.

Thick blood films, prepared in the manner described, were
examined from 265 persons. Trypanosomes were found in only
eight of them by this method, while they were found in forty-seven
by gland puncture, in ten by the examination of thin smears, and
in five by the examination of cover-slip preparations. All the
thick films in infected cases were examined during half an hour at
least. One film of blood in which trypanosomes had been found
by thin smears was examined without result for three-quarters of
an hour and another one was examined in the same way for an
hour. Three thin smears were examined without finding trypano-

somes in a case in whom the parasites had been found by the thick
film method.

D. Centrifugalisation of the blood.

The blood of only three persons was centrifugalised. Ten c.cm.
of blood was drawn from a vein in each case and centrifugalised
three times; over an hour and a half was spent in the preparing and
examining of from two to four cover-slip preparations from each
person. Trypanosomes were found in none of them, although they
had been previously found by gland puncture in two of them.

E. Auto-agglutination.

When it was first described, it was decided that auto-
agglutination was not always present in trypanosomiasis, but that it
often did occur in persons and in animals who were suffering from
that disease, and it was concluded that though auto-agglutination

201

had no diagnostic value it frequently encouraged a successful
search for trypanosomes in persons in whom they were not at first
found.

A good deal of attention has been given to auto-agglutination
recently24._ For this reason the cover-slip preparations made from
350 persons, whose glands were punctured, were examined for
auto-agglutination. These persons have been classified, in Table I,
according as trypanosomes were found in them or not. They are
further divided into four classes according to the absence of
auto-agglutination, or according to the amount of it which was
present. An inspection of the table shows that well-marked
auto-agglutination may be present in persons who are not infected
with trypanosomes, and also that some degree of auto-agglutination
is very constantly present in persons who are infected with
trypanosomiasis, although it may be absent altogether from some
of them. It is worth noting that trypanosomes were probably
present in two of those persons in whom the parasites were not
seen, although auto-agglutination was well marked; most of our
cases were only examined once and the presence of a slight
temperature with enlarged glands and auto-agglutination makes it
probable that trypanosomes would have been found in these two
cases had the search for the parasites been persevered in.

TABLE I

Marked Slight Very slight | No
| agglutination | agglutination agglutination | agglutination

Trypanosomes not present _...| II 37, 59 176

Trypanosomes present ...

|

F. Gland palpation and puncture.

The classification of persons with enlarged cervical glands which
was proposed in the Congo Free State has been retained. This
classification has been adhered to because it seems to be a reasonable
one and a useful one. It is reasonable because nothing can be
clearer and less ‘ mysterious’ than reference to actual measurements,
and because it can be appreciated by every doctor; many physicians

262

have never seen ‘ familiar objects,’ such as pigeon’s eggs, filberts and
pfennig pieces, but every medical man should have a very accurate
knowledge of the length of one centimetre. It would be misleading
to divide glands into puncturable and unpuncturable, because after
a little practice, anyone can draw gland juice from glands which he
would, at first, have thought unpuncturable. It is a_ useful
classification because the results of our examinations show every
negro with ‘+’ glands, without some evident cause, is, almost
invariably, a case of trypanosomiasis. This is especially so if the
enlarged glands have the thickly fluctuating consistency to which
many observers have alluded (Grey and Tulloch’, page 61). Asa

c

rule, only a few of those classified as ‘ + —’ have trypanosomes,
while almost none of those classed as ‘ + — —’ have been found to
be infected. These points are illustrated by the following table of
those with enlarged glands seen among the 12,298 persons who were

palpated in the Gambia.

Taste IT
Degree of glandular | + +— | +-#—— | Total
enlargement |
| | SS
| |
Seen ee an as a 56 136 | 2102+ 2294+
Punctured wee Sot are 36 63 233 | 332
Trypanosomes found ... ses 36 28 4 | 68

It will be noticed that trypanosomes were found in the gland
juice of all of the thirty-six ‘+’ cases. The parasites were also

c

found in twenty-eight out of sixty-three ‘+ —’ cases; they were
also seen in four out of 233 ‘+ — —’ cases. The number of cases
classed as ‘ + —’ and‘ + — —’ in whom trypanosomes were found
is, proportionally, much larger than it was in the Congo?.

It will be noticed from Tables II and III that there were a
considerable number of infected persons among those who were
classified as‘ + —’ and‘ + — —’. This is to be explained, in part
at least, by our having classified those in whom there was a difference
between the degree of enlargement present in the posterior triangles
of the neck according to the triangle which showed the lesser
enlargement. Consequently, of the four positive cases who had

263

“+4 — —’ glands on one side, two had ‘ +” glands on the other
side of the neck, and two had ‘ + —’ glands.

The posterior cervical glands were taken as an index of the
general enlargement of lymphatic glands which occurs in human
trypanosomiasis, just as in the Congo, because they are the groups
least exposed to the usual casual causes which produce enlarged
glands.

In order to ascertain whether the enlargement of lymphatic
gland groups was general in persons infected with trypanosomes,
and in order to ascertain whether enlarged glands frequently
occurred from other causes than trypanosomiasis in the Gambia, all
of the gland groups of 312 persons were palpated. Dr. Hopkinson
states? that the Jolloffs have enlarged glands more frequently than
members of any other tribe. We did not notice that difference, but
it was apparent that the Foulahs, and the few Jolahs whom we saw,
had fewer persons with enlarged neck glands that had the
Mandingoes and Jolloffs who lived near them.

As a rule, all early cases of trypanosomiasis had a considerable
degree of general glandular enlargement, but there were six early
cases who had one or more gland groups, usually the axillary,
epitrochlear or sub-maxillary, not at all or only slightly enlarged.
A little watching, however, would probably have discovered signs
in at least one of these cases, who had a temperature of 100° F.,
which would have caused it to be called a well advanced case; it is
well known that the size of the lymphatic glands diminishes in
advanced trypanosomiasis, and that the value of gland palpation
and gland puncture is least in just those cases which can be
recognised more easily by clinical, rather than by laboratory
methods.?

An inspection of the results of the palpations (Table III) shows
that children, boys especially, are most likely to have enlarged
glands; this is probably because of their lack of personal
cleanliness.

Boys often have more or less generalised skin disease, usually
‘craw-craw,’ they very frequently have a good deal of scurf, and
almost always a certain amount of tartar. The frequency of foul
mouths and of a high grade of pyorrhoea is very great among the
Gambian natives of all ages and classes. All these things often

264

caused enlarged glands; the slightly enlarged posterior cervical
glands of a large number of boys were certainly due to scurf;
while enlarged sub-maxillary glands, in persons who had no other
eroups enlarged, were usually due to an infected mouth. Lice, and
cuts or scratches on various parts of the body were also frequent
causes of enlarged glands. A considerable degree of enlargement
of the groin glands was almost universal among men.

Four or five of the persons in whom trypanosomes were found
had evident reasons for their enlarged glands; so the presence of an
adequate explanation, such as a dirty, crust-covered head, for
enlarged cervical glands by no means excuses their not being
punctured. This is especially so since lack of personal care is very
usual in established trypanosomiasis.

It is worth repeating—‘ until the contrary is proved every native
with enlarged glands must be suspected of trypanosomiasis.’

Table III shows the incidence of enlarged glands and of
trypanosomiasis among 9,069 natives who have been classified
according to their sex and age in order to ascertain whether any
class in the Gambia is especially liable to be infected with
trypanosomiasis.

Up to about fourteen, native boys are still children, after that
they become men and work in the fields. After forty-five, as a rule,
men have acquired a competence which allows them to rank as
leaders in the direction, rather than in the execution of affairs. Up
to twelve, girls are children, but after that they commence to spend
the whole of their time in women’s work. After forty, women are
usually scarcely strong enough to do the hard work which falls to
women of middle age. It is for these reasons that the divisions
according to age have been made at the years specified in the table.
An inspection of the table shows that approximately an equal
number of males and females were examined.

There were eleven cases of trypanosomiasis in male children and
five in female; but there were twenty-two cases of trypanosomiasis
in adult males and twenty in adult females. There were no cases of
trypanosomiasis in aged persons of either sex. These differences are
not large ones, but they indicate that if any class in the Gambia
is especially affected by trypanosomiasis it is that class which is
most exposed, by reason of its occupation, to the bites of tsetse-flies.

Py. “

265
Taste III
ee = 3 a : 4
Persons examined Male 4714 Female 4355
= Totals
Age o-14 | 14-45] 45-X | 0-12 12-40] 40-X
Size of glands Seen gee sod KS 13 I 4 15 fe) 43.1 43
a Punctured ae Bat weak. ° 2 ie) fe) 27
Positive 5a 3 | 12 o | 2, fe) ) 27
| |
SER) Bae seals 22 On) e2es| 24 2} 8] 118
—_ Punctured sed | 9 ° | 7) 18 ° 56
Positive ey 5 Sic ik ke. 3 12 fe) 25
|
Seennuy -24 <5 | OSOM Waa 54. | 407} 317 29 | 2088 | 2088
(aia as <i Be | ;
4 445 Punctured ....|_-107 32 o Pll as 23 oO] 193 |
|
| Positive Af Sa aaa a} “po I ° 4
ho ce a tf ee co Dh" lal pa (ae
’ |
[pScenls eee. -.-| ILLg | 1734] 435 | 975 | 1927.| 630 | 6820 | 6820
_—— ss ——
— | Punctured —... 7 4 ° BA tes se ° 18 |
Positive Py fc) | ) ° | ° fe) fe) fo)
[Ra 2d Sw (ee be
Totals’ ..2|"2024' | 2200 490 | 1411 | 2283] 661 | go69
j Much the same result is obtained in Table IV, where all the

cases of trypanosomiasis seen by us in the Gambia are classified
in the same way, according to age and sex. For the purposes of
this table all persons with ‘+’ glands whom we were unable to
puncture are considered to be infected. It is justifiable to do so
because trypanosomes were found in all of the thirty-six ‘ + ’ cases
who were examined by gland puncture. It will be seen from this
table that there are, still, more cases of trypanosomiasis among
the male children, while the number of cases in adults is about

ve

266

equal, and there are no cases in the aged. Results, very similar to
these, were obtained by Kinghorn and Montgomery in Rhodesia.!!

TaBie IV
Persons examined Male Female
Age 0-14 14-45 45-X O-12 12-40 | 40-X | Totals
Early cases | <.. — eaefe Gn 27 ° 60 | 24 ° 68
Clinical cases 5c ao 2 4 ° fe) 5 wy aie
Persons with + glands who |
were not examined... 6 7 ° | 22 || 6 oO | 2a
Potal’” |... re 19 38 fo) 88 35 fe) 100
|

Table V gives a list of the towns in which natives were palpated,
together with the results of the examinations. Its interest is chiefly
local; it may serve as a basis for future examinations undertaken to
ascertain whether trypanosomiasis is increasing or decreasing in the
Gambia.

A comparison of this table with the map shows that no district in
the Gambia is especially affected by trypanosomiasis. Towns, such
as Mandinaba, Demban Kai and Essau, which are placed near the
water have, as is always the case in such towns, an unusually large
percentage of their population infected.

Sometimes natives say that more persons die of sleeping sickness
during the rains than in the dry season. This probably means only
that more persons weakened by trypanosomiasis die of intercurrent
infections at that time of the year because of the hardships caused by
the unfavourable climatic conditions.

It is regrettable that lack of time and the reluctance of natives to
be examined prevented the examination of all the ‘ +’ and ‘ + —’
cases. Many more ‘ + — —’ cases were seen than were examined;
but the. great majority of those whose cervical glands were
puncturable have been examined. It would have been impossible

to puncture a gland in the posterior cervical triangles of very many

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273

of those who have been classified as ‘+— —’. Four ‘+ — —’
cases whose neck glands were too small to be punctured were
punctured in other groups; trypanosomes were found in none of
them. Neither were parasites found in three cases whose neck
glands were much enlarged from tuberculosis and contained
necrotic material.

G. Pulse and temperature.

It has been noticed frequently that a rapid pulse and a slight
degree of occasional fever are often early symptoms of human
trypanosomiasis. It was, consequently, thought that the presence
of both, or either, might be of value in encouraging the search for
trypanosomes in early cases of trypanosomiasis in whom the
parasites could not be found at a first examination. The pulse rates
and temperatures were, therefore, taken in 281 persons whose gland
juice, or blood, was examined for trypanosomes. Cover-slip
preparations, smears, and thick films were made from the blood of
all these persons. It soon became evident that an increased pulse
was of little value because the nervousness of the natives on account
of the examination often ran up the pulse rates, in even healthy
adults, to 100, or more. This was especially true of the children;
some of them had never before seen a white man. Our records
show many children, and a few men and women, who have normal
or only slightly elevated temperatures, and pulse rates of 120 or
even 130 beats to the minute. Examination of the blood has shown
that some of these persons had malaria; we believe that in most
of them the rapid pulse was simply caused by apprehension. As
a rule, there was a distinct increase in the pulse rate of the
sixty-one infected persons examined to 100, 120, or more, and that
with temperatures of only 99°F. This was not always the case,
for one man of thirty-five, with trypanosomiasis, had a pulse rate
of 63 and a temperature of 99° F. when he was first seen; others
had pulse rates of 78, with temperatures between 99° F. and 100° F.

It has long been known that an isolated observation of an
abnormally high temperature in a case of trypanosomiasis does not
bear a very definite relation to the probability of finding
trypanosomes in the peripheral circulation of that case. In the
present series there was, almost always, a slightly elevated

274

temperature, between 99°F. and 100°F. in those in whom
trypanosomes were found. Two persons who had no malarial
parasites in their blood, had temperatures of over 100° F.;
trypanosomes were seen in the blood of only one of them.
Apparently healthy persons, especially young adults and children,
were often seen in whom neither malaria parasites, trypanosomes,
nor any obvious cause for fever could be found, who had
temperatures varying between 99°F. and 100°F. We have no
explanation to offer. We do not think that the natives’
temperatures were inaccurately recorded because of the high
temperature of the air which often reached 105° F. or 107° F. The
thermometer was always carefully cooled to below 96°F. with
water before being used; oral temperatures were always taken, and
the native was made to keep his lips tightly closed so long as the
thermometer was in his mouth.

VI. THE METHODS OF DIAGNOSING TRYPANOSOMIASIS

DISCUSSED AND COMPARED

One object in initiating a comparative series of examinations,
by different methods, for the presence of trypanosomes in a series
of 283 persons, was not so much to determine which was the most
efficient method, but which was the most effectual. It was not
wished to determine by which method the largest number of cases
could be detected by spending unlimited time in _ repeated
examinations. It was wished to determine which method would
discover the largest proportion of cases in the shortest time. For
that reason a limit was set on the length of time to be spent in
examining preparations made by each of the methods employed.

One cover-slip preparation of blood and one of gland juice were
usually examined from each case; if more were examined the fact
has been noticed in the comparison of results. About fifteen
minutes are required in which to properly examine a 3 inch square
cover-slip preparation of blood for living trypanosomes; the same
length of time is needed for examining a similar preparation of
gland juice. Preparations of gland juice can be perfectly well
stained first and examined later for trypanosomes; all of ours were
examined in fresh cover-slip preparations because we believe it to
be the easier and the quicker method; we have never examined
gland juice in hanging drops.

a

279

Because the examination of fresh preparations requires only
fifteen minutes, it was determined to spend only fifteen minutes in
searching a smear or a thick film. This limit has been rigidly
observed in all our cases save in the examination of preparations
coming from cases in whom trypanosomes had been found by
gland puncture. These were searched, unless trypanosomes were
found sooner, for half an hour at least.

Taste VI

| Gland Puncture | Thick Films | Thin Blood smears Coverslip preparation

Single | Repeated Single Repeated Single _Repeated Single Repeated

examina- meant examina-| examina-| examina-| examina- examina- examina-
tion | tion tion tion tion tion tion tion
Trypanosomes
found... 48 me 2 ; I 9 ° 5 I
| — — — i — —- a
Total —...| 50 8 | 9 6
|

This table is a record of the simultaneous examination of 283
persons, selected by gland palpation, by gland puncture, by thick
blood films, by thin blood smears, and by fresh coverslip
preparations of blood. The successful examinations made by each
method are sub-divided so that it can be seen whether a more severe
examination than that ordinarily used was employed; it must be
understood that, usually, more than the allotted length of time
was spent in searching for trypanosomes by each method when they
had been found previously by some other method.

Because of the great length of time which it requires, attempts
to centrifugalise the biood were soon abandoned, and the only
methods used in each of the 283 cases, compared in Table VI, were
a cover-slip preparation, a smear, a thick film of blood, and the
examination of a fresh cover-slip preparation of gland juice. An
inspection of this table proves that, in the Gambia, gland puncture
is by far the most successful of the methods we compare. It is
consequently a very valuable diagnostic method. It is doubly
valuable because of its simplicity and because of the rapidity with
which it can be employed. The chief value of smears and of thick
films of blood is that they provide a means by which specimens,
taken when there is no opportunity of examining them, may be

276

preserved to be searched for trypanosomes later on. Since, at least,
three or four times the amount of blood required for a smear is
used in making a thick film, parasites certainly ought to be
found more often in thick films than in smears. Some observers
have found thick films more useful than gland puncture in finding
trypanosomes in some cases, especially in those cases from whom
the parasites have disappeared after treatment. We have very
little confidence in this method. A trypanosome may lie,
unrecognisable, at the bottom of some of the heaps of débris which
remain, and stain, in every thick film. Many of the trypanosomes
found in thick films are only partially stained, sometimes almost
nothing of the parasite can be seen beyond a nucleus and a
blepharoplast. A trained microscopist who has examined many
thick films may always be able to recognise such trypanosomes
quickly, others can not. The time required for the preparation of
a thick film is also an objection to the method. All of these reasons
led to the abandonment of the dehaemoglobinised thick film
method when it was first tried by us in 1903. We were searching
then for a rapid routine method of recognising trypanosomiasis and
we decided that the centrifugalising and fresh examination of
small quantities of blood was a much better method of examining
it. At that time, it was also decided, from the examination of a
very considerable number of smears and cover-slip preparations,
taken at the same moment, from animals and men infected with
trypanosomes, that the parasites could be found more easily, living
and moving, in fresh preparations than dead and motionless in
stained ones. Attempts to make living trypanosomes, in cover-slip
preparations of blood, more conspicuous by vital staining were also
abandoned, among other reasons, because most aniline dyes
evidently hastened the death of the trypanosomes; neutral red was
the least harmful of those which we tried. In view of our belief
that the examination of cover-slip preparations is a better method
than the examination of smears, the figures given for each method
in Table VI and in the paragraph describing the findings obtained
by the examination of cover-slip preparations are surprising to us.

Gland palpation is very simple. Large neck glands can be
recognised by any intelligent negro, and the persons possessing
them can be brought to a doctor for examination by any native

Sy

policeman. Gland puncture is not a difficult manoeuvre. With the
assistance of an orderly, capable of boiling a syringe, gland
puncture can be done and the whole of the specimen examined in
less than twenty minutes. From our experience in the Congo, in
Sierra Leone, and in the Gambia, we can only conclude that giand
palpation, followed by gland puncture is by far the most effectual
means of finding trypanosomes in, at least, those early cases of the
disease seen by us.

The shortcomings of gland palpation and puncture have always
been very evident. Very early cases in whom glandular
enlargement has not appeared, and late cases from whom it has
disappeared may be missed by it; the missing of the latter group
of cases is not serious since they are usually easily recognised by
gross clinical signs. The missing of the first group is serious. At
present there is no means of determining how large a proportion
of the cases of trypanosomiasis present in a community will be
missed by gland palpation and gland puncture; that cases will be
missed is abundantly shown by work done in the Congo?. It was
shown there that a man without enlarged glands might have
trypanosomiasis in his cerebro-spinal fluid, although he seemed
quite healthy and although parasites were not found in his blood;
it was also shown that trypanosomes might not be found in the
gland juice of a small percentage of persons although they were
present in the finger blood. Many observations made since then
have shown the same thing; but even if an appreciable percentage
of cases is missed by this method that is no reason why the method
should not be used in attempts to check the disease by the restraint
and treatment of the exceedingly considerable percentage of cases
which can be detected by it. It is difficult to estimate the number
of cases of trypanosomiasis which will remain undetected by gland
palpation and gland puncture, because there is no certain method
of recognising the disease. Its absolute diagnosis rests upon the
demonstration of the parasite causing it and, in our hands, the
method of examination which we wish to control has been much
the most efficient of all the methods at present available for
finding trypanosomes; until more perfect means of diagnosis are
devised, the only certain way of determining what proportion of
cases of trypanosomiasis are missed by gland palpation and

278

puncture would be to keep a substantial number of persons who have
been examined by the method under observation for a considerable
period in a locality where they would not be exposed to re-infection
and where it would be possible to re-examine them at intervals.
The Gambia does not offer these conditions perfectly, but it does
so to a considerable degree; and it is hoped that the results of
future observations, made on those persons, with slightly enlarged
glands, in whom we found no trypanosomes, may make it possible
to form an estimate of the proportion of infected persons whom
the examination of glands failed to detect there. It was attempted
to keep track of the natives with enlarged glands who were
examined in the Congo®. It is impossible to draw any certain
conclusions from the reports which have been received concerning
them, because many of them are mising, and becatse they inhabited
areas where sleeping sickness is endemic. Nevertheless, an
examination of the figures suggests that a larger number of those
with enlarged glands, in whom trypanosomes were either not found
or not looked for, ultimately died of sleeping sickness than would
have been expected had they been entirely healthy persons.

We do not anticipate that the proportion of infected persons in
the Gambia in whom trypanosomes have not been found by gland
palpation and puncture will be a large one, if only for the
following reasons. The efficiency of gland examination depends
upon the selection of persons with enlarged glands for puncture.
All of those with much enlarged ‘+’ glands are almost always
infected ; in the Gambia trypanosomes were found in thirty-six out
of thirty-six. Parasites are frequently found in those with
moderately enlarged ‘ + —’ glands by a single examination (in
the present instance twenty-eight out of sixty-three were infected);
if such persons were detained for examination they would be
examined, frequently, over a period of some weeks before being
allowed to proceed?. It is very probable that, in this way,
some of them would be shown to be infected. Always, very few
cases are found among those with glands that are very slightly
enlarged, ‘ + — —’; in the Gambia there were only four cases
among 233 persons examined (see Table II). All four of these
cases were persons who had moderately enlarged ‘ + —’ glands on
one side of their neck; consequently, if they had been detained as

77)

suspected cases, they would have been repeatedly examined before
being passed as probably uninfected persons. If these four cases
be subtracted, 229 persons with little more than normal glands
remain; their glands were punctured and their blood was examined
in cover-slip preparations, in smears and in thick films. None of
them were found to be infected. Of course the result would have
been more convincing had the examination been often repeated,
if the blood had been centrifugalised, and if susceptible animals
had been inoculated from all these persons; but the examination
which they did receive was not an entirely insignificant one, and
we believe that the immediate future will not show that many
individuals among these 229 persons had trypanosomiasis when
they were examined.

The glands of two persons, one a ‘ +’ case, the other a ‘ + —’
case, in whom trypanosomes were found by examining the blood,
were not punctured; it is probable that gland puncture would also
have detected the parasites. In one instance trypanosomes were
found in a blood smear from a ‘ + —’ case in whom parasites were
not found by a single perfect examination of gland juice. We
regret that most of our blood smears and thick films were not
examined until our return to England, and that it was consequently
impossible to examine these persons by gland puncture until we
were satisfied that trypanosomes could be found in them by this
method of examination.

It must be remembered that trypanosomes can be found in the
lymphatic glands of any group, and that the femoral or axillary
glands can often be punctured when those of the posterior cervical
triangles are too small to be examined. The continued observation
of a series of suspected or actual cases of trypanosomiasis, in whom
trypanosomes can not be found by gland puncture, is greatly
needed; it might throw interesting light upon the course and
development of the disease.

The use of gland palpation and puncture was first urged as the
basis of measures intended to check the spread of trypanosomiasis
because of figures obtained, in the Congo Free State, from the
simultaneous examination by different methods of several hundred
persons; 250 of them were cases of trypanosomiasis. The work
which led to these results was the direct outcome of an observation

280

made by Greig and Gray, that trypanosomes were present in the
glands of persons with trypanosomiasis. We had long been
searching for a rapid method of diagnosing the disease, and the
idea at once presented itself that such a method might be found in
gland puncture!’. In the hope of finding a part of the body in
which the trypanosomes occurred in larger numbers than in the
blood, a series of preparations had been taken immediately after
death from the organs and body-fluids of many persons and animals;
in only one case, examined within half an hour after death, were
living trypanosomes, found in the glands?5. This observation is
an interesting one, since it emphasises, and may have some relation
to the fact that trypanosomes sometimes die very quickly in
preparations of gland juice. Consequently, preparations obtained
by the puncture of glands must be examined as soon as they are
made.

Methods exactly similar to those used in the Congo have been
employed by us, or by one of us, in the Gambia and in Sierra Leone
with the same results. Similar results have been obtained by many
persons in many parts of Africa; yet, it is true that gland
palpation and puncture have failed to detect cases of
trypanosomiasis in Ashanti, on the Gold Coast, in Nyasaland!8, and
elsewhere. As far as the Nyasaland cases are concerned it will be
interesting to observe whether human trypanosomiasis there, which
is said not to be caused by that parasite, runs the same course as
the disease produced by Trypanosoma gambiense.

VII. THE PRACTICAL APPLICATION OF GLAND PALPATION
AND PUNCTURE

Although we have urged, and do still urge, that gland palpation
and puncture should be made the basis of measures enforced, in
areas, where human trypanosomiasis exists or threatens to become
endemic, with the object of collecting natives for treatment and
isolation in some type of restricted settlement; we do not, in any
way, urge that this method should be used in the maintenance and
administration of such measures to the exclusion of all other
methods of diagnosing trypanosomiasis. It is a very efficient
method of diagnosis; in our experience it is the most efficient one.

281

It is almost the quickest of the methods in individual examinations,
and by it a large percentage of infected persons can be weeded out
from a whole population infinitely more quickly and more cheaply
than can be done in any other way. It is possible that other
methods might detect a slightly larger percentage of infected
persons, but economic exigencies—expense—will frequently make
it impossible to employ enough trained physicians to carefully
examine every native in a district by the tedious, though possibly
more efficient, methods of centrifugalisation of the blood; the
expense of maintaining a staff of physicians and native assistants
capable of examining the native population in that district by
gland palpation and puncture would be very much less.

No one can deny that gland palpation and puncture form a
very efficient method of diagnosing trypanosomiasis; in our hands
it has been the most efficient of all methods, although it does
sometimes fail. It would be regrettable if the extravagant claims
which have been made for the method should lead to a reaction by
which its real value might be obscured.

In our experience it has not be a difficult method to employ.
Natives whose glands are punctured feel nothing after the prick
of the needle and, which is almost as important, they can see
nothing. Once or twice whole villages have become frightened and
refused to be examined; but that has been very unusual. Tact and
a generous distribution of sweets, beads, kola nuts and small
novelties has usually successfully overcome all distrust!4.

In practice, we believe that much can be done in many parts of
Africa to control trypanosomiasis if measures suggested by a
knowledge of the incidence of the disease are enforced. That
knowledge, in our opinion can be gained most quickly and easily
by gland palpation and puncture. Just as the French Commission,
the German Commission, Broden and Rodhain and others have
observed, it was seen in the Congo that some cases—especially very
early ones and late ones—are missed by the examination of single
preparations of gland juice, or of even several preparations taken
during a period of a few days. Consequently, when it is possible,
gland palpation and puncture must be supported by other, though
less efficient, methods.

The presence of auto-agglutination, of increased pulse-rate and

282

of heightened temperature in persons, apparently otherwise healthy,
will always be suspicious and, as is suggested by the paragraphs
dealing with these signs, be sufficient to cause the examination of
persons possessing them to be persevered in. But, none of these
signs have any diagnostic value in themselves. The value of
examining the blood for trypanosomes by inspection and by animal
inoculation is admittedly very great, and these devices must be
employed where it is necessary to do so.

In our opinion the most convenient and most effectual order for
the routine examination of persons for trypanosomiasis is gland
palpation and puncture, cover-slip preparation of blood,
centrifugalisation of the blood, thick blood films, blood smears and
animal inoculations. Auto-agglutination and clinical signs are
valuable, but they are merely suggestive.

VIII. NATIVE TOLERANCE OF TRYPANOSOMIASIS

An inspection of Tables II and IV shows that we saw no cases
of enlarged glands, nor of trypanosomiasis, in persons past middle
age. In reviewing these tables, it was suggested that this absence
of cases might be explained in part by the lack of exposure to
infection of those past middle age, because of the occupations
which they follow; but, it may be that this lack of cases is due to an
immunity acquired later in life. The idea is not a new one! 14 21,

The fact that trypanosomiasis has been present in the Gambia
and elsewhere on the West Coast of Africa for many years, in
places where Glossina palpalis exists, without assuming the
epidemic form which it has taken in the Congo Free State and in
Uganda, of itself, suggests that the West Coast natives may have
acquired some immunity to it. In the Gambia the customs of
natives, almost none of whom were riverine, has doubtless much
to do with preventing the spread of trypanosomiasis; but there
seems to be something more than that.

There are many records, some based altogether on the
observations of Europeans, others based on observations of natives
and of Europeans, of persons who have lived for four or more
years after they probably became infected by trypanosomes; these
records prove that persons may have a ‘tolerance’ for trypanosomes

283

and live comparatively healthily though infected by them. This fact
must be remembered in appreciating the results of treatment, for
it must be asked whether an improvement in the condition of a
patient has been due to the drug administered or whether the
treatment has merely coincided with the commencement of a period
of tolerance in which the parasites are not necessarily destroyed,
although the effect produced on the patient by them almost
disappears.

Little is known of the reasons which may cause trypanosomiasis
to assume an acute phase. In experimental animals it is known
that an intercurrent infection may determine a sudden multiplication
of trypanosomes. It may be that some similar factor which
‘produces a lessened resistance’ might be the cause of an acute
phase of trypanosomiasis in persons who had previously had a
chronic form of the disease.

Almost nothing is known of the outcome of those cases of
trypanosomiasis in whom parasites are present, although there are
almost no symptoms. It is not known whether they recover, nor, if
they die, for how long the disease may run a chronic course.

It may be suggested that the overwhelming preponderance of
middle-aged persons in our infected cases might be explained by
the chronic nature of the infection which allowed those infected in
youth to live on to middle-age, still infected, but tolerant of their
infection. In that case the absence of cases after middle-age might
be the expression of an immunity acquired as the result of a
preceding persistent infection.

Observations on these points are needed badly; it is to be hoped
that many of those who were found to be infected in the Gambia
by this expedition can be followed. It is worth noting that all of
the six persons who were found to be infected in the Gambia in
1902 were dead in 1906.

IX. RECOMMENDATIONS

It is not our intention to propose a scheme for the prevention of
sleeping sickness in the Gambia. To do so is the province of the
Resident Medical Officers, and it is they who will decide which of
the measures, which have been employed elsewhere in the prevention

284

of sleeping sickness, are most applicable to the situation in the
Colony of the Gambia.

There are, however, a few very evident improvements to be
made which may be mentioned. The situation of the town of
Essau, where five per cent. of the population are infected with
trypanosomes should certainly be changed. The town is at present
placed at the side of a swamp and is almost enclosed on two sides
by mangroves. As a result Glossina palpalis is often seen within
the village. Demban Kai, where 4°3 per cent. of the population are
infected is situated almost as badly. It should be moved.
Mandinaba where three out of twenty-two persons were probably
infected, might, also, be moved.

There are many fords and river-side washing places which are
closely enclosed by mangrove bush and by other thick vegetation.
Tsetse-flies are always present in such places; consequently the
natives who frequent them are constantly bitten, and it would be
well if all the thick brush about fords, wharfs, or places used for
washing by women, could be cleared for a distance of at least 150
yards on every side. The strip of bush just to the south of the
town of Bakau, at Cape St. Mary, swarms with Glossina palpalzis.
The thick undergrowth should be cleared from this bush, and that
especially if it is determined to establish permanent European
quarters at the Cape. At present this bit of jungle is a menace to
the neighbouring native town and to everyone who passes through
it.

The growing of rice should be forbidden in places where the
fields are in swamps near the river, where Glossina palpalis exists;
most of the rice-fields in the Gambia are of that sort. It is possible
that ‘mountain rice’ (O7vyza sativa, var.), which will grow in
comparatively dry places, might be substituted in the Gambia for
the present variety, which can only be grown in marshes. Even if
‘upland’ or ‘mountain rice’ cannot be grown, the prohibition of
the cultivation of marsh rice would not be a great hardship. Many
natives, for example, those to the north of the Niumi Forest, eat no
rice and they, like many others, look on it as a luxury rather than
a necessity. Many of those who grow rice have eaten it all within
three or four months of the harvest time, and for the rest of the
year live on other grains. Others eat it regularly twice, or even

285

three times a week. Very few of the towns use maize as much as
they might do and, possibly, it might be substituted for rice
extensively.

We believe that much can be done to improve the situation in
the Gambia by such methods and by the isolation and treatment
of infected persons who would be detected, largely by systematic
gland palpation of the whole population. There is very much in
the description of the preventive measures employed in the Congo
Free State which seems to be good (Sleeping Sickness Bulletin,
No. 26, p. 193). The clauses which forbid the concealment of
cases, make notification of cases compulsory, and make traders and
other employers of labour responsible if infected persons with
enlarged glands are found among their employés, seem to be
especially praiseworthy.

The examination of the population and the treatment and
maintenance of the cases detected would be placed under the
direction of, probably, two medical officers appointed for that
purpose and assisted by native orderlies and inspectors. From our
talks with the natives, we do not think that it would be very
difficult to persuade them to send cases of trypanosomiasis, at least
those in whom symptoms are evident, to villages established for
observing and treating them. It would be comparatively easy to
get the natives to come to them if two such villages were established
in the Gambia, one near the sea coast, and a second up the river,
probably at McCarthy Island. The establishment of such villages
would be worth while, if only for the sake of the observations
which it would be possible to make from them on the course of the
disease in persons sent toe them. It would be worth while also, from
the patients’ point of view, for it does seem certain that, in some
instances, early, radical and persevering treatment may cure
trypanosomiasis.

The physicians attached to the sleeping sickness isolation
villages would have opportunities for studying many interesting
points which can only be investigated by those resident in the
Colony for considerable periods. Not the least of these would be
the careful examination of a large number of native and wild
animals in order to ascertain whether Trypanosoma gambiense
occurred naturally in any of them.

286

Xx. CONCLUSIONS

1. Gland palpation and puncture is, by far, the most
effectual of the procedures, employed by us in the Gambia, for the
diagnosis of human trypanosomiasis.

2. At least 08% of the population of the Gambia are infected
with trypanosomes.

3. Measures designed to control human trypanosomiasis may
be usefully instituted in the Gambia; they should include a
continued examination of the whole population, the establishment
of villages for the isolation, observation, and treatment of cases,
and the appointment of a special staff for the administration and
execution of these projects.

XI. REFERENCES

1. Dutron anp Topp. First Report of the Expedition to Senegambia. Memoir XI of the
Liverpool School of Tropical Medicine, 1902.

2. Durron anp Topp. Gland Palpation in Human Trypanosomiasis. Memoir XVIII of
the Liverpool School of Tropical Medicine, 1906.

3- Topp. A review of the position of Gland Palpation in the Diagnosis of human trypano-
somiasis. Journal of Tropical Medicine and Hygiene. August 1, 1908.

4. Hoprxtnson. Sleeping Sickness Bureau Bulletin. No. 21, p. 381.

5. Diagnosis of Human Trypanosomiasis. Bulletin of the Sleeping Sickness Bureau, No. 2,
p- 53- Sleeping Sickness Bureau, Royal Society, Burlington House, London, W.,
England.

6. Report on Human Trypanosomiasis from Leopoldville. Bulletin No. 5, p. 190.

7- Symptoms of Human Trypanosomiasis. Bulletin No. 9, p. 330.

8. Report of the Commission to April, 1909. Bulletin No. 10, p. 397.

g- Report of the Coramission 1906-1908. Bulletin No. 10, p. 355.

o. Report of the Commission 1906-1907. Bulletin No. 11, p. 420.

1. Second Report on Human Trypanosomiasis in North-Eastern Rhodesia and Nyasaland.
Bulletin No. 12, p. 467.

12. Diagnosis of Human Trypanosomiasis. Bulletin No. 14, p. 57.

13. Nyasaland Sleeping Sickness Diary. Bulletin No. 17, p. 181.

14. Report on Sleeping Sickness in the Volta District. Bulletin No. 18, p. 222.

15. Sleeping Sickness News from the Gold Coast. Bulletin No. 19, p. 257.

16. Sleeping Sickness News, Gold Coast. Bulletin No. 21, p. 378.

17. Durron anp Topp. Gland Puncture in Trypanosomiasis. Memoir XVI, Liverpool

School of Tropical Medicine, 1904.

18. Sleeping Sickness News from Nyasaland. Bulletin No. 22, p. 415.

19. Report on Sleeping Sickness in Northern Rhodesia, 1910. Bulletin No. 23, p. 36.

zo. Report of the Sudan Sleeping Sickness Commission. Bulletin No. 24, p. 85.

21. Report on Human Trypanosomiasis in Ashanti. Bulletin No. 25, p. 133-

22. KINGHORN AND Montcomery. Report of the Sleeping Sickness Expedition to the

Zambesi, 1907-08. Annals of Tropical Medicine and Parasitology, Vol. II, No. 2.

23. Sleeping Sickness News, Northern Nigeria. Bulletin No. 5, p. 204. See also Bulletin

No. 21, p. 385.
24. Yorke. Auto-agglutination of Red Blood Cells in Trypanosomiasis. Bulletin No. 24,
Pisa
25. Durrdh, Topp anv Curisty. Human Trypanosomiasis in the Congo. Memoir XIII,
P- 42, 1904.
26. KincHorn. Bulletin No. 16, p. 147.

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287

THE MECHANISM OF THE PRODUC-
TION QF SUPPRESSION OF URINE IN
BLACKWATER FEVER

BY

WARRINGTON YORKE

AND

RALPH W. NAUSS

(From the Runcorn Research Laboratories of the Liverpool School
of Tropical Medicine).

(Recezved for publication 17 June, 1911)

Suppression of urine is a by no means infrequent sequel to an
attack of blackwater fever. Owing to the extreme gravity of this
symptom—responsible as it is for a great proportion of the fatal
cases—the mechanism of its production is a subject of considerable
practical importance. It is unnecessary here to refer in detail to
the work of previous authors, as a full account of the bibliography
of the subject is given in the papers of Werner* and Barratt and
Yorket. The work of these authors and of de Haan} upon the
condition of the kidneys of blackwater fever patients who have
succumbed from suppression of urine has demonstrated the
existence of granular material in the lumen of the renal tubules,
and these workers infer that the suppression of urine is of
mechanical origin, dependent upon the occlusion of the tubules by
granular plugs. On the other hand, a nervous inhibition of
glomerular secretion is regarded by A. Plehn§ as the essential
factor in this condition. Again, nephritis has been mentioned by
some writers as playing an important part in its production.

* ‘Ueber die Nieren beim Schwarzwasserfieber,’ Archiv. fiir Schiffs- und Tropenhygiene, 1907,
Band XI, Bicheft 6.

t ‘An investigation into the Mechanism of Production of Blackwater,’ Annals of Tropical Medicine
and Parasitology, 1909, Vol. III, No. 1.

} ‘Die Nieren beim Schwarzwasserfieber,’ Archiv. fiir Schiffs- und Tropenhygiene, 1905, S. 22.

§ ‘Die Nieren beim Schwarzwasserfieber,’ Archiy. fiir Schiffs- und Tropenhygiene, 1903,
S. 270.

288

For many years it has been known that the injection of such
poisons as glycerine, pyrogallic acid, toluylen-diamin and
haemolytic serum frequently produces haemoglobinuria and
sometimes leads to suppression of urine. It is doubtful whether the
condition produced by such injections can be regarded as at all
comparable with that occurring in blackwater fever, since it is
highly improbable that the poisonous effects of the drugs in question
are limited to the red blood cells.

Barratt and Yorke* observed that intravenous injection of an
isotonic solution of haemoglobin made from the animal’s own
erythrocytes was quickly followed by the appearance of
haemoglobinuria. The injections resulted in an increased flow of
urine. Sections of the kidneys of these animals showed the
presence of a certain amount of granular material in the lumen of
some of the tubules. In none of the animals was there any tendency
towards anuria.

Whether the occurrence of haemoglobin free in the blood plasma
and its subsequent elimination by the kidneys is sufficient to produce
suppression of urine through mechanical blocking of the renal
tubules, or, whether a nephritis, or nervous inhibition of glomerular
secretion, is to be regarded as the essential factor in the development
of this condition in blackwater fever is a question, therefore. which
has not, as yet, been definitely decided. It was with the object of
throwing further light upon this point that our investigations were
undertaken.

Our experiments were, with certain modifications, carried out
along lines similar to those employed by Barratt and Yorke, 1.e.,
the production of experimental haemoglobinuria in rabbits by the
intravenous injection of solutions of homologous haemoglobin.

Technique. An isotonic solution of haemoglobin was made as
follows:—A rabbit was bled from the external jugular vein and
the blood collected in oxalated saline solution. The red blood
cells, separated from the plasma by centrifugalisation, were
washed three times in normal salt solution and then laked by the
addition of distilled water. Sufficient sodium chloride was now
added to render the solution isotonic and the precipitated stromata
thrown down by means of the centrifuge. The clear solution of

* Loc: Cite

——( itt

289

haemoglobin was then withdrawn. The percentage of haemoglobin
present in the solution was estimated, in terms of human red blood
cells, by means of a von Fleisch] haemoglobinometer. As it was
undesirable to bleed the experimental animal to an undue degree, the
haemoglobin solution was prepared in part from the animal’s own
red blood cells, and in part from those of other rabbits. In order
to facilitate the collecting of specimens of urine the bladder was
opened supra-pubically, a cannula inserted and the wound closed by
sutures. The rabbit was then placed in a specially constructed box
which allowed of moderately free movement, but was sufficiently
narrow to prevent the animal from turning round. A rubber tube
attached to the glass cannula passed through a hole at the end of
the box into a capsule in which the urine was collected. The
solution of haemoglobin, warmed to 37°C., was now slowly
injected into one of the veins of the ear. After an interval of two
or three minutes to allow of the haemoglobin solution being
thoroughly distributed throughout the circulation, a sample of
blood was removed from the other ear and the degree of
haemoglobinaemia estimated. The urine in the cannula was
observed to be tinged with haemoglobin a few minutes after the
injection. From time to time the urine was removed from the
capsule, its volume measured and the amount of haemoglobinuria
estimated.

Our earlier attempts to produce anuria by this means were
invariably unsuccessful. Injection of the haemoglobin solution was
followed by an increased flow of urine. This was probably to be
explained by the diuretic action of the sodium chloride solution in
which the haemoglobin was dissolved. After a time the diuresis
subsided, but, as a rule, it lasted until the haemoglobinuria was
distinctly decreasing in amount.

In later experiments we endeavoured to reproduce more closely
the conditions obtaining in blackwater fever in man. In this disease
the blood is anaemic and the patient is more or less collapsed. It
appears probable that a decreased filtering force in the glomerull,
resulting from a low blood pressure, may play a not unimportant
part in the production of anuria in these cases.

With this consideration in view we decided to reduce the blood
pressure of our experimental rabbits, partly by keeping the animals

290

on as dry a diet as possible for twelve hours previous to the
injection of haemoglobin, and also during the experiment, and
partly by bleeding the animal to a moderate degree from the
external jugular vein.

In order to obviate as far as possible the diuretic action of the
sodium chloride, the strongest obtainable solution of haemoglobin
was employed. Such solutions were prepared by dissolving the
washed erythrocytes in the minimum quantity of distilled water
necessary to produce complete laking.

As a result “of these precautions we succeeded in producing
suppression of urine in a number of animals.

Condition of the blood during experiment. Only a trace of
haemoglobinaemia was detectable in the blood of the animals at
the commencement of the experiment before injection of the
haemoglobin solution. As a rule, bands of oxyhaemoglobin were
just visible in a column 20 mm. high corresponding to an amount
of haemoglobin equal to less than o'1 per cent. The amount of
haemoglobin injected into the circulation at one time varied from
I°5 to 13 grammes. Examination of the blood from time to time
showed that the amount of haemoglobinaemia gradually decreased.
The rate of disappearance was not constant, however, but occurred
more rapidly at first and then gradually became slower, whilst the
last traces of haemoglobin solution persisted for many hours.
Moreover, it was occasionally observed that when a second or third
injection of haemoglobin solution was made in the same animal
after an interval of six to twelve hours a high degree of
haemoglobinaemia persisted for a considerably longer period than
that resulting from the first injection, even though a greater
quantity of haemoglobin was introduced on this occasion than on
the second. This point is well illustrated in Table 11, where, after
the first injection the haemoglobinaemia within five hours fell from
12°6 to 2°8 per cent., whilst, after the second injection it only
decreased from 9'7 to 5°25 per cent. during a period of nearly
sixteen hours.

The significance of this observation is not very obvious. As
only a comparatively small proportion (Table 1) of the
haemoglobin circulating in the plasma is eliminated through the
kidneys into the urine, the greater portion must be broken up in the

“=

291

body. It has been shown by Tarchanoff* that the intravenous
injection of solutions of haemoglobin in dogs is followed by a
considerable increase in the formation of bile pigment. Moreover,
the work of Simpsont has demonstrated that in those cases of
malaria in which there is reason to believe that considerable
destruction of red blood cells is occurring, the amount of faecal
urobilin is greatly increased. It appears probable, therefore, that

Taste 1.—Comparison of the amount of haemoglobin injected intravenously with
that excreted in the urine.

Amount of haemo- | Amount of haemo-

No. of globin injected globin excreted Ratio of amount
Experiment intravenously in urine injected to amount Remarks
g. g. excreted in urine
I 3°4 0-72 reg Urine contained
haemoglobin when
animal died.
2 162 0°97 Ipeys) 5
3 29°4 53 136
4 29°8 6°34 1:5
5 16:0 1°45 ria
6 41-0 2°8 ae es
7 23°7 1°15 Tes 2 Suppression of
urine
8 28-2 2°66 ips es
9 20'°8 I°5 1:14 Fe

a large proportion of the haemoglobin introduced into the
circulation is got rid of by the liver, and that after a time the
liver cells lose to a certain extent their capacity for eliminating from
the blood stream large quantities of haemoglobin, and hence the
rate of disappearance of haemoglobinaemia may be in the later
stages much retarded.

*‘Ueber die Bildung von Gallenpigment aus Blutfarbstoff,’ Pfliiger’s Archiv., Band IX, S. 53
und 329.
+‘ On Haemoglobin Metabolism in Malarial Fever,’ Roy. Soc. Proc., 1g11, Vol. LXXXIIT,
P17}

292

In spite of the fact that in several of our experiments very large
quantities (as much as 41 grammes) of haemoglobin were introduced
into the circulation, we never observed any icteric condition of the
skin or conjunctivae. On the other hand, the faeces were usually
found to be semi-fluid and green after two or three injections of
haemoglobin.

Owing to the fact that a certain amount of blood was withdrawn
from the animal, and that this was replaced by haemoglobin
solution, haemocrit records made during the experiments showed,
as a rule, a progressive diminution of the volume of red blood cells
as compared with that of the plasma. This decrease in the relative
volume of the erythrocytes was, however, much more marked in the
earlier experiments than in those in which the animal was kept on a
dry diet and in which exceedingly strong solutions of haemoglobin
were injected.

Condition of urine during experiment. ‘The urine in the glass
cannula was found to be tinged with haemoglobin five to ten
minutes after the intravenous injection of the haemoglobin solution.
Estimation of the percentage of haemoglobin passed in the urine
showed that although a high degree of haemoglobinuria was quickly
reached, nevertheless there existed no definite relationship betwen
the concentration of haemoglobin in the urine and that present in
the blood plasma, a very high percentage of haemoglobinuria being
frequently observed after injection of comparatively small amounts
of haemoglobin. As a rule, the maximum percentage of
haemoglobinuria was not developed at once, but occurred some time
(two to four hours) after the injection. A further point of interest
is that after a second or third intravenous injection of haemoglobin
the concentration of haemoglobin in the urine did not reach so high
a level as that following upon the first injection, although in some
instances the amount of haemoglobin circulating in the blood plasma
was considerably higher.

It is clear that the percentage of haemoglobin present in the |
urine at any given time would of necessity depend upon several
factors—firstly, the amount of free haemoglobin in the blood
plasma; secondly, the rate at which it passed through the renal
epithelium; and, thirdly, the amount of water which the kidneys
secreted. When a comparison of the volume of urine passed and

a95

the percentage of haemoglobinuria is made at regular intervals
after the intravenous injection of haemoglobin solution, it is found
that the latter varies to a certain extent inversely with the former,
i.e., the more urine voided the lower the percentage of haemoglobin
which it contains. Although the diuretic action of the sodium
chloride solution is sufficient to account for the fact that the
maximum degree of haemoglobinuria is not developed for some time
after the injection, yet it affords no adequate explanation of why,
after the second or third injection of haemoglobin, the percentage
- of haemoglobinuria is much lower than that following the first
injection, even though in this case the blood plasma contained less
free haemoglobin than in the former. It cannot depend upon the
fact that the haemoglobin was diluted by a greater secretion of
water, as, after the second or third injection, the amount of urine
voided was distinctly less than earlier in the experiment. The only
possible explanation is that either less haemoglobin was being
excreted by the renal epithelium or that the haemoglobin was being
held up in the renal tubules. This point will be returned to later
in considering the microscopical appearances of the kidneys.

Although the first few specimens of urine examined after
intravenous injection of haemoglobin solutions were perfectly clear
without any deposit on centrifugalising, later specimens were turbid
and contained a considerable quantity of solid material.
Frequently, soft red masses of a gelatinous consistency closely
resembling clots were passed. Microscopical examination of the
deposit in the earlier stages showed that it consisted almost entirely
of granular débris and casts. The clot-like masses themselves were
composed of granular débris held together by mucoid material.
The individual granules were at first of small size, although later
in the experiment casts consisting of very large granules (3-4 “) were
frequently seen. They were of a brownish red colour, similar to
that of red blood cells in urine, and were obviously derived from
haemoglobin. In addition to this granular material there were
also large flattened epithelial cells originating from some portion
of the urinary tract below the kidneys.

Later, the character of the deposit changed considerably. In
addition to the granular material large numbers of renal epithelial
cells—isolated and also in the form of epithelial casts (Fig. 1)—

204

were found. Both the free cells and also those of the casts were
frequently loaded with fine brown granules. Occasionally, a
complete cast of the epithelium lining a portion of a renal tubule
was seen in the form of an epithelial cylinder filled with granular
material. On a few occasions red blood cells were found and,
rarely, well marked red cell casts (Fig. 1).

Fic. 1. Solid material passed in the urine of Rabbit 8 on the third day, during the period
of almost complete suppression of urine. A—Epithelial cast. B—Finely granular
cast in which are incorporated epithelial cells. C—Red blood cell cast. D—Finely
granular cast. E—Cast consisting of coarse granules. F—Isolated epithelial cells.
Magnification 300 diameters.

If no marked degree of suppression supervened, the urine
cleared up after a certain time, depending upon the amount of
haemoglobin injected, and the deposit of granular material and

m1 =.

295

epithelium gradually diminished, until, finally, the urine became
normal. In several cases the flow of urine was greatly diminished
or ceased altogether for a period. In Experiment 9 the animal
developed complete anuria lasting over sixty hours; subsequently
the secretion of urine gradually became re-established. In these
cases (Rabbits 7, 8 and g) the urine was greatly altered. No
haemoglobinuria was observed. On acidifying with acetic acid and
boiling, a dense white precipitate of albumen appeared. This was
readily distinguishable from the dark chocolate precipitate which
results when urine containing haemoglobin is boiled. On
centrifugalisation there was a large deposit consisting of granular
casts, some of which were built up of granules of very large size
(3-4 #), and enormous numbers of more or less degenerated renal
epithelial cells and casts. This condition of the urine persisted until
the animal’s death.

Condition of the animal during the experiment.—Intravenous
injection of large quantities of haemoglobin was in our experiments
frequently followed by unfavourable symptoms. A considerable
proportion of the rabbits injected died—some of them after even
comparatively small doses (3'4 grammes). As a rule, the animal did
not appear to be much affected during the first hour. Later,
however, it became decidedly irritable, the slightest stimulus
producing considerable reaction. Shortly before death the animal
suddenly developed violent convulsions. Almost all the rabbits
seemed to be more or less collapsed at first, but if they survived
the injection longer than two hours they generally quickly
recovered their normal condition. We were unable to discover the
cause of death in those animals which died in conyulsions. Careful
post mortem examination conducted immediately upon the animal’s
death failed to reveal any signs of intravascular clotting either in
the large veins, the heart, or in the pulmonary arteries.

Those animals which developed suppression of urine displayed
characteristic symptoms. Chief among these were loss of appetite,
rapid emaciation and considerable thirst (when once suppression of
urine was established ordinary moist food was given). Death
occurred in these animals two to eight days after the development
of suppression of urine.

Condition of the kidneys. In considering the conditions

2096

obtaining in the kidneys of these rabbits it is desirable to divide
the experiments into two classes; firstly, those in which there was no
indication of suppression of urine; and, secondly, those in which
there was a more or less marked diminution in the amount of urine
passed.

The kidneys of the first group varied considerably, both
macroscopically and microscopically, according to the amount of
haemoglobin solution injected and also according to the length of
time which elapsed between the intravenous injection of

Tasre 2.—Weight of Kidneys in experimental and normal rabbits

{
No. of | Amount of | No. of hours | Weicut or Kipnrvs,
times haemo- | animal lived me
No. of | Weight of | haemo- globin after first injec-| :
Experi- rabbit, | globin solution tion of Remarks
ment g. solution | injected haemoglobin
was g. solution Right Left
injected
— ! —————— = SS. ae —
1,400 | I 2:5 5 57 6:0
Le 1,420 I 4:9 1°3 73 7:0
Hi 1,980 I 3°4 3°5 7:0 72
III* 1,860 I 8-6 7 Wen 7e7/ Was
1Vve 1,570 2 11:3 22 | 6:8 65
2 1,650 2 16:2 20 | 6°3 6-2
3 1,750 4 294 | 38 67 68
4 1,920 4 37°38 44 100 100
5 2,870 5 16°3 98 he O26 10°5
6 1,630 8 410 70 | 10°35 1O"4
a | 1,235 7 23°97 68 } II55 10°75 Suppres-
8 | 1,740 3 28-2 94 | ro 10°75 sion of
9 | 1,960 3 20°8 217 \| x45T5 13°8 urine
Normal ...| 1,550 — — — 6:0 611
Normal ...| 2,300 _ — — 2 7-4

haemoglobin and the removal of the kidneys. The kidneys of those
animals which died after a single injection were but slightly altered
in appearance. They were congested and of a dark brown or
chocolate colour, but were not definitely enlarged (Table 2).
Microscopically, a number of the convoluted tubules were seen to
contain brownish coloured plugs. These were also present to a
less extent in the tubules of the sub-cortical zone. There was no
trace of solid material in the meniscus of Bowman’s capsules nor
in the collecting tubules of the papilla (ducts of Bellini). No
dilatation of the tubules or glomeruli was observed.

* These experiments are not included in Tables 3-11.

297

A much more striking picture was afforded, however, by those
kidneys which were removed after several injections of haemoglobin.
Here these organs were distinctly enlarged and heavier than those
of normal rabbits (Table 2). As before they were of a dark
chocolate colour. On section the cortex was found to be thicker than
normal, and the whole kidney was seen to be traversed by dark
lines, giving it a markedly striated appearance. Frequently these
dark coloured streaks were grouped in fan-shaped areas (Fig. 2),
whilst the aspect of the intervening spaces was normal. The
microscopical appearances presented by the kidneys were very
characteristic. A large number of the convoluted tubules and tubes
of Henle were filled with plugs of granular material, and here and
there epithelial cells were found intermingled with the granular
casts. The casts extended down into the large collecting tubules
of the papilla. As before, no deposit was found in the meniscus
of Bowman’s capsules. Many of the convoluted tubules and tubes
of Henle were distended and the epithelium in places had broken
away from the basement membrane.

The kidneys of the animals belonging to the second group—
those in which total or partial suppression of urine occurred—were
profoundly changed. They were of a dark brown colour and
greatly increased in size (Fig. 2) sometimes weighing more than
twice as much as those of a normal rabbit (Table 2). On section
they were found to be markedly oedematous. The cortical and
sub-cortical zones were greatly thickened. Dark striations were
visible, but they were not nearly so distinct as in the kidneys
belonging to the previous group, and the appearance was confined
chiefly to the sub-cortical portion of the pyramid. Microscopical
examination showed that the whole structure of the kidney was
radically altered. The tubules of the cortex and sub-cortical region
were enormously dilated and many of them contained casts of
granular material and epithelium in various stages of degeneration.
The epithelium lining the tubules was in many places exceedingly
thin, and in others had completely disappeared.

Scattered here and there between these large dilated tubules
were small islands of tubules tightly packed together, so that their
lumina were completely obliterated. Some of the glomeruli were
enormously distended and others, like certain of the tubules, so

298

crushed as to be scarcely recognisable. The large collecting tubules
were also much involved. In many places the epithelial cells had

broken away from the basement membrane and were either lying

A

8
UP
G

Fic. 2. Diagrammatic representation of the appearances found on section of —A, normal

kidney ; B, kidney of an animal which had received several injections of haemoglobin ;

C, kidney of Rabbit 9, in which there was complete anuria for 60 hours. Magnification
- 1*5 diameters.

loose within the tubule or had entirely disappeared. In other
tubules the epithelium was intact, but the lumina were filled with

299

masses of epithelial cells and granular débris. Occasionally, a
collecting tubule was found in which the lining epithelium was
normal and the lumen entirely filled by a mass of granular material
and isolated epithelial cells surrounded by a ring of epithelium
evidently derived from a higher portion of the same collecting
tubule.

As to the exact nature of the granular casts existing in the renal
tubules and subsequently voided in the urine, it is difficult to speak
with certainty. When examined in the fresh unstained condition
their colour resembled closely that of the red blood cells. Moreover,
they stain with various dyes (eosin, van Gieson, iron alum
haematoxylin) in a manner precisely similar to the erythrocytes.
There appears to be no doubt but that they are derived from
haemoglobin, and we are of opinion that these casts are composed
of minute droplets of haemoglobin (or of a very closely allied
substance) held together by mucoid material. Of course this
statement refers only to the casts existing in the renal tubules and
urine examined within a few hours of the injection. The casts
examined at a later period of time (four to eight days) after
the haemoglobin injection, were obviously different. The material
had in places changed to a light brown colour and no longer stained
well. In other places it had become in part crystalline. We were
unable to obtain the iron reaction with ammonium sulphide and
ferrocyanide of potassium, probably because the decomposition of
the haemoglobin had not progressed sufficiently far. This reaction
was obtained by Werner in several cases of blackwater fever in
man.

We have hitherto avoided any reference to the question as to
which portion of the renal tubule is responsible for the elimination
of haemoglobin. As we intend to discuss this question more fully
in a future communication, we shall content ourselves here with
stating that as a result of our observations we are of opinion that
the haemoglobin does not pass through the glomeruli, but that it is
excreted by the epithelium of the renal tubules, more especially by
that of the convoluted tubules.

If the information obtained from examination of the urine be
considered in conjunction with that derived from a study of the
pathological condition obtaining in the kidneys, one is enabled to

300

form a fairly definite conception as to the effect which intravenous
injection of haemoglobin has upon the renal secretion of an
otherwise normal animal. The immediate result of such injections
is a diuresis, dependent upon the action of the sodium chloride
solution used for dissolving the haemoglobin. At the same time
the renal epithelium commences to excrete haemoglobin into the
tubules. At first this is quickly carried away by the rapid secretion
of watery urine from the glomeruli. The result is a considerable
flow of clear urine containing haemoglobin in solution. As the
diuresis subsides the haemoglobin becomes deposited to a certain
extent in the tubules. At this stage the urine is less in amount and
contains a quantity of solid material consisting chiefly of granular
casts and débris. Still later, especially when the degree of
haemoglobinaemia is high, epithelial cells are shed from the lining
membrane of the renal tubules. This is probably dependent upon
one or both of the following factors. In the first place it is
possible that the excretion of large quantities of a foreign
substance like haemoglobin may be attended by injurious effects
upon the renal epithelium. In the second place the presence of
casts in the lumen of the tubules would tend to damage the lining
epithelium mechanically, either by friction as the plug was forced
down the tubule owing to the wis a ¢ergo of the glomerular
secretion or by causing a complete or partial block of the tubule,
and, as a result, dilatation of that portion above the plug, and
consequently separation of the epithelium from the basement
membrane (Plate XIII, figs. 3 and 4). This stage represents the
commencement of the development of suppression of urine.
Gradually, as haemoglobin continues to be excreted by the renal
epithelium more and more tubules become occluded. The situation
in which complete blocking of the lumen first occurs appears to
be, as a rule, the narrow tubules of Henle. Coincidently with the
plugging of the tubules there is a marked diminution in the amount
of urine voided and also of the haemoglobin it contains. The
condition steadily progresses, until, finally, complete anuria
(Case Q) results. After cessation of the flow of urine has lasted for
a certain time a gradual re-establishment of urinary secretion occurs.
The urine is loaded with albumen and is exceedingly turbid. On
centrifugalisation a large deposit, sometimes equalling as much as

301

one-fourth of the volume of urine, is thrown down. This deposit
consists of granular casts, epithelial casts, granular débris and
degenerated epithelial cells. Urine of this nature continues to be
passed until the animal’s death. During the period of total or
partial anuria marked changes occur in the kidneys. Owing to the
continued secretion of the glomeruli those portions of the tubules
above the plugs become enormously distended with fluid. As a
result, the epithelium lining these portions becomes extremely thin
and in places entirely disappears. The dilated tubules press upon
adjacent tubules, so that on microscopic examination the latter
appear of small size and their lumina are completely obliterated
(Plate XII, fig. 1). After a time, owing to gradual disintegration
of the casts plugging the tubes, the urine commences to escape into
the ureter once more. The serum proteids and red blood cells pass
through the damaged epithelium and those portions of the tubules
devoid of epithelial cells, with the result that the urine is loaded
with albumen. Although the casts may be to a great extent washed
away in the course of the next few days, yet the renal tubules
remain dilated and the serum proteids continue to escape into the
urine until the animal’s death, which in these cases is probably due
to uraemia.

CONCLUSIONS

It is evident from these experiments that, under certain
conditions, the mere passage of haemoglobin through the kidneys of
a healthy animal is sufficient to cause suppression of urine owing
to occlusion of the lumen of the renal tubules by plugs of granular
material derived from the haemoglobin. The process is considerably
facilitated by any factor which tends to lower the blood pressure
of the amimal, and as a result the secretion of water by the
malpighian capsules. On the other hand, when the blood volume
of the animal is kept up, as in Case 4, by the injection of saline
solution (that of the haemoglobin solution which in this case was not
particularly strong) and by feeding the animal on moist food, a
large amount of haemoglobin (38 grammes) may be injected without
any tendency to suppression of urine.

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DESCRIPTION OF PLATES

The sections were stained by Heidenhain’s iron alum
haematoxylin method and the figures drawn with an Abbé camera
lucida.

PLATE XII

Fig. 1.—Section of renal cortex of Rabbit 9. Many of the
convoluted tubules are greatly distended and in some
the lining epithelium is considerably flattened.
Scattered between the dilated tubes are islands of
tubules tightly packed together, so that their lumina are
completely obliterated. Two Bowman’s capsules are
seen in the section. A number of the dilated tubules

contain plugs of granular material. Magnification 240
diameters.

Fig. 2.—Transverse section of renal pyramid (sub-cortical portion)
of Rabbit 6, showing many dilated tubules (tubes of
Henle and collecting tubules) completely plugged with
granular material. Magnification 350 diameters.

PLATE Xil.

A. M. B., del.

&

Ba Essleriine : ast lozi To *
Saiedbllos ai cic Fr 7

a ~ ; ‘ . 7

313

ON THE RELATION OF THE ORGANIC

PHOSPHORUS CONTENT OF VARIOUS

DIETS Oo) DISEASES, OF _NULRITION,
PARTICULARLY BERI-BERI

BY
Ge. te Stee SON, bA., Wb.; B.SC.,

AND

ESV EDI, NEAL? B.SC.
PART) I.

(From the Laboratories of Tropical Medicine, and of
Bio-Chemistry, Liverpool University.)

(Received for publication 10 June, 1911)

In November, 1910, Professor Ross called our attention to Fraser
and Stanton’s work on The Etiology of Beri-beri (1910), and asked
us to enquire into the subject with a view to further researches on
similar lines and at the same time supplied us with a large sample
of a rice, the use ef which as a diet had been associated with an
outbreak of Beri-beri.

Shortly after we had commenced our work the monograph of
Schaumann (1910) appeared in the Archives for Ship- and Tropical-
Hygiene, and this work was found to be so complete and exhaustive
that further investigation of the main facts seemed superfluous, and
we turned our attention to confirming some of the newer facts and
to further investigations of points—mainly of a chemical nature—
arising out of his results.

Schaumann’s work is so important that it seems advisable to give
here an abstract of the more important sections of his monograph,
which should be consulted for full details and also for the exhaustive
bibliography.

After a historical review of the previous theories of the causation
of Beri-beri and of the work of himself and others refuting these
conceptions—e.g., a specific infection, contamination from without

314

by toxic bodies, or by autogenous development of such bodies in the
diet, etc., he proceeds to the consideration of the work which con-
nects ‘ Tropical Beri-beri’ with the use of highly prepared rice—e.g.,
rice in which the husk and outer layers of the grain have been
removed by the process of milling—and of that which connects ‘ Ship
Beri-beri’ with the use of similar rice as the staple diet, or with the
use of preserved foods and other restricted or unsuitable diets.

Braddon (1909) first called attention, in 1901, to the fact
that Beri-beri was prevalent in those Eastern tribes and races, e.g.,
Chinese, who used a highly-milled rice as their staple diet, but did
not occur among races such as the Tamils, who cured their rice in
such a way that the pericarp was not removed from the grain in the
process of milling; Fletcher (1907) confirmed these observations,
showing that the substitution of whole rice for polished rice in the
dietary of the inmates of an asylum abolished Beri-beri, while the
subsequent reversing of the diet led to an outbreak of the disease.
Since 1907 polished rice has been abolished finally from the dietary
of the inmates and Beri-beri has ceased, though previously
extremely prevalent and causing many deaths. Fraser (1909)
obtained similar results with gangs of labourers inhabiting separate
compounds, and others have added further confirmation. The
practical application of these observations by hygienists in our
Eastern Possessions, in the Japanese navy and army, etc., has
resulted in a great diminution of the incidence of the disease.

Turning now to another side of the question, Schaumann refers
to the experimental production of polyneuritis in fowls; Eijkman
(1897), in Batavia in 1896, discovered that fowls fed entirely on
any variety of polished rice became lame and ultimately died, and
demonstrated lesions in the peripheral nerves and anterior horn
cells of the dead animals. On whole rice, however, no such
symptoms were developed, and addition of the polishings to the
prepared rice prevented their onset.

He further proved a similar difference between barley and
prepared barley, and finally discovered that rice, barley and rye are
no longer adequate after heating for a time to a temperature of
120° C., and birds fed on the above fall ill and die with typical
appearances of peripheral neuritis. For diets so treated the name
‘denaturised’ is used.

315

Grijns (1908), in Batavia and Holland, confirmed Eijkman’s
results, and showed the development of polyneuritis in fowls when
polished rice, sago and tapioca formed the sole diet, and that the
same train of symptoms arose on a diet of flesh which had been
heated to 120° C.

He discovered that the addition of small quantities of ‘Katjang-
idjo’ beans (Phaseolus radiatus L.) rendered safe a diet previously
injurious, and these beans also lost their preventive power if heated
to 120° and further demonstrated that the power of a rice to produce
Beri-beri varied directly with the degree of preparation, that ts
with the extent to which the pericarp (or silver skin) was removed
in milling.

Axel Holst and Froélich (1907), working at Christiania with
special reference to Ship Beri-beri and to scurvy, carried out a series
of researches on pigeons and guinea-pigs, using various limited diets.
Their results in the main confirm those of Eijkman and Grijns, both
for prepared rice and barley and for heated foodstuffs. They demon-
strated, in addition to the polyneuritis, marked emaciation in the
birds and animals used, and also oedema of the subcutaneous tissues
and muscles; further in mammals they found changes in the gums
accompanied by loosening of the teeth.

In addition they examined various sorts of bread, and found that
wheat bread was harmful both to pigeons and to guinea-pigs—bread
baked with yeast, however, was much less harmful than bread baked
with baking-powder. It should be noted that guinea-pigs are much
less resistant than fowls to such restricted diets and that restriction
of these animals to one sort of grain, whether ground or not, is
invariably deleterious ; rarefaction of the bones, haemorrhages in the
neighbourhood of the epiphysial line, degenerations of bone marrow,
and sponginess of the gums are often produced.

Holst further determined the amounts of peas, unshelled barley,
etc., which required to be added to diets in order to prevent the onset
of symptoms, and also calls attention to the fact that preserved
meat has often been denaturised by heating to 120°.

Fraser and Stanton (1910) further confirmed these observations
with regard to the association of polished rice and polyneuritis.

Schaumann (1908) and Fraser and Stanton (1909) (independ-
ently) discovered that diets which produced peripheral neuritis

316

were invariably poor in phosphorus, and that substances (e.g., rice
meal, Katjang-idjo), which have the power of preventing the
development of neuritis, are, on the contrary, rich in that substance ;
indeed that the smaller the percentage of a diet in phosphorus the
greater is its influence in producing Beri-beri in man, or neuritis in
fowls.

Schaumann thought that the active principle containing phos-
phorus was probably nucleic acid, and Grijns, following up this
hypothesis, experimented with the nucleo-proteins of Katjang-idjo ;
no curative effect was observed with nucleins extracted by alkali,
but some slight postponement of death was observed in neuritic birds
treated with the phosphorus-containing extract obtained with hot
water.

Schaumann further gives exhaustive tables of the composition of
various food stuffs—grains (fresh and prepared), peas, beans, potatoes,
meat, etc., both in the fresh and dried states, showing their composi-
tion with reference to protein, carbohydrate and fat, and in particular
their various phosphorus-containing compounds—inorganic, phytin-
like compounds, nucleins and phosphatides—and discusses the
present state of our knowledge of the metabolism of these bodies.

Schaumann next proceeds to a critical review of the various
theories of the etiology and points out that neither place, climate,
nor season appear to be of importance, and that no specific organism
has been isolated though many bacteriologists, and even Koch
himself, have made careful investigations; Fletcher and Fraser also
carefully excluded the question of infection since they showed that
whether Beri-beri was present or absent among the inmates of a
building or compound it could be banished by feeding with partly
milled rice or called forth by feeding on wholly milled rice.

Braddon, with respect to Beri-beri, and Eijkman, with reference
to Polyneuritis in birds, at first assumed that some toxic agent was
present in the kernel of the grain which gave rise to Beri-beri and
that the antidote to this toxin was present in the pericarp, and that
so no bad effect followed feeding with the whole grain; but, if the
pericarp were removed, the toxin already present or elaborated during
digestion was free to exercise its effects. Others assumed that the
fully shelled rice became contaminated more easily by toxic bodies
(e.g., arsenic) and so caused ill effects, but Fraser and Stanton showed

317

that polished rice was equally deleterious when freshly ground as
when it had long been exposed to the risk of contamination.

In spite of careful chemical investigations it has proved impossible
to isolate any toxic body from polished rice, from denaturised (heated
to 120°) flesh, or from other substances which cause Beri-beri or
Polyneuritis; Fraser and Stanton further showed that sera, flesh,
and other products of neuritic fowls caused no deleterious symptoms
when administered orally or by injection to sound animals.

Schaumann then considers the theories which ascribe the develop-
ment of Beri-beri to a faulty nutrition, and he calls attention to the
theories of Nocht (1908); the latter ascribes both the multiform
manifestations of Tropical Beri-beri and the usually milder Ship
Beri-beri (which appears to be somewhat allied to scurvy), to dietetic
errors; and lays special stress on the fact that it is not a question
of defect of the main components of food stuffs—proteid
carbohydrate and fat—but of some subtle defect of the less known
constituents—enzymes, complements, compound proteids, etc.

Schaumann himself, however, is inclined to consider the substance
or substances of importance to be organic compounds of phosphorus.
He resolved first of all to investigate the possibility of the neuritis
being due (1) to the development of oxalic acid by fermentation or to
acidosis from deficiency of alkaline salts in the grain; (2) to the
removal of ferments or other thermolabile substances by milling or
‘denaturisation, and (3) to investigation of the influence of phos-
phorus compounds, organic and inorganic.

He points out that the exact chemical nature of the phosphorus
compounds in food stuffs and the body is but imperfectly known, that
the methods of separation are but approximate and uncertain in many
cases, and that the isolated products are probably modified by the
processes of extraction, and so differ in physiological effect from
their precursors.

Subject to modifications necessitated by the light gained in the
process, he resolved to try diets, as polished rice and denaturised
foods, which were known to produce neuritis, etc., and see whether
any favourable results are obtained by the administration of such
diets, with the addition of :—

1. Known organic and inorganic compounds of phosphorus.

2. Small amounts of substances rich in phosphorus, e.g., rice

318

meal, yeast, Katjang-idjo, wheat meal, testicular extract, or extracts
from roe, etc.

3. Phosphorus compounds isolated by him from articles of food,
e.g., yeast, Katjang-idjo, etc.

Special stress was attached to investigations with denaturised
foods in which the defect was probably simpler than in milled foods,
and Schaumann also planned to investigate microscopically and
chemically the nerves and other tissues of the animals experimented
on, and to carry out chemical investigations on fresh and preserved
foods—foods from Beri-beri ships (and moulds, etc., contaminating
them)—and also on the phosphorus excretion in faeces and urine of
birds and Beri-beri cases.

Schaumann’s first results show that excess of oxalic acid (or other
toxic products) and deficiency in autolytic enzymes can play no part
in the etiology of Beri-beri nor of Polyneuritis in fowls, and further
that the fault of diet lies neither in deficiency of proteins nor of
inorganic salts, since addition of these substances to a diet which
gives rise to Polyneuritis has no protective influence, and also since
the diets which Fletcher, Fraser and Ellis found to cause Beri-beri
contain respectively 92, 103 and 99 grams of protein per man per
day, a figure quite sufficient to maintain equilibrium, especially con-
sidering that Fletcher and Ellis were dealing with inmates of
institutions for mental disorders and not with men engaged in hard
manual labour. Turning now to phosphorus compounds, Schaumann
gives exhaustive details of the various organic and inorganic com-
pounds present in foodstuffs, and in the various organs of the body
with their characteristics and the methods of extraction, etc. He
then passes to a consideration of phosphorus metabolism; an adult
man at work requires about two grams of phosphorus daily on
mixed diet, while a dog seems to require less than 0'5 gram per
day; birds in some instances require much larger amounts than
mammals of the same size.

Passing on, he considers the metabolism of each of the better
known classes of organic phosphorus compounds, phosphatides,
nucleoproteins, phytin, etc., and also inorganic phosphates, giving
an account of their absorption, assimilation, retention and excretion.

He calls attention to the fact that phosphorus is present in
especially large amounts in the organs whose functions are most

319

complicated and important; further, the main organic phosphorus
compounds are in large part assimilated by the alimentary tract as
organic compounds, without the previous splitting off of the phos-
phoric acid, and the organs of man and the higher animals have
further a marked power of storing excess of such compounds and of
drawing on this store at times when these are deficient in the diet.
He quotes Miescher’s well-known work on the increase of salmon
roe at the expense of the phosphorus of its muscles, and suggests
that pregnant females may store up phosphorus to be subsequently
given out in the milk. In addition to this, new-born animals usually
possess a store of phosphorus proportionate to the length of time
which will pass before they can forage for themselves (as is known
to be the case also with iron). In times of hunger the animal
organism is more economical of its phosphorus than of any other
inorganic constituent, and seems to have a more urgent need of its
phosphorus than even of its proteids.

Albu and Neuberg (1906) consider that it has been practically
demonstrated that the body has not the power to synthesise the
organic phosphorus compounds necessary for the life of the cells
from phosphorus-free proteins and inorganic phosphates, du¢ ¢hat
animal life is dependent for these substances (as for proteins and
carbohydrates) on the synthetic powers of the vegetable kingdom.

Schaumann next resolves to attempt as far as possible experi-
mentally to determine the way in which an absence or deficiency of
each or all of these compounds in the diet influences the animals, as
Liebig has worked out the influence of salts on the growth of plants.
The problem is much more difficult in the animal kingdom, however,
since the substances are of great complexity, present great variety in
each class, and are not only difficult to isolate but undergo important
modifications in the processes of separation. These difficulties may
lead to failure or to the results obtained experimentally being
wrongly interpreted.

All earlier observers of neuritis, etc., in animals had considered
that the causation of the lesion was to be attributed to some poisons
(toxin, oxalic or other acid), which developed endogenously or
exogenously in the nutriments producing them, and which could be
neutralised by certain (equally hypothetical) anti-bodies which are
present in certain food-stuffs, e.g., rice meal, Phaseolus radiatus,

320

etc. Fraser and Stanton (1909) have called attention to a parallelism
between the lack of phosphorus in the food and the onset of Beri-beri
in men and of neuritis in cattle, and in that have followed the
suggestion made by Schaumann in 1908.

Schaumann’s earlier experiments were not strictly conclusive as
showing that lack of organically combined phosphorus was the most
important and indeed the only factor in the etiology of Beri-beri,
and his further experiments are planned with a view to attempting
to find such conclusive proof.

Researches on Pigeons. Polished rice (20 grams per bird per day)
forms, in the majority of cases, the basis of the food. It is used in
the form of a pap or porridge, to which various amounts of the
different substances can be added, with a view to determining their
protective power; in other cases barley-or barley bread was used as
a basis, but these experiments have to be viewed independently of the
rice experiments if the average time required for the development of
neuritis, etc., is to be taken as a measure of the possession or lack
of protective power.

Well-grown and well-nourished pigeons are found to be the most
suitable for experiment, young animals and fancy breeds being less
serviceable. Birds are specially suitable because their metabolism is
very regular, and deficiency in the food is rapidly indicated.
Further, the curative influence of protecting substances on sick birds
is much more rapidly noticeable than on mammals.

Control experiments with pigeons showed that death occurred in
the average period of thirty-five days on polished rice or rice bread ;
the birds exhibiting more or less severe appearances of lameness and
losing 41 per cent. of their original weight.

If the rice is previously thoroughly extracted with cold water, the
birds die much faster, in an average period of twelve days; extraction
removed only 3°43 per cent. of the original proteid but 36 per cent.
of the original phosphate; a difference of proteid per bird per day
which would be unlikely to so hasten the end.

Non-protective Substances. Addition of dried egg-albumen, of
albumen metaphosphate, calcium glycero-phosphate, and of
inorganic salts (with or without phosphates), had no marked
influence in either direction, as will be seen from Table I.

Nucleic acid from yeast prepared for commercial purposes

321

(scientific and medicinal) appeared to have a very unfavourable
influence, but a specially manufactured sodium nucleate, carefully
prepared to break up the molecule but little, seemed to have a slightly
favourable influence. The birds only lived a few days longer than
on rice alone, certainly, but the lameness was not marked.

Protective Substances. The meal or bran from the outer parts of
grain which is removed in milling, however, had a very different
influence when added to the polished rice pap used for feeding.

With two grams of rice meal (P,O, 3°8 per cent.) added to the
pap for the day’s ration, the birds remained fit and strong (for
seventy-two days), and indeed gained in weight—1'5 gram per bird
per day scarcely sufficed to keep them strong, and with 1 gram loss
of weight commenced.

Equally favourable was the influence of the addition of wheaten
bran, which contains 1'1 per cent. of P,O,. Four birds each received
2°5 grams of this per day, with the usual ration of rice pap. They
lost some 20 per cent. of their weight, though otherwise remaining
well for twenty-eight days; the ration was raised to 5 grams each
per day, but as the birds did not recover their initial weight it was
raised fourteen days later to 7°5 grams, and the birds in the next
twenty-five days not only reached their original weight, but actually
added a further 10 per cent., and were perfectly fit and active.

In a further set of five pigeons, two grams per bird per day of
dried brewer’s yeast (P,O. 4°2 per cent.) was added to the diet of
rice pap; the birds remained well, but in two months lost a little
(3°5 per cent.) of their weight. The allowance of yeast was reduced
to one gram per bird daily, and except for the loss of a further 3°5 per
cent. of their weight, they were at the end of a month apparently
just as well as at the beginning of the experiment three months
earlier.

Schaumann now compares these three ‘ protecting’ substances as
regards the phosphorus and proteid contents of the daily allowance
needed to keep a pigeon well and maintain its weight, and it will be
noticed that the phosphorus contents almost coincide.

Substance Daily allowance (gram) P,O; content (gram) | Protein content
| (gram)
Meat. oa 1°5 0.063 0°55
Wheat bran... 50 0055 o-72
Rice meal ya 1°§ 0'057 016

322

Curative Effects. In addition to trying the protective influence
of these various substances when added to the polished rice diet
from the beginning of the experiments, Schaumann also tried their
power of curing pigeons which were already suffering from neuritis
and in many cases were apparently very near to death.

In strange contrast to the fact that it exercised no protective
power when administered in the diet from the beginning, is the effect
of nucleic acid prepared from yeast when administered to birds
already lamed by neuritis. Of fourteen cases in which this was
administered (forced feeding), six died before the nucleic acid had
passed out of the crop; in five cases with repeated forced feeding
with nucleic acid and rice, some improvement manifested itself, but
was only transitory, and the birds died in five days—the lameness
which at first lessened having again returned.

In the remaining three cases the result was even better; rice
pap containing 3°5 per cent. of yeast nuclein and of dry egg-albumen
was repeatedly administered to one bird, which-had lost 25 per cent.
of its weight and was very lame and unable to move after fifty days’
feeding on rice meal with egg-albumen; the next day the bird was able
to walk with considerable agility, to use its wings in normal fashion,
and to feed itself, and had lost the continuous convulsive movements
of head and limbs that had previously troubled it. Twenty-four
hours’ further treatment, and the bird was apparently fully recovered
and able to walk and even fly. Six days later it was killed and no
degenerated nerve fibres could be found.

Similar results were obtained in the case of a second bird, which
was not killed, however, and so no examination of the nerves was
possible. The third bird apparently completely recovered on a
similar treatment, and was able to fly on the third day of
treatment. It was killed, and the usual appearances were found
after death—oedema of the muscles of the limbs, other muscles with
diffuse haemorrhages, numerous nerve fibres with typical
appearances of degeneration in the sciatic nerve. In the upper limb
nerves no degenerated fibres were found, however, and the general
condition of nutrition appeared good.

Dried pressed yeast is even more powerful a curative agent than
yeast nuclein. Unless the bird dies before the yeast has passed from
the crop, its administration results invariably in recovery if a
sufficient amount be given.

—"

323

Schaumann has tried this in a large number of birds, small
amounts (0-1 to 0-3 gram) have no effect, but one gram a day is quite
sufficient. In birds not severely lamed, and able to feed themselves,
recovery is fairly rapid. On addition of yeast to the diet, the
symptoms of lameness disappear in a day or two, the weight regains
rapidly its level on mixed diet, and the degenerated nerve fibres
progressively diminish in number and in the amount of degeneration.
(Only a few typically degenerated fibres were found in a bird killed
on the sixth day, and after fourteen days no degenerated fibres were
found, but a few still show ‘ foam-structure,’ the earliest stage of
degeneration, the last of regeneration.)

Schaumann takes for illustration in his text one of these birds,
which had lost 40 per cent. (120 grams) of its weight on an exclusive
diet of polished rice and was so severely lamed as to be on the point
of death; 3 grams of dried rice pap with 1 gram of yeast was
forcibly administered (the dried yeast contains 4°25 per cent. P,O.).
The bird could walk fairly well in twenty-four hours, and after a
further administration of I gram of yeast with its rice, was again
able to fly. Schaumann gives photographs of this bird taken on these
three days, and the improvement is most marked, especially as this
bird had (also the first one mentioned as cured with yeast nuclein,
photographs of which are also reproduced) convulsive movements
of the head and limbs, accompanied by spasm of limbs and neck
muscles (leading to retraction of the head), a condition which is of
even more immediately fatal import than a severe degree of
lameness.

After these two days the bird was able to feed itself, and received
rice pap with addition of 5 per cent. of dried yeast. Its improve-
ment was marked (‘visible’), and in twelve days added 44 per cent.
to its weight (80 grams).

Katjang-idjo beans (P,O, 1°08 per cent.) exert an excellent
curative effect; lame birds are sufficiently restored in forty-eight
hours to run and fly, and their weight rapidly increases—(30 per cent.
in eleven days, 17 per cent. in four days). The amount of the bean
required is about 1°3 gram per day for each bird.

Dry yellow peas were almost as effective as Katjang beans,
curing five birds very rapidly.

Many organic phosphorus-containing extractives of plants and

324

tissues were also used for curative purposes. Nucleic acids from yeast
have already been referred to and will be remembered to have some
power.

Yeast lecithin appeared to have no curative influence, but seemed
to have some protective effect, for one pigeon was still alive after
eight weeks’ feeding with rice pap to which 0°5 gram of yeast
lecithin per day was added, and though it had lost some weight no
lameness appeared.

Ovolecithin, tried in one case, improved the condition of the
lamed bird at first, but the good effect was not permanent, and
Katjang-idjo lecithin similarly seemed to have an initial but only
transient curative effect.

Commercial phytin and protylin were tried and found to be
valueless.

Schaumann separated several extracts from Katjang beans—the
pepsin-hydrochloric acid extract contained a large proportion of the
phosphorus and was a powerful curative agent. Administration of
one gram per day for two days, followed by o'5 gram for seven days,
completely restored one markedly paretic pigeon; and a similar
result was obtained with a body of the phytin group extracted from
this bean, two lame birds being obviously better the day after its
administration, but continuation of the treatment did not avail to
completely restore the birds, or indeed to keep them alive.

Testicular extract has a distinct protective power; two birds to
which 1°5 grams per day each was given with the rice pap lived
respectively forty-eight and seventy-five days, on the average twice
as long as on rice alone. Both showed great loss of weight (54 per
cent.) and neuritis, though one was lame for a short time only, and
the other lived for thirty days after symptoms of lameness had
appeared. The same extract administered to a paretic bird improved
its condition and materially delayed its death (probably by about
thirteen days).

Schaumann here calls attention to the complete ‘protective’ and
‘curative’ influence of this extract in the case of dogs.

Some signs and symptoms are present in all the birds which are
receiving polished rice alone or with non-protective additions.
Diarrhoea with thin execretae is one of the most noticeable, the colour
of the stools being decidedly green—the stools contain only about

325

25 per cent. of the phosphate present on a full mixed diet. Loss of
body weight is also constant, varying in amount from 25 to 54 per
cent. In nearly all cases the birds showed loss of appetite in the
later stages, remained sitting and were unable to fly.

The differences in duration of life were very considerable, not
only with different nutriments (non-protective), but even in the same
series—the greatest variation in the same series was from twelve to
sixty-one days.

Equally marked were the differences in the degree of incapacity
which developed before death. In rare cases scarcely any weakness
of the legs was observable even just before death occurred, and the
birds might even fly shortly before the sudden onset of death. In
other cases all grades of lameness of legs and wings may appear,
some are quite unable to fly, and can only move with difficulty,
walking uncertainly, stumbling and falling forward.

Irregularity and galloping of the heart beat occurs in a fair
number of lame birds.

The same variability was shown in the degree of degeneration
of the nerves of different birds of the same series when examined
after death.

Schaumann states that the grade of lameness is no measure of
the amount and erade of degeneration in the nerves; marked lame-
ness with slight degeneration, or slight lameness with marked
degeneration being both observed.

In one case no difference could be made out in the degree of
degeneration of the two sciatic nerves of a pigeon, one excised when
markedly lame, the other obtained by killing the bird when
apparently fully restored by two days’ feeding on Katjang beans.
In other birds, which were killed after being apparently completely
restored from their lameness by one or two feeds of curative
material, the nerves were obviously very degenerated in spite of the
fact that the paresis had completely disappeared.

Schaumann suggests that the phosphatic bodies may in some way
serve as sources of energy (physical or chemical) in the nervous
system, and refreshment of the supply enables the central nervous
system to overcome the hindrance of the degenerated nerves.

The researches on pigeons have been given in some detail, and
we propose to only briefly note those carried out on other animals.

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Guinea-pigs kept on polished rice or maize, with or without the
addition of yeast, die during the fourth week; the loss of weight is
between 20 and 40 per cent., and though lameness is not noticeable
there are signs of early degeneration in the nerves. In addition,
haemorrhagic lymphadenitis may develop in animals on yeast. In
many animals, also, rarefaction of bones (osteoporosis) was very
evident, and the bones might be as thin as paper.

Rats were kept on a diet of egg-albumen, starch (potato), sugar
and inorganic salts, with or without phosphate (albumen-metaphos-
phate); death occurred in about 4o days without apparent lame-
ness; on fine rye bread, denaturised by heating with soda, they die
in 63 days (average), losing 47 per cent. of their weight.

Rabbits.—Maize, and maize with egg-albumen, is inadequate:
death occurs in 45 days, with slight degeneration, and the loss of
weight is about 30 per cent; other symptoms are lameness and loss
of reflexes in the hind limbs, loss of hair, loss of appetite and
activity ; they die of exhaustion or convulsions. One strong rabbit
lived after ninety-seven days’ feeding on rice and egg-albumen.

Maize with the addition of 4 per cent. of dried yeast is fully
adequate and maintains the animals in normal health.

Katjang-idjo and peas rapidly restore animals which have become
paretic—_the lameness vanishing and the body weight rising rapidly.

An Ape, which thrives on rice pap, currant bread, nuts and fruit,
died with paresis and loss of nearly 30 per cent. of its weight after
seventy-four days’ feeding on pap from polished rice previously
extracted with water. There was little typical degeneration, but
nearly all the nerve fibres showed ‘ foam-structure.’

In the next set of animals, denaturised horse flesh was used as
the basis of diet. The flesh is heated in an autoclave to 120° C. with
dilute soda-solution—the alkali being afterwards neutralised with
hydrochloric acid. The nuclein molecule is probably split in the
process. The proteids are not apparently affected. (Neither
ammonia nor sulphur bases are formed. )

Rats, on denaturised flesh, become paretic in about a month, but
the illness does not progress, and the experiments were abandoned
after three months or more. Loss of weight, about 5 per cent.

Cats..—Denaturised flesh: death in about 50 days; loss of
weight, 30 per cent. Jameness present before death -slight nerve
changes demonstrated.

328

Dogs.—Denaturised flesh (about 1 kg. a day); loss of weight,
25 percent; death, 50 days (average). Severe lameness starting in
the hind limbs is the first symptom, and progresses to complete
incapacity ; death occurs with convulsions. In one case there was
sponginess and haemorrhages in the mouth and gums, with
ulceration; nerve degeneration is only slight. One dog lived for
fifty-five days and was kept quite fit and well by the addition to the
diet of four grams a day of testicular extract; in the earlier stages
with larger amounts (six grams) of extract he put on additional
weight to the extent of I1 per cent. (1,200 grams), showing that the
horse flesh is not deficient in caloric value, but the testicular
extract (P,O. 3°36 per cent.) supplies some essential substance. The
testicular extract was replaced on the 56th day by 5 grams of yeast,
with the result that the dog gained 1,150 grams (15 per cent.) in
weight in thirty-one days and was very well and active.

Though dogs die in 50 days on the average on denaturised flesh,
this one lived three months in full health, when some five grams a
day of these substances (rich in phosphorus) were added. He was
now kept on denaturised flesh alone, and in 50 days had lost 2,550
grams and was severely paralysed. Four days later, when death was
apparently imminent (the pulse was 270), ten grams of fat-free yeast
were administered—the dog improved and was able to move in
twenty-four hours (pulse now 100). For three further days five
crams of yeast were given and the dog had completely recovered and
was visibly fatter.

Similar rapid cure was obtained in other cases with testicular
extract—one animal gained a kilogram in six days after treatment
was begun.

Schaumann emphasises the fact that the symptoms and changes
in these animals are similar to those found in sailing ship Beri-beri,
and that in patients with that disease, as in the animals, the reaction
to a more suitable diet is exceedingly prompt.

Tropical Beri-beri differs more particularly in the length of time
required for recovery, which is often many months.

By feeding a goat on rice and maize till lameness and loss of
weight were marked, and repeatedly reviving it with Katjang beans
and yeast, either in single doses or continued for short periods,
Schaumann prolonged an experiment for six months. The goat

ao

was at the end very weak and paralysed, and was slow in reacting
to curative treatment. The animal was killed and extremely careful
examinations made of its organs. Degenerative changes were present
in the nerves and some wasting was also shown in the columns-—
no alteration of the cellular elements was demonstrable. Some
alterations were detected in the vagi, and the muscles were
oedematous and markedly rich in nuclei.

The appearances and symptoms were suggestive of those of
Tropical Beri-beri.

Schaumann gives full details of his various experiments on
animals, with an account of the results of the post mortem and
microscopic findings and tables of averages. He also gives the
results of exhausitve analyses of all the substances used for feeding
in these experiments, with special references to the amounts and
characteristics of the different classes of phosphorus compounds
present in them, and to various extracts obtained by solvents
(acids, alkalies, etc., peptic and pancreatic digestion).

He draws the following conclusions from his experiments on
animals.

CONCLUSIONS

I. Food stuffs which lead to the development of Polyneuritis in
animals are characterised by a low content of phosphorus or of
certain organic compounds of phosphorus. This may be either
fundamental or be caused by artificial processes.

II. Animals are not protected from the ill effects caused by such
diets by the addition thereto of proteids, inorganic salts, inorganic
phosphates, or the synthetic organic compounds of phosphorus
(calcium glycero-phosphate, albumen-metaphosphate).

III. The addition of certain substances, rich in organic phos-
phorus, to such diets exercises both a protective and a curative effect.
Yeast, rice meal, wheat bran, peas, Katjang beans, and testicular
extracts are the chief substances with this power. Carnivora and
herbivora, however, react rather differently to testicular extract, the
former are completely protected, the latter only in a less degree.

IV. Artificially separated organic phosphorus compounds of
various kinds, prepared from these natural protective substances,
exercise only a moderate and transient influence. Such compounds

bee

include yeast nucleic acid, phytin-lke compounds from Katjang,
phytin from rice meal, and possibly certain phosphatides.

V. Apparently the protective or curative effect of these sub-
stances is dependent not on any one of their organic compounds of
phosphorus, but on the collective effect of a number of these.
Animals do not apparently possess the power of forming the organic
phosphorus compounds necessary to their economy from inorganic
phosphates by their own metabolism, but are dependent for their
provision on the plant world, as they are for other classes of food-
stuff (e.g., protein and carbohydrate).

VI. The metabolism of phosphorus and nitrogen stand in close
relationship to one another.

VII. Spontaneous or experimental polyneuritis in animals
appears to be a disease of metabolism, attributable to the lack of

some specific organic phosphorus compounds whose identity is still
uncertain.

SAILING SHIP BERI-BERI

Turning now to the subject of Ship Beri-beri, Schaumann points
out that this disease generally occurs on sailing ships on the return
voyage, that it does not usually attack the majority of the crews, and
that change of diet, particularly fresh meat and vegetables, exercises
a rapid and complete curative effect. (Crews are mainly Europeans.)

In his tables are given the logs of the voyages of ten sailing
ships, the incidence of Beri-beri among their crews, the provender
and its appearance, and the men’s statements as to the cooking and
palatableness of the various articles of diet. In a second set are
analyses of the various foodstuffs from the ships and others of fresh
foodstuffs for comparison; these show the alterations in proteid,
etc., due to preservation and long storage, more especially with
reference to their phosphorus contents; further examinations were
made of the substances after cooking, again with special reference
to the behaviour of their phosphorus and eatability; the moulds
present were also examined. In other tables are given the results of
analyses of the excretae of Beri-beri patients and the results of
experiments on animals with the provender from the ships, etc.

He notes first that the phosphorus excretion of the patients is

331

exceedingly low (50 per cent. of normal), and that it rapidly rises on
phosphorus rich diets but some retention of phosphorus occurs.

He points out that the various customary articles of diet carried
on sailing ships fall into two groups: rice, white bread, and potatoes,
etc., in the one group, poor in phosphorus, are known to give rise to
neuritis in animals.

Of the other group of substances, rich in phosphorus, the salt meat
has been shown to lose nearly 50 per cent. of its phosphorus by the
combined action of the pickle lye and of the water used in boiling,
K6nig (1904). Schaumann’s results confirm this loss in the meat
from the Beri-beri ships, and further show the presence in the lye of
purin bases, which must arise from breakdown of nucleo-proteids.

The peas and other legumes are found to be usually mouldy, are
hard and resistant to cooking even with soda. They are frequently
quite uneatable, and many Beri-beri patients turn against them, and
Schaumann further shows that their phosphatides s¢em to have
undergone considerable changes.

Preserved vegetables carried on one ship were very rich in
organic phosphorus, and had been found to be extremely valuable in
the treatment of the sailors stricken with Beri-beri. Preserved meat
is also rich, particularly in lecithin phosphorus. *

Schaumann considers that the cause of Beri-beri on ships lies
in the lack of organic phosphorus compounds in the diet of the
sailors. This is especially noticeable on the return voyage (especially
in ships loading home from nitrate and guano ports where fresh meat
and vegetables are not obtainable), in part owing to the sailors being
partly driven to bread, rice, etc, through the mouldiness of the peas
or their hardness from keeping (rendering them impossible to soften
even when boiled in soda), or the decay and smell of the meat. To
this is added in other cases the loss of organic phosphorus in the
salt meat due to the action of the lye, and the similar loss in the
leguminous foods owing to standing or to boiling with soda.

He calls attention again to the affinity between scurvy and ship
Beri-beri (vide also his animal experiments), and suggests that scurvy
may be found to be due to use of stale vegetables, and Beri-beri to
use of rotten or stale flesh—the important factor in each being lack
of organic compounds of phosphorus.

| Schaumann does not appear to note that this article must often be denaturised by heating
to 120° in preserving. (Horst, loc. cit.)

532

As a curative agent, he says all sailors know the value of fresh
meat and vegetables. As a preventive agent, he suggests that it
might be possible to carry dried yeast, rice meal or testicular extract
for use when the ordinary diet becomes inadequate, and especially
lays stress on the probable value of requiring all ships engaged on
voyages where fresh provender may be difficult to obtain, to carry a
supply of Katjang beans in sealed (sterilised) cases. In the future,
he hopes a more active principle requiring but small bulk may be
isolated.

Sailing ship Beri-beri is a disease of metabolism dependent on
lack of organic phosphorus in the atet.

TROPICAL BERI-BERI

Schaumann’s observations on this disease are in part based on
the conclusions of workers in the East, and in part on clinical and
therapeutic observations on patients in the Hospital at Hamburg.
Tropical Beri-beri contrasts with sailing ship Beri-beri in being
chiefly observed among the Chinese stokers, trimmers, etc., of steam-
ships, who cook for themselves and keep to their accustomed dietary
even when on board. The dietary is usually frugal, considering the
hard work they perform.

Fletcher and others have shown that Tropical Beri-beri is con-
nected with the long continued use of a predominating amount of
polished rice in the diet (uncured rice is used synonymously with
polished rice). Grijns and others have shown the same connection of
polished rice with polyneuritis, and rice meal or its alcohol extract
(Fraser and Stanton) prevents the occurrence. Breaudat (1910) has
demonstrated that rice meal (the part removed in polishing) has a
similar curative and prophylactic influence in Tropical Beri-beri.

Analyses of the total mixed diets of Fraser, Fletcher and Ellis
show that they are quite ample in carbohydrate, proteid and fat, and
in caloric value for the requirements of men, judging by ordinary
physiological standards, and the development of Beri-beri cannot
depend on lack of these substances. On the contrary, the diets con-
taining polished rice and causing Beri-beri contain only about two-
thirds of the phosphorus requirements (4-5 grams) of a man, even

955

if a high average value is taken as a basis of calculation, and must
often be less than this figure. The process of cooking may further
reduce this. The pericarp which forms the difference between Ber1-
beri rice and wholesome rice is specially rich in organic phosphorus
and in fat, but contains no other peculiar substances as far as can be
determined. Grijns showed that the fat is not the important
principle, and so we are left only with the phosphorus compounds.

Schaumann gives analyses showing that the urinary excretion of
phosphorus in patients with Beri-ber1 is much below the normal
(average 50 per cent.), and this 1s accompanied by an almost equal
diminution of urinary sulphur and nitrogen. Previous observers
found even larger differences (70 per cent.). Addition to the diet of
substances rich in phosphorus leads to a marked rise in the excretion
of all these bodies.

Aron (1910), by careful experiments on the metabolism of a
Beri-ber1 patient extending over four weeks, showed that he was
unable to obtain sufficient phosphorus and nitrogen on the diet used
before the attack to meet his requirements, which were higher than is
normal. Remedy of the defects resulted in rapid cure. Aron further
showed that outbreaks of Beri-beri on ships often occurred about
six or eight weeks after the substitution for that previously used of
a rice with a much lower phosphorus content——possibly only one-third
of the previous, the rest of the diet remaining unaltered.

Hirota (1900) has shown that nursing children (in Japan) often
develop severe Beri-beri two or three weeks before their mothers, and
improve rapidly on change of milk. This is associated with a great
diminution of the phosphorus (especially in organic combination) of
the milk, and would seem to be associated with phosphorus hunger
in the mother, resulting in a retention and sparing of phosphorus.

Hulshoff Pol (1910) has demonstrated that Katjang beans form,
with regard to polyneuritis in animals, a certain and rapid cure for
Beri-beri, and extract of the beans is equally efficacious. He further
showed that addition of a similar amount (150 grams) to the dietary
of various pavilions in an asylum absolutely abolished Beri-beri ;
vegetables were less effective. The staple diet in the asylum consisted
of polished rice.

This has been confirmed by many others, and though the chronic
lesions of Beri-beri usually disappear very slowly under treatment,

334
occasionally the paralysis disappears with the startling rapidity
observed in the experiments on animals.

Schaumann and Werner tried the therapeutical effects of phos-
phorus-rich compounds on Tropical Beri-ber1 and obtained improve-
ment by the use of yeast, nucleic acid, testicular extract, etc., though
the improvement was not so marked as in polyneuritis in animals,
owing to the more advanced lesions in these cases requiring longer
for regeneration. (Fat-free yeast and carefully prepared nuclein
should alone be used.) Katjang beans, peas, and also the peptic
extract of Katjang are also very valuable.

On these grounds it appears clear that Tropical Beri-beri
resembles experimental Polyneuritis and Ship Beri-beri in being due
to lack of organic phosphorus in the diet, but 2¢ appears to be due to
a chronic deficiency of long duration, with severe deep-seated
lesions requiring a long time to cure. The experimental neuritis in
a goat already described is analagous to Tropical Beri-beri. The
other experimental cases and Ship Beri-beri, on the contrary, are
due to a sudden large deficiency of organic phosphorus, and the
lesions, though severe, are not deep-seated, and are rapidly recovered
from.

In the majority of cases, Berz-berz is due to a gross deficiency of
the organic phosphorus in the dtet. In other cases the differences
may be individual! in the patient, since some individuals require much
larger amounts than others, and others again may be unable to absorb
and assimilate the compounds, though present in the diet. Schau-
mann quotes the case of a German colonist, who, after being very
ill with malaria on the Amazon, developed Beri-beri on the voyage
home though on an ample diet ; in this case the intestinal absorption
appeared faulty, as there was a great deficiency in urinary, and a
very large increase in the faecal phosphorus.

Occasionally, epidemics of Beri-beri appear to be due to a
bacterial infection of the gastro-intestinal tract, either through the
catarrh interfering with absorption or to the bacteria (or their
products) absorbing or splitting the organic phosphates before they
could be absorbed.

Tropical Beri-beri is a disease of metabolism due to the amount
of organic phosphorus compounds assimilated being below that
essential for the human organism.

This is in the majority of cases due to deficiency of organic phos-

335

phates in the diet, but may in occasional cases be due :—(a) to
deficient absorption of organic phosphates by the alimentary tract,
or (6) to bacterial infection possibly splitting the phosphates before
absorption or interfering with the absorptive power of the intestine.

CONCLUDING CONSIDERATIONS

A whole cycle of other diseases in all probability have a similar
etiology to that of Beri-beri, more especially Ship Beri-beri.
Scurvy has already been conclusively shown to be of this nature by
a whole series of observations, and to all appearance Infantile
Scurvy, Rickets, and Osteomalachia are also included. Pellagra and
the form of malnutrition described by Czerny and Kellner in
artificially fed children may be further examples.

The suspicion arises that all these diseases may be due to
deficient assimilation of organic phosphorus, but it is difficult to
understand how a similar cause can produce the different effects
shown by the symptoms in these diseases.

It has already been shown that the animal organism is not
adapted to build up organic phosphorus compounds for itself, but
depends for these on the plant kingdom, and it is probably equally
unable to form compounds of one group from those of another (e.g.,
nucleo-proteids from phosphatides).

Since different groups play different roles in the economy of man,
it is certain that deficiency of each group will have its own special
effect, and in addition this will be complicated by an associated
influence on the general metabolism; for example, inorganic com-
pounds would react on the alkaline earths of the bones, nucleo-
proteids would react on the proteins, etc.

Individual differences would further intervene to complicate the
picture, more especially age differences, and infancy, childhood,
puberty, pregnancy and old age would, in particular, have a far-
reaching influence.

So by deficiency of single groups, or of combinations of groups
of phosphorus compounds, various symptom complexes—different
diseases—may be originated. Thus lack of nucleo-proteid in adults
is probably connected with Beri-beri; in children a similar deficiency
might give a different disease: deficiency of phosphatides may give
yet a third, a combination of the two will give yet another picture.

336

Application of the principles already applied to Beri-beri in this
paper may bear fruit also in these diseases, though only after long
and complicated researches may the answers be obtained.

Schaumann hopes that his speculations may lead to encouraging
further researches along systematic lines. While quite recognising
that they are but hypotheses resting on slight grounds, yet the
advance of science and medicine would be but slow were it not
largely aided by experimenters, who have striven with all the
means in their power to prove or disprove theories based on even
slighter grounds; and Schaumann has felt it right not to conceal
his speculations, as they may yet bear fruit.

We feel that this monograph is so thorough and complete, and so
well thought out, as to deserve communication at considerable length,
especially as recent occurrences have shown us that it, like
the previous researches of other workers on similar lines, has not
become as widely known in physiological, scientific and medical
circles in this country as it would had it not been published in a
journal mainly concerned with Tropical medicine. We think that all
readers will agree with us that Dr. Schaumann is to be congratulated,
not only on his important contribution to our knowledge, but on its
arrangement and its literary merit.

To our nation, with its wide shipping interests and tropical
possessions, it is the more important, and we must congratulate our-
selves that it is largely based on the work of British medical men
and scientists in our Eastern possessions. Their work has already
had widespread influence in checking the heavy incidence and
mortality of Beri-ber1.

In 1910, Fraser and Stanton published a further paper carrying
on their work on these lines, and have further confirmed the associa-
tion of organic compounds of phosphorus with the deficiency of
diet causing Beri-beri. |

Our own researches as far as they have extended are in full
agreement with those of Schaumann, though results have as yet only
been obtained with our experiments on pigeons.

We can confirm the unfavourable influence of polished rice,
steamed rice, and steamed barley fully; and the protective influence
of whole rice, whole barley, rice meal, yeast and Katjang-idjo.
The curative effects of yeast in pigeons severely affected with

337

neuritis were even more marked than we had expected, and we were
astonished at the rapidity and completeness with which the birds
recovered.

It was not our intention to communicate any of our results till
considerably more experiments and analyses had been concluded, nor
indeed to abstract previous work at such length as we have done.

The great public attention, however, which has been directed for
some months to the question of our bread, led us to call attention to
this literature in a letter to the British Medical Journal of May 6th,
and now to communicate in detail those of our results which bear on
this problem. We had so many enquiries for references that we
thought it right to quote the previous work at some length, since
the problem of the influence of rice in Beri-beri is so closely allied
to that of a standard bread.

It is interesting to remember that the Germans refer to Rickets
as the English disease, and to reflect that it is far more common in
this country than it is in Germany, and further that the Highlanders
and our Irish peasants are in large measure free from it.

Yet’ the children of all these races are brought up largely on
similar diets (excepting such as are bottle-fed from the start). In the
poorer classes of all, milk forms some part of the diet ; in the peasant
class it is usually good, in the town children it is often, however,
neither abundant nor containing cream. Besides this, the children
get their main nutriment from the national bread and from rice.
Polished rice is used in all the nations, but the Highland child gets
porridge from oats, the Irish child potatoes, the German rye bread,
and the English child white wheat bread.

The organic phosphates are undoubtedly present in the oatmeal
(0-9 per cent. P,O.) of the Highlander, and in the potatoes (unless
deeply peeled); in the rye bread the organic phosphorus appears
to be diffused through the whole grain (P,O. 1 per cent.), and even
fine rye bread does not originate polyneuritis in fowls (Holst) or
in rats (Schaumann).

In the fine English white wheat bread, however, the phosphorus
has been removed with the bran, and is used largely (as is rice meal)
as one of the best possible foods for fattening cattle. White wheat
bread (P,O, 0-2), as Holst has shown, causes polyneuritis in fowls
and Schaumann shows the existence of the important protective
phosphates in the wheat bran.

a)
335

Our own researches fully confirm Holst’s results; indeed, the
effects were more markedly deleterious than he found. The bread
was a white flour bread, guaranteed to be made from the finest white
flour, unbleached and unadulterated. Well nourished pigeons when
limited to this bread devoured it greedily, but failed to flourish.
Diarrhoea, and loss of weight, early commenced; listlessness and
lameness followed shortly after. The first bird died on the 15th
day, and others on the 106th and 20th days, showing marked degenera-
tive changes in their peripheral nerves. Several were revived when
extremely weak and nearly ready to die, showing severe lameness
and, im some cases, the convulsions and retraction of the head
described by Schaumann ; but the average duration of life (allowing
one or two days for the revived birds) was 29 days, and the average
loss of weight 26 per cent.

Far different is the picture on Standard or whole-meal bread.
The birds continue active and well, maintain their weight, clean and
plume themselves; they remain able to fly and walk, and at the end
of seven weeks were all perfectly well and had, on the average,
gained 8 per cent. of their original weight. On whole-meal bread
in two cases, pairing occurred; one pair successfully hatching the
two eggs (though the diet was changed to a mixed diet during the
sitting); the other pair were put on white bread, broke the eggs
and began to go downhill.

These results are given more completely in the tables at the end
of this article, and appear to us, in conjunction with Holst’s, Leonard
Hill’s (B.M.J., April 30), and Schaumann’s, to fully confirm the
claims of the advocates of Standard or whole-meal bread.

We have seen that some degree of rickets is almost universal
among the children of our poorer classes, who, in addition to lack of
sunlight and fresh air, live largely on white wheat bread, and that
it 1s not so prevalent in those nations whose children eat porridge
(oatmeal 1 per cent. P,O.), or rye bread, with a higher content of
organic phosphorus compounds. We know that they often get little
else but poor milk and margarine (P,O. 0°03 per cent.), which may
also be deficient in similar compounds, and we see that marasmus,
diarrhoea, oedema of the limbs, spasm and convulsions (especially
tetany) are common to rickets and to the experimental neuritis of
pigeons and animals.

It seems to us that Schaumann’s hypotheses are not likely to long

339

lack justification with regard to this disease and also to its close
ally, infantile scurvy, though the symptom complex is probably com-
plicated by secondary effects on digestion and on the metabolism of
lime and proteid.

It may be objected that our white bread is baked with yeast and
so the missing organic phosphates are compensated for, but the
amount of yeast used is very small and probably insufficient (Holst’s
pigeons died on yeast bread towards the end of three months; more
slowly, it is true, than on bread baked with baking powder (average
40 days), but none the less surely). And it must further be remem-
bered, that much of the bread now sold is made with baking powder
and not with yeast, and so a further factor making for deficiency is
introduced. Failures of absorption, bacterial infections, and other
internal disorders no doubt play their part as in Tropical Beri-beri,
but it may well be that success of the present agitation for a whole-
meal bread will have a wide reaching effect on the betterment of
the physique of our nation, in lowering our death rate and in lighten-
ing the overcrowding of our hospitals.

The following tables include the results of experiments referred
to in the text. Some of the series are merely confirmatory of the
results of previous observers; others refer to the experiments with
white and Standard bread. The results of curative treatment with
yeast and other substances are also briefly alluded to, and in a final
table the results of analyses (E. S. E.) of various substances used are
given.

We hope in a future contribution to give the results of our attempt
to isolate the active principle, whether it be one of the organic
phosphorus compounds or a substance which associates itself with
these in its reactions, as do ferments with nucleo-proteids. We wish
at the present time to express our indebtedness to Professor
Sherrington, Professor Moore, Miss Tozer, and others for their
kindness in advising and assisting us in various chemical and neuro-
logical problems that have confronted us.

In conclusion, we wish to emphasise the great importance of
these investigations to a country such as this with wide Shipping and
Colonial interests, and hope that an appreciation of these facts will
lead to the adoption of the necessary additions, where such diets are

largely used, as has already been done in the Straits Settlements
with striking results.

342

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degeneration could be discovered in small cutaneous nerves, in the
anterior and posterior spinal roots, in the vagi, or in the vestibular
nerves in spite of careful search in several cases. Nor did any
degeneration appear to be present in the spinal cords.

SERIES | and F

The rice and barley grain used in experiments A and E was
also used in these series, but was previously exposed to a temperature
of 120° C. for two hours in an autoclave.

The birds on steamed rice failed very rapidly, marked convulsive
neuritis occurring on the 16th and roth days respectively, and the
birds would certainly have died in the course of forty-eight hours.
Forced feeding with ordinary brewers’ yeast (and rice) was instituted,
and in both instances the birds were free from convulsions and able
to walk in twenty-four hours, and in a further day appeared quite
normal.

Professor Sherrington kindly examined one of these birds for us
before and after the first twenty-four hours of yeast feeding. Before
treatment started it could barely stand and held the head completely
retracted ; on attempting to walk it became convulsed and turned a
series of back somersaults till brought up by some obstruction; the
wing reflex (flap reflex) was absent, and the eye and head reflexes
were abnormal. The next day it could walk and fly readily, the
wing reflex and head reflexes were normal, and all signs of
labyrinthine trouble had disappeared. The third bird became very
weak on the 24th day, and was barely able to move or feed itself.
It rapidly recovered on yeast feeding and change of diet. In
Table I, in reckoning the probable day of death, an ample margin
has been allowed.

The birds on denaturised barley failed slightly less rapidly, the
first died with convulsive seizures on the 22nd day, the last on the
45th day. One was restored on the 28th day when very near death
by yeast feeding, which was continued for three days. It failed again
on the barley diet and died on the 44th day.

SERIES m

Shows the absolute adequacy of a diet of denaturised rice, with
the addition of one gram dried yeast per bird per day.

345
SERIES n

Shows that the protective influence of the yeast is entirely
destroyed by heating to 120° C. The birds showed typical neuritis
and other symptoms usually associated. Two rapidly recovered on
treatment with ordinary yeast.

PERCENTAGE OF P,0,;IN VARIOUS DIETS USED :—
Percentage of P,O;

Polished rice 6 samples oh ae Ase see 0:26
Rice meal 4 o is Bee ran Boe 2-75
Uncleaned rice 3 ” O61
Dried yeast 4 a se oe see va 4:03
Katjang beans 2 e nap #60 oat aoe 0°95
Barley grain 3 = $e sie oer st 0-92

REFERENCES

Apu anv NevserG (1906). Phys. und Path. des Mineralstoffwechsels, Berlin.

Aron (1910), Berl. klin. Woch., No. 21, p. 995-

Brappvon (1909). Brit. Med. Journ., p. 1007.

BreaupaT (1910). Bull. Soc. Path. Exot., Jan. 12, p. 13.

E1jkMaN (1897). Virchow’s Arch., CXLVIII, p. 523.

Frercuer (1907). Lancet, June 29, No. 4374, p- 1776.

Fraser AND STANTON (1909). Arch. f. Schiffs- und Trop.-hyg., XIII, Beiheft 6, p. 82.
Fraser (1909). Lancet, XXVI, 4459, p- 451-

FRASER AND STANTON (1910). Lancet, No. 4515.

Grins, G. (1908). Geneesk. Tijdsch. v. Ned. Ind., XLVIII, 5. pp. 680-704.

Hirota (1gow). Zentralbl. f. inn. Med., p. 273.

Hoist, Axet aNpD Fréxicu (1907). Journ. of Hyg., Oct., VI, 5.

Hutsuorr Pot, J. (1910). Arch. f. Schiffs- und Trop.-hyg., XIV, Beiheft 3.

Konic (1904). Chemie der Menschlich. Nahrungs- und Genussmittel, Berlin.
Nocut, B. (1908). Arch. f. Schiffs- and Trop.hyg., XII, Beiheft 5, pp. 115-130.
Scnaumann, H. (1908). Arch. f. Schiffs- und Trop.-hyg., XII, Beiheft 5, pp. 137-157.
Scuaumann, H. (1g09). Arch. f. Schiffs- und Trop.-hyg., XIII, Beiheft 6, 82.
ScuauMANN (1910). Arch. f. Schiffs- und Trop.-hyg., XIV, Beiheft. 8.

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Volume V December, 1911 No. 3

ANNALS

OF

TROPICAL MEDICINE AND
PakAsSlTrPOLOGY

ISSUED BY

gaa LIVERPOOL SCHOOL®@F TROPICAL MEDICINE

Editor

Prorrssor §1rk RONALD ROSS, Mayor I.M.S. (Rert.), D.P.H., F.R.C.S.,
Disexe bie ob RS. hiCoR:

In Collaboration with

J. W. *W. STEPHENS, M.D., Canras., D.P.H.
R. NEWSTEAD, M.Sc., A.L.S., F.E.S., Hon. F.R.H.S.
j. L. TODD, B.A., M.D., C.M. McGill, D.Sc., Liv.
H. WOLFERSTAN THOMAS, M.D., C.M. McGiil.

ANTON BREINL, M.U.Dr.

2IAWUWA

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34:7

TABLES OF STATISTICAL ERROR”

BY
PROFESSOR SIR RONALD ROSS, K.C.B, F.RS.,

AND

WAETER STOTT,

HONORARY STATISTICIAN, LIVERPOOL SCHOO! OF TROPICAL MEDICINE

(Recetved for publication 24 October, 1911)

CONTENTS
I. ExPLaNaTION
1. Proportionately Small Samples ...... page 347
2 ebhe (Corcecta Procedures crs.-ch-n-ser- els 3ks
3. Proportionately Large Samples ...... ,, 356
fomebninesr or One) Class scaecnsae sackets A
II. Tastes

I. EXPLANATION

1. PROPORTIONATELY SMALL SAMPLES

These Tables have been specially constructed for practical use in
sanitary, pathological and clinical work.

Suppose that we are studying things of any nature, such for
example as men, cases of sickness, insects, leucocytes, trypanosomes,
seeds, stones, etc, and wish to ascertain what proportion of all of
them belong to a particular class, that is, have a given characteristic.
Then we can answer this question with absolute certainty only in one
way, namely, by examining the whole number of such things that
exist in the region under consideration. But this is generally
impossible; and we must, therefore, content ourself with examining
only as many of the things as we can—ascertaining what proportion
of these possess the given characteristic, and thence zxferring what
probable proportion of the same things in the region under

consideration belong to the same class.

* To be obtained as a separate publication for two shillings and sixpencs, postage
included, from the Clerk of the Laboratory, School of Tropical Medicine, University,
Liverpool, All rights reserved,

348

For€xample, if we wish to know with certainty how many people
of any natonality have blue eyes, then we must examine all the
people of thatnationality. But this will be impossible. Hence we
must examine 10,0F 100, or 1,000 or more of the people; ascertain
how many of ‘ese haveblue eyes; and then izfer from this sample
what proportion of the whole iation are /zke/y to have them.

Everyone knows, of course, fiat (provided the methods of
examination are always equally careful anl.trustworthy) our estimate
is more likely to be near the truth if we examima large sample than
if we examine only a small one-—that is. if we examnz_ many of the
things than if we examine only a few. For instance, it vould be
absurd to attempt to estimate the proportion of persons with blueyes
by examining only five or ten persons. We should come nearer>y
examining one hundred or one thousand, and so on; but we shoulc
reach absolute certainty only by examining all the people in the
country. The important question now arises: How many of the
things must we examine in order to become reasonably sure that
our result is within a gzven percentage of the exact truth? The
labours of mathematicians enable us to answer this question, and the
following Tables enable us to answer it in most cases without
calculation. We must begin by dealing with the case in which the
total number of things is so large that we cannot take for a sample
more than a very small proportion of that total number. Hence the
heading of this section is Proportionately Small Samples. .

First, however, we must understand what exactly we mean by
the phrases ‘reasonably sure’ and ‘a given percentage of the
truth.” Both these phrases contain ideas of degree. Thus, if we are
carrying out a strict scientific enquiry, we may wish to be able to
say that the betting (or probability, as it is called) is 99,999 to 1 that
our result is within one per cent. of the truth. We must then
consult Table A 1, under the second column. Or it may suffice to
say that the betting is 99,999 to 1 that our result is within five per
cent. of the truth; and we then consult the same table under the
sixth column. But it may happen that we can afford to be content
with a lower degree of probability than this—it may suffice to say
that the betting is only gg to 1 that our result is within 3 per cent. of
the truth; and we then look at Table A 4, under the fourth column.
Lastly, we may be allowed to content ourself with an ‘even chance’ or

——

———

349

‘toss up '—that is, a probability of 1 to 1; and for this we consult
Table A 6.

The reader will now be easily able to understand the tables. The
figures in the heading of each give the degree of sureness (so to
speak) with which we may rely upon the inferences drawn from the
table. Thus the odds are 99,999 to 1 that the inferences to be
drawn from the first table are sound; or, in other words, out of
100,000 trials of the table only one is likely to be wrong. Table
A 5, however, is likely to be wrong once in ten trials; and Table
A 6, once in two.

The percentages at the head of each column give the percentage
of ‘ statistical error ’—that is, the amount by which the truth is likely
to diverge from our observed result. Thus, if our result is 70 per
cent., and the statistical error is 5 per cent., then the odds (as
denoted by the figures 99,999 to 1, etc.) are that the truth will lie
anywhere between 70 + 5 per cent. and 70 — 5 per cent.—that is,
between 75 per cent. and 65 per cent. If, however, the statistical
error is only one per cent., then the truth is likely to lie between the
narrower limits, 71 per cent. and 69 per cent. If the observed
result, #/us the error, exceeds 100, or if the observed result, minus the
error, is less than 0, we conclude that the number of things hitherto
examined is not yet large enough to yield a useful result.

The figures down the first (left hand) column of each table refer
to the percentage of the observed result up to 50 per cent.; and the
figures on the same line in the body of the table give the total number
of things which must be examined in order to ensure that that result
will lie within the percentage of error given at the head of the
corresponding column, with the degree of probability given at the
head of each table. Thus, if the observed result is 43 per cent., we
must examine 47,780 things to ensure that the odds are 99,999 to 1
that the truth lies between 44 per cent. and 42 per cent. But
we need examine only 1,328 things to ensure that the betting is
99,999 to 1 that the truth lies between 49 per cent. and 37 per
cent. We must examine 26,541 things to ensure, with a probability
of 999 to 1, that the truth lies between 44 per cent. and 42 per cent.
(Table A 3); but we need examine only 415 things to ensure, with a
probability of 9 to 1, that the truth lies between 47 per cent. and 39
per cent. (Table A 5); and only 185 things that, with a probability
of g to 1, it lies between 49 per cent. and 37 per cent.

359

If the observed percentage is over 50, we subtract it from 100
and obtain the required figures for the remainder. For example, the
figures for observed percentages of 70 per cent., 79 per cent., and 85
per cent. are precisely the same as those for 30 per cent., 21 per cent.
and 15 per cent.

On inspecting the tables we see at once that the figures diminish
rapidly in successive tables, and also in successive columns (from left
to right). From these observations we gather, as we might have
expected, that the number of things required to be examined
diminishes (a) with reduced probability of correctness, and (4) with
increased statistical error.

The figures increase as we descend the columns. That is, the
number of things required to be examined increases as the observed
percentage rises from 1 per cent. to 50 per cent.; but after that (by
the rule just given) it diminishes as the observed percentage continues
to rise from 50 per cent. to 100 per cent.

The tables are calculated for only six degrees of statistical error,
namely, from I per cent. to 6 per cent. But it 1s easy to obtain the
figures for any required degree, simply by dividing those in the
column for 1 per cent. error by the square of the required degree.
Thus for a probability of 99,999 to 1, and an observed percentage of
43, we must examine 478 things for a statistical error of 10 per cent.,
and 4,778,000 things for a statistical error of 1-10th per cent. It
will be seen that the columns for 2 per cent., 3 per cent., etc., follow
this rule.

It is also easy to obtain approximately the figures for fractional
observed percentages, such as 2’5 per cent., or 27°3 per cent., because
the increase in the number of things required to be examined is,
roughly, proportional to the increase of the observed percentage.
Subtract the next lower from the next higher figure in the table;
multiply the remainder by the decimal fraction, and add the result
to the next lower figure. Thus in Table A 1, in the column for
I per cent., the figure opposite 2°5 per cent. observed percentage will
be about 4,747; and opposite 27°3 per cent. will be about 38,686.
But such refinements will rarely be needed.

We are often obliged to examine such a small number of things
that the error is evidently greater than the 6 per cent. calculated for
in the Tables, and we may wish to know exactly how much it 1s. In

351

this case proceed as follows:—-Select the degree of probability
required, and in the appropriate table look out the observed
percentage actually obtained. Take the number opposite to this in
the column for 1 per cent. error, and divide it by the number of things
actually examined. The error will be the square root of the quotient.
Thus, suppose that we have examined only 300 leucocytes, and have
found 45 per cent. of these to be mononuclears. The number we
ought to examine for a I per cent. error at a betting of 9,999 to 1 is
37,459. The ratio of this number to 300 is 125; and the square root
of this is 11°2. Hence the statistical error of our work at this
betting is 11°2 per cent. But 6,697 leucocytes would have sufficed
at a betting of only 9g to 1. The ratio of this to 2:00) 1S ‘22° 32"
so that the statistical error at 9 to I is only 4'7 per cent——as could
have been roughly inferred from Table A 4. .

The reader should note the large number of things which must
be examined before a result can be obtained to any high degree
of probability and within narrow limits of error ; and he will doubtless
remember many confident assertions based upon much smaller
samples.

EXAMPLES

1. How many persons of one nationality must be examined
before we can assure ourselves, to a probability of 99,999 to 1, that
from 65 per cent. to 67 per cent. of all the nation do not possess
blue eyes? Awswer: 43,744.

2. How many of a patient’s leucocytes must be examined before
we can bet 999 to 1 that between 42 per cent. and 4o per cent. of
all his leucocytes are mononuclear ?* Awzszwer: 26,194.

3. How many of his leucocytes must be examined before we can
bet aboud 100 to 1 that 69 per cent. to 65 per cent. of all his leucocytes
are ‘polynuclear’? Answer: 3,068.

4. How many of his leucocytes must be examined before we can
bet 9,999 to 1 that his eosinophile leucocytes number between 1'5 per
cent. and 2'5 per cent. of his total leucocytes? Answer: 11,868
(Multiply figure in column for 1 per cent. in Table A 2 by 4).

5. On examining 100 of his leucocytes we find that 7 per cent.

* Always supposing that the leucocytes are evenly distributed throughout the
circulation.

352

of them are large mononuclears. How many more leucocytes
must we examine before betting aéowt 10,000 to 1 that the same
percentage holds for all the leucocytes in his body within an
error of 3 per cent.? Answer: gg5.

6. On examining 65 case-records we find that 13 of the patients
died. How many more case-records must we examine before betting
QQ to 1 that the case-mortality of the disease is between 19 per cent.
and 21 per cent? Awmswer-: 10,551.

7. Our new line of treatment has cured one out of five cases of a
hitherto incurable disease. How many more cases must we treat
before betting 999 to 1 that we can cure between 15 per cent. and
25 per cent. of all cases? Answer; 688.

8. On examining nearly 2,000 of a patient’s red corpuscles, we
find that 8 per cent. of them are nucleated, and bet 9 to 1 that the
' percentage of nucleated red-corpuscles in his whole body is—what ?
Auswer. Between 7 per cent. and 9g per cent.

Q On examining 220 things, we find that 31 of them belong to
a particular class. What is the statistical error at a probability of
gg to 12 Answer: 6 per cent.

10. On examining 138 leucocytes we find that 15 per cent. of
them are large mononuclears. What is the proportion of large
mononuclears in the whole body, at a betting of about 1,000 to 1?
Answer: Anything between 5 per cent. and 25 per cent. (Find the
figure for 10 per cent. error.)

11. On examining 500 malaria parasites, we find that 14 per
cent. of them are sexual forms. What is the betting that the
statistical error is about I per cent.? Amswer,; An even chance.

12. In the same case, what is the betting that the error is not
greater than 4 per cent.? Answer; gg to I.

13. Out of 200 leucocytes we find 23 per cent. to be mononuclears.
What is the statistical error at a betting of 999 to 1? Answer: 9°79
IgE

200

14. Next day, in the same patient, out of the same number of
leucocytes, we find 4o per cent. to be mononuclears. What is the
statistical error at the same betting? Answer. 11'4 per cent.

15. Can we bet g99 to 1 that there has been an increase of
mononuclears in this case? Amswer, No, because the errors overlap ;

per cent. (Square root of

= 7

453
that is, the difference between 23 and 40 is less than the sum of 9'79
and I1°4.

10. Can we bet 99 to 1 that there has been an increase?
Answer, Yes, because with this lower degree of probability the sum
of the errors, namely 7°7 and 8'9, is less than the difference between
the observed percentages, 23 and 4o.

17. Working at a probability of g99 to 1, we find that out of
100 leucocytes two are eosinophiles. What is the error? Amswer:
Between 4 per cent. and 5 per cent. What are we to conclude?
Answer: That we must examine more leucocytes until the error is
at least less than the observed percentage.

18. On examining 136 things we find about 10 per cent. to
belong to a particular class. What, roughly, is the error at a betting
of 9,999 to 1? Answer: 10 per cent. (Find the square root of the
quotient of 13,621 divided by 136.)

ig. On examining 200 things we find 80 of them belong to a
particular class. What is the error at a betting of g9,999 to 1?
Answer; 15°3 per cent. (Divide 40,785 by 200, and find the square
root of the quotient.)

20. Working at a probability of 9,999 to 1, and an error of I
per cent., how many things must we examine in order to assure an
observed percentage of 41°72? Answer: About 36,789.

1. THE CORRECT PROCEDURE IN PRACTICAL WORK

The Tables will, then, be of practical use in many kinds of sanitary
and medical work; as, for instance, in estimating the frequency of
death, or of some symptom in a given disease; or of some symptom,
such as enlargement of the spleen or rickets, in a population; or in
making differential counts of leucocytes in a patient, or of colonies
of bacteria growing on a plate culture. But an examination of
the Tables will convince us that the procedure now generally adopted
in attempting such estimates is very faulty, because observers seldom
trouble much regarding that all-important point, the szze of the
sample—that is, the total number of things which they must examine
in order to obtain a sufficiently correct result. Certainly, often
(though not always), they recognise that the sample must be large ;
but they usually fix its size quite arbitrarily—as, for instance, when
they say beforehand that 200, or 500, or 1,000 leucocytes must be

354

examined for differential counts. This, however, may lead to the
most untrustworthy results, because, as we have seen, the size of the
required sample is not fixed, but depends on several factors,
including the observed percentage of things of the particular class—
that is, the very percentage which we are seeking to ascertain. We
cannot, therefore, fix the size of the sample beforehand, but must do
so as we proceed in the work.

The size of the sample depends upon three factors, namely :- -

(1) The degree of sureness which we have to attain ;

(2) The percentage of statistical error, or degree of accuracy,
which may be allowed ; and

(3) The observed percentage of things of the particular class
which we are endeavouring to ascertain.

The correct procedure is, therefore, as follows :—

(1) First decide definitely as to the degree of sureness which must
be attained, and the percentage of error which may be allowed.
These will depend upon the importance of the work and the time
which we can devote to it. For strict scientific or large sanitary
investigations we may require a very high degree of sureness, say,
99,999 to 1, and very small limits of error, say 1 per cent. And this
will be specially the case when we have to compare resulis obtained
at different times; as, for instance, when we wish to know whether
the mononuclear leucocytes increase with the progress of a disease
(see examples 13-16), or whether an epidemic is diminishing. Here it
is absolutely essential that the statistical errors obtained at the two
different times are not large enough to overlap. On the other hand,
we may often be permitted to adopt lower degrees of sureness and
high percentages of error, especially when we are merely seeking
some corroborative evidence or when differences between successive
estimates are so large and striking that even a large percentage of
error cannot mislead the judgment. Here, as in regard to the
following paragraph, we must often be guided by the progress of the
work. But, as soon as we decide upon these points, we can
determine which table, and which column in that table, are to be
used:

(2) Secondly, before fixing upon the size of the sample, we
should endeavour to obtain by trial a rough estimate of the observed
percentage of things of the particular class which we are studying.

—

355

Suppose, for example, that we have to make a differentia! count of
leucocytes. Then we do not wish, on the one hand, to allow too
much statistical error, or, on the other hand, to waste time over
examining too large asample. Suppose, first, that the 9 to 1 Table
is sufficient. | Begin by examining 100 leucocytes. Suppose that
40 per cent. of these are mononuclears. Then we can see at once
from the Table how many leucocytes must be examined to give a
reliable result at that observed percentage. If a 6 per cent. error
will suffice, we anticipate that we shall require to examine only 81
more leucocytes. If a 2 per cent. error must be obtained we shall
have to examine 1,524 more leucocytes. As we now proceed in the
task we shall find that the observed percentage changes considerably
when we have examined 200, 300, 400 leucocytes, and so on (we
should calculate the percentage, not for each successive batch of 100
leucocytes, but for the total number examined from the beginning).
Finally, when about 1,400 leucocytes have been examined, we
anticipate that we are approaching the required limit (for 2 per cent.
error). Suppose that at 1,559 leucocytes the observed percentage
stands at just about 36 per cent——thus agreeing with the Table.
We then stop; having obtained a 30 per cent. ratio, with a betting
of 9 to 1, and a statistical error of 2 per cent. That is, the probability
is 9 to I that the truth lies between 38 per cent and 34 per cent.;
and we have not wasted time in reaching this result.

If, however, we require high degrees of probability, or iow degrees
of error, or both, there will be little use in attempting the preliminary
rough estimate by a small sample of only 100 leucocytes, and we
had better take for it at once 500 or 1,000 or more as the case might
be.

(3) Of course, in differential leucocyte counts we often possess
beforehand some inkling of what observed percentage we are to
expect. Thus, the eosinophiles are generally few in number, and
the ‘polynuclears’ numerous; and we judge roughly regarding the
size of the sample accordingly. The same thing usually happens
in other kinds of enquiry.

(4) It is a great mistake to suppose that a large sample will
compensate for inaccurate working. These Tables are based on
the supposition that each thing examined has been accurately
assigned to its proper class. Things which cannot be certainly

356

assigned to their proper class must be rejected; but the number of
them must be noted, and the proportion which they bear to the
whole number of things must be afterwards determined, with
estimates of probability and error, by precisely the same methods
as those described. They constitute, in fact, a third class by
themselves.

(5) If the things under study can be divided into three or more
classes, determine separately the proportion of each class to the
whole. This does not necessarily require different series of
investigations. | We simply extract the figures from the records ;
but care must be taken that the samples are sufficient for each class
by itself.

(6) If while examining successive samples of things (such as
leucocytes) we find that the observed percentage in each sample in
succession tends always in one direction, that is, either to increase or
to decrease, then we may suspect that some influence other than
mere chance is at work. The number of successive samples required
to verify such a suspicion will depend upon the nature of the material,
and might be large; but in many cases if the observed percentage
always increases, or always diminishes, in at least five successive
samples, then we may have grounds for further enquiry upon the
point, or for reference to a trained statistician.

(7) The probability or degree of betting which is generally
accepted by statisticians as amounting almost to certainty is 49,999
to 1. The figures for this can be obtained by multiplying the
corresponding figures for a betting of 9,999 to 1 (Table A 2) by the
factor 1°189;. or, what is nearly the same thing, by increasing the
figures in Table A 2, by 20 per cent.

(8) Great care must always be taken that samples are chosen
really at random, and are not selected with any conscious or
subconscious bias.

3. PROPORTIONATELY LARGE SAMPLES

We have hitherto dealt with Comparatively Small Samples—that
is, with samples which are small compared with the total number of
the things in existence. For instance, if we are studying all the
people in a country or all the leucocytes in a patient’s body, we shall
seldom be able to examine more than a very small proportion of

a

357

these people or leucocytes. But there are cases when the total
number of things is not so very large that we cannot examine a very
considerable proportion of them. Suppose, for instance, that we
wish to ascertain the proportion of children with enlarged spleen,
not in the whole world, but in a large village ; and suppose that there
are 200 children in the village, but that we have time to examine
only 50o0f them. Here the sample is comparatively large, being one
quarter of all the children in the village. Or suppose that we can
examine 180 of the children; here the sample is so large that it
approaches the whole number of things under study (i.e., the children
in the village). Obviously, on examining these large samples we
shall approach much nearer to the exact truth than would be
anticipated from the Tables. In the second case, for instance, we
should have to examine only 20 more of the children in order to
obtain absolute certainty (provided that the examinations are careful
enough). Hence, clearly, the Tables must be corrected for the case
of Large Samples. A very suitable and easily applicable method
for this purpose is to multiply the statistical error given in the
Tables by the Factor.

where xz is the number of things in the sample, and .V is the total
number of things under study.

Examining this Correction Factor, we see that when x is very
1

small compared with J, the term becomes so small that it

she

may be neglected; so that the Factor now becomes the square root
of unity; that is, unity. This multiplied into the statistical errors
given in the Tables does not modify them at all—so that the Tables
are then quite correct. Here we have, of course, the case of
Comparatively Small: Samples, where V is a very large number,
such as all the people in a country or all the leucocytes in a person.

Again, if z=WN, that is, when the sample includes all the things
n—I
N-1
zero. This multiplied into the statistical errors makes them vanish.
In other words, there is no statistical error because we have reached

in existence, the term equals unity, and the Factor becomes

certainty.
Between these values the Factor is a vulgar fraction, which can

355

easily be calculated. Thus, if # =50 and \’=200, the Factor is the
square root of a5 ; that is, 0°868. This reduces the statistical

error, but not much. If xz = 180, the Factor is the square root of
aa ; that 1s, 0o-317—which reduces them considerably.

If the total number of things is at all considerable—say over 20-—

then the Factor becomes nearly the same as the square root of
iM

—- In this case we can give a table of the various values of

. ; n
the Factor which correspond to the various values of ,: In the
ye

following Table the proportion of things examined to total things,

1 ~ ° ’
vy: JS given in percentages, and the corresponding values of the

Factor are put below :

Taste B
nt
00 = 5 10 15 20 25 30 35 40 45 50
Factor = 097 095 0:92 O89 087 084 O81 O77 O74 O71
100 ~ = 55 60 65 7O 75 80 85 go 95 I00
Factor = 067 0:63 0°59 0°55 0°50 0745 0°39 0°32 0:22 O00

Thus, if we examine half the total things, the statistical errors are
reduced to about seven-tenths of the values given in Tables A. It
will be seen that this Factor makes little difference in the statistical
error unless the number of things examined is more than one-tenth
of the total.

EXAMPLES

21. Nine out of 145 children in a village are absent. On
examining the remainder we find 14 (say 10 per cent.) of them with
enlarged spleen. What is the error at a betting of 9,999 to 1?
Answer: 2°5 per cent. (The Factor is 1/4. See also Example 18.)

22. There are 1,000 people on an island. Out of 100 of these
60 are found to be affected with filariasis. What is the statistical
error at a betting of 9 to 1? Answer: About 7°7 per cent.

23. Out of 884 people in a town, three-quarters are examined,
and 73 of these are found to be in bad health. How many of all
the people in the town are likely to be unwell? Answer: Between
Q per cent. and 13 per cent., at a betting of 999 to I.

—a

359

4. THE NUMBER OF THINGS OF ONE CLASS EXISTING
IN A GIVEN AREA, BULK, OR TIME

Suppose that we wish to know the number of things of one kind
contained in a given area, bulk, or time. Then the only way to
ascertain this with certainty is to count all the things. Suppose,
however, that we have no time for this, and must content ourself with
counting the things in a measured sample, and then estimating from
this observed result the most probable number of the things in the
whole area, bulk or time. The question then arises: How large
must the sample be in order to reduce the statistical error, with a
given degree of probability, to below a given percentage?

For example, suppose that we wish to know how many separate
stones there are in a million cubic feet of gravel. We cannot count
them all, and must, therefore, content ourself with counting how
many there are in a sample of, say, one, two or more cubic feet.
In how many cubic feet, then, must we count the separate stones
in order to be able to calculate the total with the required
degree of accuracy? Or suppose that we wish to know how many
leucocytes or parasites there are in the total blood of a patient, then
in how much of his blood must we count these objects in order that
the most probable truth will le between sufficiently narrow limits ?
Shall we take one, two, or more cubic millimetres of his blood for
our sample ?

First, in order to use the sampling method at all, we must
know that the things are equally distributed throughout the
area, bulk, or time. If this is not the case, we cannot know that our
sample accurately represents the whole material. [or example, it
would be useless to attempt to estimate the population of Britain
by counting all the people in one-tenth of the area of the country
because the population is not distributed equally at random
everywhere, but is gathered specially into certain districts and cities
according to certain economic laws. Similarly, we cannot estimate
by taking samples of the peripheral blood how many blood parasites
there are in the whole body unless we know that these organisms
do not collect specially in certain parts of the circulation.

But—it may be asked -if the things are equably distributed,
what further trouble will there be? We have only to count the

360

number found in any sample, and then to multiply that figure by
the total number of samples contained in the whole area, bulk, or
time. If the stones are equally distributed in a million cubic feet
of gravel, then there will be exactly a million times as many in the
whole mass of gravel as there are in one cubic foot. But this is
not so. It may be that 4y chance the stones in the first cubic
foot taken as a sample are exceptionally large, and therefore
are exceptionally few. Or it may happen by chance that the
trypanosomes in a first. sample of blood are exceptionally
numerous, or exceptionally few, as the case may be. We shall then
form a totally wrong estimate if we trust merely to the simple but
untruthful method just mentioned.

To obtain accurate estimates by any method in cases like these
may require the services of a trained statistician, and also, often,
a special study of the kind of material under consideration—
especially to ascertain whether the things are really equably
distributed. But for the purposes of this Article the following
Table will often be useful, because it serves to give some idea of
the number of things which must actually be counted if they are
equably distributed throughout the whole area, bulk, or time.

Taste C
PROBABILITY | SratisticaL Errors

= Beets). i= rt tn ile, ) ues) Se
| 1% | 2% | 3% | 4% | 5% | 10% | 20%

I tol | 4,550 1,138 506 285 | 182 46 Il

g tor | 27,057 6,764 3,006 | 1,691 | 1,082 271 68

gg to 1 66,350 16,588 7 a72. | 4,147 | 2,654 664 166

999 to I | 108,284 27,071 12,032 6,768 | 4,332 1,083 271

g999 to x |, 151338 | 37,835 |. 16,815 95459 6,053 1,513 378

99,999 tor | 194,938 | 48,735 21,660 12,184 | 73797 1,949 487

t

Suppose, for example, that a cubic foot of gravel has been found
to contain 3,000 stones, then the most probable number of stones in
a million cubic feet of the same gravel will be 3,006 millions, within
an error of 3 per cent., and at a betting of gto 1; that is, we may

361

bet 9 to 1 that the most probable total number of stones in the
million cubic feet will lie between about 3,096 and 2,916 millions.
Or suppose that 271 trypanosomes have been found in one cubic
millimetre of blood in a patient weighing 64°74 kilogrammes
(142 lbs, or about ro stone English), who should contain about
3,000,000 c.mm. of blood altogether; then we may bet 9 to 1 that
the most probable number of trypanosomes in the whole of his
blood will le between 894,300,000 and 731,700,000 (10 per cent.
error). And if we have counted 4,200 leucocytes in the c.mm. of
blood, we may bet 9,999 to 1 that his total blood contains between
13,350 and 11,844 millions of these cells (6 per cent. error).

As stated in Section 1 (page 350) if we wish to know the number
of things to be counted in order to yield an error within more than
5 per cent. we have only to divide the figures under the 1 per cent.
column twice over by the required percentage (that is, by the square
of the required percentage). Thus, for an error of 10 per cent. we
divide by 100; and for an error of 31°6 per cent. we divide by 1,000.
For example, if we find 27 malaria crescents in I c.mm. of the same
patient’s blood, we may bet g to 1 that he contains between about
106 and 56 millions of crescents altogether (always provided that
they are equably distributed in the blood).

The above Table is only for proportionately sinall samples, as
defined in Section 1 (page 348); that is, for samples which are
small compared with the total mass of material. When the sample
is more than about one-tenth of the total material we should use
the Correction Factor of Section 3 (page 357) for proportionately
large samples. Thus, if 1,500 leucocytes have been counted in one-
quarter cmm. of blood, and we wish to calculate the number in
I c.mm., then the error by the above Table is about 10 per cent. at
a betting of 9,999 to 1. But by the Table on page 358 the Correction

. n
Factor is 0°87 when N equals one-quarter, or 25 per cent. Thus

the error is not 10 per cent., but 87 per cent.; and we may bet
9,999 to 1 that the number of leucocytes in 1 c.mm. of blood is
between 6,522 and 5,478. But for the total blood content of
3,000,000 c.mm. the error of 10 per cent. must be maintained,
because the one-quarter c.mm. is now a ‘proportionately small
sample. This gives the number of leucocytes in the whole body
as most probably lying between 19,800 and 16,200 millions.

362

We can use the Table in another way. Suppose that we have
counted 664 things ina sample. Then the error is Io per cent. on
a probability of 99 to 1. Hence we can bet 99 to 1 that all counts
in future samples of the same size will lie between 730°4 and 5976—
assuming that the sample is proportionately small. This way of
stating the case avoids the necessity of determining exactly the size
of the sample compared to the whole material. In blood counts,
for instance, we are concerned with the number of things in unit
of blood rather than in the whole body, and we often wish to know
whether this number is increasing or diminishing. If the number
of things in a second sample is outside the limits of error declared
from the first sample, we may assume, at the appropriate probability,
that there has been an increase or decrease, as the case may be.
If otherwise, the difference may be due merely to chance in the
taking of the samples, and not to any real change in the total number
of things in the whole body.

EXAMPLES

24. We have counted 4,250 red corpuscles in one-thousandth of
a cubic millimetre of blood. What is the most probable number in
one cubic millimetre, at a betting of 99 to 1? Answer: Between
about 4,420,000 and 4,080,Ccoo.

25. How many may we expect to find in a second sample of
the same size, at a betting of 999 to 1? Answer: Between about
4,403 and 4,037.

20. A week ago we found 490 red corpuscles in one-ten-

thousandth of a c.mm. of a patient’s blood. To-day we find only

294 in the same sized sample. May we bet 99,999 to 1 that there
has been a decrease? Amxswer: No, the errors overlap. May we
bet g to 1 that there has been a decrease? Azswer: Yes.

27. Blood has been diluted 100 times. In ;3sth of I c:mm.
of the mixture we found 500 red corpuscles. How many do we
expect to find in 1 c.mm. of the blood at a probability of g99 to 1?
Answer: 5,000,000, with an error of 14°7 per cent.

28. We have found 553 things in a sample. In how many out
of 100 similar samples of the same material should we expect to find
an error greater than 7 per cent.? Awxswer: In ten.

29. A newly-appointed official finds that he is obliged to write

_—_

363

150 letters during his first week of office. How many letters should
he expect, at a betting of 999 to I, to have to write at the same rate
every week in the future? Azswer: 150, with an error of 26°87
per cent.

30. We have found one filaria embryo in 1 cmm. of blood.
What may we infer regarding the total number in the whole
circulation of 3,000,000 c.mm.? Azswer,: The odds are 1 to 1 that
the error is less than 67°5 per cent., and that the total number of
embryos in the circulation lies between 5,025,000 and 975,000. In
one out of two such cases the error may exceed this amount.

364

Il. TABLES

At. 99999 to I

Errors
2% 3 % 4 % 5 % 6%
483 215 121 78 54
956 425 239 153 107
1419 631 355 227 158
1872 832 468 300 208
2315 1029 579 371 258
2749 1222 688 440 306
3173 1410 794 508 353
3587 1595 897 574 au?
3992 1774 998 639 444
4387 1950 1097 702 488
4// a 7195 764 53%
5147 2288 1287 824 572
5512 2450 1378 882 613
5868 2608 1467 939 652
6214 2762 1554 995 691
6550 2912 1638 1048 728
6877 | 3057 1720 1101 765
7194% ||» 03497 1799 1151 800
7500 | = 3334 1875 1200 834
7798 | 3466 1950 1244 867
Bo8s | 3504 2022 1294 899
8363 3717 2091 1339 930
8631 3836 2158 1381 959
8889 | 3951 2223 1423 988
9138 4062 2285 1463 1016
9377 4168 2345 1501 1042
9606 4270 2402 1537 1068
9825 4367 2457 1572 1092
10035 4460 2509 1606 1115
10234 4549 2559 1638 1138
10425 4634 2607 1668. 1159
10605 | 4714 2652 1697 1179
10776 4789 2694 1724 1198
10936 4861 2734 1750 1216
11088 4928 2772 1774 1232
11229 4991 2808 1797 1248
11360 | 5049 2840 1818 1263
11482 5104 2871 1837 1276
11592 5152 2898 1855 1288
11697 | 5199 2925 1872 1300
11789 | 5240 2948 1887 1310
11872 | 5277 2968 1900 1320
11945 | 5309 2987 IgI2 1328
ee 5337 3003 jie 1335
12062 | 5361 3016 1930 1341
12106 5381 3027 1937 1346
12140 5396 3035 1943 1349
12165 | = $407 3042 1947 1352
12179 | 5413 3045 1949 1354
121th) ee 3046 1950) ee

A2. 9999 to I
Errors
I % | 2 % 3 % | 4 % 5 %
i — [ee See

I 1499 375 167 | 4 60
2 2967 742 330 1} 186 119
3 4404 1101 490 276 WATE
4 | 5812 1453 646 | 364 | 233
5 7189 1798 799 | 450 | 288
6 8536 2134 949 534 342
7 9852 | 2463 1095 616 | 395
8 11139 | 2785 1238 | 697 446
9 12395 3099 1378 | 775 | 496
10 13621 3406 1514 852 545
II 14816 | 3704 1647 926 | 593
12 15982 | 3996 | 1776 | 999 | ~640
13 C7117 4280 1902 | 1070 685
14 18221 4556 2025 0) 1139 | 29
15 19296 4824 2144 | 1206 | 772
16 20340 5085 2260 | ie 814
17 21354 50 | 2578. ~ (| 335. || 855
18 22338 5585 | 2482 | 1397 894
19 23291 5823 2588 | 1456 932
20 24215 6054 | 2691 | Ist4 | 969
21 25107 6277 | 2790 | 1570 1005
22 25970 6493 | 2886 | 1624 1039
23 26802 6701 2978 1676 1073
24 | 27605 6902 3068 1726 1105
25 28376 7094 BUSS gl kya P1135
26 29118 7280 3236 | 1820 | 1165
27 29829 7458 | 3315 1865 | 1193
28 | 30510 7628 3390 1907 1221
29 31160 7790 3463 | 1948 | 1247
30,02 31781 7946 3532, | 1987 | 1272
Se 26} (32372 8093 3597 | 2024 | = 1295
32 32932 8233 3660 | 2059 1317
33 33461 8366 3718 | 2092 | 1339
34 33960 8490 3774 2123, | 1359
35 | 34430 8608 3826 Zr52 {| 1378
36 34868 8717 3875 2180 | 1395
; (a ey 8820 3920 2205 | I411
38 | 35655 8914 | = 3.962 22290-1427
39 36004 goo! 4001 2250 | 1441
40 36322 go81 4036 2270 | 1453
‘41 36609 9153 | 4068 2289 1465
42 | 36866 9217 | = 4097 2305 | 1475
43 37093 9274 4122 2319 1484
44 heenilnee, goe3 | 4144 2331 Agg2
45 | 37459 9365 | 4162 23422-1499
46 37593 9399 | 4177 2350 1504
47 37699 9425 4189 2357 1508
48 * 37774 9444 4198 2301 1Sil
49 37820 9455 4203 2364 | 1513

2° hn} al £365.

30

366

A 3. 999 to I
Errors

2% 3 % 4% | 5 %
268 120 67 | 43
531 236 ce | 85
788 35% 197 127
1040 463 260 167
1286 572 322 206
1527 679 | 382 245
1763 784 | 441 282
E993 886 | 499 319
2218 986 555 «| 355
2437 1083 610 390
2651 1178 663 424
2859 1271 715 458
3062 1361 766 490
3260 1449 | 815 522
3452 1535 863 553
3639 1617 gio 583
3820 1698 955 611
3996 1776 999 640
4167 1852 1042 667
4332 1925 1083 693
17 1995 1123 739
4646 2065 1162 744
4795 2131 1199 767
4938 2195 1235 791
5076 2256 1269 813
5209 @315 1303 833
5336 2372 1334 854
5458 2426 1365 | 874
5574 2478 1394 892
5685 2527 1422 gIo
5791 2574 | 1448 927
5891 2618 | 1473 943
5986 2661 1497 958
6075 | 2700 | = 1519 972
6159 | 2738 1540 986
6238 2773 1560 998
6311 2805 1578 1010
6378 | 2835 1595 1021
6441 | 2863 1611 1031
6498 | 2888 1625 1040
6549 2911 1638 1048
6595 | 2931 1649 1055
6636 | 2949 1659 1062
6671 2965 1668 1068
6700 2978 1675 1072
7250 | 2989 1682 1076
6744 | 2997 1686 1079
6757 3004 1689 1081
6766 3007 1692 1082
6768 __ 3008 © 1692 1083

— |
Sewnw OW) Orn~I mee | =

13934
ao
map rhe)
14672
14890
15096
15287
15467
15633
15787
15925
16051
16164
16263
16349
16422
16482
16530
16562
16582
16588

99 to I

367

Errors

COI Otis WN

At O40 I
ERRORS

re) ae ll ae oe eee
268 67 30 17 | 11 | 8
531 133 59 34 ve 15
788 197 88 50 | 32 | 22
1039 260 116 65 42 29
1286 322 143 81 , 52 36
1526 382 170 96 62 | 43
1762 441 196 Ill 7s a 49
1992 498 222 125 80 | 56
2216 554 | 247 139 89 62
2436 609 271 153 98 68
2649 663 295 166 106 | 74
2858 715 318 179 is, || 80
3061 766 341 192 123 86
3258 815 362 204 131 gI
3450 863 384 216 139 96
3637 gio 405 | 228 146 102
3818 955 425 239 153 107
3994 999 444 250 160 III
4164 1041 463 261 167 116
4330 1083 482 271 174 121
4489 1123 499 281 180 125
4643 1161 516 291 186 129
4792 1198 533. | 300 192 134
4936 1234 549 309 198 138
5074 1269 564 318 203 141
5206 1302 579 326 209 145
5333 1334 595 334 ee ez4
5455 1364 607 341 219 152
5571 1393 619 | 349 223 155
5682 1421 632 356 228 158
5788 1447 644 362 232 161
5888 1472 655 368 236 164
5983 1496 665 374 240 167
6072 1518 675 380 243 169
6156 1539 684 385 246 171
6234 1559 693 | 390 250 174
6307 1577 701 | 395 253 176
6375 1594 709 399 255 178
6437 1610 716 403 258 179
6494 1624 722 406 260 181
6545 1637 728 410 262 182
6591 1648 733 412 264 184
6632 1658 737 415 266 185
6667 1667 741 417 267 186
6697 1675 745 419 268 187
6721 1681 747 421 269 187
6740 1685 749 422 270 188
6754 1689 Fist 423 271 188
6762 1691 | 752 423 271 188
6764 1691 | 752 423 271 188

1% Bos

I 45° 11
2 8g 22
3 Hs 33
+ nes 44
5 | 216 54+
6 257 64
7 296 74
8 335 84
9 373 O3
10 409 102
II 445 III
12 480 120
13 515 129
If 548 137
oe) 580 5
16 611 153
17 642 160
18 671 168
es 70 £75
20 728 182
21 755 186
22 781 195
23 806 201
24 | 830 207
25 853 215
26 875 219
27 897 224
28 gI7 229
<2 EY i 234
a p55 239
31 973 243
32 ae 247
33 1006 251
34 1021 255
35 1035 #59
36 1048 262
37 1060 265
38 1072 268
39 1082 271
40 1092 273
41 | 1100 275
42 / 1108 277
43 L115 279
44 | 1121 280
1 1126 281
46 | 1130 282
47 1133 283
48 1135 284
49 | 1137 284
fo =| _‘1137 284

A 6.

369
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371

NOTES ON SOME BIOOD PARASITES
IN. REPTILES

BY

Dr. HARALD SEIDELIN
(Recerved for publication 15 July, 1911)
I
Filaria (Mikrofilaria) tmperatoris (d’Hérelle and Seidelin, 1909)

Together with F. d’Heérelle I published, in 1go09, a short note
on two mikrofilariae from the blood of two different snakes from
Yucatan, México. We hoped to be able to make further
observations on the subject, but so far I have not, for my part, had
any opportunity for studying new cases, and probably shall not
obtain it soon. I have, therefore, re-examined the specimens from
the one case, which belonged to me, and propose now to give a
somewhat more detailed description of the parasite. The
observation is necessarily incomplete, as I have only had at my
disposal dried and stained films, and, moreover, adult filariae were
not found. The description and figures may, however, facilitate
the identification 1f some other observer should meet with the same
parasite.

The blood films were obtained from a mesenteric artery of a
Boa imperator which was brought, badly injured but yet with signs
of life, to the laboratory. They were fixed in methyl alcohol and
stained with Giemsa’s stain. As the preparations were only
intended to contribute to an investigation of the frequency of
haemogregarines they were laid away to be examined later, and thus
the opportunity was lost of examining the parasite zz vivo and of
obtaining films for wet fixation.

The parasites were fairly numerous, numbering about seventy in
a slide smear. Their general form and structure correspond well
to the description and figures of other blood filariae by Annett,
Dutton and Elliott (1901), Looss (1905), Manson (1907), and others.
The long, slender body has a blunt anterior extremity—the head—
and a rapidly tapering tail. The surface of the body shows in
some portions a delicate, transverse striation (fig. 1). The head’

572

forms a direct prolongation of the body, and is provided with a fine
mouth-opening, situated somewhat to the (ventral) side; besides
numerous small reddish dots no other definite structures are
observed, especially no ‘spicule’ or ‘prepuce.’ Beginning at the
neck and continued throughout the body are seen larger granules,
which have the aspect and staining properties of cell nuclei. Several
hundreds of these granules are present, and they are situated in
several longitudinal rows which are, however, interrupted at two or
three places. The first interruption seems to correspond to the
so-called nerve spot, in which no structure is seen; the second is
occupied by the ‘excretory cell,’ an ovoid body containing a clear
space, the outlines of which are nearly concentric with those of the
‘cell.’ The excretory pore is not seen. The third interruption
probably contains the ‘genital cell,’ but such a body cannot be
distinguished. The last few granules in the tail are situated in a
single row.

The most peculiar feature of these mikrofilariae is that each
embryo is situated in an envelope which is generally egg-shaped,
but sometimes more oblong. Only in a few cases is the embryo
observed free, but then the empty envelope is seen in its immediate
vicinity so that the liberation would seem to have taken place
artificially, by the spreading of the blood. The envelopes stain a
pale blue, and do not show any structure. In their interior the
parasites occupy different positions, being sometimes curled up in
a spiral (fig. 2), and sometimes nearly straight (fig. 3); between
these two extremes all intermediate positions are seen. One
cannot help being impressed with the view that individuals like the
one depicted in fig. 2 are embryos enclosed in their egg-membranes.
On the other hand, the aspect of individuals like fig. 3 resembles
considerably that of an ordinary mikrofilaria in its sheath, although
the envelope does not adapt itself to the shape of the worm. As
a whole the different forms observed correspond closely to the
figures given by Manson (1883), as illustrating the transformation
of the egg membrane into a sheath, and it would, therefore, seem
that our observation supports the view advocated by that authority
upon the process, a view which has not been universally accepted.
Such huge bodies as the envelopes would, of course, if comparatively
‘rigid, be unable to circulate through the blood-capillaries of man;

J

how far they might do so in those of a snake, I do not know, since
the blood was, as it has been said, taken from a mesenteric artery,
a vessel of a large diameter.

The principal dimensions of the worm and its envelope, as
measured on dried and fixed films, are as follow (average of
various individuals) : —

Total length of body, 167 pu.

Width of body, 4 pu.

Distance from anterior extremity to nerve spot, 45 p.

Distance from anterior extremity to excretory cell, 65 p.

Distance from anterior extremity to genital spot, 128 p.

Diameter of granules, 1°3 m:

Long diameter of excretory cell, 2°4 p.

Short diameter of excretory cell, 2 u.

Length of envelope, 115 # (maximum 142“, minimum 86 ,).

Width of envelope, 34 /.

LS)

In the Boa zmperator neither adult filariae nor embryos seem to
have been observed before, but in the Boa constrictor three filariae
have been described: Féelaria boae constrictoris, Leidy, 1850;
F. bispinosa, Diesing, 1851; and FP’. mucronata, Molin, 1858. They
are stated to occur ‘between the muscles of the ribs and_ the
integument,’ ‘in cavo abdominis’ and ‘in cavitate thoracis and
vasa majora’ respectively. In no case are embryos mentioned.

Filarial embryos in the blood of other reptiles have been briefly
described by Rodhain (1906) and mentioned in a footnote by

Prowazek (1907).
REFERENCES
Annetr, H. E., Durton, J. E., anv Ertiorr, J. H. (1901). Report of the Malaria Expedition
to Nigeria, II, Liverpool Sch. Trop. Med. Memoir IV, pp. 1-92.
Dirsine, C. M. (1851). Systema Helminthum II, Vindobonae, p. 278.

pb’ Hérevte, F. anv Semperin, H. (1909). Sur deux microfilaires du sang des serpents. C. R. Soc.
Bul., LXVII, p. 409.

Leivy, J. (1850). Description of three filariae. Proc. A.N.S. Phil., pp. 117-118. [Reprinted
in Leidy, J. (1904). Researches in helminthology and parasitology, pp. 40-41].

Looss, A. (1905). Filariasis, in Mense’s Handbuch der ‘Tropenkrankheiten, I, pp. 157-155.

Manson, P. (1883). The Filaria sanguinis hominis, London, p. 25.

Manson, Sir P. (1907). Tropical Diseases, 4th Ed., Lond.

Mot, R. (1858). Prospectus helminthum quae in prodromo faunae helminthologicae Venetiae
continentur. Wien, p. 31.

Prowazek, S. vy. (1907). Untersuchungen iiber Haemogregarinen. Arb. a. d. Kaiserl. Gesund-
heitsamte, XXVI, p. 35.

Ropnatn, J. (1906). Filaire infectant le sang chez l’Agama Colonorum dans I’Ubangi. Central-
blatt }. Bakt. u. Parasitenk., XLII, pp. 545-546.

I]

Haemogregarina imperatoris

In the same blood-smears a haemogregarine was observed which
probably represents a new species, since it shows some peculiar
morphological features, and, moreover, differs from most other
known haemogregarines by the deleterious influence which it exerts
upon its host-cells.

The infection was an abundant one, and the elements observed
belong evidently to different phases of evolution; but the scarcity
of the material at my disposal precludes a full consideration of
these questions. ,

The intracorpuscular parasites which appear least advanced in
development are nearly cylindrical bodies with slightly rounded
extremities; their length is somewhat less than that of a normal
erythrocyte. The protoplasm stains very feebly, and contains only
a few reddish granules (Giemsa stain) or no granules at all. The
nucleus is generally situated in the central portion of the body, and
shows a dense chromatin reticulum, but no definite structural
elements can be distinguished. A capsule seems always to enclose
the parasites, but it is not always well defined (fig. 16).

In the somewhat larger forms, the granules in the protoplasm
have increased both in number and size and, when prominent, stain
exactly like chromatin (figs. 17 and 18). The granules show a
more or less irregular distribution, and two or three of them may
be exceptionally large (fig. 19), but no one can be pointed out with
certainty as blepharoplast or other definite structure. Besides the
large granules others may be present which are fine, dust-like, and
occasionally very numerous. Granules are as a rule not mentioned
in the description of haemogregarines, but those here described
evidently correspond to the volutin-granules of Reichenow (1910).
The protoplasm shows, moreover, in the advanced stages, a diffuse
colouring, pale blue or sometimes more pink. The nucleus is
similar to that of the smaller forms, but it is more often
eccentrically situated, and there can frequently be seen one or two
nucleoli in its interior (fig. 20). The chromatin of the nucleus
sometimes forms well delimited large granules, which are united
only by delicate filaments (fig. 21), and in a few cases a formation

375

of definite chromosomes seems to have taken place, eight such being
distributed in two rows of four each (fig. 22); some more advanced
stages of division are also observed (fig. 23). The capsule is in
the larger forms fairly well defined, and its extremities are often
delimited from the central portion by delicate lines which are
stained red (figs. 17 and 25); such lines are considered constant by
Sambon (1908-9), but they are seldom mentioned or figured by
other authors. I have seen them frequently in other haemogre-
garines also, but by no means constantly, and never so sharply
defined as they appear in Sambon’s somewhat schematic figures.
If this author is right in describing them as lines of cleavage, it is
readily understood that they may be inconstant.

In their most advanced stages the intracorpuscular elements
attain almost the dimensions of a normal erythrocyte (figs. 24 and
25). They still conform essentially to the description already
given, but in some individuals one extremity is very slender and
bent against the body in an acute angle (fig. 26).

A number of parasites belong to a different type. They are
long and slender, with one blunt extremity, and the other slowly
tapering. Being doubled up they easily find room in the
erythrocytes, although they are much longer than their host-cells ;
sometimes the extremity of the tail is again bent in a sharp angle
against the body. The protoplasm stains dark blue or violet, and
contains a number of fine and occasionally a few coarse granules.
The nucleus is situated near the bend, in the anterior half of the
body; it is approximately quadrangular, and occupies the whole
of the width of the body. It is sharply limited and deeply
stained (fig. 27). The long: forms are in this case always intra-
corpuscular; in the blood of other snakes, however, I have
repeatedly seen them free in preparations which were taken from
the living animal and immediately fixed. [ emphasize this,
because Flu (1910), Reichenow (1910), and others state that these
elements are always intracorpuscular, normally, and become free
only under abnormal conditions, as when the blood is_ being
preserved outside the body.

These long elements have, according to Lutz (1901), and most
other authors, no direct connection with the more oval forms; the
former are derived from mikrozoits, the latter from makrozoits.

376

Reichenow (1910), however, asserts that the long forms are at a
later stage of the evolution transformed into oval bodies, the
extremities being drawn in when the parasites are preparing to
divide. All schizonts are said to undergo such a transformation,
and it would seem not at all unlikely that some of our larger forms
belong to this group, the significance, especially of the large oval
elements with short slender tail (fig. 26), becoming intelligible if
they are considered as transitional stages. It is, of course, possible
to adopt this view with regard to the larger forms only; the small
oval parasites must have a different origin, and would, according
to Reichenow, represent gametes, but this view does not agree with
the presence of mitotic figures in several of them. The distinction
between male, female and indifferent haemogregarines which is
made by many authors depends, however, to a large extent on
analogy, and cannot be too carefully considered before being
accepted. In the present case no such differentiation can with
certainty be made.

In this case no small parasites were seen free in the blood-
plasma, except the one depicted in fig. 28, which appears to have
just escaped from its host-cell. An erythrocyte is also shown in
fig. 29, which evidently has harboured a parasite. The
possibility cannot be denied that these appearances may have been
produced artificially. Another very interesting individual is seen
in fig. 30; it consists of a large body with faintly blue-stained
protoplasm and numerous chromatin-granules, whilst no well
defined nucleus is seen. At one extremity of this body is seen
another structure, much smaller and very slender, half inside, half
outside the larger one; the smaller element is wholly chromatin-
stained. The whole appearance suggests strongly the penetration
of a mikrogamete into a makrogamete. The possibility of
conjugation taking place in the body of the snake is admitted by
Hartmann and Chagas (1910). So far very little is known about
the sexual phase of the snake-haemogregarines and about the
allied subject of their transmission; with regard to Haemogregarina
stepanowz of tortoises, Reichenow (1910) has shown that the sexual
phase is found in a leech.

A few words should now be said about the effect of the
haemogregarines on the erythrocytes. In the case of the smaller

S07

parasites their host-cells do not show any important alterations,
excepting a slight increase in size and a dislocation of the nucleus.
Corresponding to the somewhat larger forms, the erythrocytes are
not only enlarged, but often differentiated into a pale peripheral
portion and a more deeply staining zone surrounding the parasite.
The corpuscles which harbour the most advanced stages of parasites
are of enormous size, their length being about twice the normal;
they have apparently lost all their haemoglobin, and have become
mere shadows, the outlines of which it may be difficult to
distinguish. In a few instances numerous fine red granules are
seen in the decolourized erythrocytes, perhaps representing remains
of the broken-up haemoglobin, but closely resembling the
fine granules of the parasites (fig. 25). The nuclei become
disfigured by compression and their structure more dense, but no
fragmentation is observed, as has been described by Marceau (1901)
and others. This deleterious influence on the erythrocytes
constitutes a considerable difference from what is the case in most
haemogregarine-infections, in which as a rule no alteration of the
host-cells is observed, or (in the case of Karyolysus sp.) only of the
nucleus. Prowazek (1907), however, gives figures (from lizards)
very similar to our own.

Besides these parasites ] must briefly mention other bodies
which are of interest, as they may easily be mistaken for free
forms of haemogregarines. In fact, I am not absolutely sure that
they are normal blood elements, but there is a great probability in
favour of their being so. I refer to the bodies depicted in
figs. 4-11 on Plate XIV, which are characterized by a large, well-
staining nucleus, a nearly unstained protoplasm, sometimes very
scarce, and a well-defined red border-line. Two nuclei may be
present, as in fig. 11, but no mitotic figures are seen. These
corpuscles are similar to haemogregarine-merozoites, as figured by
Reichenow (1910) and others, but on the other hand they bear a
striking resemblance to some figures which are given by Werzberg
(1910). This author has found such bodies in the blood of
Tropidonotus natrix, and in Chamaeleon sp., and is inclined to
consider them as thrombocytes, though he leaves room open for
doubt. On examining, here in England, the blood of a
Tropidonotus natrix, 1 have also found similar cells, and _ this

378

circumstance, of course, confirms the identity of those seen by
\Werzbere and by myself. One considerable difference seems,
however, to exist, namely, in the staining of the nucleus. The
figures of Werzberg show a very pale nucleus, whilst my
preparations, both from 77. matrix and especially from Boa
imperater, show a deep staining of the nucleus. This may be due,
perhaps, to the different methods of staining, Werzberg having
used Pappenheim’s so-called Unna-Ziehl stain, and I the Giemsa
stain; but as other nuclei, especially of the erythrocytes. in
Werzberg’s figures are deeply coloured, the explanation is not quite
satisfactory. However, it may be admitted that the bodies are
probably of the same nature, and are constituents of the blood ; this
is so much the more likely, as, in 77. matrix they do not show any
movements in fresh preparations, as haemogregarines probably
would do. It then remains to consider what their real nature may
be. In both cases the border lines stain red; this seems by the
Unna-Ziehl stain to indicate a relationship to the erythrocytes, as
the cytoplasm of these elements takes the same colour. But in my
specimens the red is not like the eosin colour of the erythrocytes, it
resembles much more the azur-eosin stained chromatin. By other
methods, it does not give the reaction of either. It does not stain
like chromatin with iron-haematein, nor does it take up eosin, like
haemoglobin; in both cases it remains unstained. Moreover, it
retains its colour badly; in Giemsa-preparations it is only visible
in dry smears when no differentiation is employed. Subsequent to
the differentiation with acetone which becomes necessary when the
‘wet method’ is used, no red colour is to be found. By this
differentiation the nuclei of these bodies are also often to a large
extent decolourized, though all the surrounding erythrocyte-nuclei
are deeply stained. In fresh preparations it is in most cases difficult
to observe more than the nuclei of these cells, but occasionally the
border-line appears quite sharp, and the protoplasm very slightly
granular. Moreover, their protoplasm appears to be very fragile,
or at least very plastic, since the bodies in the stained preparation
present very irregular and varied forms. The border-line is
evidently but loosely connected with the cytoplasm, since it often
becomes separated from its periphery and takes on different
positions, sometimes so peculiar that a certain resemblance to a

579

flagellum is produced (figs. 4, 7 and 8). How far these characters
are artificially produced by the preparation of the films it is
difficult to say, since an exact observation of the bodies was not
possible in the specimens which were examined in the fresh state
or treated by ‘ wet’ fixation and staining, as above-mentioned.

Similar bodies, but without a definite border-line, were found in
the blood of other snakes in Yucatan; haemogregarines were also
present. Only in the case of the Tvofpzdonotus natrix have no
intracorpuscular parasites been detected. No relationship is
apparent to other blood elements; at present these bodies must
be classified as ‘thrombocytes,’ a special kind of cells, probably
corresponding to the platelets of higher animals.

Another peculiar kind of cell, the significance of which has not
been determined, is represented in figure 12. They give at first
view the impression of leucocytes, but a more close inspection shows
them to be enclosed in a large, faintly eosin-stained protoplasmic
body, similar to that of an altered erythrocyte. Their outlines are
nearly circular, they have a granular, dark-staining protoplasm,
and an eccentrically situated dense nucleus. It cannot be
determined if these bodies are of a parasitic nature, or if they
represent a stage of evolution of certain blood-elements.

REFERENCES

Fru, P. GC. (1910). Uber Haemogregarinen im Blute Surinamischer Schlangen. Arch. f.

Protistenk., XVIII, pp. 190-206.

Harrmann, M., ann Cuacas, C. (1gto). Vorlaufige Mitteilung iiber Untersuchungen an

Schlangenhaemogregarinen. Arch. f. Protistenk., XX, pp. 351-360.

Lutz, A. (1go1). Uber die Drepanidien der Schlangen. Centralbl. f. Bakt. u. Parasitenk.,
Abt. I, XXIX, p. 390.

Marceau. (1gor1). Note sur le Karyolysus lacertarum. Arch. de Parasttol., 1V, pp. 135-142.

Prowazex, S. v. (1907). Untersuchungen iiber Haemogregarinen. Arb. a. d. Kaiserl.
Gsndbtsamte., XXVI, pp. 32-36.

Retcnenow, E. (1910). Haemogregarina stepanowi. Arch. f. Protistenk., XX, pp. 251-350.

SamBon, L. W. (1908-9). Haemogregarines of Snakes. ‘fourn. Trop. Med. and Hyg., XI,
pp- 355 and 374, XII, pp. 22, 38 and 70.

Werzeerc, A. (1910). Uber Blutplattchen und Trombozyten. Fol. baematol., X, pp. 301-
390 (p. 368).

380
III

Amoeboid parasites in the blood-plasma of a Lizard

In the blood of a lizard (Lacerta sp.) from Yucatan, which has
not been identified, I observed amoeboid parasites in the plasma,
while no intracorpuscular forms were seen. Similar known parasites
are, at least in some stages of their development, intracorpuscular.
Individuals of very different sizes and shapes were observed but all
seem to belong to the same kind of organism. The differences
correspond probably to different evolutionary phases, although of
course the results cannot be entirely relied upon to show exact details
considering the technique employed (dry smears, methylalcohol
fixation, Giemsa stain). I shall consequently not attempt a detailed
description, much less classification, but only shortly state with the
aid of illustrations what I saw.

A large number of minute bodies are seen with a fine chromatin
‘dot and a blue protoplasm which is occasionally solid, but as a rule
forms a more or less delicate ring around a comparatively large
vacuole (fig. 31).

From this stage all subsequent gradations in size are met with
(figs. 32-40) until the largest forms which consist of a protoplasmic
body the size of which approximates to that of an erythrocyte, and
which shows a granular structure and contains numerous small
vacuoles and very often one or two larger ones. In most cases one
nucleus is present ; it is always small and consists of a central darker,
and a peripheral less intensely stained portion; often a second, still
smaller chromatin-stained element is also present at a considerable
distance from the nucleus (fig. 36), and occasionally two nuclei of
about equal size are seen (fig. 33); when the latter is the case the
cytoplasm shows a certain symmetry and we possibly have to do with
a divisional phenomenon. This would also agree with the regular,
rounded shape of such elements, but, on the other hand, they are
not of particularly large dimensions. In several instances no
definite nucleus is present, but several small chromatin-granules.
Many of the parasites show pseudopodia in different numbers and
of different shapes. The fixation has probably given them a more
rigid aspect than they would have had if examined in the living
state.

381

Forms like those shown in figs. 41 and 42 may possibly be
distorted amoebae, although the possibility of different kinds of
parasites being present must also be considered.

It is remarkable that this extracorpuscular parasite appears to
have a more pronounced effect upon the erythrocytes than
intracorpuscular parasites often have. Many erythrocytes are
metachromatic and vacuolated, and in places where they are closely
grouped together the plasma shows a faint eosin staining, a sign of
haemolysis having taken place. Related to this phenomenon is
probably another which was also observed in this case, namely, the
presence of numerous pigmented leucocytes, as shown in the figs. 13,
14 and 15. I can find no mention of these in works on
haemogregarines or other intracorpuscular parasites of reptiles.
Nor have I observed them in such infections; besides in the case
here reported I have only met with pigmented leucocytes once in a
lizard, but I have no notes on the presence of parasites in that case.
The aspect of the pigment-granules resembles that of malarial
pigment, but no statement as to its nature can be made. One would
naturally be inclined to connect the pigment with the destruction
of haemoglobin; it may be pointed out, however, that Downey
(1910), who recently has mentioned the existence of similar pigment
in phagocytes from the lympho-renal tissue of a fish (Polyodon
spathula) describes in detail the transformation, not of the
cytoplasm of the erythrocytes, but of their nuclei, into pigment.
Here is evidently a point which, for general pathological reasons,
deserves furtlier investigation.

REFERENCE

Downey, H. (1g1o). Phagocytosis of Erythrocytes, etc. Fol. haematol., 1X, pp. 81-86.

382

DESCRIPTION OF PLATES

All figures have been drawn with Abbé’s apparatus, using
Zeiss’s 3 mm. apochromatic objective and, in the figs. 4-42,
compensating ocular 12; for fig. 1 compensating ocular 8, and for
figs. 2 and 3 compensating ocular 4 has been used. The
magnifications are respectively 1300, 850 and 450 linear. Most of
the figures have been executed by Mrs. Margrethe Seidelin.

PLATE XIV
Figs. 1-3. Filaria-embryos.
Figs. 4-11. Various forms of ‘ Thrombocytes.’

Fig. 12. Leucocyte-resembling cell in large protoplasmic body; to
the left a lymphocyte.
Figs. 1-12 from the blood of Boa imperator.

Fig. 13. Pigmented mononuclear leucocyte.
Fig. 14. Pigmented leucocyte with two nuclei.

Fig. 15. Pigmented and unpigmented leucocyte fusing together
Figs. 13-15 from the blood of Lacerta sp.

XIV

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at

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PLATE XV
Figs. 16-30. Haemogregarines in blood of Boa zmperator.

Figs. 16 and 17. Young forms with pale protoplasm and few

granules.

Figs. 18-20. Larger forms with more deeply stained protoplasm
which contain numerous large granules; in fig. 18 three
such granules are very prominent.

Fig. 21. The chromatin in the nucleus forms regular granules.
Figs. 22 and 23. Mitotic figures.

Figs. 24 and 25. Large forms in enlarged and decolourized
erythrocytes; in fig. 25 and 26, especially in the first,
numerous fine red granules are seen in the cytoplasm of
the erythrocyte.

Fig. 27. Long, slender form.

Fig. 28. Parasite escaping from host-cell.
Fig. 29. Empty host-cell.

Fig. 30. Large free form; conjugation ?
In Figs. 19, 20, 25 and 26 cleavage lines are seen.

Figs. 31-42. Parasites from the blood of Lacerta sp.

In Figs. 35, 39 and 40 pseudopodia are seen. Figs. 41
and 42 distorted amoebae or flagellata.

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385

SOME EXPERIMENTS ON LARVICIDES

BY
Sik>RE ROSS, K.C.B:,, FR Ss
AND

Roe. LOTS M.A., B.Sc.
(Receeved for publication 17 July, 1911)

At present the substance in most general use as a larvicide is
petroleum, which acts by forming a thin film on the surface of the
water, thereby drowning the larvae when they rise to the surface to
breathe. In the case of enclosed bodies of water, such as wells and
tanks, this usually acts admirably at first, but in the course of a
short time the film, unless very thick, gets broken. This happens
much more readily in pools or stagnant places on river banks where
mosquitos breed. The film of petroleum in these cases soon gets
broken or blown to one side sufficiently to allow the mosquitos to
lay their eggs, and the larvae to develop into mosquitos. Moreover,
the presence of grass or weeds on the banks of these pools prevents
the formation of an unbroken film over the whole surface, and this
permits the reproductive processes to go on more or less
uninterruptedly.

Petroleum has other disadvantages arising from its
inflammability and its liquid nature, which render it less convenient
for transit.

An ideal larvicide would appear to be a solid substance which
kills larvae even when used in the form of an extremely dilute
solution. This would be convenient for use, as no great weight of
material would have to be carried from place to place. The
solution, when diluted to the maximum point at which it will still
kill larvae in a reasonable time must also be harmless to men and
domestic animals if the water is liable to be used for drinking
purposes. Should there be no chance of the water being drunk,
however, a stronger solution may be used, and the larvae killed
more quickly.

We have tried a number of solutions as larvicides, and an

386

aceount of the results obtained with those substances which we
thought might be most suitable is given in the following pages.
Most of our experiments were carried out with the larvae of Calex
pipiens, about fifty larvae being used in each case.

We first tried the effect of a larvicide recommended by
Le Prince, quoted by Ross (1910), prepared by dissolving resin in
crude carbolic acid, and treating the solution with caustic soda.
We found it difficult to obtain a proper emulsion without sediment,
however, as the resin did not dissolve readily in the carbolic acid.
This larvicide has been used with success in America, where it is
said to cost only sevenpence a gallon, the average mixture
containing 300 gallons crude carbolic acid, 200 pounds resin, 30
pounds caustic soda.

Our results with this larvicide are as follows :—

One part larvicide in 500 parts of water killed 80 per cent. of
the larvae (Culex pipiens) in two hours, and the remainder in
three and a half hours.

One part larvicide in 1,000 parts of water killed 70 per cent.
of the larvae in four hours, and most of the remainder by next
morning (eighteen hours). One larva, however, lived in this solution
for twenty-two hours.

One part larvicide in 2,000 parts of water killed about 30 per
cent. of the larvae in six hours, but the rest of the larvae were killed
very gradually, some surviving for about fifty hours.

We used the different ingredients in the proportions mentioned
above in the preparation of this larvicide, the value of which seems
to depend very largely upon the composition of the crude carbolic
acid used. Le Prince found this larvicide to act more quickly and
at a greater dilution than we did, perhaps because he employed a
stronger carbolic acid from which to make the final product.

We next tried an emulsion prepared by the ‘ Sanitas’ Company,
Limited, called ‘Sanitas-Okol.’ This appears to contain a large
proportion of phenols and allied compounds, and when much
diluted mixes well with water.

We obtained the following results with ‘ Sanitas-Okol,’ using
Culex pipiens larvae.

One part ‘Sanitas-Okol’ to 600 parts of water killed all the
larvae in fifteen minutes.

— A

387

One part ‘Sanitas-Okol’ to 1,000 parts of water killed about

' 40 per cent. of the larvae in fifteen minutes, and the rest in

seventy-five minutes. Many of the pupae were nearly ready to
hatch out into mosquitos, at which stage we frequently found them
to be more resistant to the action of larvicides, perhaps because they
are then less firmly attached to the outer shell, which prevents the
solution reaching their tissues so readily.

One part ‘ Sanitas-Okol’ in 2,000 parts of water killed 50 per
cent. of a young lot of larvae in sixteen minutes, and the rest in
twenty-eight minutes.

One part ‘ Sanitas-Okol’ in 2,500 parts of water killed all the
larvae except one in seventy minutes, the last being dead in ninety
minutes.

One part of ‘ Sanitas-Okol’ in 5,000 of water killed 40 per cent.
of the larvae in seventy minutes, and the rest in two and a half
hours.

In another experiment this dilution (one in 5,000) killed 50 per
cent. of the larvae in one and three-quarter hours, and all but three
in three and a half hours, the last three being dead in about five
hours.

One part ‘ Sanitas-Okol’ in 10,000 parts of water killed all the
larvae except two in six hours, the last two dying in the course of
the night.

With one part ‘ Sanitas-Okol’ in 20,000 parts of water about Io
per cent. of the larvae lived for twenty-one hours, while two larvae
were still alive after another twenty-four hours.

We have also tried the effect of ‘Sanitas-Okol’ on the larvae of
Anopheles bifurcatus, and found as follows :—

One part ‘ Sanitas-Okol’ to 5,000 parts of water killed eighteen
larvae out of twenty in two and a quarter hours, the other two living
for three and a quarter hours.

One part ‘Sanitas-Okol’ in 10,000 parts of water killed
twenty-three larvae out of twenty-six in five and a half hours, and
the rest in about eight and a half hours.

It will be seen that ‘ Sanitas-Okol’ acts very powerfully as a
larvicide, and can be used quite satisfactorily in dilutions up to
I in 10,000. It is also quite non-poisonous at this great dilution.

In view of the fact that mercuric chloride acts so well as a

388

germicide, we thought it well to try the effect of solutions of this
compound on Culex larvae.

One part mercuric chloride in 2,000 parts of water only killed
the larvae extremely slowly, one larva living for three days.

One part mercuric chloride in 1,000 parts of water killed all the
larvae except one in eighteen hours.

We carried out no further experiments with mercuric chloride,
as it is evidently unsuitable as a larvicide from its poisonous nature
and comparatively feeble action on larvae. It would also be too
expensive to use on a large scale.

The same objections can be taken to the use of copper sulphate,
which failed to kill all the larvae in two days, even when used in
the form of a solution containing I part of copper sulphate to 500
parts of water. A_ solution of cupric hydrate in ammonia,
containing the same proportion of copper as mentioned above, had
no better effect as a larvicide, although one part of ammonia
solution (34 per cent.) to 125 parts of water killed all the larvae in
twenty minutes.

Oxalic acid was not found to act powerfully on Culex larvae.
A solution containing one part of the acid in 1,000 parts of water
killed the larvae slowly, one surviving eighteen hours, and when
one part of oxalic acid in 2,000 parts of water was used Io per cent.
of the larvae lived for about thirty hours.

It may be of interest to add that saponin (one part to 400 parts
of water) was found to have no effect on larvae. While we had
scarcely considered it in the light of a possible larvicide, we thought
it worth while to try the effect of keeping larvae in such a solution,
in view of the haemolytic action of saponin on the blood of higher
animals.

We have finally turned our attention to potassium cyanide as
a larvicide. As this substance is very much more poisonous than
any others we had employed we did not use solutions as dilute as
one in 1,000, which would be unsafe should there be any possibility
of any water thus treated being drunk.

The following results were obtained with potassium cyanide,
using Culex pipiens.

One part cyanide in 26,000 of water killed all the larvae in
less than two hours.

389

One part cyanide in 58,000 of water killed all the larvae in
three and one-third hours.

One part cyanide in 106,000 parts of water killed all the larvae
in five hours.

In another experiment with nearly the same strength (one in
110,000), all the larvae except two were killed in five hours, the
last two living about seven and a half hours.

One part cyanide in 240,000 parts of water killed all the larvae
in the course of a night (less than sixteen hours from the time the
cyanide was added).

One part cyanide in 303,000 parts of water killed about 50 per
cent. of the larvae in six and a half hours, and all except one in
twenty-two hours.

We have not carried out any experiments with weaker solutions
of potassium cyanide than last mentioned, as we did not consider
it advisable to use solutions in which larvae can live for a longer
period than about eighteen hours, and a solution of one part
cyanide to 300,000 parts of water was found by repeated
experiment always to satisfy this condition, though in some cases
individual larvae survived about twenty-four hours.

We have tried the effect of a solution containing one part
potassium cyanide to 300,000 parts of water on two lots of
Anopheles larvae, and in both cases about 80 per cent. of the larvae
were killed in less than eight hours, and the rest in from twelve to
fifteen hours.

Strychnine in dilutions greater than one in 50,000 parts of
water appeared to have no effect on larvae. As we had obtained
much more satisfactory results with potassium cyanide we did not
try solutions of strychnine stronger than the above.

We have been unable to discover any other substance whose
potency as a larvicide approaches that of potassium cyanide. This
compound also possesses the advantage of being easily carried
about and of being comparatively cheap. Its one drawback lies
in the fact that it is extremely poisonous, and it is apt to undergo
partial hydrolysis with the production of a certain proportion of
free prussic acid.

On the other hand the use of potassium cyanide can be restricted
to stagnant water which is not used for drinking purposes, while

39°

it must be remembered that water containing cyanide in the
proportion of one to 250,000 or 300,000 would have to be consumed
in very. large quantities before having any deleterious effects.

We have endeavoured to find the most convenient form in
which to use potassium cyanide as a larvicide. It is, of course,
most conveniently carried about in the solid form, but if a piece be
simply thrown into a pool of water it sinks, and although it is
very readily dissolved the solution may not diffuse rapidly through
the whole pool. Accordingly, we tried mixing the cyanide with
a floating soap, both in the form of powder, and compressing the
mixture into pills or tablets. These will still float, provided that
the proportion of cyanide to soap is not too high, and that too
great pressure is not used in the preparation of the tablets. In the
latter case the resulting tablets are too dense, owing to there being
but little air retained in the substance, and consequently they sink.
The advantage of using these floating tablets is that the cyanide
soon dissolves out, and the solution being heavier than water
diffuses comparatively rapidly through the whole mass of the
water, and thus practically none of the larvae escape exposure to
the action of the drug. Other substances of a light and porous
nature may, of course, be used instead of soap as a medium for
the preparation of floating tablets containing the requisite amount
of cyanide. These substances should preferably be insoluble, or at
least of a comparatively inert nature.

A convenient size of tablet is one containing three or four
grains of cyanide, which will suffice for twelve or sixteen gallons
of water. These should be sealed up in tins of about 100
tablets, and, of course, all precautions must be taken to prevent
any danger of tablets being eaten by children or others.
Instructions ought also to be given that no water which is to be
used for drinking purposes is to be treated with cyanide. With
these precautions there is practically no danger of any harm
resulting from the use of potassium cyanide in this form as a
larvicide. It is unnecessary to refer here to previous experiments on
larvicides, as these are familiar to all students of the subject.

REFERENCE

KKOss, Sik RK. Prevention of Malaria (John Murray), section 43.

be

AN INVESTIGATION OF THE EFFECTS

PRODUCED UPON” THE: EXCRETION

OF URINARY PIGMENTS BY SALTS
OF QUININE

BY

W. M. GRAHAM, M.B.

(DIRECTOR, MEDICAI. RESEARCH INSTITUTE, LAGOS, S. N.)
(Received for publication 27 June, 1911)

In view of the preponderating rdle attributed to salts of
quinine in the production of blackwater fever, it has been evident
to me for some time that useful information might be secured by
examining the action exerted by these salts upon the blood of a
healthy adult, living in a malarial country, who had become
accustomed to their prophylactic use. It was possible that the
action which these salts exerted upon the healthy differed only in
the amount of the reaction from that which they exert in a case
of disease. In the healthy, the action exerted would be more easily
observed in the absence of the various complications introduced into
such an investigation by an attack of malarial fever.

Then, the fact that the effect produced upon the urinary
pigments by a prophylactic dose of quinine might be used as an
index to that exerted on the blood suggested itself, and an
examination of the effect of a single dose of the drug was
undertaken.

The subject of the experiment was a healthy European adult,
long resident in West Africa, where, on several occasions in previous
years, he had suffered from attacks of malarial fever (malignant
tertian).

The investigation was conducted as follows :—All urine passed
between 9.30 p.m. (bedtime) and 7.30 a.m. (breakfast) was
collected in a clean beaker for examination. This period of ten
hours was selected because of its convenience, and because during
this period the environmental conditions would remain fairly

392

uniform; for no food or fluids would be taken, the temperature
in bed would be nearly uniform, and the amount of sweating would
be less subject to alteration by changes in temperature, amount of
exercise, amount of clothing, or from leaving the shade of the
house for the higher temperature out of doors.

An attempt was also made to keep the dietary as uniform as
possible during the period of experiment. No fluids were taken
except at meals, and then in equal measured amounts. The bowels
were naturally opened twice a day, but there was no diarrhoea. The
occupation was sedentary, a short walk being taken daily at
sundown.

On the first day of the experiment all urine passed between
9.30 p.m. and 7.30 a.m. was collected to serve as a control,
representing the normal excretion uninfluenced by the action of a
salt of quinine. On the following night, at 9.30 p.m., a dose of
fifteen grains of quinine hydrochloride (B. W. & Co.’s tabloids)
was taken with six ounces of water, a similar amount of water
having been taken on the first night and on all the subsequent nights
of the experiment. All urine passed in the ten hours from
9.30 p.m. to 7.30 a.m. on the seven subsequent days was collected
and a preliminary examination of the amount, colour, specific
gravity, and reaction, was made.

It was then prepared for the photographing of its absorption
spectrum in the following manner. The urine collected each
morning was poured into a tall measuring cylinder, covered and
placed before a well-lighted window for two hours, so as to ensure
the complete conversion of urobilinogen to urobilin. Then 100 c.c.
of this urine was placed {n a beaker and rendered alkaline
by the addition of a measured amount of ammonium hydrate
(NH,OH). The precipitated phosphates were removed by filtration,
and to the clear filtrate a solution of zinc chloride (ZnCl,) was
added cautiously until the precipitate formed began to remain
undissolved. The urine was then again filtered, poured into a
Baly tube and at once photographed in a Hilger quartz
spectrograph by acetylene gas light projected through a quartz
condenser. By the use of a Baly tube it was possible to produce
readily on the same plate a series of ten photographs taken
through regularly diminishing depths of urine under such uniform

393

conditions that the amount of absorption shown by the different
-depths could be compared; and on different days, when dealing
with urine of varying specific gravity, a urine of low specific
gravity could be compared with one of high specific gravity without
the necessity of diluting the latter with water, a device likely to
introduce a variable error.

The photographs were taken upon the Wratton and Wainwright’s
pan-chromatic plate. This plate, though the best procurable, is,
unfortunately, very unequally sensitive to the different colours.
The ratios given by the makers are: red, 3; ; green, ;, ; blue, 3.
And this fact must be borne in mind when interpreting the absence
of any record in the blue region. This is not the only defect,
however, for these plates also show three dark bands: one between
C and D, one between D and E, and one between b and F, the
plate being evidently relatively blind in these three places. These
three bands are also present in photographs of the solar spectrum,
and are therefore not due to the use of acetylene as an illuminant.
This, of course, adds greatly to the difficulty of interpreting the
absorption bands in these three regions, but has no appreciable
effect on the steepness of the curve on each plate shown by each
complete series of ten photographs.

The photograph of each day’s urine will be seen to consist of
twelve records in series. The first eleven of the series were
photographed through a regularly diminishing depth of urine from
10 cm. to 0 cm. by steps of 1 cm. Number I! in each series
represents, therefore, the effect produced upon the plate when no
urine was interposed, the other conditions remaining constant. In
it, in one set of photographs, is shown the D, or sodium line, for
purposes of orientation. In the first series of photographs this line
is shown in the 12th record. In the second series of photographs
the 12th record represents in all the spectrum of burning
magnesium ribbon, and is intended to enable the solar line b to
be located.

The exposures given to all the records on all the plates were
accurately timed with a stop-clock and were equal. The plates
were developed with the same developer for equal periods of time.
The only variant was the temperature of the dark room, which
varied about two degrees Fahrenheit. Thus, the factors likely to
cause variation in the results, were kept as small as possible so as

394

to make the records comparable with each other. There is one
other variant, the daily average air temperature and range. I am
unable to furnish these data as I am writing this in England, where
I have no access to meteorological records. The mean temperatures
of the two months, August and September, are, however, very
much alike.

Two similar experiments were made upon the same subject:
the first from 31st July, 1910, to 7th August, 1910, inclusive, and
the second from 10th September, 1910, to 17th September, 1910,
inclusive. During both periods, the subject’s temperature remained
normal, and parasites could not be found in his peripheral blood.
Tables showing the results obtained, quantity, colour, reaction,
etc., will be found below. In each table the first item represents
the control, that is the urine collected before any quinine was
administered. I have treated it as unity, or 1%, and it is with it
that the results obtained for the seven subsequent days must be
compared. The ratios thus obtained should then be compared
with the photographic ratios.

Now, in estimating the results shown in these tables, the
following facts are important :—

1. The amount of absorption required to extinguish the
chemical or photographic effect on the pan-chromatic plate in the
blue region, is, when compared with that required in the red or
green, in the ratio of 3 : <4. A control photograph 1s, therefore,
absolutely necessary.

2. Under normal conditions the percentage of solids excreted
daily in the urine is nearly constant, but the quantity of urine
passed depending, as it does, on the water constituent, varies
greatly.

The amount of solids and pigments in urine 1s therefore usually
in the inverse ratio to the amount of water. It follows, therefore,
that if there were in these samples no abnormal increase in the
pigments, the ratio borne by the quantity of urime passed each ten
hours to the control quantity would be similar to the ratio borne
by the photographic records of each day to the photographic control.

For example:—TYaking the photographic record of the t1oth
September, 1910, as control or unity, and comparing the steepness
of the photographs of the seven subsequent days with it, a series of
ratios can very readily be prepared. Then, taking the quantity of

395

urine passed on 10th September, 1910, as the control or unity, and
comparing the quantities passed on the seven subsequent days with
it, another series of ratios can be calculated. Both these series of

ratios are shown in the following tables :—

Data Obtained in Experiment |
31—7— 10 to 7—8—10

|
Date | Quantity Colour Reaction
3I—7—I0 | 640 c.c. Yellow Acid
|
1—8—10 | 766 c.c. Pale-yellow ... 5
|
2—8—10 260 c.c. | Pale brown ...
3—8—I0 595 cc. | Yellow
4—8—10 605 c.c. *
5—8—10 660 ¢.c. $i es
6—8—10 Hgo c.c.
7—8—10 655 c.c. ee
Ratios
Date Quantity Absorption A—Q
Ratios Ratios
10 10 ae
es 10 10
12 10
1-—8—10 = — — 0°20
10 ie)
Pa 6
2—8—10 ze es + 1'00
fe) 10
9 | T4 ‘.
—— — = + 0°50
3-—-8—10 Io 10 :
. I
p26 10 24 = + 0°46
10 10
8—1 =i 2 + 0°47
°° 10 | 10 ;
10°7 II
6—8—10 ml — + 0°03
10 10
3 10°2 } 10 0'02
ae ee 10 10

396

Data Obtained in Expertment IT

10—9—Io to 17—g—I10
|

]

|

/ Tem- Sp. gr. Solids
Date Quantity Colour | Reaction Sp. gr. perature | reduced in
to 60° F. | 10 hours
= — ee es aoe
grms.
10—g—I0 | 595 c.c. | Yellow ...) Acid 1013 84° F 1021 29
11—g—10 |-- 803 c.c. | Pale yellow | 33 1010 80° F 1018 _ _| &* 33°6
12—9—10 235 c.c. | Pale brown ss 1027 84° F 1035 19°!
|
13—g—10 | ssoc.c. | Amber... =; | 1021 84° F 1029 s«23'°6
14—9—I0 | 530 c.c. | Pale amber sa 1016 84°F 1024 29°6
15—9g—I0 | sso c.c. | Bright $9 1014 81°F 1021 26'9
yellow |
16—9—-10 | 650 c.c. | Yellow , 1014 80° F 1020°6 | 3rl
17—9g—10 | 541 c.c 3 . 5 | 1015 80° F 1021°6 | 75 fs
Ratios
Date Quantity Absorption A—Q
Ratios Ratios
= 1D 10 ee
og et 13 6
a 19 | 10 vee
12—9—I2 ae = + 140
Ie 10
aaa ee) a :
eS == = + o°§0
1 —19 ue ze :
455 == 75 + 0°52
2 29 ,
Le Er mat mie a + 0°60
10°9 II
16—9—I¢ es = + ool
i7—0-—t< ZS = + O°!
ees aoe to 10 2

397

The experience gained while carrying out the previous
experiments enabled the last experiment to be performed with
greater accuracy in detail, and the data of this experiment are,
therefore, somewhat superior to those of the first experiment.

It will be seen, however, that the results obtained in both
experiments agree fairly closely in essentials. [| believe the
following conclusions may be drawn from the results obtained in
both :—

1. That a dose of fifteen grains of quinine hydrochloride causes
an early increase in the amount of water excreted in the urine.

2. That this increase is followed within twenty-four hours by a
marked decrease, which is accompanied by an increase in the
excreted pigments.

3. That these pigments consist largely of urobilin (chemical
and spectroscopic proofs).

4. That there is an approximate return to the normal elimina-
tion of water and pigment, the phase being almost completed in one
week.

Subjective Sensations

The subjective sensations following a dose of quinine are best
observed by a person who regularly takes one large weekly dose of
the drug, for then the sequence of the sensations is observed to be
very similar from week to week, and the regularity is sufficiently
great to enable the operation of causes other than quinine to be
excluded.

1. <A dose of fifteen grains of quinine hydrochloride taken on
Saturday night is followed, after an hour or so of restlessness, by
sound sleep accompanied usually by pleasant dreams. Without
quinine sleep is usually dreamless.

2. On the following day, Sunday, there is a feeling of some
exhilaration, with deafness, ringing in the ears, some tremor of the
hands, and an inclination for bodily and mental activity.

3. On Monday a certain amount of depression of spirits sets
in, which lasts through Tuesday. Deafness continues almost
unchanged, and sleep is not so sound.

4. The feeling of depression passes away by Wednesday night

398

or Thursday morning, when the aural effects have considerably
subsided.

The increasing use of salts of quinine in malarial prophylaxis
has added a continually increasing importance to the estimation of
the effects produced by them upon persons who take a daily or
weekly dose for long periods of time. The difficulties of the
subject are, however, great, for even the condition of the quinine
molecule while circulating with the blood is unknown, and there
are no convincing proofs of the mechanism by which these salts
exert their well-known destructive action on the malarial parasite.

Even the dosage is still a matter for debate, and there are no
observations yet available showing the effects exerted by prophy-
lactic doses of quinine upon the health, special senses, or excretions
of a healthy person. At present it can only be affirmed that a
European living in a malarious country such as West Africa has a
choice of two alternatives :—

1. Recurring attacks of malaria.

2. Quinine prophylaxis.

Both alternatives are harmful, and it therefore becomes simply
a question of which 1s least so.

Few experienced persons in West Africa would have any
hesitation about deciding for quinine prophylaxis, and there are
fewer still who would not wish that some other alternative were
available permitting them to choose otherwise, for this long
continued daily or weekly use of quinine produces at least in a
certain percentage of Europeans some of the following evil
effects : —

1. Diminished acuity of hearing.

2. Premature onset of presbyopia.

3. Mental depression.

4. Dyspepsia and skin affections.

So long then as the proximity of an infected native population
and of suitable anophelines makes such prophylaxis necessary for
European existence, so long the following questions will possess
both a practical and theoretical interest :—

I. At what rate is a salt of quinine excreted ?

II. Is the amount of excretion in direct or inverse ratio to
the amount of the dose?

399

III. How long does it exert an influence upon the subjective
sensations of the person ?

IV. What changes does a full dose of a salt of quinine exert
upon the urinary pigments, salts, acidity, specific gravity, etc., and
does this effect differ in healthy persons and in persons harbouring
the malarial parasite ?

No. I. The practical importance of this question will be
appreciated when the following points are considered :—

1. The whole of a dose of a salt of quinine cannot be
recovered from the excretion during the first twenty-four hours after
administration.

2. Many persons are taking daily throughout the year a dose
of five grains of quinine.

3. A cumulative action of the drug would seem to be a
necessary contingency from these premises.

4. Does the drug accumulate in the body, and, if so, how is
its cumulative action manifested ?

No. II. An answer to this question would enable the safest and
most efficient dose to be fixed.

No. ill. Answers to this question would enable an estimate to
be made of the influence exerted by idiosyncracy, and of its relation
to the effect produced upon the special senses.

No. IV. Of all these, the question of the effect exerted upon
the elimination of the urinary pigments appears to be most
important, especially the effect exerted upon those pigments derived
directly from haemoglobin; for an increased excretion in the urine
of such pigments would be likely to follow any increased destruction
of red blood corpuscles. This applies with special force to the
urobilin, the excretion of which, in both urine and faeces, is known
to be increased in diseases favouring abnormal destruction of red
blood corpuscles. A personal or partial answer to part of question
No. IV is furnished by these experiments, but further observation
made upon other persons are required to enable the amount of the
personal factor or idiosyncracy to be estimated. To exclude the
possible influence on the results of malarial infection, similar
experiments in a non-malarious country upon persons who have
never suffered from malaria are very desirable. It is for this reason
that I have explained in the present article so very fully the methods
by which my results have been obtained.

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401

THE” PASSAGE OF” HAEMOGLOBIN
THROUGH THE. KIDNEYS

BY

WARRINGTON YORKE, M.D.

(From the Runcorn Research Laboratories of the Liverpool School

of Tropical Medicine)
(Received for publication 15 September, 1911)

If an isotonic solution of haemoglobin obtained from rabbit's
red blood cells be injected into a vein of a normal rabbit, the urine
is found to be tinged with haemoglobin a few minutes after the
injection. Whilst performing experiments devised with a view to
studying the mechanism of production of suppression of urine in
blackwater fever, the opportunity was afforded of examining a large
number of kidneys removed from the animals at various periods
after intravenous injection of haemoglobin solutions.

It appeared possible that a careful study of this material might
throw some light upon the manner in which haemoglobin passes
through the kidneys from the blood stream to the urine.

Attention has already been drawn by Yorke and Nauss (1911) in
a previous communication to the fact that under certain conditions
suppression of urine follows the intravenous injection of large
amounts of homologous haemoglobin. Microscopical examination
of the kidneys in such cases shows the condition to be due to
occlusion of the renal tubules by casts. These plugs are of different
appearance, according as the kidney examined is obtained from
animals which have succumbed shortly after the injection of
haemoglobin or several days later. When seen in kidneys obtained
within a few hours of the injection they are but slightly granular
and appear to consist of a fairly homogeneous material, which,
when unstained, is of a brownish colour. When stained with various
dyes they react in much the same manner as do the red blood cells,
e.g., with eosin they stain a brownish pink and with van Gieson a
brownish red, whilst with Heidenhain’s iron alum haematoxylin
method they are dyed a dark blue-black colour.

402

The casts found in those kidneys where death occurred several
days after the intravenous injection of haemoglobin are, as a rule,
exceedingly granular. The granules vary considerably in size,
from being mere points to granules of about 2 mw in diameter. These
granular casts possess in the main the same staining characteristics as
the previous variety. Sometimes in the later stages certain of the
casts were found to have become in part crystalline.

As to the precise nature of these plugs one is unable to speak
with certainty. They are only found after intravenous injection
of haemoglobin, and from their appearance both unstained and also
after staining with various dyes there appears to be no doubt but
that they are derived from haemoglobin. Either they consist of
haemoglobin itself supported by a mucoid basis, or they are
composed of some closely related derivative. I was able to obtain
the iron reaction neither with potassium ferrocyanide and
hydrochloric acid (Berlin blue reaction) nor by first treating with
ammonium sulphide and then subsequently with hydrochloric acid
and potassium ferrocyanide (Turnbull’s blue reaction). Possibly
the failure to obtain this reaction was because the process of
disintegration had not continued sufficiently far for the setting free
of iron from albuminous combination to have occurred. It is
interesting, however, to note in this connection that the iron reaction
was obtained by Werner (1907) in the case of the casts found in the
kidneys of several human beings who had succumbed from
suppression of urine following blackwater fever. Here, however,
the individuals had survived the attacks of haemoglobinuria by
over a week. A careful examination of the exact site in which the
casts occurred revealed the fact that they were limited to the renal
tubules, and were never to be found in the meniscus of Bowman’s
capsules.

Plugs were frequently found in all portions of the tubule, with
the single exception of the glomeruli. The fact that the kidneys
removed from over thirty animals, at periods varying from one hour
to over eight days after the injection of widely different amounts of
haemoglobin, were examined, without discovering casts in the
glomeruli in a single instance, is highly suggestive that haemoglobin
does not escape through Bowman’s capsules.

The possibility suggests itself that haemoglobin might in reality

403

be excreted by means of Bowman’s capsules, but that it is so quickly
carried away by the rapid stream of water escaping from the tufts
that it is not recognised. However, even assuming Ludwig’s theory,
that the constituents of the urine are filtered through the capsular
tufts in the proportion in- which they exist in the blood plasma, to be
correct, then in certain of our experiments the glomerular filtrate
must have contained at least 8 to 12 per cent. of haemoglobin. If
such a solution of haemoglobin be spread in a fairly thick film—
considerably less, however, than the thickness of the section of the
kidney employed—on a glass slide, fixed immediately before drying
has occurred, and then stained with WHeidenhain’s iron alum
haematoxylin or van Gieson, a highly-coloured granular appearance
is produced, somewhat resembling the casts seen in the tubules.
Such appearances were never observed in the meniscus of the
Bowman’s capsule in our experimental animals. Moreover, even in
those cases in which suppression of urme had occurred and where
presumably it would be impossible for any haemoglobin which
escaped through Bowman’s capsules (sometimes in these cases
considerably dilated) to have been washed away, no casts were found
in the meniscus of the capsules.

Examination of the kidneys of three blackwater fever patients
who had succumbed from suppression of urine revealed an exactly
comparable state of affairs. Casts being constantly found in all
portions of the renal tubules, except in the meniscus of Bowman’s
capsules. The same was observed in the kidneys of dogs which
died during the passage of haemoglobin resulting from infection
with Pzvoplasma canis.

It is difficult to determine the exact portion of the renal tubule
which is responsible for the excretion of haemoglobin Presumably,
however, it is the epithelium of the convoluted tubules and possibly
also that of the tubes of Henle, as in sections of kidneys removed
within a few hours of the intravenous injection of haemoglobin the
casts are found to be limited to the cortex, and are not seen in the
large collecting tubes of Bellini. Later, however, the plugs are
found in the large collecting tubules, but in these cases they have
probably simply descended from higher portions of the tubules.

Again, it is interesting in this connection to compare the
percentage of haemoglobinuria in relation to the percentage of

404

haemoglobinaemia observed at definite intervals after intravenous
injection of haemoglobin. In Figures 1 and 2 the results of two
such observations are given. It is at once apparent from a study of
these graphs that the curve representing the percentage of
haemoglobin passed in the urine does not run at all parallel with
that representing the degree of haemoglobinaemia, for, whereas the
latter falls in a comparatively regular manner from the time of
injection until the end of the experiment, the former does not reach
its maximum until after the lapse of some hours (4 to 5) and,
moreover, the curve is irregular, neither attaining its maximum nor
falling to zero by regular gradations.

19%

x
’ Sean ag PLASMA

Soiwors? wah saa e) “ave iish) Foo arise

Fic. 1.—Graph representing the percentage of haemoglobin found in the plasma and urine
after intravenous injection of an isotonic solution of homologous haemoglobin.
The figures along the curve representing the degree of haemoglobinuria indicate
the amount of urine passed.

Although the degree of haemoglobinuria varies to a certain extent
inversely as the volume of urine passed, nevertheless, this factor
alone is insufficient to explain the phenomenon that the maximum
amount of haemoglobinuria is not attained for several hours after
the intravenous injection of haemoglobin. Assuming that
haemoglobin is filtered through the glomeruli, according to Ludwig’s
view, then one would expect that the percentage found in the urine
would depend upon the following two factors. Firstly, it would
vary directly with the degree of haemoglobinaemia, and, secondly,
it would vary indirectly with the volume of urine passed into the
bladder, or, in other words, with the amount of concentration that
occurred during the passage of the urine through the convoluted
tubules. This, however, does not appear to be the case.
Furthermore, it was observed that when several large amounts of

405

haemoglobin were injected into the same animal at stated intervals,
the degree of haemoglobinuria resulting from the last injection was
usually much lower than that following the first injection, even
though the degree of haemoglobinaemia in this case was not so great
as that resulting from the last injection. Moreover, as in these cases
the volume of urine excreted after several injections was greatly
diminished in amount, owing to partial suppression having
occurred, the lower percentage of haemoglobinuria observed could
not result from the secretion of a large quantity of watery urine
dependent upon decreased absorption of water as it passed through
the uriniferous tubules.
18 +S

7,
16

~~» GDF GD Qn oo

+——+—-+—_ 4-4
OF fa) 42 253 5k t Hh 5 54 6 O47 7z & Hours.

Fig. 2,--Graph representing the percentage cf haemoglobin present in the plasma and urine
after intravenous injection of an isotonic solution of homologous haemoglobin.
The numbers along the curve representing the degree of haemoglobinuria indicate
the amount of urine passed.

It would seem that these observations are more in harmony with
the view that haemoglobin is secreted by the renal epithelium than
that it is filtered through the glomeruli, and, that the amount of
haemoglobin eliminated into the urine is dependent upon the activity
of the epithelium lining the renal tubules.

There is, moreover, additional evidence which affords support to
this view, and that is obtained by examination of the epithelium
lining the capsules and various portions of the uriniferous tubules.

406

On examining sections of the kidneys of pups, which have died
from Pivoflasma canis during the passage of haemoglobin, fixed in
formalin and stained with Heidenhain’s iron alum haematoxylin
method, one is at once struck by the presence of large numbers of
darkly staining granules in the epithelium of the renal tubules.
These granules vary in size, some being exceedingly small and
others much larger, some of the latter being 2 to 3m in diameter.
In sections stained with Delafield’s haematoxylin and van Gieson’s
solution, the granules are of a pale brownish pink colour. These
intracellular granules resemble very closely in appearance the
granules composing the casts in the lumen of the tubules. They were
only found in the cortical region of the kidney, and appeared to be
almost entirely limited to the epithelium lining the convoluted
tubules. They were never observed in the flattened epithelium of
the glomeruli nor in the epithelium lining the collecting tubules of
Bellini.

Analogous appearances were observed in the sections of the
kidneys of the experimental rabbits, but here the granules were not
present in such large numbers as in the dogs.

As in the case of the plugs found in the lumen of the tubules, one
is at present unable to decide as to the exact nature of these
granules. There appears, however, to be but little doubt that they
are derived, in part, at least, from haemoglobin, and are connected
with its excretion through the kidney.

Attempts to demonstrate the existence of iron in the epithelial cells
lining the tubules of these kidneys were unsuccessful. Here again,
however, the explanation may be that the haemoglobin had not
sufficiently broken down for liberation of the iron from its proteid
combination to have occurred. It is suggestive in this connection to
note that Stieda (1893) has described in the kidneys of blackwater
fever patients the existence of granules in the epithelium of the
convoluted tubes, and states that these granules gave the iron
reaction.

REFERENCES
Stiepa H. (1893). _Einige histologische Befunde bei tropischer Malaria. Centralbl. f. allg.
Patholog. u. path. Anat., IV, pp. 321-331.

Werner (1907). Ueber die Nieren beim Schwarzwasserfieber, Archiv fiir Schiffs- und
Tropenhygiene, XI, Beiheft 6.

Yorke W. ano Nauss R. W. (1911). The mechanism of the production of suppression
of urine in Blackwater fever. Annals of Trop. Med. and Parasir., V, 2, p. 287.

Ha! iene Smivigy alps
‘6 peri dae pl i" ree. For t i
= hy " ;

Av aa

' eh) ee TL : f

; : i’ hi ne 7 : 1 ns

408

DESCRIPTION OF PLATE XVII

The tissues were fixed in formalin and the sections stained by
Heidenhain’s iron alum haematoxylin method.

Fig. 1.—Section of renal cortex of a pup which died from
Piro plasma canis during the passage of haemoglobinuria.
In the epithelium lining the convoluted tubules are
numerous darkly stained granules varying considerably
in size. The granules are not present in Bowman’s
capsule.

Fig. 2.-Section of renal cortex of a rabbit four hours after the
intravenous injection of haemoglobin. This kidney
presents the same appearances as are shown in Fig. 1.

PLATE XVIL.

7 =
g /, % 1d & op . y e@ ee N ¥ 6 7 a)
RAN Ly i{eg tt rhe & ar GF -2
be Ow DO O7k care he
*) . 4 fos: © ay.

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Py», - at ‘cad < % 4° ‘
oy at BE rat Pa oO a ier ee?
SMES BO Re ee .
- 3 ~ » wy a ‘ z ( 4 Oe 9%
a LP ug: “Sk:

le A.

P. P. Press, Iinp.

409
PSEUDO-RELAPSES IN CASES OF
MALARIAL FEVER DURING
CONTINUOUS QUININE TREAT-

MENT

BY
nik BONae hOSs, KC.B., F.R.S.,

AND

DAVID. THOMSON, M.B.,,CH.B., D.P.H.
(Received for publication 18 September, 1911)

There seems to be a current belief that relapses may occasionally
occur in cases of malarial fever even during continuous quinine
treatment. Caccini (quoted by Ross, 1910) states that a relapse
occurred in 15 per cent. of 1,002 cases which had quinine daily.
We cannot, however, find accurate data regarding all these
relapses, as to whether parasites were found in the blood during the
relapse, or how much quinine had been given, or for what period.
Again, from various sources it has been stated that cases of malaria
occur in the Amazon region which are resistant to quinine treatment ;
but so far as we know accurate data are not given regarding the
presence or absence of parasites during this unsuccessful treatment.

During the past two years we have been studying the effects of
quinine on malaria, employing enumerative methods by which we
constantly know the number of parasites present in the blood per
c.mm. Seventy-five cases studied in this way all showed the
remarkable destructive power of quinine towards the asexual
malarial parasites. In all cases where quinine was given in doses
of ten grains thrice daily, it was almost impossible to find asexual
parasites in the blood after three days of the treatment, no matter
how numerous the parasites were before the treatment was
commenced. In no case did we ever discover a reappearance of these
parasites while this dosage was continued.* It would appear
to us, that so far, no drug has been found with so great a curative

* Such a case hae, however, occurred while this article was in the press. It will be
reported later.

410

power in any disease, as that of quinine in malaria. In our cases of
malaria, which came from various parts of the world, including the
Amazon, it never failed to show a very remarkable and rapid
curative power. It is vastly superior to arsenic, methylene blue,
trypan blue, picric acid, etc. We would like, however, to call
attention to apparent relapses occurring during quinine treatment.
In five (three cases P. falczparum, two cases P. vivax) or 6°7 per cent.
of our cases, a sudden isolated rise of temperature occurred during
the quinine treatment, accompanied sometimes with a feeling of
cold and slight shivering. No asexual parasites could be detected
on prolonged search by thick film during these attacks of fever.
On one occasion the blood was examined by one of us and thirteen
students for over half an hour, yet no parasites either sexual or
asexual could be found. All these apparent relapses were, therefore,
non-parasitic relapses. One naturally wonders if these are
connected in any way with the malarial infection, or if it is due to
the quinine treatment, or if they are mere coincidences, the fever
being due to some other cause. We investigated the hospital
records of one hundred cases of various diseases, not malarial,
and found that similar more or less inexplicable isolated rises of
temperature occurred in 17 per cent. of them. The diseases in which
these isolated temperature rises occurred most frequently were cases
of latent phthisis, cases of valvular heart disease, Bright’s disease,
chorea, rheumatism and bronchitis, and to a less frequent extent in
various other conditions, and in one case of spastic paraplegia.
These isolated and more or less inexplicable rises of temperature,
therefore, occur in other diseases during treatment as well as in
malaria, and it seems possible that they may have no real connection
with the original disease. In some cases the temperature could be
explained by a sudden and transitory inflammation of the tonsils.
One of our cases of malaria had a slight tonsillitis during his
pseudo-relapse. Again these rises of temperature in two of our
cases did not occur on the proper day, so that they would appear not
to be malarial.

Although numerous non-parasitic rises of temperature with rigors
occur in blackwater fever, and although it has been shown by one
of us (D. T.) that, from the behaviour of the leucocytes, one might
infer that the malarial virus lingers long in the system in spite of

411

continuous quinine treatment, yet we have no proof that these
pseudo-relapses are due to the disease. Some of the relapses during
quinine treatment recorded by Caccini may possibly correspond to
the 17 per cent. of incidental rises of temperature found in cases
not malarial, during treatment in hospital. We therefore think that
accurate enumerative observations are needed before the possibility
of such relapses, and of cases resistant to quinine, can be fully

accepted.

LITERATURE

Caccint (1902). ‘Fourn. of Tropical Medicine, Nos. 8, 9, 10, 11, 12.

Ross (1g10). Prevention of Malaria, p. 138.

412
sevdo- Relafs ses During a Treatment. (Three Cases. P falciparum.)

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413
THE TRYPANOSOMES FOUND IN TWO
HORSES NATURALLY INFECTED IN
THE GAMBIA
WARRINGTON YORKE

AND
B. BLACKLOCK

(From the Runcorn Research Laboratories of the Liverpool School
of Tropical Medicine)

(Received for publication 30 September, 1911)

CONTENTS
PAGE
LORIE eu ToC: atte oa poet oS A de 2 ee 413
II. Morphology of the Trypanosomes in the Blood of the two Horses .......c..0.04. 414
III. Results-of Inoculation into Laboratory Animals ...........c.ccccecceceeeeneseevece ces 417
IV. Morphology of the Trypanosomes in various Laboratory Animals ................+. 417
Pe eNO ARAHOTLOL LUC MLV DANOSOMICH © cots 13a%s dégdy vas cuuceysssveceevsespgesronevesececsaree 428
ee MMEEIEVIOLIGING LEV EANGEOINED «orto. paseds-cascrPavsecroperétcorneesecescacassccoccsvsesseves 429
LTE SU To ee ae ae A Be Bo i a ee ee Le 8 A Fos) hee oe ie 432

I. ORIGIN OF THE STRAINS

In June, 1911, we were enabled to obtain trypanosomes from
two horses, naturally infected in the Gambia. For the first strain
we are indebted to Professor Todd, who kindly sent to Runcorn a
polo pony, Horse A, with a natural infection. Trypanosomes were
found in the blood of this animal for the first time in March of this
year. For the second strain we are indebted to Sir John
MacFadyean, who kindly supplied us with a mouse infected from a
horse, Horse B, brought to him from the Gambia by Mr. Foster,
F.R.C.V.S. By the courtesy of the latter we were permitted to
make films from the blood of this horse on its arrival in Liverpool.
In this animal the first history of illness dates from about the
middle of April, 1911, and trypanosomes were found in its blood
for the first time on the 6th of May.

414

Il. MORPHOLOGY OF THE TRYPANOSOMES IN THE
BLOOD OF THE TWO HORSES

The parasites found in films made from the blood of the two
horses immediately upon their arrival in Liverpool presented
remarkable morphological differences. In fact the trypanosome
observed in Horse A was easily distinguishable morphologically
from that infecting Horse B. In the former the parasites were of
considerable length, uniform, and were furnished with a distinct
free flagellum, whereas in the latter they were much shorter and no
free flagellated forms were found. In view of this observation we
decided to study in detail the parasites infecting each of the
animals.

A. Characters of the trypanosome infecting Horse A. When
examined in fresh preparations the parasite exhibited remarkable
activity, dashing across the field of the microscope in a manner
strongly suggestive of 7. vivax. In films fixed in absolute alcohol
and stained with Giemsa’s solution, the trypanosomes appear to be
remarkably uniform in length. The posterior portion of the
parasite is broad, and the body gradually tapers towards the
anterior end. The nucleus lies near the centre of the animal, but
anterior to the broadest portion of the body; it is, as a rule, well
defined, but occasionally somewhat diffuse. The blepharoplast,
round and distinct, is situated either laterally close to the posterior
extremity, or terminally; the membrane is simple and narrow. The
parasite has a well-marked flagellum, the free portion varying from
4m to-7. As will be seen from Table I, the length* of the
trypanosome varies from 171 m to 25°4 » with an average of 20°60 p.

In addition to the form just described, occasional short,
non-flagellar trypanosomes are encountered. These are, however,
exceedingly scarce, only two such being met with in 1,000
trypanosomes counted. :

* Method of Measuring. In every case the blood films were fixed in absolute alcohol and stained
with Giemsa. The parasites were drawn with the aid of the Abbé camera lucida at a magnification
of 2,100 diameters, and measured along the middle line of the body from the posterior extremity
to the tip of the flagellum. The actual length of the parasite was then determined by dividing by

the magnification. The trypanosomes were drawn as they came, only dividing forms being ignored.

Tasie 1.—Measurements of the Trypanosomes in Morse A

|
Date Animal No. measured | Maximum Minimum Average

|

June 3 | Horse A | 20 25°4 18-6 23°2
|

6 20 23°2 T4°0 1g't

9 20 23:2 17"! 20'2

II os 20 23:8 19°0 20°6

12 20 23°8 17°1 20°9

13 20 24°2 Tig 21°3
|

16 3 | 20 22°8 185 21°0
|

21 20 23'0 18°C 21°2

22 20 238 122 20°0

22 a 20 22°32 18°5 20°!

—_——___--. [=e — ——— ee ——— —

| |

Total. 5.0%: 200 25°4 11°9 20°6

B. Characters of the trypanosome infecting Horse b. We had
no opportunity of examining this trypanosome in fresh preparations
of the blood.

In stained preparations, the parasites found were invariably
short and non-flagellated, the undulating membrane being but
slightly developed, and the body of the creature prolonged to
the extremity of the flagellum. Nucleus oval and central,
blepharoplast terminal or latero-terminal. The length of 100
trypanosomes varied from 10 » to 16°9 #, with an average of 12°9 p.
No free flagellated forms were met with in the blood of this horse,
but it must be remarked that we were only in a position to examine
films made on a single day of the infection.

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417
III. RESULTS OF INOCULATION INTO
LABORATORY ANIMALS

A. Axntmals inoculated from Horse A. Two rats and two mice
inoculated from this horse, at a time when the peripheral blood
contained trypanosomes in fair numbers (three to a field, objective
DD, ocular No. 4), failed to become infected. Two of four
guinea-pigs, and two of three rabbits inoculated with the animal’s
blood became infected. Two goats showed parasites on the
eleventh and fourteenth day respectively, after inoculation. The
results of the inoculations made from these animals are given in
Table II.

As will be seen from this table, the rats and mice inoculated
directly from the horse, and those sub-inoculated with the
trypanosome after passage through a goat were all refractory. - This
applies also to those rats and mice inoculated with the parasites
after a second passage through goats. Rats and mice
sub-inoculated from a rabbit infected directly from the horse
contracted the disease, and from one of these mice a further series
of rats and mice was with difficulty infected.

In Table III are given details of our experimental inoculations
into laboratory animals.

B. Animals inoculated from Horse B. Ne understand from
Sir John MacFadyean that mice inoculated from this horse were
easily infected. From a mouse received from him infected with
this strain we found that goats, rabbits, guinea-pigs, dogs, rats
and mice were easily infected. In mice the incubation period
averaged five days and death occurred on the fourteenth day.

IV. MORPHOLOGY OF THE TRYPANOSOMES IN VARIOUS
LABORATORY ANIMALS.

Trypanosomes from Horse A. The trypanosome found in goats
resembled in every respect that occurring in the blood of the horse.
They were all long (17 » to 26°), free flagellated forms, with
the exception of very occasional short (14 # to 15 ») non-flagellated
forms. In Rabbit 1467 the parasites averaged from 20'2 4 to
21°7 # in length, until the day on which death occurred, when only
short forms of an average length of 14'7 m were encountered. In
Rabbit 1494, also inoculated directly from the horse, only short
forms, with an average length of 11°3 to 13°1 » were found.
Similarly only short non-flagellated trypanosomes were met with

418

Taste IIT.— Pathogenicity of the Trypanosome from Horse 4 in Laboratory Animals

Day on which

|
|
|

No. of Animal from parasites first | Day on which | Remarks
Experiment — which inoculated | found in blood | death occurred |
ee ae — SS eee
Horse A Unknown Unknown | June 24th, rrr}
1608 A Mouse 1535 A | 15th -- Alive on 2tet day
Goats
1485 Horse A 12th 26th
1497 ¥ 1oth 44th
1559 Goat 1497 11th -— | Alive on 6cth day
1584 Mouse 1535 8 ~ - | Alive on 46th day, parasites
| never seen
1605 Goat 1559 gth —_ Alive on 30th day
Rassits
: )
1467 Horse A | 23rd 36th
1489 -- -- Alive on rroth day ; parasites
| never seen
14.94 . | 1gth - | Alive on to2nd day
1694 Goat 1559 16th = "Alive on 32nd day
GUINEA-PIGS
} °
1490 Horse A —- 64th | Parasites never found in
| blood
1495 - 1gth 22nd
1519 5 ~- 52nd Parasites never found
1520 > 18th — | Alive on gznd day
MA 5 Fee Paes come Oe
Dogs
1524 Goat 1485 ith 20th
1553 A pe 1497 — — Alive on 55th day ; parasites
never seen
1553 B S59 —~ — _ Alive on 48th day ; parasites
never seen
1609 my 2tGos — — | Alive on 20th day ; parasites
___never seen

AQ

Tani III.—continued—Pathogenicity of the Trypanosome from Horse 4 in Laboratory Animals

| Day on which |
No. of | Animal from parasites first | Day on which | Remarks
Experiment | which inoculated | found in blood | death occurred ,

Rats
mate ee i, Pes Gaon ee ,
1465 Horse A -- gth Parasites never seen
1466 7 os | 6th .
1406 2 mati | 4th ” ”
1526A Goat 1407 = | = Alive on 65th day ; parasites
never seen
1526 B ” aie | ae ” ”
1526 C 53 a = ”
1532 A »» -- x3
1632B a ~
1534 Rabbit 1467 6th 23rd Few parasites on two days
only
1582 A Goat 1559 - 3oth Parasites never seen
1582 B 59 _ 36th *
1582 C 9 -- -= Alive on 36th day; parasites
| never seen
1582 D ” = Gas ” ’
1582 E a3 — - a ee
1582 F > _— — * 5
1586A Mouse 1535 B 25th | -- Alive on 45th day ; parasites
_ never seen
1586 B 2 _ 25th Parasites never seen
1608 B, 1 Mouse 1535 A 8th =| — Alive on 20th day
1608 B 2 - -- = Alive on 20th day ; parasites
never seen
1608 B, 3 ” — 14th Parasites never seen

1610 A Pup 1563 -— 8th a8

420

Tasve IIT.~continved—Pathogenicity of the Trypanosome from Horse 4 in Laboratory Animals

—_— 7 ro

Day on which |
No. of _ Animal from | parasites first | Day on which Remarks
Experiment — which inoculated | found in blood | death occurred

Mice

1498 A | Horse A | _ 6th Parasites never seen

1498 B = — sth ”

1527A Goat 1497 -= 8th | -
| | |

1527 B * _ — Alive on goth day; parasites

: never seen

1535 A | Rabbit 1467 18th — Killed for inoculation on 58th
day

1535 B 3 ard — Killed for inoculation on
32nd day

1583 A - | Goat 1559 7th

1583 B ¥ — | 7th Parasites never seen

1583 C ' | — — Alive on 47th day

1585 A ~ Mouse 1 535 B = 3oth Parasites never seen

1585 C = 24th ;

Taste [V.—Pathogenicity of the Parasite from Horse B in Laboratory Animals -

| {
| Day on which |
No. of _ Animal from | parasites first | Day on which | Remarks

Experiment | which inoculated | found in blood | death occurred |
5,
Goats --
1587 die Mouse 1562 A 3th «=f — ~~") Alive on 46th day
“4 : [ee Se liwterstemen es
Docs
1624 | Goat 1587 7th | — | Alive on 12th day
wh 7 oe oe eee
RaBBiTs
1603 Goat 1587 | 8th — | Alive on 30th day

Le

GUINEA-PIGS

1620 Goat 1587 8th — Alive on 12th day

alin 40 )

421

Tare IV.—continued—Pathogenicity of the Parasite from Horse B in Laboratory Animals

| Day on which |
No. of, Animal from parasites first | Day on which Remarks
Experiment | which inoculated found in blood | death occurred
|

ne ml |

Rats
1618 A Goat 1587 oth — Alive on 12th day
1618 B 8th — |
= ee ee | es
Mice
1554 Mouse (Sir J. sth 7) roth
| Mc.) |
1562 A | Mouse 1554 | 6th 14th
1562 B | 7 5th 14th
1514 Mouse 1562 A 5th | 8th
1619 A | Goat 1587 4th _- | Alive on 11th dav
| |
1619 B | os 5th as |

in the blood of infected guinea-pigs. A pup inoculated from one
of the goats showed only short (10'9 » to 18°7 #) non-flagellated
forms. Only short (96 to 18 #) non-flagellated trypanosomes
were found in the blood of a rat and two mice sub-inoculated from
one of the rabbits.

A detailed account of the measurements of the trypanosomes in
various animals experimentally infected is given in Table V.

It will be seen from this table that the parasite varies
morphologically in different animals, and sometimes even in the
same animal on different days of the infection, in a most
remarkable manner. .

Trypanosome from Horse b. The trypanosomes found in the
blood of mice infected with this strain were short (10°4 » to 18 p#)
non-flagellated, and were similar in appearance to those occurring
in the blood of mice infected with Horse A strain. Dogs
sub-inoculated from the mice contained trypanosomes varying in
length from 10 » to-49 #, and in rabbits inoculated from the same
source the trypanosomes varied from 9’5 m to 15'2 #.

In the blood of goats inoculated from the mice the trypanosomes
measured from 10'9 # to 18 p.

Table VI shows the measurement of the trypanosome in animals
infected with this strain.

422

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428

V. COMPARISON OF THE TRYPANOSOMES IN THE
TWO HORSES

The fact that the trypanosome occurring in Horse A 1s
morphologically distinct from that present in films made from the
blood of Horse B on its arrival in England—both animals being
naturally infected in the Gambia—appears to us to be one of
considerable interest. The morphological distinction, as we have
previously stated, consists in the fact that whereas the
trypanosomes infecting Horse A were almost invariably free
flagellated and uniform in length (average length 20°6 »), those
observed in the blood of Horse B were short and without free
flagellum.

A second point of interest is that the trypanosomes found in
the blood of certain laboratory animals (dog, guinea-pig, rat and
mouse), inoculated with the strain derived from Horse A, in no way
resembled the majority of the parasites present in the blood of this
horse. The trypanosome in these animals was short and
non-flagellated, and its measurements corresponded closely to those
of the parasite present in the blood of Horse B. No long free
flagellated forms were at any time observed in the blood of these
laboratory animals. On the contrary goats infected with the
Horse A strain exhibited the long free flagellated forms, and only
very exceptionally was a stumpy non-flagellar form seen. One of
the rabbits infected from this horse was remarkable in that until
the last day of the disease the blood contained, with the exception
of very occasional short trypanosomes, only long free flagellated
parasites. On the last day, however, none of the latter forms was
noticed, but a large number of the short non-flagellar trypanosomes
was present.

The question now arises, was Horse A infected with a single
trypanosome capable under certain conditions of assuming a long free
flagellum and under other conditions of existing in a non-flagellar
state? Either this is so or this animal was infected with two distinct
trypanosomes, one of which was a_ short non-flagellated
trypanosome, and the other a long free flagellated trypanosome.
The short trypanosome occasionally found in the blood of
Horse A was similar, morphologically, to the parasite infecting
Horse B. The point to be determined, therefore, is whether or no

429

the long forms infecting Horse A are of the same species as the
short stumpy forms occurring in the same horse and in Horse B.
We have endeavoured to decide this question experimentally. A
series of laboratory animals, goats, dogs, guinea-pigs, rats and
mice, inoculated with the blood of a mouse infected with the
parasite from Horse B, became easily infected, and in all these
animals the trypanosome retained its short character, no free
flagellated forms being found. Next, the long free flagellated
parasite from Horse A, as it appeared in the blood of a goat, was
inoculated into a series of rats and mice. These sub-inoculated
animals did not become infected. Again, the short forms derived
from Horse A, as seen in the blood of an infected mouse, were
inoculated into a horse, goat, rats and mice. Of these animals the
goat gave a negative result, parasites never being found in its
blood, and test animals—dog, rat, mouse—inoculated with large
amounts of its blood, not becoming infected. Some of the mice
and rats had a few parasites in the blood for one or two days only ;
the horse has had parasites for one day. In none of these animals,
including the horse, have free flagellated trypanosomes been
observed, short forms only, and these in very small numbers,
appearing in their blood. Next, certain animals—rabbits, rats,
mice—which had proved quite refractory to the parasites, long or
short, derived from Horse A were inoculated with parasites derived
from Horse B, and became easily infected. Further experiments

are being done.

VI. IDENTITY OF THE TRYPANOSOMES

During the past few years several attempts have been made, for
example, by Montgomery and Kinghorn (1908-9) and Laveran
(1911), to classify the various pathogenic trypanosomes.
Nevertheless, we are partly at a loss to assign a place in any of these
classifications to the trypanosomes with which we are at present
dealing.

We shall first consider the trypanosome found in the blood of
Horse B. An examination of it leads one to associate this parasite
with quite a definite group. Morphologically this trypanosome is
closely allied to 7. dimorphon in the sense in which the term 1s

430

used by Laveran and Mesnil. In its reaction upon laboratory
animals also it corresponds definitely with this type of trypanosome.
It falls, therefore, into that group which Bruce has designated by
the term 7. pecorum, in which he includes 7. dzmorphon (Laveran
and Mesnil), 7. congolense (Broden), 7. confusum (Montgomery
and Kinghorn), Edington’s trypanosome from Zanzibar, the
trypanosome from Chai-Chai (Theiler), and Bevan’s trypanosome
from Southern Rhodesia. We consider, therefore, that the
trypanosome in the blood of Horse B is probably 7. dimorphon
(Laveran and Mesnil).

We take next the trypanosome infecting Horse A. From a
study of the parasites in the blood of Horse A merely, one would
naturally associate the trypanosome with that group of which
TI. vivax is a type, and in which Bruce (1910) has placed the
T. vivax of Uganda, that from Togoland, the trypanosome from
Pordage’s ox, and also 7. cazalbouz. The reasons for associating
the trypanosome as it appeared in the blood of Horse A, with the
trypanosomes of this group are its extraordinary activity as seen
in fresh preparations, and its morphological appearances in stained
blood films, namely its uniformity in size, the peculiar shape of the
body, the smallness of the membrane, and the presence of a free
flagellum. The average length of this parasite is 206 w, which
is less than that given by Bruce for 7. vivax (23'7 »), but which
corresponds very closely to the length of 7. cazalboui (21 p).

The view that this long form of parasite is closely akin to the
T. vivax group is strengthened by observing the results of
inoculations into laboratory animals, because we failed to find this
form in the blood of dogs, guinea-pigs, rats and mice inoculated
with the blood of Horse A, while a trypanosome whose appearance
in fresh and stained preparations was identical with that of
Horse A, was found in goats inoculated from this animal.

Against the view that this trypanosome from Horse A is
T. vivax, we have the fact, mentioned above, that its length is less.
In addition to this, however, the fact that we succeeded in infecting
a rabbit with this form of trypanosome, militates against such a
conclusion. Bruce,* in his paper on the diseases of domestic animals

* Bruce, Joc. cit.

431

in Uganda, writes, in reference to 7. vivar, ‘This species of
trypanosome is similar to 7. xanum, in that it is only pathogenic to
equines and bovines, and has no effect on the smaller laboratory
animals.’ Moreover, Laveran* in a recent paper, states that the
rabbit, amongst other laboratory animals, is refractory to
T. cazalbouz. We recall here Bruce’s statementt+ that it is probable
that Z. vivax and T. cazalboui are the same species.

We pass now to the consideration of the short forms of parasite
found in small numbers in the blood of Horse A, and in the blood
of goats inoculated from it, and in larger numbers in the blood of
such dogs, rabbits, guinea-pigs, rats and mice as we succeeded in
infecting from the horse or goats. Seen in fresh films of the blood
of these animals, and studied in stained preparations, this parasite
has considerable resemblance to the 7. pecorum group.

Against the view that it is identical with 7. pecorum is the fact
that we succeeded in infecting guinea-pigs with the parasite.
Bruce} found that guinea-pigs were refractory to 7. pecorum, and
Theiler (1909) observed that a trypanosome from Zululand, included
by Bruce under the name 7. fecorum, was also innocuous to
guinea-pigs. Whether, however, the apparent insusceptibility on
the part of a certain laboratory animal to infection with a
trypanosome is sufficient ground for differentiating a parasite from
similar trypanosomes known to infect such animal, appears to us
to be a very doubtful question. Even the most recent literature
contains contradictions on this point, e.g., Laveran§ states that
T. vivax is pathogenic for the dog and rat. Further, attention has
frequently been drawn to the fact that the virulence of a
trypanosome can be considerably altered by passage through various
animals.

But a glance at Table II will show that the short forms from
Horse A are, up to now, at any rate, of a very low pathogenicity for
several laboratory animals, especially noticeable, perhaps, in the
case of mice, rats and dogs, and in this respect they differ entirely,
not only from the short forms of parasite found in Horse B, but

* Laveran, loc. cit.
+ Bruce, T. vivax, loc. cit.
t Bruce, T. pecorum, loc. cit.

$ Laveran, loc. cit.

432

also from our laboratory strains of 7. dzmorphon and T. pecorum.
Even those animals which became infected, with the exception of
a dog, had a chronic infection, and some appear to have recovered.

In regard then to the infection in Horse A, we find ourselves
confronted by two possibilities—

1. Horse A is suffering from a double infection. One
trypanosome resembles 7. vivax, and the other resembles the
7. pecorum group, but presents remarkable differences from this
group in its pathogenicity to laboratory animals.

or

2. Horse A is infected with one trypanosome. This
trypanosome resembles closely 7. vzvav. But if we are to accept
this view, one’s conception of 7. vivax requires to be somewhat
modified. In this connection, however, we know that Ziemann
(1905) discovered short forms occasionally in 7. vzvax infection.

VII. CONCLUSION

1. We consider the trypanosome found in Horse B to be
T. dimorphon, sensu Laveran and Mesnil.

2. The long form in Horse A appears to us to be 7. viva.

3. As regards the short form found in Horse A, we do not feel
justified at the present stage in assigning its position. It may be
a dimorphon-like trypanosome of low pathogenicity, or simply a
modification of the long parasite of Horse A.

REFERENCES TO LITERATURE

Montcomery AND KinGrorn (1908-9). On the nomenclature of the mammalian trypano-
somes observed in North-Western Rhodesia. Annals of Trop. Med. and Parasit., p. 333-

Laveran (1911). Identification et essai de classification des trypanosomes des mammiferes.
Annales de l'Institut Pasteur, p. 497.

Bruce (1909-10). Trypanosome diseases of domestic animals in Uganda. I, 7. pecorum.
Proc. Roy. Soc., B, LXXXII, p. 468.

Bruce and others (1910). Trypanosome diseases of domestic animals in Uganda. _ III, T. vrvax
(Ziemann). Proc. Roy. Soc., B, Vol. LX XXIII, p. 15.

‘Tne1Ler (1909). Sur l’existence de Trypanosoma dimorphon ou d’une espeéce voisine au Mozam-
bique et au Zoulouland. Bulletin de la Société de Pathologie Exotique, p. 39, Jan. 11.

ZIEMANN (1905). Nachtrag zum Beitrag zur Trypanosomenfrage, Centralblatt fiir Bak.,
p- 662.

DESCRIPTION OF PLATE x2VUOL

Films stained by Giemsa’s method after fixing in absolute
alcohol. Figures drawn with Abbé camera lucida, using 2 mm.
apochromatic objective and No. 18 compensating ocular (Zeiss).
Magnification reduced to 2000 diameters.

TRYPANOSOMES FROM Hovse A STRAIN

Figs. 1-3.—Parasites from Horse A.
Fig. 4.—Parasite from Goat.

Figs. 5, 6.—Parasites from Dog.
Figs. 7, 8.—Parasites from Rabbit.
Fig. 9.—Parasite from Guinea-pig.
Fig. 10.—Parasite from Rat.

Figs. 11, 12.—Parasites from Mouse.

TRYPANOSOMES FROM Horse B STRAIN

Figs. 1-3.—Parasites from Horse B.
Fig. 4.—Parasite from Goat.

Fig. 5.—Parasite from Dog.

Fig. 6.—Parasite from Rabbit.
Fig. 7.—Parasite from Guinea-pig.
Figs. 8, 9.—-Parasites from Rat.
Fig. 10.-—Parasite from Mouse.

PLATE XVIII.

9 li \ 12

10
Horse A STRAIN

a

P. P. Press, Iinp. Horse B STRAIN

435

mI EXAMINATION OF THE’ CITY “OF
GEORGETOWN, BRITISH GUIANA, FOR
THE BREEDING PLACES OF MOSQUITOS

BY

KK, - Wish NEB B.S,, B.SC.,.D-P.H.

GOVERNMENT BACTERIOLOGITST.
(Recetved for publication 1 November, 1911)

The coast line of British Guiana is really an alluvial mud flat,
characterised by its flatness and by its lowness of level, which is
four to five feet below that of the sea at high water of spring
tides. Its soil is heavy alluvial clay, mixed with marine salts and
vegetable deposits. The city of Georgetown lies at the angle of
this low-lying sea coast and the mouth of the Demerara river. As
its level is below that of the sea, the city and the cultivated lands
around it require embankments on every side. Sluices on these
embankments at suitable times of tide give vent to the surface
drainage of the land. The city is intersected by numerous trenches
and open drains, varying from two to ten feet wide, which
discharge the surface water alone. Water for drinking is rain
water collected from the roofs and conserved in large wooden vats
and iron cisterns.

There is a further water supply carried by pipes and distributed
to the various parts of the city, used mainly for fire purposes, but
also for flushing, washing stables, watering, etc. This supply is
a brown peaty water, obtained through an open trench from an
empoldered area some ten miles from the city. Sewage disposal is
mainly by cess-pits; in a few limited areas the pail system is
utilised.

In June, 1909, as an outcome of suggestions of Sir R. Boyce,
it was proposed to make a close examination of Georgetown, with
reference to the breeding places of the myriads of mosquitos always
present.

Difficulties arose as to the right of entry of the Staff of the
Bacteriological Department on premises, and it became necessary)

436

to await the passing of a Mosquito Ordinance bestowing powers of
entry; this was achieved in September, 1910.

This mosquito survey was begun in December, 1910, and was
finished in September, 1911, and was carried out by the following
persons duly authorised by the Honourable the Surgeon-
General :—The Government Bacteriologist, the Assistant Govern-
ment Bacteriologist, Dr. Duncan, and two skilled Laboratory
Attendants specially trained for the work.

In spite of the size and great extent of this city of 60,000
inhabitants, it was decided to enter, examine and report upon
all premises, lots, yards, etc., within its boundaries, and no
premises were left unvisited The parks and other open spaces,
Government buildings, barracks, etc., were visited and reported
upon. The trenches in various parts of the city and outskirts were
systematically examined as to the conditions under which they
were breeding or likely to breed mosquitos. In addition, certain
pastures and waste lands lying to the windward side of the city
were also surveyed.

Special notice was directed to the kind of mosquito larvae or
eggs found, the nature of the places in which breeding was actually
occurring, to potential breeding places (1.e., where breeding might
occur at times other than that of this visit of inspection).

Records were also made of the condition of the vats, the extent
and efficiency of screening and also of the general condition of
the yards.

During the progress of this survey, as opportunity occurred,
occasion was taken to interest the people in this subject, and
numerous (50 to 60) small lectures delivered.

Two thousand five hundred and sixty premises were entered
and examined, and of these 1,490 were found to be breeding
mosquitos at the time of inspection.

A detailed statement of the above particulars with respect to
each lot and half lot occupied has been tabulated and forwarded
to the Honourable the Surgeon-General.

The following table shows concisely the various districts of
the city, the time of the year when examined, the number of
premises, the number and percentage of those breeding mosquitos
the number of premises breeding S/egomyza fasciata, Culex
fatigans, Anopheles (Cellia) albipes and other mosquitos : —

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438

From this it will be seen that all parts of the city are affected
alike; premises of the rich and poor are equally involved.

The following table indicates the number of vats screened,
defectively screened and totally unscreened, the number of premises
with potential breeding places, and also the number of these

premises kept in a disgraceful condition : —

| Vats | Vats Vats Potential | Disgraceful
Ward | screened screened unscreened breeding yards
| well defectively places
ay eras SE Pe a — ——|—_—_ —- —_ —_— |] —_ ——- — —
Kingston .| 83 51 6 73 13
oe a |
Stabroek 103 102 8 70 6
Queenstown | 64 70 8 go 36
ion SSS ———* —
Charlestown 184 155 12 204 26
<< —— oe
Alberttown | 197 122 ) 241 8
a ee ene OT be See Sake | eee | |
Cummingsburg 646 284 13 214 18
Bourda 333 138 13 216 24
Lacytown ... 283 213 12 18S 22
Robbstown and Newtown | 43 15 2) 14 $
Werken Rust 421 170 6 275 68
Wharves, Riverside 99 20 6 28 4
Wortmanville 145 SI I 162 | 34
Railway line wal 4 5 1 76 4
2 ASS Se ed Aa a. ie Gee ee
Totals 2605 1396 88 1848 271

439

The Anopheline mosquitos (nearly always Cellza albipes,
occasionally Cellza argyrotarsis) were found breeding in nearly all
trenches, where overgrown with vegetation, both in the city and
environs. Where no vegetation was permitted no Anopheline larvae
were discovered. These mosquitos were also found breeding in
the hollowed-out stumps of trees which had been cut down in the
lots and by the roadside. Numerous Anopheline larvae may be
found during the rainy seasons, in the small grass-grown
cross-drains of the Queenstown district. They may occasionally
be met with in cocoanut shells and in the grass-grown pools and
trenches around the barracks.

The developmental forms of the various Culicine mosquitos
were most frequently met with, and the breeding places may be
summed up as follows :—

Stegomyta fasciata (syn. S. calopus) was found breeding
in vats, water barrels, tubs, tins, pots, cisterns and defective
gutters in the majority of the premises infected, including the
river wharves and the premises on the railway line. The
breeding places were always close to human habitations, and
were never found in trenches or unoccupied land.

Culex fatigans was found breeding in vats, tanks, barrels,
tubs and old tins on about one-tenth of the premises visited.

Culex confirmatus was found once in a_ pond in
Charlestown.

Melanoconion atratus was found breeding in open trenches
and ponds covered with vegetation such as in Thomas Street,
in the pools of the railway line, the ‘ Governor’s fish pond,’ and
the trenches by Kelly’s dam.

Culicelsa taenitorhynchus was a_ frequent habitant of
trenches, more especially those by the military barracks,
Kelly’s dam, and the sea wall.

Of the Aedine mosquitos only one specimen was found, viz. :
Aediomyia squamipenna. This was found breeding in trenches
and ponds covered by vegetation in Thomas Street, the ‘ Governor’s
fish pond,’ and by Kelly’s dam.

In the vats, barrels and tubs, the larvae found were almost
always Stegomyia fasciata or Culex faligans.

440

In old tins, broken crockery, bottles, calabashes, cocoanut
shells, fallen palm sheaths, etc., almost always the larvae
found were those of Stegomyza fasctata and Culex fatigans.
Occasionally, under these circumstances, Culex szmzlis, Culicelsa
taentorhynchus or Culex confirmatus were found.

In the trenches, when cleansed of vegetation, no evidence of
mosquito breeding was found, but where vegetation covered the
surface numerous larvae of Anophelines and Aedzomyza squami-
penna and Melanoconion atratus were discovered.

The ‘ Governor’s fish pond’ illustrates excellently the influence
of vegetation in giving protection to the larvae from the
ubiquitous predaceous fish (Gzvardinus poeciloides). When cleared
of vegetation, frequent examination failed to reveal larvae. The
presence of larvae was coincident with the growth of vegetation.

In the trenches and open lands to the windward side of the city,
where vegetation is associated with the water, larvae of Anophe-
lines, Culicelsa taeniorhynchus, Aediomyia sguamipenna and
Melanoconion atratus were readily found.

Those premises on which potential breeding places were
found numbered 1,848, or 70°9 per cent. of all premises visited.
While many such potential breeding places were defectively
screened vats, old pots and tins, the great majority were barrels.
On no less than 1,203 premises were one or more barrels holding
either rain water or the peaty water from the Lamaha Conservancy.
On only 11°5 per cent. of these premises was any attempt at
screening a barrel shown, and on the greater number of this small
minority the screening (generally very defective) was only
vouchsafed to one or two out of numerous barrels. —

The systematic inspection of the vats (this does not refer to
iron cisterns or tanks) shows that 63°9 per cent. were effectively
screened. One thousand three hundred and ninety-six vats had
more or less defective covers nullifying any beneficial effect of
screening. In several instances pieces of wood were used to prop
open an otherwise efficient vat cover, thus rendering the purpose of
the cover useless.

In many vats an efficient cover had been put on and then
completely neglected; the sun drying the unseasoned wood caused
warping and the production of large cracks.

441

Eighty-eight vats were found in which there was no attempt at
screening.

A most serious disadvantage under which the inhabitants of
this city allow themselves to labour is the excess of vegetation
and litter in the yards and lots. No less than 261 premises were
kept in a condition which can only be described as disgraceful.
The exclusion of the sun keeps the premises damp and dark, and
provides excellent cover and breeding places for mosquitos, rats,
and other noxious insects, vermin, etc. The exclusion of fresh air
and the general dampness encourage and aid the spread of
tuberculosis.

The extraordinary collection on some premises of old tins and
other worthless rubbish giving rise to stagnant water has to be seen
to be believed.

The city of Georgetown is richly supplied, not only with
mosquitos but also with convenient and comfortable breeding
places. It seems almost impossible to realise that during the wet
season over 70 per cent. of all premises in this city are breeding
countless myriads of these pests, and that during the dry season
nearly 60 per cent. are equally incriminated.

This state of affairs, occurring as it does in a city priding itself
on being up to date, is scarcely to be credited, and reveals the
urgent necessity for vigorous and prompt action by _ those
responsible for the health of the city.

Undoubtedly the presence of numerous unscreened barrels
contributes most to this state of affairs, and the close screening of
a barrel and the provision of a tap near the bottom should be a
sme qua non for even tolerating their existence.

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443

A CASE OF HUMAN TRYPANOSOMIASIS
IN NYASALAND WITH A NOTE ON THE
PATHOGENIC AGENT

BY
HUGH S:*STANNUS
AND

WARRINGTON YORKE
(Recezved for publication 9 November, 1911)

In October, 1908, the first case of human trypanosomiasis was
discovered in Nyasaland. A native who was examined in
consequence of his having accompanied his employer through the
Congo was found to be infected. He presented no symptoms, and
from that time to the present, during which period treatment has
been given, he has preserved excellent health. During the next year
further cases came under observation which, in the absence of any
history of infection in the then known ‘ palpalis areas’ gave rise to
suspicions as to the local conditions in Nyasaland. Investigations
carried out to determine the presence or otherwise of Glossina
palpalis in the Protectorate proved negative, and uncertainty
persisted.

In the second half of the year 1910, however, the occurrence of
a case in a European, whose movements were exactly known,
followed by the discovery of some forty cases in a localised area
in the country, has brought into relief the subject of human
trypanosomiasis in Nyasaland.

For reasons which the above remarks make obvious, the case of
this European is now published in some detail, and the question of
the identity of the pathogenic agent dealt with in view of the
fact that a trypanosome derived from a case of sleeping sickness,
contracted in North-Eastern Rhodesia, has been shown by Stephens
and Fantham (1910) to present certain morphological peculiarities,
and also by Yorke (1910), a pathogenicity in experimental animals,
which differentiate it from all known strains of Trypanosoma
gambiense.

44

The history of the patient zs as follows.

P. R., aged 60, male, of Beaufort West, Cape Colony, came to
Nyasaland on a shooting trip; he landed at Chinde, and travelling
by the Zambezi and Shiré rivers, passed up through Blantyre on
July 27th, 1910, on his way to Angoniland, arriving at Manda on
July 1st, where he remained eleven days. On July Ist he travelled
to Mpastso, via Mpunzi and Dedza, thence to Mt. Dzobwe, forty
miles to the westward, and back again; leaving again on the 19th
he went to Mpinzi, remaining in the neighbourhood of the Diampwe
river until the 23rd, but reaching Nkoma the following day. Here
he remained till the 28th, when he proceeded to Mvera, where he
again spent some days. Here, on August 13th, he saw a case of
sleeping sickness, a native whom he photographed as he lay in the
open, but did not approach within a few yards of him. He left
Mvera for the lower country towards the shores of Lake Nyasa for
the shooting. On the 14th he was at Maganga’s village; the 15th
and 16th were spent in the Patsanjoka dambo, a large area of partly
uninhabited country, but containing much game. On the 17th he
was at Nsarula, a village on the Lintipi river. During these three
days all the party were bitten by tsetse flies (species unrecognised),
but the patient suffered most severely owing to an exposed neck.
A return was made to Mvera on the 18th, and the next day he went
on to Kongwe, where he complained that the bites on the neck were
painful, he, however, continued his journey the following day
(20th), and reached Nambamba on the 22nd. During the journey
he had not appeared very well, and on the day of arrival at
Nambamba gave up shooting owing to the pains in the neck. The
following day his neck was- examined by one of his companions,
who found a ‘lump about the size of a shilling, rather light in
colour surrounded by a dark purple ring,’ in the sub-occipital region
where pain had been complained of; at the centre of the lump was
what appeared to be a puncture due to the bite of a fly. On the
24th, at Sante, on the Bua river, pain in the neck was shooting up
into the head, causing severe headache. The lesion on the neck
was ‘hard as if an abscess were developing,’ so poultices were
applied ; the temperature was found to be 102'5° F. The following
day the swelling of the neck had extended; the face was puffy,
speech impeded and the temperature remained at 103° F:- On the

445

28th the lump had disappeared, but the oedema persisted. The
temperature varied between 102° and 104° F. during the next few
days, and the condition remained unchanged. On the last day of
August the blood was examined, and trypanosomes found in large
numbers. An injection of six grains of atoxyl was immediately
given (11 a.m.), followed twelve hours later by a critical rise of
temperature to 108° F., and then a fall to 99°F. the following
morning, when atoxyl (six grains) was again given.

Progress of case.

Rapid improvement took place to commence with, and by the
end of a few weeks patient was able to sit up all day and walk a
little; the appetite was good, and there was little in the way of
symptoms; constipation, an old trouble, was marked.

On September 24th there was an attack of adenitis, involving
the posterior cervical glands of both sides, which responded to
treatment by the application of Linimentum A.B.C.; one large
gland, about the size of a filbert-nut, persisted for one week. There
were several recurrences of this adenitis during the next two months.

A characteristic rash made its appearance on several occasions,
the first was on the 78th day, lasting two or three days. The
temperature showed a marked periodicity during the earlier part of
the illness; it rose on Tuesday of each week and remained between
102° and 104°F. till Thursday, followed by a period of normal
temperature till the following Monday; later the maxima showed a
steady fall and the apyrexial periods became progressively longer,
two days low fever and five days free in each week. The
accompanying temperature chart shows these points.

Preceding or accompanying the rises of temperature there were
constitutional symptoms, the patient feeling ill and bilious, ofter
with vomiting. Anaemia and emaciation, general cachexia, were
progressive though the mental condition remained good. With the
constitutional symptoms patient retired to bed, but at other times
sat up in a chair.

Such is a short account of the case up till the time he left the
country on January 13th, 1911, for South Africa, where he died in
April of the same year.

One fact is worthy of special note, and that is the time which
elapsed between the patient being bitten by tsetse fly and the onset

446

of symptoms which practically fixes the incubation period at between
six and ten days.

The treatment given was as follows:—For the first five weeks
six grains of atoxyl were injected intra-muscularly on Thursday and
Friday of each week. For a second period of five weeks three grains
of atoxyl were given every 3rd and. 4th day. From the 11th to the
18th week soamin (ten grains) was administered on two successive
days in every other week, and perchloride of mercury once in the
intervening weeks. As the injections of mercury were very painful,
and were not followed by any improvement, they were discontinued
and soamin alone administered in ten grain doses every Thursday
and Friday.

Blood. On the first occasion when the blood was examined
trypanosomes ‘swarmed,’ the field resembling the blood of a rat
heavily infected with 7. gambiense. Their numbers diminished
with treatment, and on some occasions none were found in the
course of a fairly quick examination. Though it was impossible
to make careful enumerations of trypanosomes, still it was noticed
there was a certain periodicity in their numbers corresponding to
the temperature curve, fewness being associated with the apyrexial
intervals, larger numbers with the rises in temperature.

Morphological features of the parasite in the blood of the
patient. Unfortunately, the material at our disposal was rather
limited, consisting of a slide of the blood made on August 31st, the
day on which the disease was first diagnosed, a couple of slides
made on November 21st, and one on January 4th, when the patient
passed through Zomba on his way to Chinde. The slide made on
August 3Ist contained numerous trypanosomes, and was sent to the
Sleeping Sickness Bureau and examined by Sir David Bruce, who
found that the parasite did not differ in any way from the Uganda
T. gambiense (1910).

In the specimens prepared on November 20th and January 4th
trypanosomes were more scanty, and the parasites could not be
distinguished in any way from T. gambiense. The parasite
presented the characteristic dimorphism, slender forms with long
free flagella, short stumpy forms with no free flagella, and
intermediate forms being found.

The Morphology of the parasite in animals experimentally

$47

infected. The parasite was also studied in the blood of several
experimental animals, and the results, in short, communicated by
us (IQII) to the Royal Society.

An English rabbit, bred in Nyasaland, previously shown to be
free from any trypanosomal infection was infected with the
trypanosome by subcutaneous inoculation with a small quantity of
the patient’s blood, taken on the 135th day, and subsequently
sub-inoculations were made into a monkey (Cercopithecus sp.) and a
goat. The animals were kept in fly-proof cages. The rabbit and
monkey both became heavily infected and exhibited numerous
parasites in the peripheral blood, whereas in the goat trypanosomes
were only occasionally found in small numbers.

Examination of the parasite in the blood of the rabbit and
monkey at once revealed the same morphological peculiarity which
was observed by Stephens and Fantham in the trypanosome obtained
from a case of sleeping sickness contracted in the Luangwa valley
of North-East Rhodesia, i.e., among the stout and stumpy forms
some had the nucleus at the posterior (non-flagellar) end (Plate
XIX, figs. 5-12 and 14-16). When the parasites were numerous it
was found that these posterior nuclear varieties formed from I to 4
per cent. of the total number of trypanosomes present. Posterior
nuclear forms were only observed when the blood contained fairly
numerous parasites. They measured 17 to 22 » long.

The other parasites found were indistinguishable from
T. gambiense and exhibited the usual dimorphism. The cytoplasm
of many of the parasites observed in the blood of the monkey was
vacuolated in a remarkable manner, sometimes as many as five or
six large clear vacuoles were seen in a single parasite (figs. 3, 4, 5
and 9). In many of the parasites the cytoplasm contained large
coarse granules. The posterior extremity of many of the parasites,
especially those in which the nucleus was situated posteriorly,
presented a blunt ‘cut away’ appearance. Parasites similar to those
described by Stephens and Fantham as ‘snout’ forms were likewise
observed, but they did not appear to be a prominent feature. After
finding these posterior nuclear forms in the blood of the rabbit and
monkey, we re-examined carefully the slide of the blood of the
patient himself, made on August 31st, at a time when the parasites
were numerous. A prolonged search failed to reveal the presence of

448

any typical posterior nuclear forms, but several dividing forms were
seen, in one of which the nuclei were situated close to the
blepharoplast (figs. 16 and 17).

Pathogenicity. Unfortunately, absence of laboratory animals
prevented the investigation of this point. The three animals
(rabbit, monkey and a goat) inoculated with the strain, by one of us
(H. S. S.) in Nyasaland, were all easily infected.

In the case of the Rabdit the temperature rose before the
completion of the sixth day, and trypanosomes were found for the
first time in the peripheral blood on that day. The animal was
listless and showed a disinclination to feed. The temperature
reached a maximum of 105°F. on the r1oth day and then fell,
reaching 100°F. on the 12th day, rising again immediately
afterwards. There was a second fall on the 23rd day and again a
rise till the death of the animal on the 27th day. Loss of appetite,
rapid emaciation and anaemia were noted, with onset of paralysis
during the twenty-four hours preceding death, the muzzle resting on
the ground, then involvement of fore-quarters and stertorous
breathing; other symptoms noticed, usually seen in_ rabbits
suffering from trypanosomiasis, were oedema of face and purulent
discharge from the nose and ears. During the last six days
trypanosomes were present in considerable numbers.

Monkey. Inoculated from the rabbit. Parasites appeared in the
peripheral blood on the 7th day. During the next week
trypanosomes were present in considerable numbers, but later they
were scanty though persisting till the time of its death, on the 58th
day. There was an irregular pyrexia, the temperature varying
between 99° and 102° F. Emaciation and anaemia were progressive
though appetite was well maintained, loss of hair about the eyebrows
was noticed, and though towards the end it became very quiet, it
was not somnolent, death was preceded by eight hours of coma.

There was distinct auto-agglutination of the red blood cells.

Goat. Inoculated from the rabbit also, developed the disease.
Trypanosomes found in the blood on the 15th day and on
subsequent occasions, but always in very small numbers until the
death of the animal on the 28th day.

Symptoms were intermittent pyrexia, extreme wasting, anaemia,
oedema of the face and, towards the end, paresis of forequarters.

449

The rapidity of the course which the disease took in this animal is
worthy of remark, and is in accordance with the observations of one
of us (W. Y.) working with the parasite obtained from a case
of trypanosomiasis infected in the Luangwa valley.

Conclusions. As a result of our observations we are of opinion
that the trypanosome in question is not 7. gambiense. On the other
hand this trypanosome resembles very closely 7. rodesiense, and is
probably identical with it.

The disease was contracted in a district (Dowa sub-district of
Central Angoniland) where Glossina palpalis has never been found,
but where Glosszna morsttans is known to exist in large numbers.
It appears probable, therefore, that this trypanosome (7.
rhodesiense) is a distinct species which is capable of transmission by
some other agent than Glossina palpalis, probably Glossina

morsitans.

LITERATURE

STEPHENS AND FanTHaM (1910). ‘On the Peculiar Morphology of a Trypanosome from a
Case of Sleeping Sickness and the Possibility of its being a new species (T. rhodesiense).’
Proc. Roy. Soc., B, Vol. LXXXIII, p. 28, and Ann. Trop. Med. and Parasit., IV, pp.
343-350:

Sleeping Sickness Bulletin’ (tg1o), Vol. II, No. 21, p. 346.

Strannus, H. S., anp Yorke, W. (1911). *'The Pathogenic Agent in a case of Human
Trypanosomiasis in Nyasaland.’ Proc. Roy. Soc., B, Vol. LXXXIV, pp. 156-160.

Yorke, W. (1910). * On the Pathogenicity of a Trypanosome from a case of Sleeping Sickness
contracted in Rhodesia.’ Ann. Trop. Med. and Parasit., Vol. IV, pp. 351-368.

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DESCRIPTION OF PLATE XIX

Drawn with Abbé camera lucida, using 2 mm. apochromatic
objective and No. 18 compensating ocular (Zeiss). Magnification
2150 diameters.

Figures drawn from parasites in the blood of the monkey,
except when otherwise stated.

Figs. 1-4. Forms with the nucleus median. Figs. 1 and 2 show
line connecting blepharoplast with nucleus; in figs. 3
and 4 marked vacuolation of cytoplasm is seen.

Figs. 5-12. Forms in which the nucleus is seen to become
gradually more posterior until it lies on a level with
the centrosome (fig. 5 from patient’s blood, fig. 8 from
rabbit’s blood).

Fig. 13. Division form with nucleus median (from _patient’s

blood).

Figs. 14-17. Division forms with one or both nuclei posterior
(figs. 16 and 17 from patient’s blood).

Stannus & Yorke.

PLATE XIX

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EB Wileon, Cambridge

453
A SECOND SERIES OF EXPERIMENTS
DEALING WITH THE TRANSMISSION
OF GOITRE FROM MAN TO ANIMALS

BY

ROBERT. McCARRISON, M.D., R.U.L, -M.R.C.P. (Lonp.);

CAPTAIN, INDIAN MEDICAL SERVICE,
AGENCY SURGEON, GILGIT, KASHMERE

(Recezved for publication 21 October, 1911)

OBJECT “OP “THE: RESEARCH

In a former series of experiments, the results of which were
communicated to the Royal Society in June (1911), and published
in full in the ‘Annals of Tropical Medicine and Parasitology’
(1911), it was shown that the thyroid glands of goats, fed on water
infected with the faeces of goitrous individuals, underwent in
50 per cent. of cases an increase in size and presented the following
changes on microscopical examination : —

(1) A marked dilatation of the vesicles with colloid substance,
and a thinning of their walls.

(2) A flattening of the epithelial lining of the vesicles.

The object of the present research was :-—

(1) To repeat the experiment of feeding goats on a water highly
contaminated with the faeces of goitrous individuals.

(2) To ascertain the effect on the thyroid glands of animals of
cultures of micro-organisms grown from the faeces of goitrous
individuals, when administered by the mouth.

(3) To ascertain the effect on the thyroid glands of animals of
such cultures of micro-organisms when administered by the mouth
in association with an artificial water containing the carbonates of
lime, magnesium and sodium.

(4) To ascertain the effect on the thyroid glands of animals of
the above-mentioned carbonates when administered alone.

The experiments were carried out during the months of January
to May, 1911. The animals employed were goats and dogs.

454

EXPERIMENT A.—The administration to goats of water
contaminated with the faeces of goitrous individuals.

The method of preparing this water has been detailed in a former
communication?: a pure water was allowed to percolate through a
mixture of sterilized soil and faeces, and collected in a stoppered
bottle. Twelve female goats, aged two years, were confined in a
pen on a goitre-free soil, and provided with this fouled water as the
only drinking water for a period of 107 days (from 20th January to
6th May, 1911) The animals were liberally fed on a ration
consisting of one pound of grain and as much lucerne grass as they
could eat. All other possible sources of infection were rigidly
excluded. It had been observed in my former experiments that the
goats employed lost in weight; I, consequently, added the grain to
the ration in the case of the animals of the present experiment. At
the end of 107 days seven of the twelve goats were taken at random
and killed. Their thyroid glands were removed, weighed, and
subsequently examined microscopically. Manual examination of
the thyroids of these animals during the course of the experiment
showed that in about 50 per cent. of cases the glands had increased
in size, and were readily palpable. But it was observed that the
size of the organ fluctuated considerably ; at one examination it was
easily felt and its whole outline could be clearly appreciated, while
at another it was felt with difficulty.

The following table shows the weights of the thyroid glands as
compared with the body weight of the animals :—

}

_ Proportionate weight of
No. Weight of animal | Weight of thyroid thyroid to weight of

| animal

I 42 lbs. 2:8 gms. 1:74.65

Il 42 lbs. 3°O gms. 1:6,999
Iil | 38 lbs. / I*3 gms. 1:14,600
IV | 55 lbs. 3:1 gms. 1:8,800
wi 45 lbs. 2°5 gms. 1:9,000
VIII | 46 lbs. I-4 gms. 1:16,400
Vv 28 Ibs. 1-7 gms. 1:8,000

455

The average weight of a normal goat’s thyroid gland is, in
Gilgit, ;i;th part of the body weight. It will be observed that
three of the above animals (Nos. 1, 2 and 7) had thyroid glands
considerably larger than normal. The increase in size was not so
marked as in the case of my former experiments, where four out of
six goats were found to show an increase in size of the organ varying
from ;th to jth part of the body weight of the animal.
This difference may be attributed to the fact that the animals in the
former experiments were not so well fed, and, consequently, Zos¢ in
weight, while those of the present series gained in weight. It will
further be observed that two other goats (Nos. 3 and 6) had thyroid
glands considerably less in weight than normal, while these organs
in the remaining animals (Nos. 4 and 5) showed no marked deviation
from the normal size.

The histological appearances of the glands of these animals
varied very considerably : —

Nos. 4 and 5 showed no appreciable deviation from the normal
type. A small area of parathyroid-like tissue was seen in the
thyroid of No. 5.

Nos. 3 and 6 differed from the normal type in so far that the
vesicles were somewhat smaller and the cells lining the vesicles were
higher (low columnar) than is the case in the normal gland.

Nos. 1 and 2 were identical in appearance. The _ vesicles
(fig. 3) were on the whole larger, more irregular in shape, and the
total amount of colloid was greater than in the case of the normal
gland (fig. 1). The epithelium lining the vesicles was somewhat
higher (fig. 4) than in the normal gland (fig. 2). The thyroid in these
cases did not present the same degree of dilatation and irregularity
of the vesicles, nor the marked flattening of the epithelium, which
was observed in the case of my former experiments (fig. 5) (1911).
Indeed, apart from the increase in size of the organs, it cannot be
said that the histological appearances differed to any very marked
extent from the normal gland.

No. 7.—The histological appearances of this organ revealed a
considerable degree of hyperplasia. The vesicles were small, the
amount of colloid comparatively scanty, the lining epithelium was
columnar and the vessels of the gland were dilated. The gland was

456

markedly more cellular than ay, other in the present experiment
(figs. 6 and 7).

The net result of this experiment, therefore, Z die of seven
thyroid glands examined three (or 42 per cent.) were larger than
normal as determined by weight. Of these, two showed in their
histological appearances no very marked variation from the normal
type, while the third showed a considerable degree of hyperplasia.
Two other glands were in all respects-normal, while the remaining
two were considerably smaller than normal, and showed a more
cellular structure and a higher type of epithelium lining the vesicles
than is normal.

EXPERIMENT B.—The administration to goats of ‘cultures. of
bacteria grown from the faeces of goitrous individuals :

Four female goats, aged two years, were housed and fed in a
manner identical with those in Experiment A. They were provided
with a drinking water of known purity, which, as an additional
precaution, was boiled. The animals were given cultures of bacteria,
grown fromthe faeces of goitrous individuals The experiments
lasted 108 days (20th January to 7th May, 1911). During the first
month the animals were given, on alternate days, 48-hour cultures
on Musgrave’s agar of such organisms.as had grown on this medium.
At this stage of the experiment no attempt was made to purify the
growth. Musgrave’s agar tubes were inoculated direct. from the
faeces, incubated, and sub-cultures from the resultant. growth were
made on Musgrave’s agar. After forty-eight hours’ incubation an
emulsion of the bacteria present was made in distilled’ water, mixed
with milk, and introduced into the stomach of the animal through a
funnel. The contents of one tube of a 48-hour culture was given-to
each animal every other day. The organisms present in..such a
cuiture were mainly of two classes: (1) Organisms of the coli group,
and (2) a spore-bearing organism, the characters of which will be
subsequently described. At the end of the second month a pure
culture of the spore-bearing organism referred to was obtained from
the faeces of a horse which was suffering from recent goitre, and
from the 61st day of the experiment onward this organism alone
was given—one tube of a 48-hour growth to-each.animal.on-alternate
days. The-animals were all. killed. on the - 7th May, their thyroid
glands removed and subsequently examined microscopically. The

457

following table shows the weight of the thyroid gland in each case
relative to the body weight of the animal :—

pofsine |

Weight of Weight on I Be
: g nerease A weight o
No. eel bes 7th May, in weight, Pyrat ct thyroid to
20th Jan., cee Tek ib thyroid Pala
=n Ibs. in lbs. in lbs. | body weight

of the animal

I 42 54 12 Io gm. 1:27,000
II 48 65 17 2°3 gms 1:13,000
Ili a 40 3 2°O gms | 1:10,000
IV 32 41 9 1°6 gms | 1:12,000

It will be observed from the table :—

(1) That all the animals increased in weight. In case No. 2 the
imcrease in weight is very marked. In case No. 3, in which the
thyroid is of normal weight, the animal increased only three pounds in
weight.

(2) That in three cases out of four the thyroid gland was
considerably smaller than normal.

There was, therefore, in these animals no evidence of any
enlargement of the thyroid gland (goitre). But well-marked
histological changes were observed in all animals in this experiment
in which the thyroid showed any marked deviation from the normal
weight.

No. 1.—The animal increased 12 lbs. in weight. The thyroid
gland was almost three times smaller than normal. The following
changes were observed on microscopical examination: the vesicles
were round or oval, lined with cubical or low columnar epithelium ;
the colloid was small in amount, areas of parathyroid-like tissue were
seen, wholly cellular, showing an absence of colloid, and merging into
the vesicular structure of the rest of the gland; this ceilular structure
was observed to form about one-half of the total area of the section ;
the capillaries and vessels of the stroma were dilated; there was an
increase of the connective tissue stroma of the organ, especially
around the blood vessels, the walls of which appeared somewhat
thickened.

458

No. 2.—The animal increased 17 lbs. in weight. The thyroid
gland was smaller than normal. The following changes were
observed on microscopical examination: vesicles were almost wholly
absent or were filled with round, imperfectly-staining cells and
cellular débris. Where vesicles were seen they were lined with
irregular, high columnar epithelium, the lining being often
incomplete in parts. Stainable colloid was wholly absent. The
stroma was increased markedly in amount and formed a network,
the meshes of which were filled with round cells, many of which
stained imperfectly, and cellular débris. The capillaries of the
organ were not noticeably altered. The large central artery
appeared dilated, though its walls were not thickened (figs. 8 and
g). The appearance of this organ and the great increase in weight
of the animal are suggestive of a commencing myxoedema.

No. 3.—The animal increased 3 lbs. in weight during the
experiment. The thyroid gland was of normal weight. The
histological features of the organ were normal. There was a small
circumscribed area of parathyroid-like tissue in the centre of the
gland.

No. 4.—The animal increased g lbs. in weight during the course of
experiment. Microscopical examination of the thyroid gland
showed it to be rich in colloid, the vesicles rather larger and more
irregular than normal. A small cyst was present. No other
abnormalities were noted.

The net result of this experiment, therefore, is that of four goats
three had thyroid glands smaller than normal, in two of
which histological changes were marked. These changes were in
one case those of an active hyperplasia, while in the other they
amounted to a fibrosis and cellular degeneration of the organ. The
fourth gland was in all respects normal.

EXPERIMENT C.—The administration to goats of cultures of
bacteria, grown from the faeces of goitrous individuals, together with
a known quantity of the carbonates of lime, magnesium and sodium.

The conditions of this experiment were identical with those of
Experiment B. The same cultures were employed and administered
for the same length of time and in the same way. But one hour
previous to the administration of the cultures the four female goats
employed in the experiment were given a solution of 5 grains each of

459

the carbonates of lime, magnesium, and sodium. My object in giving
the animals these salts was to ascertain whether they exerted any
influence in favouring the development of a goitre in the animals to
which the bacterial cultures were being given. In certain localities
where the drinking water contains large quantities of lime and
magnesium, individual goitres are, as a rule, larger than in other
localities where the water is less hard, and though these metals are
now known not to cause the disease, yet it is possible they may exert
an influence of a secondary importance in the production of the
malady. In the present experiment it was thought that the
administration of these carbonates might, by increasing the alkalinity
of the intestinal contents, favour the deveiopment of the bacteria
administered by the mouth, these bacteria having been cultivated on
an alkaline medium. The experiment was carried out concurrently
with the previous one, and lasted the same length of time.

Three of the four goats were killed, their thyroid glands
removed and weighed, and subsequently examined microscopically.
The following table shows in each case the weight of the thyroid
gland relative to the body weight of the animal : —

Proportionate
| Weight of Weight of Increase weight of
No. animal on animal on in weight Weight of thyroid to
2oth Jan., 7th May, in lbs. thyroid body weight
in Ibs. in lbs. of the animal
I 444 55 10} I°5 gms. 1:18,200
II | 41 45 14 1°45 gms. 1:16,000
lil 373 45 7% 2"I gms. 1:10,630

The histological appearances of the glands of these animals were
as follows :—

No. 1.—-There was an increase in the connective tissue stroma ;
colloid was scanty; vesicles were rounded or oval, and either filled
with cells or lined with a high columnar epithelium. The vessels of
the stroma were not noticeably hypertrophied, though some were
dilated. These features indicated a well-marked hyperplasia (figs.

10 and 11).
No. 2.--The microscopical appearances were the same as in the

460

previous case, but the vesicles were larger, and the cells lining them of
a lower columnar type, while the colloid was rather more plentiful.

No. 3.-The gland showed little deviation from the normal type.
The colloid was plentiful. The vesicles were here and there lined
with a low columnar epithelium. An area of the parathyroid-like
tissue was present. Serial sections in this, as well as in other cases,
showed that the cellular parathyroid-like area gradualiy merged into
the vesicular structure of thyroid tissue (fig. 12), from which it
appeared to differ in no essential, except in the absence of formed
vesicles and colloid.

The net result of this experiment, therefore, is that of three goats
two had thyroid glands which showed marked degrees of hyperplasia,
while the glands were considerably smaller than normal All these
animals increased considerably in weight under the conditions of the
experiment.

EXPERIMENT D.—The administration to goats of the carbonates
of calcium, magnesium and sodium.

In this experiment four female goats of the same age as those in
the preceding experiments were employed. The conditions of the
experiment were in all respects identical with those of B and C, except
that in this case the animals were given only the carbonates of
magnesium, calcium, and sodium in doses of five grains of each of
these salts every other day. The experiment was carried out
concurrently with the preceding one, and was intended mainly as a
control to it. On the expiration of 107 days two of the four goats
were taken at random and killed, their thyroids removed, weighed,
and subsequently examined microscopically. The following table
shows in each case the weight of the thyroid gland relative to the
body weight of the animal :—

| | :
: : _ Proportionate
eer S Mg = Increase or Weight of ——-~weight of
No. Bk zs eri Ai on | decrease in thyroid in | thyroid to
ar ees a Ib ays in weight gms. | body weight
pated ae in lbs. of animal
Loaf i Po are ore el oie
| | |
I 27% 25 | — 2} I'§ gms. | 1:8,400
II

373 43 + 5% 22 gms. —‘1:9,700

461

The histological appearances of the thyroid glands of these
animals were as follows : —

No. 1.—No deviation from normal could be observed.

No. 2.—In this case the colloid was comparatively scanty, the
vesicles were small or oval, and lined with low columnar epithelium.
The stroma around the vessels was increased, though not markedly so,
around individual vesicles, and stretched into the gland, giving it under
a low power a lobated appearance. The vessels were not noticeably
dilated or hypertrophied.

The net result of this experiment, therefore, is that one of two
goats showed a slight degree of hyperplasia of the thyroid gland.

SUMMARY OF RESULTS

If the results of these four experiments are compared, several
broad differences will be noted :—

(1) In those animals which drank only highly faecal polluted
water for over three months there was a tendency on the part of the
thyroid gland to be larger than normal (3 cases out of 7).

(2) In those animals which were fed on cultures of bacteria from
the intestines of goitrous individuals there was a tendency on the part
of the thyroid gland to be smaller than normal, and this tendency
appears to be well marked (5 cases out of 7). The diminution in size
of the thyroid of these animals appears also to be associated with an
increase in their body weight.

(3) In those animals which drank a highly faecal polluted water
the histological appearances of the gland either differed in no essential
from normal, or there was evidence of an increase in size of the
vesicles, of irregularity in their shape, of a higher type of epithelium
lining the vesicle, and of a total increase in the amount of colloid
present.

(4) in those animals which were fed on cultures of bacteria from
the intestines of goitrous individuals, a marked tendency to hyper-
plasia was observed. The cells lining the vesicles were in a large
proportion of the cases columnar in type, colloid was scanty, and
there was evidence of an increase in the connective tissue stroma of
the organ. In one case the stroma was so markedly increased, and
- the cells so altered as to give rise to the suggestion of commencing
myxoedema.

462

(5) A slight hyperplasia was also observed in one of two goats
to which only carbonates of magnesium, lime and sodium had been
given.

It appears, therefore, that a considerable hyperplasia of the
thyroid gland may occur under various conditions, as it is present in
one or more cases in each of the foregoing experiments. But so
marked are the histological changes in some of the thyroid glands of
the goats of experiments B and C, and so striking is the contrast
between them and the glands of normal animals, and of the goats of
the other experiments, A and D, that one is led to attribute these
changes to the action of the bacteria administered. The cases are,
however, too few to admit of more than this general conclusion being
drawn ; and this conclusion must be subjected to the test of further
experiment on a much larger number of animals than were employed
in the present instance.

The results of feeding goats on faecal polluted water was in the
present series not so marked as in my former experiment. But here
also three goats out of seven showed an enlargement of the
thyroid gland, as determined by weight. The structure of these
glands, however, did not show the same degree of dilatation and
distension of the vesicles with colloid, nor was the thinning and
irregularity of the walls of the vesicles so marked, or the epithelial
lining so flattened as in the thyroid glands of the goats of my first
series of experiments. The results, nevertheless, are on the whole
similar to those obtained in my former experiments.'?

I have alluded to a spore-bearing bacillus, which was isolated in
pure cultures from the faeces of a goitrous horse, and which was
constantly present in the cultures from the faeces of goitrous
individuals, as having been employed in experiments B and C. Iam
indebted to the Director of the Pasteur Institute of Kasauli and to
the Assistant Director for the following description of this organism.
It is a rod-shaped bacillus, varying from 2 to 4 » in size, which
does not retain the stain by Gram’s method, but stains well with
Carbo-Fuchsin, Leishman’s, and other stains. The bacilli vary in
size and thickness, and some of them contain a lighter unstained area,
situated usually at the centre, but sometimes towards the periphery of
the organism. The growth on Musgrave’s agar shows, in addition to
the bacilli, numerous round unstained bodies, which are seen tc be

463

spores when special staining methods are employed. In some of the
bacilli spore formation is also seen. The organism is very actively
motile in young cultures. Plate cultures on agar and Musgrave’s
medium appear as small, round and opaque white colonies. The
deeper colonies are irregular, with crenated margins, and are bluish
white in colour. The growth is more profuse on alkaline (+1) agar
than on Musgrave’s medium. On agar slopes (+1) a profuse opaque
growth, white lead in colour when viewed from the surface, and
faintly brown in colour with transmitted light, is seen after twenty-
four hours.

The organism ferments glucose, laevulose, mannose, and
galactose, but not dextrose, mannite, lactose or maltose. It produces
no curdling or acid in litmus milk, and grows profusely in broth,
forming a white scum on the surface. It does not liquefy gelatine in
stab cultures; in this medium it forms a button-like growth into the
medium, with both superficial and deep gas production. On potato
there is a profuse brownish growth. The organism is not killed by
0'5 per cent. carbolic acid for twenty-four hours in an incubator, nor
by 60° C. for half an hour. It is not destroyed by boiling at 94° C.
for five minutes. Half to one c.c. of a living culture injected into
guinea-pigs produced no immediate ill effects. Larger quantities of
the living culture injected into dogs, kids, and goats gave a similar
result.

A young dog, weighing 9 lbs. 4 ozs., was given nine Musgrave’s
agar tubes of a forty-eight hour culture of this organism. Two hours
later the dog was observed to be making violent efforts to vomit, but
without result. About six hours later the animal became stiff and
unsteady on his limbs. On the following day he was observed to
have developed pronounced clonic spasms of the muscles of the limbs
and tail. He was then unable to stand, and when propped up his
legs gave way under him. These symptoms persisted, the animal
lost consciousness, a blood-stained discharge from the mouth, nose,
and anus appeared on the fourth day, and the animal died on the
ninth day of the experiment. The thyroid lobes were removed, with
two enlarged lymph glands in their vicinity. Two agar (+ 1) tubes
were inoculated with the blood-stained fluid which escaped from the
cut surface of the lymph glands. After twenty-four hours’ incubation
several colonies of the spore-bearing organism above described were

464

present in one of the tubes. The other tube remained sterile. The
thyroid gland of the animal was about one-third of the size of another
dog, which weighed 10 lbs. 6 ozs., and which was killed for purposes
of comparison. The organ on microscopical examination was found
to be almost wholly cellular, with an occasional vesicle of normal
appearance scattered here and there through the section. The
connective tissue stroma appeared to be increased, and formed a well-
marked network, the meshes of which were filled with round cells, and
with no attempt at the formation of vesicles. Colloid was wholly
absent except in the few scattered vesicles. The central artery of the
gland appeared considerably thickened. The appearances seen are
shown in fig. 13, magnified about 500 diameters. Fig. 14 shows the
thyroid of the ‘bazaar’ dog which was killed for purposes of
comparison. The magnification is in both cases the same.

Two other dogs were subsequently given thé same number of
tubes of a forty-eight hour culture of this organism on Musgrave’s
medium, but the animals remained to all appearances quite healthy.
They were, unfortunately, not killed.

If the micro-photograph of this animal’s thyroid is compared with
that of goat No. 2 B (figs. 8andQ), which was fed on cultures of this
organism for a period of two months, a striking resemblance will be
observed in the two cases. There is the same increase of fibrous
tissue, the same cellular structure of the giand, broken up into
columns of rounded cells by the network of stroma, the same absence
of colloid, complete in the case of the goat, partial and limited to a
few scattered vesicles in the case of the dog. Fig. 13 also resembles
very closely that of a dog’s thyroid figured by Mr. Edmunds in his
work on: ‘The Pathology and Diseases of the Thyroid Gland”
(fig. 19, p. 36); this figure shows the changes in a dog’s thyroid
‘four days after partial removal.’ Mr. Edmunds, in speaking of the
changes which take place in the thyroid as a result of removal of the
parathyroids, refers to this particular case as one in which, so far from
the thyroid increasing in size, it appeared to have been smaller than
normal. The small size of this dog’s thyroid and of the goats’
thyroids in my experiments B and C is very striking

That the experiments detailed in this paper have yielded striking
results there is no doubt, but I do not at this stage of my observations
propose to do more than record them. The number of animals

465

employed was too small to permit of definite conclusions beirg drawn,
but the results here recorded are sufficiently marked to justify the
belief that experiments conducted on a larger scale, and on the same
lines, would yield valuable information. In connection with these
experiments, I would draw attention to a paper which I read before
the Royal Society of Medicine on the subject of the ‘Vaccine
Treatment of Goitre,’ and in which I detail the results of treatment of
incipient goitre by vaccines prepared from coliform bacilli,
staphylococcus, and the spore-bearing organism described in this
paper.

REFERENCES

1. McCarrison, R. (1gtt). Proc. R. S., B, Aug. 18, LXXXIV, 570, pp. 155-156.
—_—— re Ann. Trop. Med. & Parasitol., Aug. 1, V, 2, pp. 187-198.
Epmunps, W. The Pathology and Diseases of the Thyroid Gland.

iS)

we

Fig.
Fig.

Fig.

Fig.

Fig.

Fig.

I

Ww

+

OV

466

DESCRIPTION (OF PLALES

PLATE XX
Thyroid gland of normal control goat. x 100.
Thyroid gland of normal control goat. x 500.

Thyroid gland of goat No. 2 (Experiment A). The
vesicles are more irregular in shape and the total amount
of colloid is greater than in the normal gland. x 100,

Thyroid gland of the same animal magnified 500 times.
The micro-photograph shows the more irregular shape of
the vesicles and the higher type of the epithelium lining
the vesicles than in the case of the normal gland.

Thyroid gland of goat, showing dilatation and
irregularity in shape of vesicles, thinning of vesicular
walls, and increase in the amount of colloid. From a
case of artificially-produced thyroid enlargement in a
goat (IQII). x 700.

Thyroid gland of goat No. 7 (Experiment A), showing
scanty amount of colloid, columnar epithelium lining the
vesicles, and dilatation of vessels. x IM.

PEATE XX

f

3 7 Zz

Picea 100)

a, ¥.% Wi
b) righ ah ry ae 4

5 eee ~
ae * — 4

Fic. 6 x 100.
P. P. Press, Imp.

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Fig.

408

PLATE XM

Thyroid gland of the same animal magnified 500 times;
showing the columnar epithelial lining of the vesicles, and
the dilated vessels in the lower part of the field.

Thyroid gland of goat No. 2 (Experiment B), showing
almost complete absence of vesicles and colloid x 100.

Thyroid gland of the same animal magnified 500 times,
showing the increase of the fibrous stroma and the masses
of cells filling the meshes of the stroma, also the complete
absence of colloid.

Thyroid gland of goat No. 1 (Experiment C), showing

vesicles lined with high columnar epithelium, and the
scanty amount of colloid. x _ 100.

Thyroid gland of the same animal magnified 500 times,
showing the high columnar epithelium lining the vesicles.

Thyroid gland of goat No. 3 (Experiment C), showing
area of parathyroid-like tissue in lower left quadrant of
field. x 100.

PLATE XE

Ric. 7 X 500: Fic. 8 x 100.

Fic. 10 « 100.

Fic 12 . 100.

.P. Press, linp.

47°

PLATE XXII

Fig. 13. Thyroid gland of a dog, showing the cellular structure of
the organ, and the absence of vesicles and of colloid.
x 500.

Fig. 14. Thyroid gland of normal ‘bazaar’ dog, showing the
normal appearance of the thyroid gland of a dog.
x 500.

PLATE XXil.

500.

Fic. 14 x

Fic. 13 x 500.

P. P. Press, Imp.

47!

A NEW BLOOD-COUNTING PIPETTE, FOR

ESTIMATING THE NUMBERS OF LEUCO-

CYTES "AND BLOOD PARASITES PER
CUBIC MILLIMETRE

BY

DAVID THOMSON, M.B., CH.B. (EDIN.) D.P.H.(CAMB.)
(Received for publication 8 November, 1911)

Prefatory Note.—This pipette* has been devised to facilitate
an extensive research, carried on during the past two years, on the
exact enumeration of parasites in the blood of cases of malarial
fever and sleeping sickness. The research was instigated and
directed by Sir Ronald Ross, K.C.B., F.R.S., and conducted with
the aid of funds supplied by the Advisory Committee of the
Colonial Office and a private fund given by Sir Edwin Durning-
Lawrence in connection with cryotherapy, at the Liverpool School
of Tropical Medicine, and at the Tropical Laboratory in the Royal
Southern Hospital, Liverpool, and has revealed a number of facts
in both these diseases.

We first attempted to enumerate the parasites in malarial fever
by finding the ratio of leucocytes to parasites in dehaemo-
globinised thick blood films (Ross, 1903). This method, however,
proved to be tedious and inaccurate, as it involved the double
process of making a leucocyte count with a Thoma Zeiss, and
finding the leucocyte-parasite ratio in a thick blood preparation.
Later, at the suggestion of Sir Ronald Ross, I commenced to
count the parasites directly in a measured sample of blood, blown
from a graduated capillary pipette and made into a thick film.
This latter method gave more accurate results, and, finally, by
devising a special pipette, I was able to enumerate quickly and with
considerable accuracy both leucocytes and parasites simultaneously
in the specimen. The references to the literature of the researches
carried on by this method will be found at the end of this paper.

* The pipettes are sold by C. Baker, 244 High Holborn, London, W.C., Price 10/6 each.

Magnified Capillary

472
I. DESCRIPTION-OF THE PIPETTE .

The pipette is about five inches long, and is made of glass
tubing similar to that used for thermometers, with a white opal
background and powerful magnifying surface. The capillary bore,
however, 1s not flattened out like that of a thermometer but is more
nearly circular in cross section. The bore is exceedingly fine and
hair-like—such that a portion of the capillary about 60 to 70 milli-
metres long has a capacity of one cubic millimetre. This cubic
millimetre length is graduated into equal parts, so that a given
fraction of a cubic millimetre of blood can be accurately blown out.
A rubber tube with a glass mouthpiece is attached to one end of
the pipette, and the other end tapers to a very fine point—which is
necessary for expelling accurate small droplets of blood (see

diagram).
tc.mm. Pipette. =1 c.mm. graduated into
8 equal parts}
—--—--——— St -—_—_ - —— Rubber Tubing
ee ee ee eee
os Se Ss eS SS er
Sage Bes Fa 2 ed ee

Ue

Glass Mouthpiece

2 °
z nat. size

The pipette possesses about the limit of capillary fineness for
blood work, because in using a capillary of less diameter it is
difficult to expel the blood (which clots quickly in so fine a bore).
When this clotting occurs it is troublesome to clear the pipette, as
there is no wire fine enough or rigid enough to penetrate it. Even
with the bore employed, considerable care and practice are required ;
but in experienced hands the pipette may be used constantly for
months without any blockage occurring. In view of the above
difficulty, I have devised a similar pipette with a larger bore and
graduated toic.mm. This latter can be easily penetrated by a fine
wire and cleaned in case of a stoppage. Such a pipette (of the
larger bore) may be preferred by beginners; but for quicker work
and for those who have had some practice, the } c.mm. pipette is
the better.

473
II. METHOD OF USING THE PIPETTE

(1) Prick the ear or finger, and allow a tiny droplet of blood to
exude. Do not squeeze (or only very little), as squeezing drives
out lymph and lymphocytes from the lymphatics.

(2) Suck the blood into the pipette.

(3) Expel the blood until the column coincides with a line on
the magnifying surface. Wipe the point of the pipette and expel
+ or } c.mm. of blood on to a clean glass slide. After this has been
carefully done, expel all the blood immediately from the pipette,
otherwise it will clot in the bore.

(4) Breathe on the measured droplet of blood on the slide,
and spread it by means of the point of the pricking needle into a
small square, about 4 mm. x 4 mm. This square should be spread
as uniformly and as neatly as possible, and takes a little practice.
A fine triangular pointed needle serves the purpose best. A certain
amount of breathing on the slide is essential to keep the blood from
drying up during the spreading process. In the case of a } c.mm.
droplet, the spread square should be a little larger—about 5 mm.
x 5 mm.

(5) Allow the little square blood film to dry in air. This
takes place in a few seconds. Fix for about two minutes in
absolute alcohol and stain with the usual blood stains. In the
case of Jenner’s or Leishman’s stain, previous fixing is of course
unnecessary. Wash gently and dry on blotting paper.

(6) If it is desired to enumerate asexual malaria parasites, the
square film should be spread over a smaller area into a thicker film
and fixed in acid alcohol (5 per cent. dilute acetic acid in absolute
alcohol) before staining. The corpuscles are dehaemoglobinised by
this procedure and do not obscure the parasites. To count leuco-
cytes, trypanosomes and crescents this dehaemoglobinisation is
unnecessary.

(7) The pipette should be cleaned and dried immediately after
use. This is done by sucking up water and expelling it alternately
several times. Repeat this process with absolute alcohol or ether,
and finally suck air through to dry the bore. By watching the bore
through the magnifying surface one can tell when it is dry. The
water and alcohol used for cleansing and drying should be free
from dust.

474

lll. HOW TO CLEAN THE PIPETTE IN CASE OF STOPPAGE

If the blood should clot in the pipette, it can often be expelled
by moistening the point of the pipette with water or by forcing
water through with a Higginson’s syringe. If this fails it may be
cleaned as follows. Heat the pipette in the Bunsen flame
gradually, but do not heat the point. Now immerse the point in
strong nitric acid, and allow to stand there till it cools. This
enables the nitric acid to get into the bore. Dry the outside of
the pipette; press a piece of indiarubber firmly against the point
and at the same time heat it with the Bunsen flame as near to the
point as possible. This procedure produces vaporised nitric acid
in the bore under high pressure. The vapour eats away the clot
and drives it upwards. The process may have to be repeated several
times if the clot is a large one. The clot may also be slowly
dissolved by completely immersing the pipette in a test-tubeful of
nitric acid. Place the test-tube in a beaker of water and alternately
boil and cool. This latter is a slower but safer cleansing procedure.
In the first method the pipette must be dry and heated gradually,
otherwise it may crack.

Sometimes the point only of the pipette becomes blocked with
material other than blood. It can easily be cleared by keeping the
point immersed in strong nitric acid for some time.

When the pipette is in constant use, it is a good practice to
suck strong nitric acid into the bore and allow it to remain there
all night. This cleans away any blood or serum which may have
commenced to accumulate in the bore or at the orifice. Before
using the pipette one should blow through it into alcohol to see if
the air bubbles through freely; as the most frequent cause of
blocking is an attempt to suck up and expel blood through a
partially obstructed orifice. The + c.mm. pipette can be cleaned
out by means of a fine wire.*

IV. METHOD OF ENUMERATING LEUCOCYTES OR PARASITES
IN THE MEASURED SQUARE BLOOD FILM

A microscope with a mechanical sliding stage and an eyepiece
having a diaphragm with a square hole is essential. For the latter

* For the pepsin method of cleaning pipettes, see Stephens & Christophers, ‘ Practical Study
of Malaria,’ third edition, page 11.

475

a circle of paper with a square hole may be fitted into the eyepiece ;
but an Ehrlich’s ocular eyepiece is better, as by means of this
eyepiece the square microscopic field can be contracted by means of
a little lever to any size required.

Place a drop of oil on the square blood film, without a cover-
glass, and place under oil immersion lens. Find the upper margin
of the square film, and by means of the mechanical stage work down,
field by field, towards the lower margin. On reaching the fifth
field from the upper margin, stop, and move the field from the
right margin of the square film to the left margin, meanwhile
counting the number of leucocytes or parasites all the way in one
imaginary band the breadth of the microscopic field. Now move
down to the tenth band from the top margin and repeat this
process, then move down to the fifteenth band and repeat again,
and finally move down to the lower margin.

Let us suppose that the number of microscopic field diameters
between the upper and lower margins is 30, and that the average
number of leucocytes or parasites in the three bands counted is
40, then the total number in the square film is 30 x 40 = 1200.
But the square film represents } c.mm., therefore the number
per c.mm. is 8 x 1200 = Q6o00. If the square film is } c.mm.,
then the number will be, of course, 4800 per c.mm.

The diagram on page 476 may indicate the method more clearly.

The greater the number of bands counted, the greater is the
accuracy of the result; but in a well-spread film where the distribu-
tion of leucocytes or parasites is fairly uniform, a count of three
bands will be amply sufficient, but one must count a varying number
of bands according to the number of parasites or leucocytes present.
Where the parasites are scarce, say under 1000 or 2000 per c.mm.
(crescents are nearly always below this number), then it is not
sufficiently accurate to calculate the number from bands, and the
total number in the whole of the square film must be counted by
means of the mechanical stage. Where the numbers are very
small, it may be necessary to take a larger specimen of blood than
4 or } c.mm. The examination of the whole square takes about
ten minutes if the objects counted are few.

Special slides can be obtained having a square 4 mm. x 4 mm.
ruled on the glass. This enables one to spread the droplet of blood

476

into an accurate square, and by finding the number of diameters
of the microscopic field across this ruled square, one obtains a
constant multiplying factor for estimating the number of leuco-
cytes, etc., in blood films spread exactly over that square.

Square Blood Film + c.mm.

oe 45 Leucocytes

aivis

ae ae cone we . .
. oP ce . . ee ae
Pa - . . - 7 Oe =
so” = oe Tae! » Y we .
“. sae ate! Fen Adis <
sist © there 54/7 5 85 -
: ’

|
Ss 35 Leucocytes
|
|
}

oo 40 Leucocytes

a ba Ge
iT,
Le

asco

»

The side of the Total Square Film is 30 times the diameter of the Square Microscopic Field.

Average number of leucocytes is 40 in a band the breadth of microscopic field.

» 40 X 30 X 8 = g600 leucocytes per c.mm.

V. SOURCES OF ERROR IN THE METHOD

I do not claim to have calculated the amount of error in this
method mathematically, but so far as I can estimate, the errors are
as follows :—

(1) With this pipette I estimate that the instrumental error in
transferring } c.mm. of blood to a slide is 5 per cent.

(2) Where only a few bands are counted across the square, there
is an error due to unequal spread of the film. The greater the
number of bands counted the smaller is the error, and where the
whole of the square is counted this source of error is eliminated
altogether. , |

477

_ (3) The error depending upon the number of objects counted
has been fully discussed by Sir Ronald Ross and Mr. Walter Stott
(1911). By referring to tables in their article, one can tell the exact
percentage of error for the number of objects counted. It appears
that to get within a statistical error of 5 per cent. one must count at
least 200 objects. (For further details see Section IV of their
article. )

VI. COMPARISON WITH THE THOMA-ZEISS METHOD

The following table gives a comparison between leucocyte
counts made simultaneously by this method and by the Thoma-Zeiss
apparatus : —

Case Thoma-Zeiss New method (average of two bands only)
Dieses re ARO cera "KGa Sar ee | eee
I 27,000 leucocytes per c.mm. 26,000 leucocytes per c.mm.
2 15,000 ” Ss 16,000
3 14,000 3 3 12,400
4 16,000 a Fr 15,500 3
5 13,000 3 5 13,600 % §
6 40,000 ” . 43,000
7 10,000 ¥ = 10,200
$ 17,000 S “ 15,100

VII. ADVANTAGES OF THE NEW METHOD

(1) Blood parasites can be enumerated. The parasites of
malaria and sleeping sickness cannot be enumerated accurately by
any other means.

(2) Only a tiny droplet of blood is required. This renders the
method very suitable for those who desire to investigate the blood
of small animals such as rats, mice, etc. It is also important where
frequent examinations are being made on one patient. The patient
does not object to the gentle prick of the needle nor to the small
amount of blood taken.

(3) No diluting fluid or special slide is required.

(4) The slides with the required square films can be stored,

478:

stained and counted at any future time when convenient. A large
number of samples can thus be taken at frequent intervals and
these can-be kept and examined at a later date.

(5) A differential leucocyte count can be made saealedeonsly4
with the enumeration.

(6) Auto-agglutination of the red cells can be detected through
the magnifying surface of the capillary.

(7) After some practice the method will be found to be
extremely rapid and convenient. | tee 7

I am indebted to Dr. J. J. Levin who kindly made the Thoma-
Zeiss counts in the above table.

REFERENCES TO LITERATURE

FantuaM, H.B. (1910). The Life History of Trypanosoma gambiense and Trypanosoma
” rhodestense as seen in Rats and Guinea-pigs. Ann. Trop. Med. & Parasitol.,
~Liverpool, IV, pp. 465-485. ;

, & Tuomson, J. G. (1910). Enumerative Studies on Trypanosoma gambiense and
Trypanosoma rhodesiense in Rats, Guinea-pigs and Rabbits. Periodic Variations Disclosed.
Ibid., IV, pp. 417-463. ,

Korxr, V. T. (1910). | Some Observations on a Case of Sleeping Sickness: Coagulation
Time of Blood, Albumoses, Choline, Cerebral Sections. Ibid., IV, pp. 325-331.
——— (1911). On the correlation between Trypanosomes, Leucocytes, Coagulation Time,

Haemoglobin and Specific Gravity of Blood. Ibid., V, pp. 127-131.
Ross, R. (1903). The Thick Film Process for the Detection of Organisms in the Blood.
Thompson-Yates & Johnston Lab. Rep., Liverpool, V, I, pp. 117-118.
,& Srotr(r1g11). Tables of Statistical Error. Ann. Trop. Med. & Parasitol., Liverpool,
V, 3: Pp- 347-369. ;
, & THomson, D. (1910). A Case of Sleeping Sickness studied by Precise Enumerative
Methods: Regular Periodical increase of the Parasites Disclosed. Ibid., IV, pp-

261-265. OH T: 3h ¢ .VUsL

———, & ——— ioe Some Enumerative Studies on Malarial Fever. Ibid., IV

" pp. 267-306. a "an

, & ———(rgro): A Case of Sleeping Sickness studied- by Precise Enumerative
Methods: Further Observations. Ibid., IV, pp. 395-416.

———, ———, &.Simpson, G. C. E. (1gt0), A Case of Blackwater Fever followed ig \a a
Peculiar Relapse without Haemoglobinuria or Detectable Plasmodia. Ibid., IV,
PP: 307-312.

_.— & Tuomson, J. G. (1910). Experiments on the Treatment of animals infected with —
Trypanosomes by means of Atoxyl, Vaccines, Cold, X Rays and Leucocytic Extract ;"
Enumerative methods employed. Ibid., IV, pp. 487-527. 3

Tuomson, D. (1911). A Research into the Production, Life and Death ak -Crescents, in
Malignant Tertian Malaria, i in Fpeated and Untreated Cases by: an. Enumerative Method.
Ibid., V, pp. 57-82. - ; Ne ie

—(1g11). The Leucocytes in. Malarial Fever: x ‘Method of Dingndatag: Malaria

long after it is apparently cured. Ibid., V, pp. 83-102.

479

SOME RESEARCHES ON THE LIFE-CYCLE
OF SPIROCHAETES

BY

H. B. FANTHAM, D.Sc., B.A., A.R.C.S.

- (Received. for publication 9 Nevember, 191 1)

CONTENTS

Pace
INTRODUCTION art Be +e ee 4 se : eee A7O
MatTertaL AND METHopDs ... 3 on ae #e = ore ne a7G
Broop-InwaBtTiNnG Sprrocuaetes: S. recurrentis, S. dutioni, S. marchouxi... 480
Division—longitudinal and transverse... i. ce “ae even AGI
Multiple transverse fission in blood of orehines me ~ ws 484

Some observations on S. duttoni and S. marchouxi
in Ticks, young and adult... ee fe ee Le an ees
SPIROCHAETES OF LAMELLIBRANCHS: S. balbianii, S. ES S. solents . 488
NOMENCLATURE $e pe oe oe . S: yo B: peut 4.02
SUMMARY ie ie +7 oe = Eas os oe ae ‘uaiOR
ADDENDUM... $6 eS hi ee ss ede = sa ee OS
REFERENCES... a x se ak wed Se oe Jes + 4096

INTRODUCTION

The elucidation of the complete life-cycle of Spirochaetes is a
matter of considerable importance from the scientific and economic
point of view. African tick fever and European relapsing fever
are due to Sprrochaeta duttoni and S. recurrentis respectively, while
S. marchouxz* has fatal effects in chickens. Spirochaetes may also
occur in the digestive tract of many hosts, as is the case in many
game birds and in a large number of molluscs. Both blood-
inhabiting Spirochaetes and those of Lamellibranchs have claimed
my attention for some years, and the following are notes supple-
menting my previous work, and contributing new items to our
knowledge of the life-history of Spirochaetes.

MATERIAL AND METHODS

The work relates to Spirochaetes of the blood, S. duttonz,
S. recurrentis and S. marchouxi ( = gallinarum),* and comparison
has been made throughout with the Spirochaetes of Lamellibranchs,

* The Spirochaete of fowls, first described by Marchoux and Salimbeni in 1903, was
named: S. marchouxi by Nuttall in a paper read on Dee. 9, 1904. It was also named
S. gallinarum by Stephens and Christophers i in a book with a preface dated Nov., 1904, but
published in 1905. — References are given on p. 496.

480

S. balbianii in Ostrea edulis and Tapes aureus, S. anodontae and
S. solenis (nov. sp., with pointed ends) from Solen ensis.

Many observations have in each case been made on the living
organisms, and confirmed later by the examination of stained prepar-
ations. For examination of fresh material, use has been made of
thermostats and warm stages kept at 37°C. and at 25°C, while
preparations have also been examined at room temperature. The
paraboloid condenser has been of service throughout, though not
indispensable. For staining, iron haematoxylin, Delafield’s
haematoxylin, gentian violet, thionin and Giemsa’s stain have been
of most use after wet fixation with osmic acid, corrosive acetic
alcohol or Bouin’s fluid. Zeiss 1/12” and 2 mm. objectives with
compensating oculars 8 and 12 have been used.

BLOOD-INHABITING SPIROCHAETES

Spirochaeta duttoni, S. recurrentzs and S. marchouxi

These Spirochaetes have long, narrow bodies with many spiral
coils. Each has a firm cuticle or periplast from which the protoplasmic

Fic. 1.—Diagram of S. duttont, showing chromatin granules, pointed ends and slight membrane
edge.
contents can be squeezed out with much difficulty, leaving the

empty periplastic sheath or cuticle behind, as was pointed out by
Stephens in 1906. A very tenuous membrane is present (Fig. 1),

481

being often so closely contracted against the body that it is almost
invisible in the living organism and in many stained specimens. The
nucleus consists of a series of bars or rodlets (‘granules’) of
chromatin distributed along the body. The structure is most
difficult of discernment owing to the minute diameter of the body,
but after prolonged staining with Romanowsky solution the body
exhibits alternate red areas of chromatin and paler bluish areas of
cytoplasm. In life the body appears homogeneous, probably owing
to the refractivity of the periplast.

The figures of the structure of S. marchouxt, published by
Prowazek in 1906, seem to me to be most accurate.

DIVISION

In the case of the above Spirochaetes multiplication in the
Vertebrate host is brought about by both longitudinal and
transverse fission. Both processes have been repeatedly observed
in life, and there has been no confusion of longitudinal division
with either entanglement forms or the flexed or ‘ incurvation’ form
of transverse division, recently described by Gross (1910) for certain
Spirochaetes of Pecten jacobaeus.

(a) (b) (c)
Fic 2.—Diagram illustrating longitudinal division. (a) Normal Spirochaete, waves passing
alternately in either direction. (4) Waves passing from split to undivided end. (c) Return

waves. Split extending.

True longitudinal division occurs in somewhat thicker individuals
found at the beginning of infection and exhibiting a perfectly distinct

482

clear body, without any entanglement or curvature on themselves.
Each organism may be in rapid backward and forward progression a
second or so before the onset of division. Rapid waves of
contraction followed by relaxation pass down the body of the
Spirochaete. A split appears at one end, and gradually widens.
The waves pass down each of the daughter forms, which diverge
from one another (Fig. 2) until they lie at an angle of 180°, when
separation occurs. At the commencement of longitudinal division
by no means could a second body, or flexion of one body simulating
such, be distinguished, no matter what form of examination were
adopted. Longitudinal division is best observed at the onset of
infection as recorded by Fantham and Porter in 1909. The
resultant forms are half the width of, but the same length as, the
body of the parent.

Balfour (19114) has recorded longitudinal division in the
Spirochaete (S. gzanulosa) of Sudanese fowls.

Transverse division, in my experience, occurs usually in straight
forms. There is no need of looping, ‘incurvation,’ ‘rolling up,’

(2) (6) (c)

Fic. 3.—Diagram illustrating transverse division. (a) Shows waves passing from either end
towards a centre ornode. (6) Shows direction of return waves outwards. Node thinner
as result of succession of waves outwards and inwards. (c) Daughter Spirochaetes moving
away in direction of the outward waves.

or other contortion-figures as a preparation for the act of transverse
division. Such contortion may occur, but my experience both with

483

these blood Spirochaetes and with those of Lamellibranchs is that
such. movements are but rarely preliminaries to division.

Ordinary straight-lying Spirochaetes, perhaps a little longer
than their fellows, divide transversely. Waves pass from each end
of the organism towards its centre, where they mutually extinguish
one another and induce return waves towards the ends. These
processes are repeated many times and the nodal point becomes
somewhat thinner, while the newly forming organisms elongate
slightly. By a final slight thinning of the node, separation is
effected (Fig. 3).

‘Delusion’ and ‘Contortion’ division. At various times
workers unable to observe longitudinal division of Spirochaetes,
partly due to the periodicity of direction of division described by
Fantham and Porter in 1909, have thought that the intertwining of
two Spirochaetes and their subsequent separation has been mistaken
for longitudinal division. Such is not the case. Large numbers
of such interlocked forms have been observed and their significance
fully realised. In such cases, at some period, two bodies are visible
at the ‘undivided’ end. Further, the series of waves in intertwined
forms is modified considerably, and the result is very unlike what
is present in true longitudinal division.

Looped or flexed forms of Spirochaetes, which may divide
transversely in some cases, have been suggested as possible
explanations of longitudinal division. Here again, the two parts of
the body of the flexed organism can be detected and also the
flexion can be witnessed and recognised for what it is (Fig. 4).

SSS

Fic. 4.—Diagram of flexed and intertwined form. Transverse division may sometimes occur
at the loop. Usually the organism uncurls and swims away. Alleged by some to be
mistaken (!) for true longitudinal division.

Any one who has carefully watched Spirochaetes in life need fall
into no such error regarding the nature of the movement. Such
flexed or ‘incurved’ individuals may become slightly thinner and
break at the point of flexion, but in my experience this is somewhat
rare. The mode of division induced by wave motion has always
been the usual method.

484

Transverse division occurs more particularly when the infection
of Spirochaetes is abundant (Fantham and Porter, 1909) and hence
is more easy of observation than is longitudinal division.

MULTIPLE TRANSVERSE FISSION OF THE BLOOD SPIROCHAETES WITHIN
THE VERTEBRATE HOST

I have observed that a very small number of S. duttonz,
S. vecurrentis and S. marchouxi, while in the blood of their
Vertebrate hosts pass through a peculiar form of asexual multi-
plication which, for want of a better term, I denote multiple transverse
fission. |The protoplasmic contents of the Spirochaete concentrate
around the chromatin masses forming a number of segments within
the periplast which acts as a sheath. A number of small,
round or oval bodies (‘granules’) are thus formed. These
may be the diameter of the body of the parent or, if they lie
obliquely or curved as they sometimes do, may exceed it very
slightly. The effect may be compared with a series of small
biconvex or spherical tabloids within a thin skin. The individual
small bodies may be compared with cocci. The periplast ultimately
ruptures at one end and the small coccoid bodies, which I designate
spores, issue into the blood stream (cf. Fig. 5, page 489).

In my opinion, this multiple transverse fission is scarcely an
essential phase of the Spirochaete in the Vertebrate host, but may
occur at the crisis, and may explain the ‘after phase.’ I regard the
phenomenon largely as an anticipation of what occurs in the
Invertebrate host, which is frequently a tick. Such resistant,
sporular ‘granules’ may occur in or on the mammalian red
cells in the case of S. duttoni or S. vecurrentis, and may be seen
sometimes apparently inside the avian red blood corpuscles in the
case of S. marchouxi. Empty periplastic sheaths, from which the
‘granules’ have issued may sometimes be seen lying in the
neighbourhood.

Balfour (1908) has stated that small ovoid bodies, or granules,
formed by Spirochaeta marchouxi occur within the blood corpuscles.
I have seen similar bodies on a few occasions. Prowazek (1906)
recorded intra-corpuscular stages of S. marchouxi, and Breinl
(1907) observed S. dzztoni forming granules in the spleen.

I have also observed in some smears of human blood from a case
which had apparently recovered from African tick fever, some

485
interesting intra-corpuscular forms of S. du¢toni. The Spirochaetes
appeared as spiral bodies with terminal swellings somewhat
resembling spermatozoa. These Spirochaetes are like forms of

S. nicollei (a variety of S. marchouxt) figured by Blanc (1911)* as
occurring in the haemocoelic fluid of Avgas persicus.

SOME OBSERVATIONS ON SPIROCHAETES (S. DUTTONI, S. MARCHOUXI)
IN TICKS (ORNITHODORUS MOUBATA,t ARGAS PERSICUS})

The foregoing is a brief account of the main results of my
investigations of certain blood Spirochaetes in the Vertebrate
host. With regard to stages of some of these organisms in the
Invertebrate there is less definite information. Dutton and
Todd, in 1905, showed that the tick Ovnzthodorus moubata was
the carrier of S#ivochaeta duttont, and they saw the passage
of the Spirochaete through the gut-wall into the body-cavity of
the tick. They also demonstrated that hereditary infection of
the ticks occurred, as did Koch. After an interval of some four
years, Sir Wm. Leishman investigated further the exact method of
transmission of S. duttont by O. moubata. Since the early part
of 1909 I have had the opportunity, both in Cambridge with
Professor Nuttall, and in Liverpool, of carrying out some
experiments confirming Leishman’s work. Hindle (1911) has also
recently confirmed the same. There is no need, then, for me to
set forth my experiments in detail, but the results of laboratory
experiments of mine may be briefly summarised as showing that
infection of the salivary glands is not the common mode of infection
(as was supposed by Koch), that the excretion from the Malpighian
tubules of the tick is infective, and passes near the end of the
period of feeding into the wound caused by the tick’s bite; that
within the adult tick the Spirochaetes undergo change, producing
small forms. Some of the Spirochaetes in the intestine of the tick
resist digestion therein to varying degrees. They may disappear as
such in a few hours after the tick has fed; they usually disappear
in a few (3 to 10) days, but may remain in the intestine, as

*It is to be regretted that in the earlier portions of Blanc’s memoir (dealing with the
structure, division and classification of Spirochaetes in general) the contents of several
important memoirs are quite overlooked, although the papers in question are listed in his
bibliography.

| The Ornithodorus came from Uganda, the Argas from Egypt.

486

Spirochaetes, for two or three weeks. This phenomenon partly
depends on the temperature at which the tick is kept, 37° C. being
an optimum for development of the Spirochaetes in the tick. Some
Spirochaetes pass through the gut-wall of the tick and reach the
haemocoelic cavity, where they may attach themselves to the
colourless corpuscles floating in the haemocoelic fluid. The
Spirochaetes' then break up by multiple transverse fission into
coccoid bodies (spores) composed of densely staining chromatin
surrounded by a thin covering of cytoplasm, like those described in
the blood. Certain Spirochaetes become intra-cellular in the
gut-epithelium and alimentary diverticula, and may produce
granules there. Ultimately some of the coccoid bodies reach the
ovaries and ova, as well as the Malpighian tubules of the tick, where
they may multiply within the cells of these organs.

STAGES IN THE TICK EMBRYO

As Leishman, Balfour, Hindle, and Blanc have recently published
their observations in some detail it is quite unnecessary for me to
recapitulate their work, consequently I will merely summarise my
results.

It has been mentioned that Spirochaetes in the tick have the power
of penetrating the gut-wall, reaching the body cavity and there in
the haemocoelic fluid and its cellular elements forming minute ovoid
or rod-like bodies. In the course of either the movement of the
Spirochaetes or of the haemocoelic fluid, the ovoid bodies reach
the ovary, where they intermingle with the developing ova, and
become incorporated with some of them. The eggs when laid may
contain these minute bodies. Recently laid eggs of Ovnithodorus
and Argas, crushed, made into an emulsion with a little sterile salt
solution, and then inoculated into mice or chickens, were not often
infective. On the other hand, when the eggs were kept in an
incubator at 34° to 37° C. for four to six days before being injected,
the experimental animals developed spirochaetosis and died in a
short time (3 to 6 days). When bacillary forms had developed
after keeping the eggs at 24° C:, the contents of the crushed eggs*

* In experiments with eggs, the contents of 6 to 12 eggs were used each time.

487

were infective in three experiments in- four fo seven days. The.
results of my microscopical examination of tick eggs are as
follows :—

1. Egg when laid shows no Spirochaetes. Extremely thin
smears show a few ovoid bodies which:are difficult of detection. .

2. Egg three to five days incubated. The embryonic
Malpighian tubes are developed. Some of the yolk is absorbed.
The ovoid bodies can be more easily detected as groups in the
Malpighian tubules. A few have begun to elongate.

3. An egg six to seven days incubated shows more organs of
the tick formed. Many of the ovoid bodies have lengthened and
become bacillary. At this stage they may rupture the cell in which
they developed, and escape into the lumen of the Malpighian tubule.

4. Owing to development of organs it is difficult to follow the
metamorphosis of bacillary or vibrio forms into fully. formed
Spirochaetes, but two methods seem possible (@) fusion of rods;
(6) elongation and growth in thickness of bacillary forms.. Probably
the latter method chiefly occurs.

5. A recently hatched infective tick contains in its gut (@) ovoid
bodies; (0) bacillary forms; (¢) a very few fully developed
Spirochaetes if kept at 35° C. for six or eight days.

STAGES IN TICK NYMPHS BORN OF INFECTED PARENTS

Nymphs of Argas persicus or of Ornithodorus moubata, born of
infected parents, usually contain coccoid bodies (spores) and
bacillary forms (which are elongating spores).

Experiments with such nymphs of O. moubata, kept at 24°C.,
show that they are capable of infecting mice with S. duttoni when
fed on them, Three-experiments positive.

_ Two experiments with two nymphs of. A. paste, born in the
laboratory of infected parents, kept at 24°C. for six days, and fed
on young pigeons, were negative. Similar results were obtained by
Brumpt and by Blanc,* but my experiments are too few for
generalisation as to the infectivity of nymphs of Argas persicus.
Again, such nymphs may possess a natural immunity, or they may
come from eggs which sid not happen to become infected, or

See Se

a * See Blanc (sons p: 102: This author doubts the connection of ‘ granules’ with the life- —
- eyele of S. micollet im Argas. See his sth paragraph on ‘p. 112. 7

488

possibly. the nymphs required. warming to a higher temperature (say
34° C.). I-had no more nymphs for further experiments at the time.

SPIROCHAETES OF LAMELLIBRANCHS

Regarding the Spirochaetes of Lamellibranchs, I have already
published several papers (1907, 1908, 1909) dealing with the
morphology and division of these organisms, and my recent results
merely confirm these. Naturally, morphological features can be
elucidated in greater detail in the case of these larger organisms than
in the case of the blood Spirochaetes. Membranes are easily seen
in life and nuclear detail can be observed by the use of the
paraboloid condenser, as has been shown by some previous workers
(Porter, 1909). In this respect I must beg to differ from Gross (1910)
in his view that Spirochaetes are enucleate.

-With regard to division, both longitudinal and transverse division
occur, and each is of the same type as seen in the blood Spirochaetes
(cf. Figs. 2 and 3), and the same description applies. In transverse
division the direct method without ‘incurvation” appears to be
common. The figures of ‘incurvation’ division published by Gross
and others, are, in my opinion, far from convincing. Movements
causing ‘deception division’ were described by Porter in 1909,
and their significance stated. Recently, a revival of these
rather old ideas has occurred, with the result that perfectly normal
methods of longitudinal and transverse division have been overlooked,
since attention was focussed on the rarer and possibly abnormal
modes. I would direct attention to a paper by Porter (1909), in
which a careful study was made of many peculiar movements of
living Spirochaetes from Lamellibranchs. Also those Spirochaetes
undergoing longitudinal division were again shown to be provided
with two membranes, produced by the splitting of the original one,
thus further confirming my work (1907-8-9). Borrel has also noted
the occurrence of a double membrane in some specimens of S.
balbiani. Further, double (that is, divided) chromatin granules
can be seen, after staining, in Spirochaetes about to divide. Such
have been figured by some authors who regard transverse division
as the only mode (cf. Calkins, 1909, p. 221).

Spirochaeta balbianu of oysters, Tapes and other molluscs,
S. anodontae and. S. solenis, n.Sp., have been under my observation

489

for some years. During this time, small ovoid bodies (‘ sporés ") of
the same diameter as the Spirochaetes have been found from time to
time (cf. Fig. 5e) and also empty sheaths, in both the crystalline
style, the intestinal contents and the water in which the Lamelli-
branchs were living. The significance of these forms was
considered, but until the actual formation of them from Spirochaetes
had been repeatedly seen (and confusion with extraneous bodies
excluded), I did not wish to publish my results. Since cross-
infection of the molluscs has been proved to occur by the Spirochaetes
swimming out of the alimentary tract and mantle cavity of the
mollusc into the surrounding water (Fantham, 1907-8), the production
of spores has not so great a significance as in the case of the blood

Fic. 5.—Shows formation of ovoid or coccoid bodies (as in S. dalbianit) within part of

A Spirochaete. All details of membrane, etc., omitted for clearness’ sake. (a) Normal

form with chromatin bars. Tenuous cytoplasm. 4) Concentration of protoplasm

round bars beginning. (c) Ovoid bodies differentiating. (d) Fully formed ovoid bodies

within periplast. (e) Periplast ruptured and degenerating. Ovoid or coccoid bodies

(spores) escaping.

forms. Bosanquet (1911) has also mentioned, in a recent note, the
formation of coccoid bodies in a preparation containing S. axodonéae.
The method of formation of spores (Fig. 5 a-e) is identical with
that seen in the blood Spirochaetes. I have seen spores issuing from

S. balbianii and S. anodontae on several occasions (Fig. §e).

49°

The. spores or coccoid bodies are probably able to withstand
conditions -unfavourable to the spirochaetiform stage of the
parasite. Spores: may also be formed in a fresh preparation of
Spirochaetes kept moist for twenty-four hours. However, I do not
consider that the coccoid. bodies are products of degeneration, as
degenerating Spirochaetes have a very different appearance.

Cross-infection by the agency-of water has been shown. I have
infected apparently clean Tapes aureus with S. balbianii by placing
an infected oyster with them. Infected Tapes placed in water with
a clean stock of Tapes result in all becoming infected. Similar
experiments with Osévea edulis, Pecten jacobaeus* and Tapes aureus
had the.same result. Sphaerium corneum has been cross-infected
with Spirochaetes from Anodonta cygnea, though with more
difficulty. Water in the aquaria or basins in which infected
individuals were placed has yielded Spirochaetes, and healthy
molluscs introduced into this water have become infected. An
intermediate host does not seem necessary for the transference of
the Spirochaetes.. Various commensals of oysters and anodons have
been examined... A¢ax boxzi, from the mantle cavities of infected
Anodonta cygnea, have been dissected, and in some of them, spores
or bodies closely resembling them, have been found. Some of these
bodies become rod-like; but as the complete metamorphosis of them
into Spirochaetes has not been observed, it is well for the present,
to consider them as under suspicion of being evolutionary stages
of S. anodontae, though they might be separate bodies.

Previously, mention has been made of attempts to disprove
longitudinal division by suggested ‘explanations’ that break down.

Much trouble has arisen in this and in other connections from a
paper by Gross (1910) on the spirochaetal parasites of Pecten. Both
Gross and those who have followed his lead, unfortunately show
a regrettable lack of knowledge of the literature on the group,
more particularly in connection with the subject of division. Had
they noted carefully the paper by Miss Porter and myself (1909),
and another dealing with movements simulating division by Porter
(1909), it seems probable that they would not have assumed that
transverse division following ‘incurvation’ had been mistaken for

“* Th ‘consequence of infection experiments, I consider that Cristispira pectinis (Gross). is
really Spirochaeta balbianii.... 2...

491

-longitudinal fission, nor that the mechanism of division could be
confused when there are such striking differences in movements.

Some writers have stated that Spirochaetes are homogeneous
and undifferentiated in structure. The instructive results of
compression of these organisms should be noted, and then the
undoubted differentiation into periplast and protoplasm no longer
presents difficulties. I have performed such experiments myself,
and by crushing S. balbianzz, the bars of chromatin issue intact
with the cytoplasm and take the characteristic coloration on staining.
The mode of formation of spores is a further indication of internal
differentiation.

Encystment of Spirochaetes has been described by several writers.
Up to the present, neither in the Spirochaetes of the blood nor in
those of Lamellibranchs have I found true encystment. Two types
of pseudo-cyst forms, have, however, been encountered : —

1. The Spirochaete becomes more closely coiled, either about
its centre or nearer one end, so that a ball-like form is produced.
This ball simulates a cyst with the body of the Spirochaete protruding

\

(a) (b)

Fic. 6.—Pseudo-cysts. (a) Ball-like coil in centre of Spirochaete, which soon uncoils.
(b) Plasmatic cyst or swelling.

492

from either end (Fig. 6a). But this form is only temporary, the
Spirochaete uncoiling after a short time and swimming away
normally.

2. Plasmatic cysts may be formed. Here the Spirochaete is
dying, and so is not normal. The periplast relaxes and the
cytoplasm tends to collect into small irregular masses or droplets,
which cause local bulgings along the body. I have not seen these
protoplasmic aggregations other than in animals in an almost
moribund condition, when the Spirochaetes, naturally, were under
unfavourable conditions (Fig. 64). One or two similar plasmatic
cysts may occasionally be found under similar conditions on a
blood Spirochaete.

NOMENCLATURE

The re-naming of the Spirochaetes of Lamellibranchs by Gross
(1910) as Cyvistispira, is, in my opinion, a mistake, is quite
unnecessary, and should be disregarded. The Spirochaete group
as a whole, (or various members of it), has received so many names
that it seems to be a mania to re-name it according to individual
fancy, as witness Borrelza, Spiroschaudinnia, Spiroftagellata,
Proflagellata, Spironemacea, and now Cristispira!

The accounts of S#irochaeta plicatilis, the type species of the
genus, given by Ehrenberg, by Schaudinn and by Zuelzer (1910)
vary so much that they cannot be reconciled, and suggest that the
authors may have dealt with different organisms. Zuelzer’s work
certainly needs confirmation. Hence, conclusions involving the
structure of S. plzcatzlzs cannot be accepted as a basis for changes in
the nomenclature of the group. It should also be remembered that
S. plicatilis is said to undergo multiple transverse fission, and in this
respect resembles the Spirochaetes of Lamellibranchs and of the
blood. It seems, then, that the new generic name C7vzstzspira 1s
unnecessary. It is much to be deplored that there are so many
attempts at fresh classifications and nomenclature before the
life-histories of the organisms are completely known.

The introduction of the term ‘crista’ for the membrane also is
unnecessary. In 1907-8, I pointed out that there was a difference
between the membrane of a Spirochaete and the undulating
membrane of a Trypanosome, for in Spirochaetes the membrane

493

does not markedly undulate. (Fantham, 1908, pp:! 32; [55,.¢58:)
Hence, I referred constantly to the structure in Spirochaetes as a
membrane only.

Quibbles as to whether bars or rings of granules of chromatin
are present in the body of a Spirochaete, also speculations as to the
exact shape of the organism need not have arisen, for the matter
was discussed fully by me in 1908, and illustrated (text-fig. 6,
p. 30) by drawings of sections of Spirochaetes cut zz stu in the
style of Anodon. I have since (1909) cut sections of infected styles
of Tapes aureus, and have figured (PI. VI, fig. 54) the sections of
the Spirochaetes therein. Careful examination of these figures
would be sufficient to settle such questions as those of shape of the
body and disposition of the membrane to the satisfaction of any
thinking person.

An interesting observation by Balfour (1911) may be noted here.
He states that ‘7veponema pallidum is a granule shedder.’ In
that case, it may be that 7. pallidum is really a member of the
genus S#irochaefa, too minute for observation of a membrane or
internal chromatin granules, and so its coils may only appear
to be fixed. Further, the ‘granules’ of Tvep. pallidum may
explain the Cytoryctes luzs of Siegel.

The numerous similarities between blood Spirochaetes and those
of Lamellibranchs, then, justify the retention of them in the same
genus. The morphology of their bodies, membranes and nuclei is
sumilar. They divide by both longitudinal and transverse division.
At one stage in their life-history they produce small, oval bodies
within their periplasts. When liberated, these oval bodies serve
either for re-infection of the same host or for cross-infection
purposes. Hence, the liie-history is on parallel lines.

SUMMARY

1. The Spirochaetes considered in this paper are S. duttoni,
S. recurrentis and S. marchouxi ( = gallinarum) among blood-
inhabiting forms, also S. dbalbiani in Ostrea edulis and Tapes
aureus, S. anodontae in Anodonta cygnea and S. solenis* in Solen
ensis. Both living and stained material have been used.

® S. solenis is about 4ou to 6ou in length in the specimens which I have measured. It is the
salt-water counterpart of S. angdontae, both having pointed ends.

494

2. True longitudinal division, as well as transverse division has
been observed in these Spirochaetes. There is a periodicity in the
division of the blood-inhabiting Spirochaetes, transverse division
occurring when the parasites are numerous in the blood, longitudinal
division occurring at the beginning and end of infection.

3. Transverse division following flexion, or ‘incurvation,’ has
been observed, but somewhat rarely. Transverse division usually
occurs in relatively straight or unflexed forms. I do not consider
that ‘incurvation’ is a necessary preliminary of transverse division.

Intertwined forms have not been mistaken for longitudinal
division.

4. The protoplasmic contents of some of the Spirochaetes of
the blood may break up into a number of small, round, or ovoid
bodies, lying loose within the periplast, which ultimately ruptures
at one end and sets them free. These minute bodies, variously
known as ‘coccoid bodies,’ ‘granules,’ or ‘spores,’ are formed at
the crisis. I doubt if these bodies represent an essential phase in the
life-history of the Spirochaetes in the Vertebrate host, but are
rather an anticipation of the similar phase in the Invertebrate
hosts of these Spirochaetes. However, occasionally ‘ granules’ may
occur inside the red-blood cells.

5. Certain S. duttonz, when ingested by Ovnithodorus moubata,
and certain S. gallinarum ingested by Argas persicus pass through
the intestinal wall of their hosts, and then form minute coccoid
bodies, spores, or ‘granules’ by multiple transverse fission. Such
granules, as well as Spirochaetes, may be found in the haemocoelic
fluid of the ticks, in the Malpighian tubules and in the gonads. _

6. Some of the Spirochaetes and spores reach the ovaries and
ova of the infected parent tick. The spores concentrate in the
Malpighian tubules of the developing embryo, which may be born
infected.

7- Many nymphs of O. moubata born of infected parents are
themselves capable of infecting. In the case of nymphs of A7vgas
persicus, although various observers have recorded negative results,
more experiments are necessary before it can be asserted that nymphs
born of infected parents are themselves not infective.

8. The main source of infection from both adult and young
ticks is the white excrement passed from the Malpighian tubules.

9. Elongation of the coccoid bodies, spores or ‘granules’ to

495

form short rods, and growth of these rods to form longer
(or vibrio) forms has been observed in the tick. In this way young
Spirochaetes are developed.

.10. The Spirochaetes of Lamellibranchs do not necessarily
depend on a carrier for change of Lamellibranch host. Cross-
infection is brought about by water, which conveys not only active
living Spirochaetes from the alimentary tract and mantle cavity of
infected molluscs to the inhalent apertures of other molluscs, but
also. coccoid bodies (spores) may be thus conveyed and cross-infect.
Coccoid bodies have been observed in process of formation in
S. balbtana and S. anodontae. (Fig. 5.)

11. The life-cycle of the Spirochaetes of Lamellibranchs and of
the Spirochaetes of the blood of Vertebrates follows a similar
course. Their morphology is much the same, allowing for
differences of size. There appears to be no justification for
separating generically the Spirochaetes of Lamellibranchs from their
allies in the blood of Vertebrates. (See p. 492.)

ADDENDUM

From a recent communication | gather that Frl. Dr. M. Zuelzer
has in the press another paper on Sfzrochaeta plicatzlis.
Unfortunately, at the time of correcting proofs of this paper,
Frl. Zuelzer’s memoir is not published, and I am unaware of her
conclusions. However, I should like to state, with all due respect
to the various authors who have written on Spirochaetes, that it
seems to me that much of the recent work on the group has been
done by inexperienced investigators who, in consequence of their
inexperience, are prone to make dogmatic and _ contradictory
statements based on slender evidence, and have a penchant for
putting forward new classifications. The literature on Spirochaetes
at present is, in consequence, in a state of the utmost confusion. As
one who has worked on many Spriochaetes in various parts of the
world since 1906, I wish to state emphatically that I do not think
serious attention should be paid to any work which does not set
forth in clear and concise language the practically complete
life-cycle of the organism under discussion, with clear and
convincing reasons for any suggested new classification and
appropriate illustrations of new features.

496
REFERENCES

This list is, of necessity, incomplete. Further references will be found at the ends of some of

the papers cited.

Batrour, A. (1908). ‘ Spirochaetosis of Sudanese Fowls.’ 3rd Report of Wellcome Labs.,
Khartoum, pp. 38-58.

Batrour, A.(1gt1a). ‘The role of the Infective Granule in certain Protozoal Diseases.’ Brit.
Med. Journ., No. 2654, pp. 1268-1269.

Batrour, A. (19115). ‘ Spirochaetosis of Sudanese Fowls.’ 4th Report of Wellcome. Trop.
Labs., Khartoum, Vol. A Medical. pp. 76-107.

Branc, G. R. (1911). ‘Les Spirochétes: Leur évolution chez les Ixodidae.’ 129 pp.,
2 plates. Paris, Jouve et Cie.

Bosanouet, W. C. (1911). ‘Brief notes on the Structure and Development of Spirochaeta
anodontae.’ Quart. Journ. Microsc. Sci., Vol. LVF, pp. 387-393, 1 plate.

Breint, A. anp KinGuorn, A. (1906). ‘ An Experimental Study of the Parasite of the African
Tick Fever.’ Liverpool School of Trop. Med., Mem. XXI.

Breint, A, (1907). ‘The Morphology and Life History of Spirochaeta duttoni.’ Ann. Trop.
Med. and Parasitol., Vol. I, pp. 435-438, 1 plate.

Brumprt, E. (1gog). ‘ Existence d’une Spirochétose des Poules a S. gallinarum dans le pays
Somali.’ C.R. Soc. Biol., t. LXVII, pp. 174-176.

Carxins, G. N. (1909). ‘ Protozoélogy,’ 349 pp. Lea & Febiger, New York and Philadelphia.
[‘ The genus Spirochaeta and allies,’ pp. 217-232. (A good account of the Spirochaete
group) |

Dutton, J. E., anp Topp, J. L. (1905). ‘The Nature of Human Tick Fever in the Eastern
Part of the Congo Free State.’ Liverpool Sch. Trop. Med., Memoir XVII.

EnrRENBERG, C. G. (1833-35). ‘ Spirochaeta, n.g., Spirochaeta plicatilis, n.sp.,’ Abh. Akad.
Berlin, p. 313.

FantuaM, H. B. (1907). ‘ Spirochaeta (Trypanosoma) balbianii, its movements, structure, and
affinities; and on the Occurrence of Spirochaeta anodontae in the British Mussel,
Anodonta cygnea.’ (Prelim. Communic.) Ann. & Mag. Nat. Hist., ser 7, Vol. XIX, pp.
493-501.

FantHaM, H. B. (1908). ‘ Spirochaeta (Trypanosoma) balbianit (Certes) and Spirochaeta
anodontae (Keysselitz); Their movements, structure, and affinities.’ Quart. Journ.
Microsc. Sci., Vol. LII, pp. 1-73, 3 plates.

FantuaM, H. B. (1909). ‘The Spirochaetes found in the Crystalline Style of Tapes aureus :
A Study in Morphological variation.’ Parasitology, Vol. II, pp. 392-408, 1 plate.

Fantuam, H. B., anp Porter, A. (1909). ‘The Modes of Division of Spirochaeta recurrentis
and S. duttoni as observed in the Living Organisms.’ Proc. Roy. Soc., B, Vol. LXXXI,
PP» 592-505:

Gross, J. (1910). ‘ Cristispira, nov. gen. Ein Beitrag zur Spirochatenfrage.’ Mittheil. zool.
Stat. Neapel., Vol. XX, pp. 41-93, 1 plate.

Hinote, E. (1911). ‘The Transmission of Spirochaeta duttoni.’ Parasitology, Vol. IV, pp. 133-
149.

LetsuMan, W. B. (1909). ‘ Preliminary Note on Experiments in connection with the Trans-
mission of Tick Fever.’ Journ. Roy. Army Med. Corps, Vol. XII, pp. 123-135.

Leisuman, W. B. (1910). ‘An Address on the Mechanism of Infection in “‘ Tick Fever” and on
Hereditary Transmission of Spirochaeta duttoni in the Tick.’ Lancet, Vol. CLXXVIII,
pp- 11-14. Also Trans. Soc. Trop. Med. & Hyg., III, pp. 77-95.

Nuttatt, G. H. F. (1904). ‘Ticks and Tick-transmitted Diseases.’ Trans. Epidemiol.
Soc. Lond., Vol. XXIV, pp. 12-26. (S. marchouxt, see table p. 16).

Porter, ANNIE (1909). ‘Some Observations on Living Spirochaetes from Lamellibranchs.’
Arch. Zool. Expér. et Gén., 5e Ser., T. III, pp. 1-26.

Prowazexk, S. (1906). ‘ Morphologische und Entwicklungsgeschichtliche Untersuchungen tiber
Hiihnerspirochaeten.’ Arb. a. d. Kaiserl. Gesundheitsamte, Vol. XXIII, pp. 554-568.
2 plates.

STepuens, J. W. W. (1906). ‘A Note on the Structure of Spirochaeta duttoni.’ Lancet, No.
4329, (Aug. 18), p. 438. c
Stepuens, J. W. W., and CurisropHers, S. R. (1904). ‘Prac. Study of Malaria, etc.’

2nd edition. London, Williams & Norgate (S. gallinarum, p. 378).

SweLrencreBeL, N. H. (1907). ‘Sur la cytologie comparée des Spirochétes et des Spirilles.’
Ann. Inst. Pasteur, Vol. XXI, pp. 448-465 and 562-586, 2 plates.

Zverzer, M. (1910). * Uber Spirachaeta plicatilis und Spirulina.’ Zool, Anzeiger, Vol. XXXV,

PP: 795°797-

497

DESMOGONIUS DESMOGONIUS,
A’ NEW SPECIES AND GENUS
OF MONOSTOME FLUKES

BY

J.W. W. STEPHENS, M.D., Cantas.

(Recezved for publication 20 November, 1911)

Some half-dozen flukes were found by Prof. R. Newstead in the
alimentary canal of an edible Nicaraguan turtle (Chelone mydas),
that died on board ship off Jamaica. They formed part of the
collection of the 23rd Expedition of the Liverpool School of
Tropical Medicine.

The colour in life was blood red. They were placed in salt
solution, as no fixative was available. On coming into my
possession they were gradually transferred to glycerine. The
following description is based on the examination of a single
specimen, as the rest of the material was lost. Though incomplete,
yet I think it is sufficient for establishing a new genus.

The body is concavo-convex, 5°2 mm. long by 1°8 mm. The
skin has no scales. It is pointed anteriorly and rounded
posteriorly, where it is furnished with two conical protuberances.
The head possesses no collar.

The alimentary tract—-Oral sucker is spherical, and has a
diameter of 0°45 mm. This is followed by a short oesophagus.
The gut caeca run almost to the extreme posterior end on either
side. They are characterized by numerous short lateral branches
internally and externally. The arrangement appears to be essentially
the same as that in the genus Charaxicephalus.

The common genital pore lies on the left side of the body about
2 mm. from the anterior extremity, just in front of the uterine

498

coils and external to the left caecum. Cirrus pouch present, lying
between the gut caeca, about a millimetre behind the posterior
border of the sucker, and followed by a curved seminal vesicle.

The testes are split wp into a number of spherical parts. These
on each side form a chain, not only external to the uterine coils,
but also to the gut caeca, and almost completely anterior to the
vitellaria with the exception of the last one or two. The right
testis is divided into eight parts and the left into seven parts.

The ovary—sSlightly to the right of the mid-line, and slightly
behind the level of the posterior vitellarian acini is apparently
globular, but no details were recognisable.

The vitellaria, situated in the posterior third of the body, lie
outside the uterine coils overlapping the gut caeca and commence
slightly anterior to and internal to the last division of the testes.
The follicles are not split up to the extent they are in
Charaxicephalus. They consist of a number of follicles to some
extent arranged in groups. The transverse vitelline duct
runs from the posterior extremity of the vitellaria on each side
obliquely towards the middle fine into a vitelline receptacle.

Eggs.—Operculate, 33 x 15 », with a tuft of long filaments at
each pole.

Looss (1902) in his key for determining the genera of the
Pronocephalidae gives the following classification : —

1. Mit 2 seitlich der Mitellinie gelegenen einfachen Hoden,
Keimstock vor ihnen (2). |

Mit 2 ebenfalls seitlch gelegenen Hoden, deren jeder in eine
Anzahl hinter einander gelegener Theilstiicke zerfallen ist;
Keimstock hinter ihnen; Darmschenkel sowohl wie der Schenckel
der Excretionsblase mit Seitenzweigen; Koérper hinten in 2 stumpf

conische F ortsatze auslauiends 3). 03.2 02 fee.2s ee Charaxicephalus.

The present genus closely resembles Charaxicephalus (1) in the
fact that the testes are split up into a number of parts, and (2) that
the ovary lies behind them. (3) In the presence of two bluntly
conical appendages posteriorly. (4) The gut caeca reach the

posterior extremity and are provided with lateral appendages
_internally. (5) The eggs are provided with a tuft of filaments.

499

It differs from it, however, in the following points :-
1. There is no collar.
2. The testes are situated external to the gut caeca and
form a chain on each side.

3. The common genital pore opens external to the gut
caecum.

4. The vitellaria occupy the posterior third (not posterior
half).

I propose,* therefore, to make for this fluke a new genus, for
which I suggest the name Desmogonius, and for the specific name
also desmogonius.

LITERATURE

Looss, A. (1902) Ueber neue und bekannte Trematoden aus Seeschildkrétén. Zool.
Jahrb., Abth. f. Syst., Bd. XVI.

* Professor Looss, who kindly examined the fluke tor me, informed me that it must
be separated from other genera of the Pronocephalidae.

500

PLATE XXIIl

Desmogonius desmogonius * 40.

sem. ves. = seminal vesicle.

c-p: = cirrus pouch.

c.g.p. = common genital pore.
r. testis = right testis.

|. testis: = left testis.

vit. = yitelline glands.

Ov. = Ovary:

PLATE XXlll.

Press, linp

Volume V February, 1912 No.

ANNALS

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TROPICAL MEDICINE AND
Peo O1OGY,

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The following courses of instruction will be given by the
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The following have obtained the Diploma in Tropical Medicine of
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Diploma in Tropical Medicine

Date of Date of

Diploma Diploma

1904 Augustine, Henry Joshua 1905 Macfarlane, Robert Maxwell
1904 Bennett, Arthur King 1905 Maddock, Edward Cecil Gordon
1904 Bruce, William James 1905 Moore, James Jackson

1904 Byrne, John Scott 1905 Nightingale, Samuel Shore
1904 Clayton, Thomas Morrison 1905 Radcliffe, Percy Alexander Hurst
1904 Dalziel, John McEwen 1905 Young, John Cameron

1904 Dee, Peter :

p4 Greenidge, Oliver Campbell 1906 Adie, Joseph Rosamond
1904 Hehir, Patrick 1906 Arnold, Frank Arthur

1904 Khan, Saiduzzafor 1906 Bate, John Brabant

1904 Laurie, Robert 1906 Bennetts, Harold Graves
1904 Maclurkin, Alfred Robert 1906 Carter, Robert Markham
1904 McConnell, Robert Ernest 1906 Chisholm, James Alexander
1904 Nicholson, James Edward 1906 Clements, Robert William
1904 Philipson, Nicholas a yaanen on

1904 Sharman, Eric Harding 190 aicanie, Norman

sgt Thonn, ais Weil oat Main coon eae
Gog Migibess Sopotem ds ancl legs tock Pailthorpe, Mary Elizabeth
1g05 Anderson, Catherine Elmslie 1906 Palmer, Harold Thornbury
1905 Brown, Alexander 1906 Pearse, Albert ay st
1go5 Caldwell, Thomas Cathcart 1906 Sampey, Alexander William
1905 Critien, Attilio 1906 Smithson, Arthur Ernest
1905 Hooton, Alfred 1906 Taylor, Joseph van Someron
1905 Hudson, Charles Tilson 1906 Taylor, William Irwin

tgo05_ Illington, Edmund Moritz 1906 Tynan, Edward Joseph

Date of

Diploma

1906 Watson, Cecil Francis

1906 Willcocks, Roger Durant
1906 Williamson, George Alexander
1907 Allan, Alexander Smith

1907 Allwood, James Aldred

1907. Bond, Ashton

1907. Branch, Stanley

1907. Collinson, Walter Julius

1907 Davey, John Bernard

1907 Donaldson, Anson Scott

1907 Fell, Matthew Henry Gregson
1907 Gann, Thomas William Francis
1907. Graham, James Drummond
1907 Hiscock, Robert Carroll

1907 Keane, Joseph Gerald

1907 Kennan, Richard Henry

1907. Kenrick, William Hamilton
1907 Le Fanu, George Ernest Hugh
1907. Mackey, Charles

1907 Maddox, Ralph Henry

1907. McCarthy, John McDonald
1907 Raikes, Cuthbert Taunton
1907 Ryan, Joseph Charles

1907 Vallance, Hugh

1908 Caverhill, Austin Mack

1g08 Crawford, Gilbert Stewart
1908 Dalal, Kaikhusroo Rustomji
1908 Dansey-Browning, George
1908 Davidson, James

1g08 Dickson, John Khodes

1908 Dowdall, Arthur Melville
1908 Glover, Henry Joseph

1g08 Greaves, Francis Wood

1908 Goodbody, Cecil Maurice
1908 Harrison, James Herbert Hugh
1908 Joshi, Lemuel Lucas

1g08 Le Fanu, Cecil Vivian

1908 Luethgen, Carl Wilhelm Ludwig
1908 Mama, Jamshed Byramji

1908 McCay, Frederick William
1908 McLellan, Samuel Wilson
1908 Pearce, Charles Ross

1908 Schoorel, Alexander Frederik
1908 Smith, John Macgregor

1908 Stewart, George Edward

1908 Tate, Gerald William

1908 Whyte, Robert

1g09 Abercrombie, Rudolph George
1g09 Allin, John Richard Percy
1g09 Armstrong, Edward Randolph
1909 Barrow, Harold Percy Waller
1909 Beatty, Guy

190g Carr-White, Percy

1909 Chevallier, Claude Lionel

1g0g Clark, William Scott

1909 Cope, Ricardo

1909 Fleming, William

1909 Hanschell, Hother McCormick
1909 Hayward, William Davey
1909 Henry, Sydney Alexander

1909

Innes, Francis Alexander

Date of

Diploma

1409 Jackson, Arthur Frame

1909 Kaka, Sorabji Manekji

1909 McCabe-Dallas, Alfred Alexander
Donald

1909 Meldrum, William Percy

1geg Murphy, John Cullinan

1909 Samuel, Mysore Gnananandaraju

1909 Shroff, Kawasjee Byramijee

1g09 Thornely, Michael Harris

1909 Turkhud, Violet Ackroyd

1909 Webb, William Spinks

190g Yen, Fu-Chun

Ig1o0 Brabazon, Edward

1910 Castellino, Louis

1910 Caulcrick, James Akilade

1910 Dowden, Richard

1910 Haigh, William Edwin

1910 Hamilton, Henry Fleming

1910 Hefferman, William St. Michael

1910 Hipwell, Abraham

1910 Homer, Jonathan

1910 Houston, William Mitchell

1910 James, William Robert Wallace

1910 Johnstone, David Patrick

1910 Korke, Vishnu Tatyaji

1910 Macdonald, Angus Graham

1910 Macfie, John Wm. Scott

1910 Manuk, Mack Walter

1910 Murison, Cecil Charles

1910 Nanavati, Kishavlal Balabhai

1910 Nauss, Ralph Welty

1910 Oakley, Philip Douglas

1910 Pratt, Ishmael Charles

1910 Sabastian, Thiruchelvam

1910 Shaw, Hugh Thomas

1910 Sieger, Edward Louis

1910 Sousa, Pascal John de

1910 Souza, Antonio Bernardo de

1910 Waterhouse, John Howard

1910 White, Maurice Forbes

1911 Blacklock, Breadalbane

1911 Brown, Frederick Forrest

1911 Chand, Diwan Jai

1911 Holmes, John Morgan

Ig11_ Jevers, Charles Langley

Ig11_ Mes, Charles Cochrane

I91t Ingram, Alexander

Ig11 Kirkwood, Thomas

191 Knowles, Benjamin

1g11_ Liddle, George Marcus Berkeley

1911 Lomas, Emanuel Kenworthy

igtt Mackarell, William Wright

1911 MacKnight, Dundas Simpson

1911 Mascarenhas, Joseph Victor -

Igt1 Murray, Ronald Roderick

1911 Oluwole, Akidiya Ladapo

Ig1I Rao, Koka Ahobala

Igt1 Sinton, John Alexander

1911 Tarapurvalla, Byramji Shavakshah

Ig11 Taylor, John Archibald

191t Woods, William Medlicott

EDITORIAL NOTICE

By order of the Committee of the Incorporated Liverpool School
of Tropical Medicine, the series of the Reports of the School, which
had been issued since 1899, were followed, from January 1, 1907,
by the Annals of Tropical Medicine and Parasitology, of which this
is the fourth number of the fifth volume.

Altogether twenty-one Memoirs, besides other works, were
published by the School since 1890, and of these ten, containing 519
quarto or octavo pages and 95 plates and figures, were published
during the two years 1904 and 1905.

The Annals are issued by the Committee of the School, and will
contain all such matter as was formerly printed in the Reports—that
is to say, accounts of the various expeditions cf the School and of the
scientific work done in its laboratories at the University of Liverpool
and at Runcorn. In addition, however, to School work, original
articles from outside on any subject connected with Tropical
Medicine or Hygiene may be published if found suitable (see notice
on back of cover); so that, in all probability, not less than four
numbers of the Annals will be issued annually. Each number will
be brought out when material sufficient for it has been accumulated.

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— a |

NOTES ON SOME BLOOD-PARASITES
IN MAN AND MAMMALS

BY

HARALD- SEIDELIN.
(Recewed for publication 15 November, 1911)

ip

In a previous paper (1910) I briefly mentioned that I had
observed, in Yucatan, various cases of non-classified fevers and that
in one case I had found, in the blood, elements of an apparently
parasitic nature. The publication of further particulars has been
delayed in the hope of receiving material from similar cases, but it
now seems advisable, although such material has not been obtained
so far, tc publish a summary of the one case observed, since it
presents some special interest, when considered in connection with
my observations (1909, 1911, I & 3) concerning the etiology of
yellow fever.

The following are the essential clinical data: —A.O., 34 years,
bullfighter, Spanish, was admitted to the lazaret in Mérida on
August 5, 1910. Febrile symptoms had been present since the day
before, and the patient, on his arrival, complained of intense
headache, nausea, and slight abdominal pain of no certain
localization. These symptoms continued, though less severe, during
the patient’s stay at the lazaret. He never felt seriously ill, even
when the temperature was comparatively high. The fever was of
an intermittent type, as shown in the accompanying chart (I), the
maximum always occurring during the afternoon or night.
Repeated examinations of the blood demonstrated the absence of
malarial parasites. No hepatic or other organic affections were
detected. The urine contained, on the fourth day of the disease, a
trace of albumin and a few hyaline casts and gave a strong
indican-reaction, but on other occasions no abnormal elements were
found, especially no bile-pigments. Jaundice was not observed.

he eighth day of the disease gave
onuclears 49°25 %, large mono-

hocytes 32 %; eosinophiles

fore the fever had subsided, but
d and to have left the town

502

CHART I

The patient left the lazaret be

nuclears and transitionals 18°5 %, lymp
he appears to have eventually recovere

the following result :—Polymorph
0°25%-

A differential leucocyte count on t

shortly afterwards.

Ut
a
TTT
Hanae

Hr nn

42 |155 |107°
39 |145 | 106°
33 |125 | 104°

7 {105 |102°

OAY OF DISEAS

27

From

, the patient being

because the possibility
ections are made by most practitioners, in

but, as no malarial parasites were
he temperature.

, however, I gram of quinine was given

j

also to be considered
to the use of quinine in yellow fever.

given to begin with,

Malarial fever was suspected,
Strong ob
the seventh day of the disease
daily, without any apparent effect on t

found, no quinine was
of yellow fever had

non-immune.
Yucatan as elsewhere,

“= +

503

On two occasions, on the fourth and eighth day, there were
observed in Giemsa preparations of the blood (dry method) elements
as those shown in Plate XXIV, figs. 1-5. They were fairly
numerous on the fourth day, but scarce on the eighth, and absent
on the ninth and tenth day.

These elements show a somewhat faintly stained body and a
darker spot, which is, as a rule, situated in or near the periphery
of the body. The faintly stained portion may be supposed to
represent the cytoplasm, though its colour is a pale purple, and not
the characteristic blue which is generally observed in protozoa; the
dark red spot has the aspect of chromatin. Some slight variations
are observed as to the shape and size of the cytoplasm, but, as a
whole, the elements are fairly uniform. Many of the elements are
apparently intracorpuscular, but it is difficult to say, whether they
are really situated inside or on the surface of the erythrocytes.
Others are extracorpuscular, either isolated or, as in fig, 5, forming
groups. The largest diameter of the bodies is about 077-11 x.

The aspect of these bodies seems to indicate their being
parasites; this impression was also that of Professor Nuttall, who
very kindly examined one of the slides with me. Other
possibilities are that they might represent nuclear granules or blood
platelets. With regard to the first possibility it may be noted that
one or two nucleated erythrocytes were observed in several of the
slides, but no transitions were seen between such nuclei and the
bodies described. The presence of a _ well-differentiated body
(cytoplasm) besides the chromatin is also a strong argument against
this possibility. With regard to blood platelets, such were seen in
all parts of the specimens, also in the vicinity of the supposed
parasitic bodies, but they differed entirely from the latter in
structure. In fact, the blood platelets present showed no peculiar
morphological features when compared with those in other blood
smears, stained according to the same technique.

The well-defined structure of the bodies seems to me to exclude
the possibility that they might be cocci.

Evidently there is no question of confusion with malarial
parasites. The bodies show a slight resemblance with Babesia and
probably also with Theiler’s (1910, 1 & 2) Anaplasma marginale,
though in the latter apparently no cytoplasm is discernible,

504.

according to Theiler, and also to Sieber (1911), and they are not
unlike the ‘ yellow fever bodies’ which I have described and believe
to be the parasite of that disease, and for which I have recently
proposed the name of Pavaplasma flavigenum (1911, 3). They
differ, however, from these bodies in being nearly uniform in size
and shape, whilst the Pavaplasma flavigenum shows considerable
variations. Other differences are that the present bodies are largely
extracorpuscular and that their cytoplasm does not show a clear
blue staining after Giemsa, as is often, though not always, seen in
the case of the yellow fever bodies. In the latter, again, double
chromatin spots are frequent, but have not been observed in the
present bodies.

Moreover, the disease in which they were found differed
considerably from the ordinary clinical picture of yellow fever. We
know, of course, that yellow fever may reveal different characters,
and it has to be considered that the patient was a non-immune in a
yellow fever country, but I would not be inclined to accept this case
as one of yellow fever without absolutely convincing proofs.

A number of cases of unclassified fevers were observed in
Yucatan by other physicians as well as by myself, and it appears
not at all unlikely that yellow fever may be one of a group of
diseases produced by blood-inhabiting parasites, which may be
different, but more or less intimately related to each other. Similar
differences might, perhaps, account tor the differences in the
clinical pictures of yellow fever in various countries, to which I have
recently called attention (1911, 2), and thus account also for some of
the obscure points in the epidemiology of yellow fever.

Should this hypothesis prove correct, the case here described
would probably belong to the same group of diseases, and its
parasite be another species of the same genus as the yellow fever
parasite. In this case a suitable name would be Pavaplasma
subflavigenum.

A type specimen has been deposited in the collection of the
Liverpool School of Tropical Medicine.

For the clinical history I am indebted to Drs. Canto and Vargas,
who kindly invited me to see the patient with them.

595
ET.

In the blood of a monkey (AZeles sp.) which I had kept for
some time in my laboratory in Mérida, Yucatan, I found an
intracorpuscular parasite, of which types are shown in figs. 6-8 of
Plate XXIV. The monkey had been subjected to other
experiments, but not to any inoculations of blood, nor to
intravascular injections. It presented an irregular fever (see
Chart II) for which the experiments undertaken did not seem to
account. When the blood parasites had been found once, repeated
examinations showed their presence on several occasions.

CRA hi

|

29 a oe a 7a
DAY OF DISEASE = 1o
Time saree 0 MORNING EVENING
Boiusiielsiry sezee 3 oii 3i7[n3i7in} _ 498
eyTePyTgt = 7 aes

+t

tH |

44h

aa
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ea
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4
eer |

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eee See
t+

008
=
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=
a
il

4

5

Bu

ae
pneu

+ t+ es fe
ht ++

tira

“Ai 4 Sa
}t e jp pt} J
Trpty tt TTT
Da
. ] TT TT]
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: THI ri z
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rrr | 7
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3

Stefi
~— >

The parasites were never numerous, and sometimes very scarce.
The only forms observed were rings with one or two chromatin
granules as shown in the plate, neither large schizonts nor gametes
were found. Puncture of the spleen resulted in the finding of a
small number of pigmented leucocytes (fig. 9) besides a few
schizonts similar to those observed in the peripheral blood.

506

This observation is, of course, incomplete, but the forms observed
were very similar to young schizonts of Plasmodium praecox, and
the parasite probably belongs to the genus Plasmodium. Similar
parasites in apes have been described by Kossel (1899), Liihe (1906),
Dutton, Todd and Tobey (1906), Halberstadter and v. Prowazek
(1907), Mayer (1907, 1908), Flu (1908), and Gonder and
Berenberg-Gossler (1908).

PRE

A disease is observed in Yucatan in imported cattle, which is
known as yellow fever of the cows. I had the opportunity of
examining blood smears from two such cases, and found small
intracorpuscular parasites, apparently a species of Babesza. No
drawings were, however, made at that time, and my preparations
have now faded to a considerable degree. The forms observed
were small solid protoplasma bodies with a single chromatin spot
or ring-shaped bodies with two or three chromatin granules. In the
smallest forms the protoplasm was extremely scarce, or seemed
entirely absent, so that only a chromatin spot was seen. No
division forms were observed.

Clinically, the disease was characterized by fever, jaundice,
oliguria and diarrhoea. The urine was only examined once, in a
fatal case on the day before death; it contained albumin and
bile-pigments, but neither haemoglobin nor blood corpuscles. The
disease was said to last for about a week in fatal cases, and anuria
to be frequent shortly before death. The mortality was said to be
very high.

On a cow which I observed shortly before it died, a few ticks
were found, as also on other members of the same herd. A specimen
was sent to Professor Nuttall, who kindly informed me that it
belonged to the species Boophilus australis, and that it had been
embodied in his catalogue under the number roto.

IV;

Trypanosoma lewist was found in about 50 % of rats examined.

507

We

Malarial parasites were found in sixty-nine cases examined in
the laboratory, besides in some private cases. The species observed
has been stated in fifty-one cases. Plasmodium vivax was present
alone in nine cases, P. malariae in two, and P. praecox in 37;

P. praecox was seen together with P. vivay in two cases, and
together with P. malariae in one case.

No case of malaria seemed to have originated inside the city
proper, and in one case only did it seem probable that infection had
taken place in a suburb, all the other patients had been exposed to
infection in the country or in other places.

REFERENCES

Dutron, J. E., Toop, J. L., and Toney, E. N. (1906). ‘ Concerning certain parasitic protozoa
observed in Africa.’ Liverpool Sch. Trop. Med. Memoir XXI, pp. 87-97.

Fru, P. C. (1908). ‘ Untersuchungen iiber Affenmalaria.’ Arch. f. Protistenk., XI),
PP: 323-330-

Gonper, R., & Berenserc-Gossier, H. v. (1908). ‘ Untersuchungen iiber Malariaplas-
modien der Affen.’ Malaria, I, pp. 47-56.

Hacnerstaprer, L., & Prowazex, S. v. (1907). ‘ Untersuchungen iiber die Malariaparasiten
der Affen.’ Arb. a. d. Kais. Ges., XXVI, pp. 37-43.

Kosser, H. (1899). ‘Uber einen malariaahnlichen Blutparasiten bei Affen.’ Zeitschr. f.
Hyg. u. Infektionskrankh., XXXII, pp. 25-32.

Luur, M. (1906). ‘ Die im Blute schmarotzenden Protozoen,’ etc., in Mense’s Handbuch der
Tropenkrankheiten, III, Leipzig.

Mayer, M. (1907). ‘ Uber Malaria beim Affen.’ Med. Klin., No. 20.
(1908). ‘ Uber Malariaparasiten bei Affen.’ Arch. f, Protistenk., XII, pp. 314-322.

Serpextn, H. (1909). ‘ Zur Aetiologie des gelben Fiebers.’ Berl. klin. Woch., No. 18, pp. 821-
823.

——— (1910). ‘Experiences in Yucatan.’ Journ. Trop. Med. Hyg., XIII, pp. 535-540.

(1g11, 1). * Protozoon-like bodies in the blood and organs of yellow fever patients.’
Journ. Path. and Bacteriol., XV, pp. 282-288.

——— (1911, 2). ‘Some differential leucocyte counts in yellow fever cases.’ Yell. Fev.
Bur. Bull., 1, 3, pp. 109-126.

(1911, 3). ‘The Etiology of yellow fever.’ Yell. Fev. Bur. Bull., 1, 7, pp. 229-268.

Sreser, H. (1g11). ‘ Anaplasma marginale.’ Rep. Govern. Vet. Bact., Union of S. Africa.
Pretoria, pp. 104-116.

Tuerrer, A. (1910, 1). ‘ Anaplasma marginale.’ (Genus nov. et species nov.) Bull. Soc. Path,
Exot., III, pp. 135-137.

——— (1910, 2). ‘ Texasfieber, Rotwasser u. Gallenkrankheit der Rinder.’ Ztschr. f. Infekt.
paras. Krankh. u. Hyg. d. Haustiere, VIII, pp. 39-62.

508

EXPLANATION OF PLATE XXIV

The figures have been drawn by Mrs. Margrethe Seidelin with

Abbé’s camera lucida. Zeiss. Apoch. Obj. Imm. 3 mm., Comp.

OCMelz:

Figs. 1-5.—Parasites from human blood. x 1300.
Figs. 6-8. —Plasmodium sp., from blood of Azeles sp. x 1400.

Fig. 9.—-Pigmented leucocyte (transitional form) from spleen of
Ateles sp. x 1400.

Plate XXIV

DO

n

THE GENUS PRISTTRHYNCHOMYTAZ,
BRUNETTI, IQIO

BY

CAriam Ww. o. PATTON, M.B., I.M.S.

AND
CAPTAIN F. W. CRAGG, M.D., I.M.S.

THE KING INSTITUTE OF PREVENTIVE MEDICINE, GUINDY, MADRAS
(Received for publication 20 November, 1911)

The genus Pristzrhynchomyia was created by Brunetti to include
a single species, /zzeata (Brunetti, 1910), which he separated from
the genus Philaematomyia, Austen, on account of certain differences
in the proboscis. The following is a portion of his original
description of the genus : —

‘With the exception of an important modification of the
proboscis, identical with Philaematomyia, Aust., the general
characters, the venation and chaetotaxy agreeing exactly.

“The two parts of the proboscis, however, are structurally
reversed, the wide basal part being fleshy and flexible, the second
part (of about equal length) being sub-cylindrical, black and
distinctly chitinized, possibly retractile to the extent of its
withdrawal partly or wholly within the fleshy basal portion. At
the end of the chitinous portion is a soft fleshy tip, the terminal
orifice being of the shape of a triangle with a rounded base (the
edges being thickened somewhat by a rim bearing the teeth). At
the apex of the triangle is a single black tooth, whilst arranged
around the orifice above are three pairs of similar black teeth.

‘Under high microscopic power the apparent “rim” of the
orifice is seen to be the base of each tooth extended considerably on
each side, so that the “rim” is not continuous.

‘The new genus is intermediate between PAzlaematomyia and
‘Musca, but the presence of the teeth suggests that it can hardly be
other than a “ biting fly.”’

510

We have examined several preparations of the mouth parts of
this fly, including one made from a co-type, identified by
Brunetti, for which we are indebted to Dr. Annandale,
Superintendent of the Indian Museum, and have found that the
description quoted above, and the figures which accompany it, are
inaccurate and misleading, and that such deviations as there are
from the type of Phzlaematomyia are differences in degree and not
in kind. The proboscis is not ‘ structurally reversed’ but consists of
a proximal portion, the rostrum, containing the fulcrum, and a
distal portion, the haustellum, which bears the oral lobes; that is to
say, the proboscis is of the ordinary muscid type, and is as
retractile as that of Musca domestica, Lin. There are five pairs of
teeth (one pair of which is rudimentary), and not three as stated by
Brunetti. The ‘rim’ is not formed by the bases of the teeth, but
corresponds to the discal sclerite of non-blood-sucking muscids. It
is obvious, from the terms used in the description, that the writer
of it is not familiar with the structure of the proboscis of a typical
Musca.

We infer from Brunetti’s paper that the description was
written after examination of pinned specimens and_ proboscides
mounted in Canada balsam without special manipulation. Under
such conditions it is extremely difficult, in fact impossible, to make
out the finer details of the parts. To study the chitinous structures
satisfactorily it is essential to clear the preparations in potash, and
to mount in balsam in varying positions. In the case of this fly,
one can, with a little care, dissect off the chitinous ring to which the
teeth are attached, and mount it flat.

The proboscis of this fly closely resembles that of
Philaematomyia insignis, Austen, which will shortly be described
in detail by one of us (F.W.C.). All the structures found in the
latter fly are represented in /zzeata, and it will only be necessary
here to indicate the points of difference between the two. The
proximal portion, or rostrum, is relatively somewhat larger than in
mmsignis; the distal part, or haustellum is, contrary to what one
would infer from the original description, considerably less densely
chitinized, and therefore less rigid. The theca is shallower, and the
thickening of its lateral margins not nearly so well marked. The
middle portion of the membrane which stretches between the two

511

lateral borders of the theca is not chitinized into a definite ‘ labial
' gutter’ such as one finds in Philaematomyia insignis and Stomoxys ;
in place of this there are two rod-like thickenings, between which
the membrane is only slightly chitinized, the whole forming a
trough to accommodate the labrum-epipharynx and hypopharynx.

The labellar rods, which are the lateral arms of the discal
sclerite, are articulated, as in zzszguis, on the ends of the labial
rods. The main portion of each is conical, the thickest part lying
in front of the end of the labial rod, the sharp internal angles
projecting inwards towards one another at the level of the tip of
the labrum. The upper ends of the rods are pointed, and diverge
widely from one another. The lower and outer angle of each of
these wedge-shaped rods is produced downwards and inwards,
and directly downwards at the tip, where it projects beyond the
axial apophysis. This downward prolongation gives attachment
to the inner ends of the teeth.

The axzal apophysis is V-shaped, its pointed apex forming the
apex of a triangle, from the sides of which the teeth appear to
arise. It is situated, however, posterior to the downward
prolongation of the labellar rods, and is not directly connected with
the teeth. The proximal ends of its arms are attached to the
labellar rods on their posterior surface.

The teeth resemble those of zzszgnzs, but are considerably more
slender and pointed. They arise from the membrane between the
pseudo-tracheae by expanded bases, the inner ends of which are
elongated and attached to the downward prolongation of the
labellar rods. The second, third and fourth teeth on each side are
approximately equal in size. The fifth is smaller, but similar, while
the first pair, which lie on either side of the tip of the axial
apophysis, are about a quarter the size of the others, and project
very little from the surface of the membrane. The ‘serrated
blades’ of Philaematomyia insignis are represented by four pairs
of spine-bearing chitinous strands. These arise from the membrane
between the base of the teeth, a little away from the distal portion
of the labellar rods. Each runs outwards parallel to the teeth, and
bifurcates in U-shaped manner at the level of the most distal portion
of the attachment of the teeth to the membrane. At the point of
bifurcation the lateral arms split up into three or four filaments,

512

which lie to a certain extent super-imposed on one another, and
are somewhat difficult to see.

The Pseudo-tracheal membrane presents no peculiarities, being
identical with that of zzszgnzs, except that the channels are a little
wider. The fourth to the seventh channels, counting from the
front, terminate between the lateral arms of the spine-bearing
strands, the filaments arising from the strand lying parallel to the
horseshoe-shaped chitinous rings of the pseudo-tracheal channels.

From the foregoing it will be seen that this fly corresponds in
all essential particulars to Austen’s description of the genus
Philaematomyia, and we are of opinion that it should be placed in
that genus, since we think that it is unjustifiable to create new
genera on minor details of structure which cannot be made out
without dissection.

One of us (W.S.P.) has bred Philaematomyia lineata from the
ege. Its breeding habits are identical with those of znszguis,
shortly to be described by us. From thirty to forty eggs are laid in
cow dung, all in one place. The eggs are slightly smaller than
those of zzszgnzs, but are otherwise similar. The larvae behave in
the same way when about to pupate, and have the same lemon
vellow colour. The puparium is similar but smaller.

Brunetti states that Dr. Annandale has frequently seen this fly
distended with blood, while feeding on cattle. We have not
ourselves observed this, although this fly is fairly common here
during the colder months. The fact that it has been taken distended
with blood is, of course, no proof that it can obtain blood
independently; it may, like Musca pattonz, Austen, and Musca
convexifrons, Thomson, suck up the blood which exudes from the
wounds made by other biting flies.

The male fly, which Brunetti does not appear to have seen, is
much like the female, but has a distinctly lighter abdomen. ft
will be described fully on another occasion.

NOTE

Since writing the above, one of us (W.S.P.) has found a new
species of Philaematomyia, the habits of which are identical with
those of zuszgnis. It is distinguished by its large size (0°7-0°8 cm.

513

long) and its coloration. There are the usual four admedian dark
stripes on the thorax, and a narrow median dark line on the dorsal
surface of the abdomen; the lateral halves of each segment are a
light olive green colour. The proboscis resembles that of
znsignis, but has five large teeth and two rudimentary ones on each
side. We propose naming this fly Phzlaematomyia gurnei, sp. nov.,
after Mrs. Patton, who was one of the first to see it. A detailed
description will be published later.

REFERENCES

Austen, E. E. (1909). New Genera and Species of bloodsucking Muscidae from the Ethiopian
and Oriental Regions in the British Museum. Ann. Mag. Nat. His. 8, iti, p. 285.

Brunetti, E. (1910). Revision of the Oriental bloodsucking Muscidae (Stomoxinae, Philae-
matomyia, Aust., and Pristirhynchomyia, gen. nov.) Rec. Ind. Mus. IV, 4, p. 50.

Crace, F. W. (1911). The Structure of the Proboscis of Philaematomyia insignis, Austen,
(Will be published in the ‘ Scientific Memoirs Series.’)

EXPLANATION OF PLATE XXV

Fig. I.—The proboscis, seen in profile, drawn from a_ potash

ck

preparation.

Fulcrum.

Membraneous wall of the rostrum.

Salivary duct (enclosed within the membrane).
Labral apodeme.

Palp.

Labrum-epipharynx.

Hypopharynx.

Labial rod.

Labellar rod.

Furca.

Fig. I].—The teeth and connected structures, seen from the front,

when extended. Drawn from a _ potash

preparation.

Axial apophysis.

Pseudo-trachea! channel.

Spine-bearing chitinous strand, representing the
serrated blades of Philaematomyia insignis.

Labellar rod.

Labial rod.

Labrum-epipharynx.

Teeth.

XXV

pede Ae

pt

eh IPE riSstOR YOR PATIL AL-
MATOMYIA INSIGNIS, Austen

BY
CAPTAIN W. S. PATTON, M.B. (EDIN.), I.M.S.,
AND

CAPTAIN F. W. CRAGG, M.D. (EDIN.), I.M.S.

THE KING INSTITUTE OF PREVENTIVE MEDICINE,
GUINDY, MADRAS

(Received for publication 27 November, 1911)

It is not a little remarkable that, although only two years have
elapsed since Mr. Austen described Phzlaematomyia insignis as a
new genus and new species, the fly has already been found to be
widely distributed throughout the East. It has been recorded from
most parts of India, from Ceylon, from Cyprus and also from
Central Africa and Socotra. Moreover, a new species, /izeata, has
been described by Brunetti (placed, erroneously, as we believe, in a
new genus Pristirhynchomyia), and we ourselves will shortly describe
another species from Madras, under the name of Philaematomyia
gurmez. It is extremely probable that the genus is a large and
widely distributed one, which has escaped the attention of
entomologists on account of its close resemblance to non-blood-
sucking muscids. One of us (F.W.C.) will shortly describe the
biting apparatus of zuszgnis, and it will then be shown that,
although the teeth are quite formidable weapons, they are so
concealed by the pseudo-tracheal membrane that even in potash
preparations a certain amount of dissection is required to expose
them. In pinned specimens it is only exceptionally that one can
see them.

These flies are of very considerable interest, on account of the
well-defined position they occupy in the muscid group. Structurally,
they are intermediate between Stomoxys and Musca, while as
regards their habits, they are intermediate between the non-blood-
sucking Musca (M. domestica, Lin., and M. nebulo, Fabr.) and

516

such flies as Musca pattoni, Austen, and Musca convextfrons,
Thomson, which have no piercing apparatus, and yet feed entirely
on blood, sucking up that which exudes from the bites of Tabanus,
Chrysops, Haematopota and Philaematomyza.

The breeding habits of this fly resemble in general those of
non-blood-sucking muscids. The eggs, fifty to sixty in number,
are laid in cow dung, the fly appearing to prefer small patches,
freshly dropped, rather than larger collections of dried dung. On
alighting, the female crawls over the surface until its finds a small
crack or crevice; the ovipositor, which is similar to that of Musca
domestica, is now thrust into the dung, the abdomen being
depressed, and all the eggs are deposited in a heap, from 1/8 to
1/4 of an inch below the surface. The process takes from six to ten
minutes.

When there are a large number of flies about, one often sees
half a dozen or more all depositing their eggs in the same spot,
their ovipositors being close to one another, while their heads are
turned outwards. When the flies have finished laying their eggs
an irregular heap of several hundreds will be found just beneath
the surface. The eggs are laid from early morning until noon,
rarely later.

Fic. 1—Egg.

The egg (Fig. 1) measures from 2 to 2°2 mm..long by 04 mm.
broad. It is of the usual muscid shape, an elongated ovoid, gently
convex along one margin and concave on the other; one end is
slightly more pointed than the other, but it has no spine. It is a
yellowish white colour and densely opaque. On the concave margin
there is a shallow groove, difficult to distinguish at the pointed end,
but widening out towards the broader end of the egg.

The larvae hatch out, through the groove, in from eight to nine
hours, that is, on the evening of the day on which the eggs were
laid. When mature, they measure about 1°25 cm., their greatest

517

breadth being about one-seventh the length. They are cylindrical,
pointed at the oral end, and are composed of twelve segments, of
which the posterior seven are of approximately equal size. They
are bright lemon yellow in colour, and on this account are readily
distinguished from other muscid larvae. All the larvae remain
together up to the evening of the second day, and then migrate,

Fig, 2—QLarya.

still in a company, from the dung, passing out from its under
surface, and burrow in the ground, under leaves, etc., to pupate.
This habit of migrating together is somewhat remarkable, and has
not been observed in any other of the Muscids we have studied
here.

The puparzum resembles that of Musca. It measures, on the
average, 0°5 cm. long by 018 broad, though there is a considerable
amount of variation in this respect. It is of a light mahogany
colour, and eleven segments can be distinguished. At the posterior
end there are two conspicuous kidney-shaped spiracles, raised
somewhat above the surface; these have characteristic markings, as
indicated in the figure, which are conspicuous on account of their
orange colour, and which are distinctive of the species.

518

Breeding takes place in Madras throughout the year. The total
time occupied is from six to seven days, varying a little according
to the temperature. The large size of the egg and the short time in
which it hatches suggest that the eggs undergo some development
before they are laid.

About eight hours after hatching the fly is ready to feed. Both
sexes suck blood, which forms their main if not their only food,
though we have seen them rubbing their proboscides on the surface
of cow dung when about to lay their eggs, in a manner which
strongly suggests that they suck up the juices from the surface,
possibly using their teeth to penetrate the crust.

Fic. 4.—Posterior Spiracles.

They feed almost exclusively on cattle and, as far as we have
observed, they only occasionally bite human beings. They do not
ordinarily exhibit any preference for any particular part of the
skin of the host, though we have found them especially attached
to the abdomen of calves which have been shaved for vaccination.
When feeding, the fly lies closely pressed against the skin of the
host, its body being parallel with the surface. They remain until
fully gorged, and are not easily disturbed; they can, in fact, be
easily picked off with the fingers. Like most blood-suckers, they
pass out a clear watery fluid, and later apparently unaltered blood,
from the anus as the abdomen distends.

From the somewhat lethargic habit of the fly, and from the fact
that it breeds rapidly and (in Madras) throughout the year, one
would expect to find that it has natural enemies, which keep down
its numbers. The chief of these is a small Hymenopteron (not yet
identified). The habit of this wasp is to settle on the dung and to

519

watch for a fly laying its eggs. Having marked a victim, it crawls
up to within an inch or two of it and then makes a short rapid
flight, settles on the fly, and after stinging it through the head
carries it away, holding it by means of its sting and its hind legs.
We have seen as many as five of these wasps on the same patch
of dung. The fly, busily engaged in laying its eggs, usually falls
a ready victim to its extremely active enemy; should it escape the
first assault, and fly away, the wasp will follow it and either catch
it as it settles on a blade of grass, or later when it returns to the
dung. The wasp also frequently attacks flies while feeding on
cattle. Unfortunately, the wasp is so small and flies so rapidly
that we have been unable to follow it to its nest.

Several small species of spider also prey on Philaematomyia,
catching them while laying their eggs. There is also a small Asilid
which has the same habit, and can often be seen to swoop down on
a fly and carry it off, grasped in its forelegs, to a neighbouring
twig, where it sucks out its juices.

Lastly, we have for several years observed a small Zachinid,
which rests on a blade of grass close to a piece of dung in which
Philaematonyia is laying its eggs. Its behaviour is very remarkable
and suggestive. It sits with its head directed towards the fly, and
every now and then darts towards it, in a very direct and
business-like manner, and at once returns to its perch. It certainly
does not catch, or attempt to catch, the fly. We have dissected
specimens of this Tachinid caught in the act, and have found that
its ovaries contain well-developed larvae, enclosed in thin
transparent membranes, through which one can see the larva making
active butting movements, as if in the endeavour to free itself.
The fly, like most of the members of that family, deposits larvae
and not eggs, and we suspect that the presence of this Tachinid
is associated with that of the Hymenopteron mentioned above, and
that the Tachinid deposits its larva on the Philaematomyza with the
intention that it shall be carried to the nest of the wasp, where it
would find ample food. We hope in time to settle this interesting
biological problem.

The simplest way of breeding Philaematomyia insignis is to
watch for a number of flies laying their eggs, and then to scoop
up the whole patch of dung and place it in a long tin tray about

520

two to four inches deep. The dung should be placed at one end of
the tray and a quantity of sand at the other. The larvae, when
about to pupate, will migrate to the sand, from which the pupae
can be removed to a closed vessel.

REFERENCES

1. Austen, E. E. (1909). Ann. Mag. Nat. Hist. (8), III, 295.

nv

Brunettt, E. (1910). Revision of the Oriental bloodsucking Muscidae. Rec. Ind. Mus.,
IV, 4, p- 59-

Crace, F.W. The Structure of the Proboscis of Philaematomyia insignis, Austen. (Will
shortly be published in the Scient. Mem. Med. Off. India, Calcutta.)

ue

4. Patron, W. S. & Cracc, F.W. The Genus Pristirhynchomyia. Ann. Trop. Med., V,
APD 5O9e514-

521

THE MEASUREMENTS OF A THOUSAND
EXAMPLES OF. TRYPANOSOMA VIVAX

BY

HanBleAGCKLOCK.: M.D.
From the Runcorn Research Laboratories.
(Received for publication 24 January, 1912)

The trypanosomes which form the basis of this communication
are derived from a strain kept up in goats, which strain was
obtained from a horse naturally infected in the Gambia. In a
previous paper dealing with the trypanosomes found in this horse,
Yorke and Blacklock (1911) acknowledged their indebtedness to
Professor Todd for his kindness in sending the animal (Horse A) to
Runcorn. As was stated in the above paper, two forms of parasites
were present in the blood of Horse A, viz., a comparatively long,
free-flagellated trypanosome, possessed of great activity of
movement, and a short non-flagellated form, of sluggish
movement. It is with the former of these trypanosomes, the long
form, that this present paper deals, and the name 7. vzvax is used
to include 7. cazalbouz, which it will be recalled Bruce (1910) says
is probably the same species.

THE SPECIES OF ANIMAL HOST CHOSEN

In making drawings and measurements of a_ particular
trypanosome, it appears desirable that all observers should, if
possible, adhere to the same species of animal host in order to
establish a ready standard for purposes of comparison. For this
reason it would have been of advantage if one could have utilised
one of the smaller laboratory animals, for example the white rat,
which has proved itself so easily susceptible to many forms of
trypanosome infection, and by means of which ready comparisons
can be made between 7. ganebiense, T. brucei, T. rhodesiense and
many other trypanosomes. But as regards the long parasite with
which we are dealing, it was found to be a matter of difficulty to
produce infection in small laboratory animals. In fact, a few

522

rabbits and white rats alone, of a large number of experimentally
inoculated animals, became infected, and the majority even of those
few recovered. So great was the difhculty encountered in this
respect that it was thought better to make use of goats, in which
animals infection was produced with great ease and certainty, and
in which after the first few passages, the long parasite alone
persisted. The short non-flagellated parasite of Horse A had died
out in the goats, as shown not only by microscopic examination of
the blood, but also by repeated inoculations (with negative
results) into a large number of small laboratory animals.

GENERAL PLAN OF MEASUREMENT

The trypanosomes were drawn and measured in small groups,
each containing twenty specimens. The number of goats from
which parasites were measured was four, which, for convenience, are
called A, B, C, D. The number of days of the disease represented
‘is twenty-two. There are thus fifty groups, each of which contains
twenty trypanosomes, drawn and measured from four goats, on
twenty-two days. The earliest day of the disease represented
among the goats is the seventeenth, and the latest is the forty-fifth.

The arrangement of the groups is made as follows :—

(1) 400 trypanosomes (twenty groups of twenty each) were
drawn on twenty separate days of the disease from three
of the goats (A, B, C).

(2) 400 trypanosomes (twenty groups of twenty each) were
drawn on one day of the disease from one goat (D).

(3) 100 trypanosomes (five groups of twenty each) were
drawn on one day of the disease from one goat (C.)

(4) 100 trypanosomes (five groups of twenty each) were
drawn on one day of the disease from one goat (D).

The reasons for adopting this plan are these :—

(1) By spreading out the first 400 trypanosomes over three
goats and twenty days of disease, and confining the
second 400 trypanosomes to one goat and one day of
the disease, one can compare two large sets of parasites -
drawn and measured under widely different conditions.

(2) By drawing and measuring two sets, each of 100
trypanosomes, drawn on a single day of the disease

523

from separate goats, one can form comparisons between
small numbers on a somewhat different basis.

(3) Finally, one can collect for comparison other sets of
100 or less spread over various animals and various days
and compare them with those given above, and can form
tables and charts to illustrate the comparative relation
of any one set to another, larger or smaller.

METHOD OF FIXING, STAINING AND DRAWING

Thin films, made from the blood of the ear, were dried, fixed
for five minutes in absolute alcohol, and stained with Giemsa’s
stain for twenty minutes*. Non-dividing parasites (taken in order
as they were found) were drawn in clear outline with the help of the
Abbé camera lucida, using a No. 18 Zeiss compensating ocular
with a 2 mm. apochromatic objective.

METHOD OF MEASURING

Measurements were carried out by Stephens’s method, which is
briefly as follows: Along the middle of a narrow strip of smooth
transparent paper a straight line is drawn. It is convenient to have
this line considerably longer than the longest trypanosome to be
measured, and terminating short of the margin of the paper. At
one extremity of the line a mark is made for identification. <A
sharp pin or mounted needle is then taken, and the marked end of
the straight line is made to coincide with one extremity of the
outlined trypanosome to be measured. Transfix the end of the
line to the extremity of the subjacent trypanosome, with the needle
held perpendicularly. Rotate the paper until the straight line lies
along the long axis of the trypanosome. Hold the paper in
position with one hand and with the other take out the needle and
pass it through the tissue paper again at the first point at which the
axis of the trypanosome begins to deviate from the straight line.
Repeat this process, following carefully every bend of the
trypanosome and keeping in the long axis of it until the opposite
extremity is reached. Hold the needle steady and place against it

* Two drops of Giemsa’s solution added to each cubic centimetre of distilled water.
A subsequent rapid wash with ro per cent. orange tannin solution gave good results for
drawing purposes.

524

a millimetre scale and read off the distance from the needle to the
marked starting point of the straight line. By this simple method
a very accurate measurement of the drawn parasite is obtained, and
from it, by calculation, the actual length of the trypanosome.

CONSIDERATION OF THE RESULTS OBTAINED

An analysis of the 1,000 trypanosomes and of the component
sets is given in Table I. From this table it will be seen that the
average measurement of the 1,000 dealt with is 21°7 w, the range
being from a maximum trypanosome of 2677 uw long to a minimum
of 15°5 « long. The trypanosome is monomorphic, all forms found
being provided with a well-marked free flagellum.

Between the averages of the two sets of 400 each, there is a
difference of only og», the first set averaging 21°4 and the
second 22°3 p.

Between the averages of the two sets of 100 each a smaller
difference is observed, the first averaging 20°9 », the second 21'5 pz.
When one comes down to the groups of twenty each, larger
variations are naturally observable.

In Table II the trypanosomes are tabulated according to their
percentage in microns under three heads, viz., those measuring less
than 20 #, those between 20 » and 23 », and those measuring 23 »
and over.

It will be seen from this table that in each set dealt with,
whether 1,000, 400, or 100, the largest number of trypanosomes
constantly lies between 20 w and 23 pn.

In Charts I, I, III, which give, for the various groups, a graphic
representation of the percentages in length, this same fact is clearly
shown.

CONCLUSIONS

(1) This 7. wax from the Gambia (Horse A) is a free
flagellated monomorphic trypanosome of an average length of
4. i

(2) The range of its extreme measurements is comparatively
small.

(3) The curve of percentage length is remarkably constant,
whether large or small numbers are dealt with.

525

Cuart I

Length in Microns

Percentage ' Percentage

1000 Trypanosoma vivax measured on 22 days in 4 Goats.

526
Curart IT

Length in Microns

Percentage | : V5 56 575 18 5119 ens adnan das 26 aS Percentage

rt Ash !
a

/

'

rn -f- U
nies Po oo

Gi Aaa Lah
opie A abide dl ain aaa

400 Trypanosoma vivax measured from 3 Goats ‘A BC on 20 days of the disease.
Dee 400 F a “A su dont, Uy on 1 day 5 es

527
Cuarrt III

Length in Microns

Percentage Percentage

100 Trypanosoma vivax measured in Goat C on 1 day of the disease.
; oe $3 9 99
~---- 100 a - .» in 4 Goats A BCD on 5 days of the disease

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528

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A
REFERENCES Fee

‘ pie
oa

Bruce and others (1910). ‘ Trypanosome diseases of domestic animals in Uganda. IL, T.
(Ziemann).’ Proc. Roy. Soc., B, Vol. LXXXIII, p. 15. ie
:

Yorke and Bracktock (rgtt). * The trypanosomes found in two horses naturally in
the Gambia.’ Annals of Trop. Med. & Parasitol., Dec., V, 3, pp. 413-

531

ENUMERATIVE STUDIES ON 7. BRUCEI

IN RATS AND GUINEA-PIGS, AND A

COMPARISON WITH 7. RHODESIENSE
AND TT. GAMBIENSE

BY

JOHN GORDON THOMSON, M.A., M.B., CH.B.
(Recezved for publicatzon 8 February, 1912)

This investigation was undertaken while I was assistant to Sir
Ronald Ross, under the Sir Edwin Durning-Lawrence Research
Fund: Already enumerative studies have been conducted by
H. B. Fantham and J. G. Thomson in animals infected with
T. rhodesiense and T. gambiense (1910-11), and a comparison was
made between these. It was pointed out that Z. rhodesiense was
more virulent than 7. gambiense, as shown by the fact that rats and
guinea-pigs lived a shorter period when infected with the former,
and it was also demonstrated that the incubation period was shorter
and the period between the heights of the crests shorter in the cases
of rats infected with 7. rhodesiense.

The following table, based on the results of Fantham and
Thomson (1910), shows this difference clearly :—

No. of Period between
Rats Incubation period Average duration the heights of
examined average, in days of life the crests
2 AST SE aE = s. vente At ose St es wate Seb tae Te!
|
T. rbodesiense ...| 22 2°9 days 11°3 days 3-4. days
T. gambiense I 4°4 days 13°8 days 4-6 days

It 1s possible, therefore, apart from morphology, to distinguish
T. rhodesiense (Stephens and Fantham, 1910) from 7. gambzense
provided a sufficient number of animals, preferably rats and
guinea-pigs, are inoculated with both strains. As a matter of fact,
however, the morphology in the case of 7. rhodesicnse in rats, as

532
pointed out by Stephens and Fantham (1910), makes the diagnosis
of T. rhodesiense very easy indeed.

I now publish four charts of animals infected with 7. d7zcez.
The method employed has already been described by D. Thomson
(1911). It is necessary in the case of heavily-infected animals to
dehaemoglobinise the slide, and spread the film over a large area.

By means of a special pipette (D. Thomson, 1911) a count was
made regularly every twenty-four hours of the number of parasites
per c.mm. of blood, and these daily counts have been represented
diagrammatically in the charts accompanying this paper.

We shall first examine the charts of Rats 52 and 23 (7. brucez).
Here we note the very rapid multiplication of the parasites. In the
case of Rat 52 the parasites rose with a rush from about 1,000 per
c.mm. to 430,000 per c.mm. in forty-eight hours, and the animal
succumbed. Again, in the case of Rat 23 the parasites rose from
under 100 per c.mm. to 490,000 per c.mm. of blood in seventy-two
hours, and the animal died shortly after. These extraordinary
numbers were never reached in so short a period either in the case
of T. rhodesiense or T. gambiense. In the case of 7. rhodesiense |
have found in a rat treated with atoxyl, and which lived fifty-one
days, that the parasites on the fifty-first day rose to one million and
a half per c.mm. (R. Ross and J. G. Thomson, 1911), but never in
the case of either 7. rhodesiense or T. gambiense have I found the
parasites in rats reach such high numbers in forty-eight to
seventy-two hours as in the case of 7. brucez.

Again, it is to be noticed that no periodic variation took place
in rats infected with 7. brucez. The rise was always continuous in
the animals observed by me. In the case of rats inoculated with
T. rhodesiense and T. gambiense, H. B. Fantham and J. G.
Thomson pointed out that a continuous rise of parasites may take
place until death in both (e.g., Rats 18-22, 30-33), but that periodic
variation also takes place in both (e.g., Rats 1-17, 23-29). So far, in
the strain of 7. drucez used here, I have been unable to obtain
periodic variation in rats, but it is quite possible that such may
occur if we could find rats of sufficient resistance.

The rats inoculated with 7. d7ucez all died within an average
period of six days. When this is compared with the average life
of rats inoculated with 7. rhodesiense and T. gambiense we have

533

a very marked difference. We conclude, therefore, in rats that
I. brucez is much more virulent’ than either 7. rhodesiense or
I. gambiense as evidenced by three points, namely :—

1. Duration of life.
Rapidity of development of parasites.

i)

3. Periodic variations.

T.brucei. 100 grams. 9.

RAT 23. T. brucei. 113 grams. 9.
ag PRE ae ee ee ee

500000
480000

460000

440000
420000
400000
380000
360000

340000
320000
300000
280000
260000
240000
229000
200000
160000
160000
140000
120000

100000

80000

GRAPHS SHOW THE DAILY RECORD OF PARASITES FOUND PER CMM. OF PERIPHERAL BLOOD.

Of course 7. bvucez is essentially an animal trypanosome, and so
might be expected to be more virulent to rats than strictly human
trypanosomes sub-inoculated into them.

We shall now examine the charts of the two guinea-pigs infected
with 7. dbrucez.

Here again, when we study the disease in 7. rhodesiense and
T. gambiense, we find that the disease runs a more or less chronic

534

course. In the case of 7. rhodesiense the average life of the
guinea-pig was fifty-nine days, whereas those infected with
T. gambiense was 111 days. The period between the crests of the
waves was longer than in rats, namely, five to eight days (H. B.
Fantham and J. G. Thomson, 1910).

Referring now to the charts of Guinea-pigs 56 and 67, infected
with 7. brace? (see charts), we find that the longest period of life
was twenty-six days, and we find again that in the case of T. brucei
the multiplication of the parasites was of much greater rapidity than
in the case of either 7. rhodesiene or T. gambiense. Thus in the
case of Guinea-pig 56 (7. drucez) the parasites in eighteen days
numbered 450,000 per c.mm., and during that time the rise was more
or less continuous. There was only one slight fall during that time,
which occurred on the twelfth and thirteenth days. The animal died
in twenty days. In the case of Guinea-pig 77 (7. brucez) we find
that on the eleventh day the parasites rose to about 350,000 per
c.mm. and then fell steadily for two days, and no parasites were
found on the thirteenth or fourteenth days, and only one or two were
seen on a film on the fifteenth day. On the sixteenth day the
parasites again reappeared and increased steadily for eleven days
until they reached over 500,000 trypanosomes per c.mm. of blood.
These numbers far exceed those found in either 7. gambiense or
T. rhodesiense. The chart of Guinea-pig 77 is of very great interest,
as it shows a distinct periodic variation, with a period of about
sixteen days between the heights of the crests. This is interesting
because of the fact that it shows on the eleventh day the animal was
able to survive a very heavy infection, the crisis being reached and a
natural recovery taking place for two days. We had evidently
resistant forms left (cf. Fantham, 1911).

For a permanent cure we must aim at the destruction, therefore,
of these resistant forms. In short, if we compare 7. brucei with
T. rhodesiense and T. gambiense in guinea-pigs we have the
following points of difference : —

1. TZ. brucet kills guinea-pigs much more rapidly than
T. rhodesiense or T. gambiense.

2. The multiplication of parasites is much more rapid, and they
reach much higher numbers in the peripheral blood than in either
T. rhodesiense or T. gambiense.

535
GUINEA-PIG 77, Rouch-waireo. Zaruces. 474 crams. o.

i209 ¢ +5 6 7 © 9 10 ii 19 13 1* 15 16 17 46 19 20 21 22 23 24 25 26
25 22) a eT ee eee j
2 a 3, BE ee ee Be Sin oa
a eS ee ee ae =i

Sot
Tas

|
|

De TeL Weve aia

ae PIG 56, 7BRvce, ee GRAMS.O.

380000] | it
360000] | si

HE

|

|

TT TET TTT

PAT AIT TALL

G OW THE DAILY RECORD OF PARASITES FOUND PER C.MM, OF PERIPHERAL BLOOD.

536

3. The periodic variations in guznea-pigs infected with T. brucei
is very different from that which takes place in the case of infections
with 7. gambzense and T. rhodesiense.

REFERENCES

FanTHAM AND THomson, J. G. (1gio-11). ‘Enumerative Studies on Trypanosoma gambiense
and I. rhodesiense in Rats, Guinea-pigs, and Rabbits; Periodic Variations disclosed.’
Proc. Roy. Soc., B, Vol. LXXXIII, pp. 206-211 (Prelim. Note); also Ann. Trop. Med.
and Parasitol., IV, pp. 417-463.

FantuamM, H. B. (rgr1). ‘ The life-history of Trypanosoma gambiense and T. rhodesiense as seen
in Rats and Guinea-pigs.’ Proc. Roy. Soc., B, Vol. LXXXIII, pp. 212-227; and Ann.
Trop. Med. and Parasitol., IV. pp. 465-485, 1 plate.

Ross, R., anp THomson, J. G. (1911). ‘ Experiments on the Treatment of Animals infected
with Trypanosomes by means of Atoxyl, Vaccines, Cold, X-rays, and Leucocytic Extract.
Enumerative methods employed.’ Proc. Roy. Soc., B, Vol. LXXXIII, pp. 227-234
(Prelim. Note); also Ann. Trop. Med. and Parasitol., IV, pp. 487-527.

StePHENs, J. W. W., anp Fantuam, H. B. (1910). ‘ On the peculiar morphology of a Trypano-
some from a Case of Sleeping Sickness, and the Possibility of its being a new species
(T. rbodestense).’ Proc. Roy. Soc., B, Vol. LXXXIII, pp. 28-33, 1 plate.

Txomson, D. (1g11). ‘ A new blood-counting pipette for estimating the numbers of leucocytes
and blood parasites per cubic millimetre.’ Ann. Trop. Med. and Parasitol., V,

Pp. 471-478.

537

A NOTE ON THE MEASUREMENTS
OF TRYPANOSOMA VIVAX IN
RABBITS AND WHITE RATS

BY

BE. BLACKEOCK, M.D.
From the Runcorn Research Laboratories
(Received for publication 8 February, 1912)

Using the strain of 7. vivax previously mentioned (vzde p. 521)
a large number of experimental inoculations into laboratory animals
was made. Details of these wili form part of a future paper
dealing more fully with further work done on the trypanosomes
infecting two horses from the Gambia previously referred to. This
note deals only with four rabbits and two white rats, which were
inoculated from goats which presented a pure infection with
T. vivax. In the case of the rabbits the incubation period varied
from eight to sixteen days, and parasites were present in the
peripheral blood for periods varying from one day (the shortest) to
ten days (the longest). In the two rats the incubation period was
five days, and parasites were found in the peripheral blood for only
one day. The greatest number of parasites found in the fresh
preparation was, in the case of the rabbits, five to a field
(objective DD, ocular No. 4), in the case of the rats one to thirty
fields.

The trypanosomes presented great activity of movement in fresh
preparations, and in stained preparations in dry films gave the
measurement results which are given below. As the rats had
trypanosomes in the peripheral blood on one day only, and in small
numbers, only a small number of specimens (fifty) could be drawn
from one rat. For comparison with these, fifty were drawn from
one of the rabbits on one day of the disease.

In the Rabbit. The average length of the fifty trypanosomes is
20°8 w, the maximum parasite measuring 23°2 #, and the minimum

17°4 M.

38

w

In the Rat. The average length of the fifty trypanosomes is
21°1 «, the maximum parasite measuring 26 », and the minimum
15 pM.

Table to show percentage incidence according to length in
microns of fifty 77ypanosoma vivae in a rabbit and fifty in a rat.

Percentage of

No. of Trypanosomes Animal Trypanosomes Trypanosomes | Trypanosomes
| measuring less = measuring measuring
than 2ou between 23u and over

2ou and 234

oO
ro)
~p
a
lon
ee
Noh

Mw NM

539

A CASE OF MALARIAL FEVER, SHOWING

A TRUE PARASITIC RELAPSE, DURING

VIGOROUS AND CONTINUOUS QUININE
TREATMENT

BY

Sim RONALD ROSS,’ K.C:B.,'F.R:S.
AND
DAVID THOMSON, M.B:, CH:B?; D:PSH:
(Recewed for publication 12 February, 1912)

In the ‘ Annals of Tropical Medicine and Parasitology,’ Vol. V,
No. 3, December 30, 1911, we described the occurrence of pseudo or
non-parasitic relapses in 6'7 % of our cases of malarial fever, during
active quinine treatment of ten grains thrice daily. We were unable
to prove that these ‘ pseudo-relapses,’ which usually took the form of
a sudden and isolated rise of temperature, had any connection with
the original fever, as similar inexplicable rises of temperature
occurred in 17 % of other diseases, during treatment in hospital.

Cases of malarial fever, resistant to quinine treatment, and which
showed relapses during the treatment have been reported to occur in
the Amazon region. During our experience of two years of careful
observations on cases of malaria, we found no case which showed
any resistant tendency to quinine, and twenty of these cases had
contracted fever up the River Amazon. We, therefore stated in our
paper (1911) that it was possible that these so-called resistant cases
might have been cases of pseudo-relapses, especially as no data had
been given with regard to the finding of parasites in them. We are
now able, however, to confirm these reports, having observed
carefully a case which showed marked resistance to quinine, and
which showed a true parasitic relapse during treatment with that
drug.

The details of this remarkable case, of which we publish a chart,
are as follows :—

540

Patient E. E., age 65. Half-caste, born in Canada. Occupation,
seaman.

History prior to admission. Patient had been to sea for
forty-five years, and had sailed to most parts of the world,
including India, Japan, etc. He had a slight attack of malarial.
fever six years ago, but it did not trouble him much. In May, 1911,
patient sailed up the Amazon river for the first time. He arrived
at Porto Vellho on 17th May, i911. There he left the ship and
took a post ashore as foreman of a gang of labourers. He got an
attack of fever on the 2nd of June, and was very ill, and states
that he had dysentery as well. He was in bed for a month and was
getting quinine thrice daily all the time. After this he remained well
for two months, during which period he had quinine, according to
his own statement, ‘only once in awhile.’ He had a second attack
of fever in September, but this attack was not quite so severe. He
then sailed down the river to Manaos and took a ship to England.
He was ill during the whole of this voyage and got quinine only
once or twice a week. He was admitted to the Tropical Ward of
the Royal Southern Hospital, under our care on the 23rd October,
1911. The duration of his illness before admission was, therefore,
159 days.

Details of case after admission. These are shown graphically on
the accompanying chart. On admission patient had fever, and the
blood examination revealed a mixed infection of benign and
malignant tertian malaria. A few crescents were present (about
16 perc.mm.) The blood showed marked auto agglutination of the
red cells and nucleated and stipuled basic red cells were numerous.
The haemoglobin was only 40%. There was no appreciable
enlargement of the liver or spleen. The patient was very weak and
somewhat emaciated, and had a tendency to be slightly delirous and
incoherent in his speech.

Quinine hydrobromide in liquid form was administered in doses
of ten grains thrice daily, by mouth. This reduced the asexual
parasites to below the detectable limit in thin films, in five days,
that is, about two days longer than usual. This dosage of quinine
was continued for seventeen days, during which period the
temperature remained normal. From the 3rd till the gth November
the blood was not examined; on the 10th November, however,

65 P faleiparum + P. Vivax. (Amazon)

$41

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D

comag if seensng de aw Peel awe ereens imtoy ha] (09) Yom

SSS

——

542

a rise of temperature having been noticed, the blood was examined
rapidly and no_ parasites were observed. Thinking that the
temperature was one of those pseudo-relapses which we had noticed
to occur before, during quinine treatment, we stopped the
administration of quinine for a few days. As the temperature,
however, persisted and showed a true malarial type, the
blood was again examined carefully from the 14th November
onwards. Asexual parasites, malignant and benign, were
found, as indicated on the chart, and, moreover, crescents began
to appear. Quinine was again given, as before, on the 14th
November, and the patient by this time was very ill and
slightly delirious, and seemed to have difficulty in articulation. He
commenced to pass his urine involuntarily. On the 21st November,
the fever showed no signs of abating, and on the 22nd November,
therefore, thirty grains of quinine bihydrochloride were injected
intramuscularly in addition to the usual thirty grains of quinine
hydrobromide given by mouth. In addition to this, twelve grains of
methylene blue were given daily in pill form. This combined
treatment reduced the asexual parasites below the detectable limit
in three days, and the crescents were reduced to 20 per c.mm. in
fourteen days. The patient improved very rapidly, and was no
longer confined to bed after the 10th December. On the 13th
December the methylene blue was stopped and the quinine reduced
to twenty grains daily. He left hospital on the 20th December.

Urine anaylsis. On the supposition that the quinine may not
have been properly absorbed from the digestive tract, on the 2Ist
November a twenty-four hours’ specimen of the urine was examined
by Dr. G. C. Simpson to estimate the quantity of quinine excreted.
It was found to contain sixteen grains. The patient was, therefore,
excreting thirteen grains daily out of the thirty grains administered
daily by the mouth. This is about the usual amount and showed
that the quinine administered was being efficiently absorbed.

A twenty-four hours’ specimen of urine was again examined by
Dr. Simpson on the 20th December, during treatment with twenty
grains daily. The amount recovered in the urine was six grains.
This patient was, therefore, absorbing his quinine efficiently, so
that we are forced to conclude that this case of malaria (mixed
infection) showed a most unusual resistance to thorough and
continuous quinine treatment.

543

Before concluding we would like to remark that, though this
patient was a weak old man of 65, yet in spite of a practically
continuous treatment with quinine (thirty grains daily) for fifty
days, he had no symptoms of deafness, nor did he complain of any
of the symptoms of quininism. It has been our practice to give
every case of malaria coming under our care ten grains of quinine
thrice daily for a period of three weeks, and out of 200 cases treated
in this manner, during the past two years, we have had few or no
complaints of quininism produced by this so-called severe treatment.
During such treatment the patients improve in health most
markedly. They gain weight rapidly, and the haemoglobin
percentage rises very quickly. They have always been able to hear
well, and during convalescence they were able to work well in the
ward. It is, however, not advisable to inform the patient as to
the quantity of quinine that is being administered. It would appear
that the majority of patients felt no more inconvenience from doses
of thirty grains daily, than from doses of ten grains daily, after che
first few days of administration.

CONCLUSIONS

(I.) A case of malaria, mixed infection (malignant tertian and
benign tertian), contracted at Porto Vellho, River Amazon, has
come under our care, shewing most unusual and marked resistance to
quinine, and also a true parasitic relapse during thorough treatment
with that drug.

(II.) Patients can tolerate, with very little discomfort, much
more quinine than is generally supposed, especially when they are
not aware that they are receiving heroic treatment.

REFERENCES TO LITERATURE

Ross & D. THomson (1911). ‘ Pseudo-Relapses in Cases of Malarial Fever during Continuous
Quinine Treatment.’ Ann. Trop. Med. and Parasitol., Dec., V, 3, pp. 409-412.

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fie UNIVERSITY: PRESS OF LIVERPOOL

Publications of the
Liverpool School of Tropical Medicine

MEMOIR I

Malarial Fever: its Cause, Prevention, and Treatment (1903). Con-
taining full Details for the use of Travellers, Sportsmen, Soldiers, and Residents in
Malarious Places. By Ronatp Ross, C.B., F.R.S., F.R.C.S. With Frontispiece
and Plate. 8vo. 2s. 6d.

MEMOIR II

Report of the Malaria Expedition to Sierra Leone (1899). By
RonaLD Ross, D.P.H., M.R.C.S., H. E. ANNetr, M.B., D.P.H., and E. E. AusTEN.
Being a full account of the first expedition of the School, and containing besides
much matter relating to the parasites of malaria, to the gnats which carry them, and
embodying some previous observations of Major Ross in India. Illustrated by four
maps and five full-page collotypes. Quarto. Price 21s. nett. ;

MEMOIR III

Report of the Malaria Expedition to Nigeria (1900). By H. E. Annevr,
M.D., D.P.H., the late J. Everett Durtron, M.B., Ch.B., and J.-H. Etrviorr, M.D:
Part 1. Malarial Fever, etc. Giving a full account of the expedition, with numerous
views in the text, charts, maps, and two plates, and containing much matter of
general importance. Quarto. Price ros. 6d. nett

MEMOIR IV

Report of the Malaria Expedition to Nigeria (1900). By the same
authors. Partir. Filariasis. Containing many new observations upon Filariae of
Birds, with numerous illustrations and nineteen plates, five of which are coloured
and give the microscopical anatomy of the head of Anopheles costalis (by the late
Dr. Dutton). Quarto. Out of print.

MEMOIR V, PART «1
First Progress Report of the Campaign against Mosquitoes in
Sierra Leone (1901). By Major R. Ross, F.R.C.S., D.P.H., F.R.S., dated 15th
October, 1901, giving details of the commencement of the Campaign, with a letter
from Dr. DanieELs regarding the results arrived at to date. S8vo. Price Is.

MEMOIR V, PART 2

Second Progress Report of the Campaign against Mosquitoes in
Sierra Leone. By M. Locan Taytor, M.B., dated 15th September, 1902. 8vo.
Price Is.

MEMOIR VII

Report of the Yellow Fever Expedition to Para (1900). By H. E.
Duruam, M.B., F.R.C.S., and the late WALTER Myers, M.B. (Dr. Walter Myers
died of Yellow Fever whilst serving on this expedition). Quarto. Out of print.

i

MEMOIR VIII

Report on the Sanitary Condition of Cape Coast Town (1902).
With suggestions as to improvement of same, by M. Locan Taytor, M.B., Ch.B.
S8vo. Out of print.

MEMOIR IX.

Report on Malaria at Ismailia and Suez (1903). By Ronarp Ross,
F.R-CS., DP AL. FF ELS., C.B: i8vo: ‘Out of print

MEMOIR X

Report of the Malaria Expedition to the Gambia (1902). By the late
J. E. Dutton, M:B., Ch.B. Quarto. Price 15s. nett.

MEMOIR XI

First Report of the Trypanosomiasis Expedition to Senegambia
(1902). By the late J. Evererr Dutton, M.B., Ch.B., Vict., and Joun L. Topp,
B.A., M.D., C.M., McGill. Quarto, Price 15s. nett.

MEMOIR XII

The Anti-Malaria Measures at Ismailia (1904). By RupBerr Boycg,
M.8.,°F.R:S. Price 1s.
MEMOIR XIII

Reports of the Trypanosomiasis Expedition to the Congo (1903-1904).
By the late J. Everetr Dutton, M.B., Joun L. Topp, M.D., and CuTHBERT
Curisty, M.D. Quarto. Price 21s. nett.

MEMOIR XIV

Report on the Sanitation and Anti-Malarial Measures in practice in
Bathurst, Conakry, and Freetown (1905). By Ruserr Boyce, M.B., F.R.S.,
ARTHUR Evans, M.R.C.S., H. HERBERT CLARKE, M.A., B.C., Cantab. Quarto.
Eight plates. Price 5s.

MEMOIR XV

General Sanitation and Anti-Malarial Measures in Sekondi, the
Goldfields, and Kumassi, and a comparison between the conditions of
European residence in India. By Lieut.-Colonel Gites. Quarto. -Price 7s. 6d.
nett.

MEMOIR XVI

Trypanosomes, Trypanosomiasis, and Sleeping Sickness: Pathology
and Treatment. By H. Wotrerstan Tuomas, M.D., McGill, and Anton BREINL,
M.U.Dr., Prag. Quarto. 6 plates (5 coloured) and 7 charts. Price 12s. 6d, nett.

Gland Puncture in Trypanosomiasis. By the late J. Evererr Durton,
M.B., Vict., and Joun L. Topp, B.A., M.D., McGill. Two charts and one figure.

MEMOIR XVII

The Nature of Human Tick-Fever in the Eastern part of the Congo
Free State. By the late J. Everett Dutton, M.B., and Joun L. Topp, B.A,
M.D., McGill. Quarto. With map, four plates, and nine temperature charts.
Price 7s. 6d. nett.

On the External Anatomy of Ornithodoros moubata. By R. NEwsrTeap,
A.L.S., F.E.S. With two plates.

i

MEMOIR XVIII

I. Gland Palpation in Human Trypanosomiasis ;
AND
II. The Distribution and Spread of ‘Sleeping Sickness’ in the Congo
Free State. By the late J. Everett Durron, M.B., and Joun L.
ae B.A., M.D., C.M., McGill. With four maps (2 colours) and four
plates. j
III. A New Dermanyssid Acarid. By R. Newsreap, A.L.S., F.E.S., and
Joun L. Topp, B.A., M.D., C.M., McGill. With one plate.
IV. Another New Dermanyssid Acarid. By R. Newsreap, A.L.S., F.E.S.
With one plate.
V. Anatomy of the Proboscis of Biting Flies. By J. W. W. STEPHENs,
M.D., Cantab., and R. Newsreap, A.L.S., F.E.S. With six plates.
Imp. 8vo. Price 7s. 6d. nett
MEMOIR XIX

Yellow Fever Prophylaxis in New Orleans in 1905. By RuBERT
Boyce, M.B., F.R.S. Imp. 8vo. Maps and six plates. Price 5s. nett.

MEMOIR XX

I, La prophylaxie de la Malaria dans les principaux postes de l’Etat
Indépendant du Congo. By the late J. Everetr Dutton, M.B.,
and JoHNn L. Topp, B.A., M.D. With four maps and four illustrations.

II. The Animal Reactions of the Spirocheta of African ‘ Tick Fever.’
By Anton BreEINnL, M.D., and A. Kincuorn, M.B.
III. The Specific Nature of the Spirocheta of African ‘Tick Fever.’
By Anton Breint, M.D. Imp. 8vo. Price 5s. nett.

MEMOIR XxXI

I. The Runcorn Research Laboratories of the Liverpool School of
Tropical Medicine. With five plates.

II. An Experimental Study of the Spirochaete of the African Tick
Fever (Spirochaeta duttoni). By Anton Breini, M.U.Dr., and
ALLAN KinGHorRN, M.B. With one figure and ten charts.

III. A Note on a New Spirochaete found in a Mouse. By the same
authors. With one figure.

IV. Comparison between the Trypanosomes Present by Day and by
Night in the Peripheral Blood of Cases of Human Trypano-
somiasis. By the late J. Evererr Durron, M.B., Joun L. Topp,
B.A., M.D., C.M., and E. N. Tosey, A.B., A.M., M.D.

V. TheLesions in the Lymphatic Glands in Human Trypanosomiasis.
By R. Howarp Mote, M.D. With two plates.

VI. Concerning certain Parasitic Protozoa observed in Africa. By
the late J. Everetr Dutton, M.B., Joun L. Topp, B.A., M.D., C.M.,
and E.N. Toney, A.B.,A.M., M.D. With two figures and one coloured

late.

et 3 Bitacapts to Cultivate Spirochaeta duttoni. By Lewis A. WILLIaAMs,
M.D., D.P.H., and R. StENHOUSE WILLIAMS, M.B., D.P.H.

VIII. Attempts to Transmit Spirochaetes by the Bites of Cimex
lectularius. By ANTON Breini, M.U.Dr., ALLAN KinGHorn, N.B.,
and Joun L. Topp, B.A., M.D. Imp. 8vo. Price 7s. 6d. nett.

ill

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. Vol. TI. No: 1.

Insects and other Arthropoda collected in the Congo Free State.
By R. Newsreap, A.L.S., F.E.S., the late J. E. Dutton, M.B., and
Joun..L. Topp, B.A., M.D., C,M., McGill, Six plates,

Description of Two New Species of African Ticks. By H. Neumann.

On some Parasites in the Museum of the Liverpool School of Tropical
Medicine. By A. Looss. Three plates.

The Presence of Spirochaeta Duttoni in the Ova of Ornithodoros
Moubata. By Capt. R. MARKHAM CarTER, I.M.S. One plate.

A note on the Therapeutics of Trypanosomiasis. By Brnjamin Moore,
M.A., D.Sc., M. NIERENSTEIN, Ph.D., and Joun L. Topp, B.A., M.D.,
C.M., McGill. Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY? | Vol. I... No. 2.

An Automatic Oiler for the Destruction and Prevention of Mosquito
Larvae in Cesspools and other collections of Water. By E. H. Ross,
and H.C. Ross. One plate.

The Anatomy of the Proboscis of Biting Flies. Part II, Stomoxys. By
J. W. W. STEPHENs, M.D., and R. Newsteap, A.L.S., F.E.S. Eight plates.

Trypanosome Transmission Experiments. By the late J. EvErerr Durron,
M.B., Joun L. Topp, B.A., M.D., C.M., McGill, and J. W. Hanineron,
M.D., McGill,

Cattle Trypanosomiasis in the Congo Free State. By the late J. EvEREtTT
Dutton, M.B., Joun L. Topp, B.A., M.D., C.M., McGill, and ALLan
KincHorN, M.B. Two plates and six charts.

Concerning the Treatment of Experimental Trypanosomiasis. By
BenjAMIN Moore, M.A., D.Sc., M. NIERENSTEIN, Ph.D., andJoun L. Topp,
B.A., M.D., C.M., McGill. Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. (Volz I. 2 No.3:

Concerning certain Parasitic Protozoa observed in Africa. By the late
J. Everett Durton, M.B., Vict., Joun L. Topp, B.A., M.D. McGill, and
E. N. Tosey, A.B., A.M., M.D. Harvard. Thirteen plates.

Yaws. By C. W. Brancu, M.B., C.M. (Edin.)

A Description of some Gold Coast Entomostraca. By W. M. Granam,
B.A., M.B.

Notes on Dr. Graham’s Collection of Cyclopidae from the African Gold
Coast. By G. Stewarpson Brapy, M.D., LL.D., D.Sc.,F.R.S. Four plates.

On the Morphology and Life History of Spirochaeta duttoni. By
ANTON Breinit, M.U.Dr. (Prag.) One plate.

The Cytology of the Trypanosomes. Part I. By J. E. Sarvin-Moorg,
A.R.C.S., F.L.S., F.Z.S., and Anton Breint, M.U.Dr. (Prag.) Five plates.

Imp. 8vo. Price 7s. 6d. nett

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY; ‘VolpTo: No; 4.

Observations on the so-called ‘Canary Fever.’ By C. E. Waker.

Gontribution a l'étude de Porvocephalus moniliformis. Par A. BRoDEN et
J. Ropuain. One plate.

On the Habits, Life-Cycle, and Breeding Places of the Common House-
Fly (Musca domestica, Linn.). By R. Newsreap, A.L.S.,F.E.S. Six plates.

Some Notes on the Morphology of Spivocheta duttoni in the Organs of Rats.
By J. J. van Locuem, M.D., Amsterdam.

Malaria and History. By W. H.S. Jones, M.A.

Two New Human Cestodes and a New Linguatulid. By J. W. W.

STEPHENS, M.D., Cantab., D.P.H. One plate.
Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. Vol. II. No. 1.

Atoxyl and Trypanosomiasis. By Sir Rupert Boyce, F.R.S., and ANTON
BreEINL, M.U.D. Prag.

My Experience of Trypanosomiasis in Europeans and its Treatment

by Atoxyl and other drugs. By Sir Patrick Manson, K.C.M.G., F.R.S.

Imp. 8vo. Price 7s. 6d. nett

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY.” Vol? IT Nie? 3.

Reports of the ‘Sleeping Sickness’ Expedition to the Zambesi for
the years 1907-1908. By A Lian KincHorN, M.B., and R. Eustace
Montcomery, M.R.C.V.S. One map.

A Report on Trypanosomiasis of Domestic Stock in North-Western
Rhodesia. By R. Eustack Monrtcomery, M.R.C.V.S., and ALLAN
KinGHorNn, M.B. One map and ten charts.

Report on the Work of the Greek Antimalaria League during the
year 1907. By M. Hapjrmicna.is, President, and JEAN P. CaRDAMATIS,
General Secretary. Imp. 8vo. Price 7s. 6d. nett

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY.» ‘Voli lle’ No.-2

A Peculiar¥IntralobularftCirrhosis of the Liver produced by the Protozoal
Parasite of Kala-azar. By Lronarp Rocers, M.D., F.R.C.P., B.S.,
F.R.C.S., I.M.S. One coloured plate.

What is ‘Schistosomum mansoni’ Sambon 1907? By Dr. A. Looss.

The Prevention of Dengue Fever. By E. H. Ross, M.A Smee
L.R.C.P. Lond.

The Life History of Trypanosoma lewisi. By J. E. Satvin-Moore, A.R.C.S.,
F.L.S., F.Z.S., Anron Brerimnt, M.U.Dr. Prag., and Epwarp HInNpDLE,
A.R.C.S. Lond. Four plates.

Notes on the Effects of Therapeutic Agents on Trypanosomes in respect
to (a) Acquired Resistance of the Parasites to the Drug, and
(b) Changes in Virulence of the Strains after Escape from the
Drug. By Benjamin Moore, M.A., D.Sc. (R.U.I.), Maximrtian NIERENSTEIN,
Ph.D. Berne, and JoHn LancELot Topp, B.A., M.D. McGill, M.R.C.S.

Observations on the Acidity and Alkalinity of the Blood in Trypanosome
Infections. By M. NierensTEIn, Ph.D.

Contributions to the Morphology and Life History of Piroplasma Canis.
By Anton BrEINL, M.U.Dr. Prag., and Epwarp HInpte, A.R.C.S. Lond.
Four plates.

Comparative Chemotherapeutical Study of Atoxyl and Trypanocides.
Part I. By M. NigeRensTEIN, Ph.D.

On Three New Species of Culex collected during the Antimalarial
Campaign in Mauritius in 1908. M.D’ EMMEREZ DE CHARMoY.

Imp. 8vo. Price 7s. 6d. nett
vi

ANNALS OF TROPICAL MEDICINE AND
PARASEPOLROGY Vol) Ile ANot @.
Concerning the Treatment of Experimental Trypanosomiasis. Part II,

By Benjamin Moore, M.A., D.Sc., M. NigRENSTEIN, Ph.D., and Joun L.
Topp, M.D.

An Unusual Case of Goundou. By Dr. R. W. Orpen. One plate.

Sub-Drainage as Applied to the Anti-Malarial Campaign on the Isthmus
of Panama. By Henry Simms.

A New Culicid Genus. By F. V. THEopatp, M.A.

The Inflicted Talipes of the Chinese. By Frank Jeans, M.A., M.B.,
B.C. Cantab., F.R.C.S. Eng. Two plates.

Contribution a l’étude de Porocephalus moniliformis. Par A. BRopEN et
J. Ropuain.

A New Human Nematode, Strongylus gibsoni,n. sp. By J. W. W. STEPHENS,
M.D. Cantab. Two plates.

On the Supposed Occurrence of Filaria immitis in Man. By J. W. W.
STEPHENS, M.D. Cantab.

A New Porocephalus (Porocephalus cercopitheci, n. sp.). By ANTON BREINL,
M.U.Dr. Prag., and Epwarp Hinp ez, A.R.C.S. Lond.

Comparative Chemo-therapeutical Study of Atoxyl and Trypanocides.
Part II]. By M. NigRENsTEIN, Ph.D.

Chemical Notes on Atoxyl. By M. Nierenstein, Ph.D.

Note sur le rdle des Tabanides dans la Propagation des Trypanosomiases.
Par Le Dr. EDMonD SERGENT. Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. Vol. II. No. 5.

On the Nomenclature of the Mammalian Trypanosomes observed in
North Western Rhodesia. By R. Eustace Montcomery, M.R.C.V.S.,
and ALLAN KINGHORN, M.B. Toronto.

Experiments on the Combined Atoxyl-Mercury Treatment in Monkeys
Infected with Trypanosoma gambiense. By Anton Breint, M.U.Dr. Prag.

Drugs from the Congo. By Prosper H. Marspen, F.C.S.

A Gregarine Parasitic in the Dog-Flea, Ctenocephalus serraticeps. By
E. H. Ross, M.R.C.S. Eng., L.R.C.P. Lond.

The Action of Aryl-Stibinic Acids in Experimental Trypanosomiasis.
By Anton Breini, M.U.Dr. Prag., and M. NIERENSTEIN, Ph.D. Berne.
Short Note on the Mechanism of Haemolysis in Piroplasmosis canis.
By Anton Brent, M.U.Dr. Prag., and H. E. Annet, M.D., D:P.H.

Gland Puncture in the Diagnosis of Animal Trypanosomiasis. By
R. Eustace Montcomery, M.R.C.V.S., and ALLAN KinGuorn, M.B. Toronto.

Observations on the Hooklets of Cysticercus cellulosae in Man. By J. W. W.
STEPHENS, M.D. Cantab, D.P.H. Imp. 8vo. Price 7s. 6d. nett

vil

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY) Wolk TIEIA Mie 1.

An Investigation into the Mechanism of Production of Blackwater.
By J. O. WakeELIN Barratt, M.D., D.Sc., Lond., and WarriInGTon YoRKE,
M.D., Liverpool.

Imp. 8vo. Price ros. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY.,)..Vol, III.....Ne: 2.

On the Flagellates occurring in the Intestine of Glossina Palpalis and in the
Intestine and Proboscis of Glossina Morsitans. By ALLAN KINGHORN,
M.B., Toronto, and R. Eustace Montcomery, M.R.C.V.S.

Second Report on Human Trypanosomiasis in North-Eastern Rhodesia
and Nyasaland. By ALian KincHorn, M.B., Toronto, and R. Eustace
Montcomery, M.R.C.V.S.

A further Report on Trypanosomiasis of Domestic Stock in Northern
Rhodesia (North-Eastern Rhodesia). By R. Eustacrk Montcomery,
M.R.C.V.S., and ALLAN KinGcHorN, M.B., Toronto. Two Plates and Map.

Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITEFOLOGY.’ Vol. IIT. Nora

Sanitary Measures and Malaria Epidemics of Athens. By Jouw
CarpDaAMATIS. ‘Three plates.

An Account of a Form of Splenomegaly with Hepatic Cirrhosis,
Endemic in Egypt. By H. B. Day, M.D., B.S., Lond., M.R.C.P., and
A. R. Frrouson, M.D., C.M. Five plates.

Bio-Chemical and Therapeutical Studies on Trypanosomiasis. By
ANTON BrEINL and M. NIERENSTEIN.

Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY ?"Vol? MI, "Nei 2:

Reports of the Twenty-first Expedition of the Liverpool School of
Tropical Medicine. Jamaica, 1908-1909: Medical and Economic
Entomology. Part I.—Ticks and other Blood-sucking Arthropoda.
By Ropert Newsreab, M.Sc., A.L.S., &c. Three plates.

Reports of the Twenty-first Expedition of the Liverpool School of
Tropical Medicine. Jamaica, 1908-1909: Malaria. By W. T. Prout,
C.M.G., M.B., Ch.M. Three plates.

Imp. 8vo. Price 7s. 6d. nett.
Vili

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY? Vol TIL. oNd;.6.

Observations on the Life History of Trypanosoma lewisi in the Rat Louse
(Haematopinus Spinulosus). By ANToN BreINL and Epwarp HInpLe.
Two plates.

On the Variation of the Haemolytic Complement in Experimental
Trypanosomiasis. By Warrincton Yorke, M.D.

Acute Craw-Craw. By R. H. Kennan, M.D., D.T.M. Two plates.

A Preliminary Note on the Prevalence of Mosquitoes in Cairo and its
Enivirons.” By F. C. Wirucocks:

The Effect of Mosquito Larvae upon Drinking Water. By RusBeErT
Boyce, F:R.S,, and F; C.. Lewis.
Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. Vol. IV. No. 1.

The Sanitary Conditions and Diseases Prevailing in Manaos, North
Brazil, 1905-1909. By H. Worrerstan Tuomas, M.D., C.M. (McGill).
Plan and chart.

The Pathological Report of a Case of Césophagostomiasis in Man.
By H. Wotrrerstan Tuomas, M.D., C.M. (McGill). Seven plates.

Etude Zoologique de L’Q@:sophagostome de Thomas. Par A. RAILLiet
et A. Henry. One plate.

“Mossy Foot” of the Amazon Region, an infective verrucotic
condition affecting the skin of the upper and lower limbs. By
H. Wo.FersTAN Tuomas, M.D., C.M. (McGill). Three plates.

Guarana. By Prosper H. Marspen, F.C.S. One plate.
Some of the Chemical Constituents of Guarana. By M. NierensteEIn, Ph.D.
Yellow Fever. By H. Worrerstan Tuomas, M.D., C.M. (McGill).

The Mosquitos of the Amazon Region. By R. Newstrap, M.Sc.. A.L.S., etc.,
and H. Wo.Frerstan Tuomas, M.D., C.M. (McGill). One plate.
Imp. 8vo. Price ros, 6d, nett

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. Vol. IV. No. 2.

Parasitic Granuloma. Conditions allied to Oriental Sore occurring in Egypt.
By A. R. Fercuson, M.D., and Owen Ricnwarps, F.R.C.S., M.Ch., Oxon.
Four plates.

Contribution a L’Etude du Porocephalus armillatus. Par A. BRopEN et
J. Ropuarn.

On the Absence of a Vesicant in the Ether Extract obtainable from
Mosquitos. By J. O. WAKELIN Barratt, M.D., D.Sc., London.

Factors in the Transmission and Prevention of Malariain the Panama
Canal Zone. By S. T. Dartine, M.D.

Preliminary Experiments on the Effect of Cold on various Diseases
in Small Animals. By Professor Major Ronatp Ross, C.B., F.R.S., and
Major C. L. Wixtiams, I.M.S. (Retired).

Malaria Prevention in Jamaica, By Sir Ruperr Boyce, F.R.S. Two plates.

On some Species of Cyclops and other Entomostraca collected by
Dr. J. M. Dalziel in Northern Nigeria. By G. StrEwarpson Brapy,
M.D., LL.D., D.Sc., F.R.S. Three plates.

A New Anopheline from the Federated Malay States. By Matcorm
Watson, M.D.; D.P.H.

On the Occurrence of Schizogony in an Avian Leucocytozo6n, L. lovati,
Parasitic in the Red Grouse, Lagopus scoticus. By H. B. FantrHam
D.Sc., Lond., B.A., Cantab., A.R.C.S. One plate.

A Case of Sleeping Sickness Studied by Precise Enumerative Methods:
Regular Periodical Increase of the Parasites disclosed. By Professor
Major RonaLtp Ross, F.R.S., and Davip THomson, M.B., Ch.B., D.P.H.

Imp. 8vo. Price 7s. 6d. nett.

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY... Vol. iV. Wa. 2

Some Enumerative Studies on Malarial Fever. By Major Ronatp Ross,
F.R.S.; and Davin Tuomson, M.B., Ch.B., D.P.H.

A Case of Blackwater Fever followed by a Peculiar Relapse without
Haemoglobinuria or Detectable Plasmodia. By Major R. Ross, F.R.S.;
D. Tuomson, M.B., Ch.B., D.P.H.; and G. C: E. Simpson, B.A., B.Sc.,
F.R.C.S. One chart.

On Haemoglobin Metabolism in Malarial Fever. By G. C. E. Simpson,
BA, BiSagC:S.

Some Observations on a Case of Sleeping Sickness: Coagulation Time
of Blood, Albumoses, Choline, Cerebral Sections, By Visxunu T.
Korke, M.R.C.P. (Edin.), D.T.M. (Liverpool), L.M.&S. (Bombay).

Some Observations on Malaria in Relation to Splenic Enlargement
and the Treatment of the Crescentic Stage. By Dr. N. F. SuRvEyor,
M.A., M.D. (Bom.), M.R.C.P. (Lond.)

On the Peculiar Morphology of a Trypanosome from a Case of Sleeping
Sickness and the Possibility of its being a New Species
(T. rhodesiense). By J. W. W. StepHens, M.D. (Cantab), D.P.H.; and
H. B. Fanruam, D.Sc. (Lond.), B. A. (Cantab). One plate.

On the Pathogenicity of a Trypanosome (T. rhodesiense, Stephens and
Fantham) from a Case of Sleeping Sickness Contracted in Rhodesia.
By WarRINGTON YorKE, M.D.

On Three New Species of the Genus Glossina, together with a Description
of the hitherto Unknown Male of Glossina grossa, Bigot. By RoBERT
NEWSTEAD, M.Sc., A.L.S., &c.

Descriptions of a New Genus and Three New Species of Anopheline
Mosquitos. By R. NewstTeap, M.Sc., A.L.S., &c.; and H. F. Carrer.
Two plates Imp. 8vo. Price 7s. 6d. nett.

x:

ANNALS OF TROPICAL MEDICINE AND
PARASITOLOGY. Vol. IV. No. 4.

A Note on the Pathology of Lesions of the Cornea and Skin in Animals
Experimentally Infected with T. rhodesiense. By Warrincton YorKE.
M.D. ‘Two plates. i

A Case of Sleeping Sickness Studied by Precise Enumerative Methods:
Further Observations. By Major Ronatp Ross, F.R.S., and Davip
Tuomson, M.B., Ch.B., D.P.H. One chart.

Enumerative Studies on Trypanosoma gambiense and Trypanosoma rhodesiense
in Rats, Guinea-pigs, and Rabbits; Periodic Variations Disclosed.
By H. B. Fantuam, D.Sc., B.A., and J. G. THomson, M.A., M.B., Ch.B.
Eight charts.

The Life-History of Trypanosoma gambiense and Trypanosoma rhodesiense as
seen in Rats and Guinea-pigs. By H.B. Fanruam, D.Sc. Lond., B.A.
Cantab., A.R.C.S. One plate.

Experiments on the Treatment of Animals Infected with Trypanosomes
by means of Atoxyl, Vaccines, Cold, X-rays and Leucocytic
Extract; Enumerative Methods Employed. By Major R. Ross, C.B.,
F.R.S., and J. G. THomson, M.A., M.B., Ch.B.

Auto-agglutination of Red Blood Cells in Trypanosomiasis. By
WARRINGTON YorKE, M.D. :

On a New Genus of Culicinae from the Amazon Region. By R. Newsreap,
M.Sc., &c., and H. F. Carter.

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Further Experimental Researches on the Etiology of Endemic Goitre.
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plates.

Non-ulcerating Oriental Sore: the Cultural Characteristics of the
Parasite as compared with a New similar Parasite in Erthesin«
fullo (Thumb), a Pentatomid Bug. By Captain R. Markuam Carrer,
I.M.S. Two plates.

Infantile Leishmaniasis (Marda tal Biccia) in Malta. By A. Critien, M.D.,
D.S., D.T.M. (Liv.)

I—A Research into the Production, Life and Death of Crescents in
Malignant Tertian Malaria, in Treated and Untreated Cases,
by an Enumerative Method. By Davin Tuomson, M.B., Ch.B. (Edin.),
D.P.H. (Camb.). Seven charts.

II.—The Leucocytes in Malarial Fever: a Method of Diagnosing
Malaria long after it is apparently cured. 3y Davin THomson,
M.B., Ch.B. (Edin.), D.P.H. (Camb.) Eight charts.

Note upon Yellow Fever in the Black Race and its bearing upon the
Question of the Endemicity of Yellow Fever in West Africa.
By Sir Rupert Boyce, F.R.S., M.B. Two charts.

On the Amoebae Parasitic in the Human Intestine, with Remarks on
the Life-cycle of Entamoeba coli in Cultures. By H. B. FantHam
D.Sc. Lond., B.A. Cantab., A.R.C.S.

Some Further Observations on the Tsetse-fly, described in these
Annals as Glossina grossa, etc. By Ropert Newsteap, M.Sc. A.L.S., Ete.

On the Correlation Between Trypanosomes, Leucocytes, Coagulation
Time, Haemoglobin and Specific Gravity of Blood. By Visunu
T. Korke, M.R.C.P., D.T.M.

x1

ANNALS OF TROPICAL MEDICINE AND
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Note on Tropical Diseases in Southern Italy. By Professor UMBERTO GABBI,
(Rome).

The papatacs Flies (Pilebotomus) of the Maltese Islands. By R. Newsveap,
M.Sc., A.L.S.,. &c. . Three plates.

The Experimental Transmission of Goitre from Man to Animals. By
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Reducing Action of Trypanosomes on Haemoglobin. By Ratpxw W. Nauss

and WARRINGTON YORKE.

The Anti-Malarial Operations at Ismailia. By J. W. W. SivepHens, M.D.
(Cantab.). Two maps.

On Some New Species of African Mosquitos (Culicidac), By R. Newsreap,
M.Sc., A.L.S., &c., and Henry F. Carter. One plate.

The Diagnosis and Distribution of Human Trypanosomiasis in the
Colony and Protectorate of the Gambia. By Joun L. Tonp, M.D.,
and S. B. WorBacH, M.D. One map.

The Mechanism of the Production of Suppression of Urine in Black-
water Fever. By Warrincron YorKE and Ratpu W. Nauss. ‘Two plates.

On the Relation of the Organic Phosphorus Content of Various Diets
to Diseases of Nutrition, particularly Beri-Beri. By G. CE.
SIMPSON, .B.A., M.B.,.B.Sc., and E. S. Eprz; M.A, B.seye Farts

ANNALS OF TROPICAL MEDICINE AND
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Tables of Statistical Error. By Professor Sir Ronatp Ross, K-C.By Paks
and WaLTER Srort.

Notes on some Blood Parasites in Reptiles. By Dr. Haratp SEIDELIN.
Two plates.

Some Experiments: on Larvicides. By Sir R. Ross, K.C.B,, H RiSgeage
E. S. Epre, M.A., B.Sc. :

An Investigation of the Effects produced upon the Excretion of Urinary
Pigments by Salts of Quinine. By W. M. Granam, M.B. Onze plate.

The Passage of Haemoglobin through the Kidneys. By Warrinetron
YorKE, M.D. One plate.

Pseudo-Relapses in cases of Malarial Fever during Continuous Quinine
Treatment. By Sir Ronatp Ross, K.C.B., F.R.S., and Davin THomson,
ME: Chobe, wOsP He One chart,

The Trypanosomes found in Two Horses Naturally Infected in the
Gambia. By Warrincron Yorke and B. BLackLockx. One plate.

An Examination of the City of Georgetown, British Guiana, for the
Breeding Places of Mosquitos. By K. S. Wise, M.B., B.S B.Seg
D.P.H. Chart and plate.

A Case of Human Trypanosomiasis in Nyasaland with a Note on the
Pathogenic Agent. By Huan S. Srannus and WarriNGTON YORKE.
One plate.

A Second Series of Experiments dealing with the Transmission of
Goitre from Man to Animals. By Rosert McCarrison, M.D., R.U.I.,
M.R.C.P. (Lond.) Three plates. ;

A New Blood-counting Pipette, for estimating the numbers of Leuco-
cytes and Blood Parasites per cubic millimetre. By Davin THomson,
M.B., Ch.B. (Edin.) D.P.H. (Camb.)

Some Researches on the Life-cycle of Spirochaetes. By H. B. Fanrnam,
D:Sc.5/B. A. seat

Desmogonius desmogonius, a new Species and Genus of Monostome Flukes.
By J. W. W. SrepHeNs, M.D. (Cantab.) One plate.

X11

ANNALS OF TROPICAL MEDICINE AND
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Notes on some Blood-Parasites in Man and Mammals. By Haratp
SEIDELIN. Plate XXIV. Two Charts. ;

The Genus Pristirhynchomyia, Brunetti, rg10. By Captain W. S. Parton, M.B.,
I.M.S., and Captain F. W. Crace, M.D.,1.M.S. Plate XXV.

The Life History of Philaematomyia insignis, Austen, By Captain W.S. Parron,
M.B. (Edin ), I.M.S., and Captain F. W. Cracc, M.D. (Edin.). I.M.S.

The Measurements of a Thousand Examples of Trypanosoma vivax. By
B. Bracktockx, M.D. Three Charts.

Enumerative Studies on 7. brucei in Rats and Guinea-Pigs, and a
Comparison with 7. rhodesiense and T. gambiense. By JoHN Gorpbon
- THomson, M.A., M.B., Ch.B. Four Charts.

A Note on the Measurements of Trypanosoma vivax in Rabbits and
White Rats. By B. Biacxiock, M.D.

A Case of Malarial Fever, showing a True Parasitic Relapse, during
Vigorous and Continuous Quinine Treatment. By Sir Ronatp Ross,
K.C.B., F.R.S., and Davip THomson, M.B., Ch.B., D.P.H. One Chart.

TEXT BOOK

The Practical Study of Malaria and other Blood
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