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6

MiCIOrOPY RBOIUTION TBT CHART

(ANSI and ISO TEST CHART No 2)

A /APPLIED IIVMGE Ine

^^ 1653 East Mo,r ^tffet

B^S Rochester. Neo Tofh !4'i)09 USA

'■Jg (716) 482 - 0300 - Phone

SaSS {7t6) 288 - 5989 - Fa«

FEB 17 1921

DOMINION OF CANADA

DEPARTMENT OF AGRICULTURE

HEALTH OF ANIMALS BRANCH

DOURINE IN CANADA

1904-1920

HISTORY, RESEARCH
AND SUPPRESSION

BY

E. A. WATSON. V.S., (Qpt. C.A.V.C.)

CHIEF ANIMAL PATHOLOGIST

b 19.1
•CZ12L cj

OTTAWA
THOMAS MULVET
WINTER TO THE KING'S MOST EXCELLENT MAJESTY

1920

DOMINION OF CANADA

DEPARTMENT OF AGRICULTURE

HEALTH OF ANIMALS BRANCH

DOURINE IN CANADA

1904-1920

HISTORY,

RESEARCH AND

SUPPRESSION

BY

E. A. WATSON. V.S.. (Qpt. C.A.V.C.)

CHIEF ANIMAL PATHOLOGIST

OTTAWA

THOMAS MULVEY

PRINTER TO THE KINGS MOST EXCELLENT MAJESTT

1920

INTRODUCTION.

Canadian experience with dourine extends over a p* riod ,>( fifteen years and covers
the study and suppression of a rnre and serious disease hitherto unknown in Pui.ada.

Although the disease is one of the oldest rreord^xl in v-terinary literature and
has been known to the Arabs an.l in certain countries of Asia and Africa for centuries.
it is only within the past twenty-fivo years tlu.t the nature of it has come to bo
recognized and understood. The blood para-ite Tnipanosoma cquiperdum. seen for
the first time by Uouget, in 1894, was for a hm^ time cont^'sU-d by leading veterin-
arians of Europe as beinp the specific axuM! of dourine, and the question was still
in dispute ten years Inter when the disease made its first appearance in Canada.

It was then recognized by Inspector Burnett (1904), and immediately afterwards
by Dr. Rutherford, then Veterinnry Dir.'ctor G.n.nil, who. as n result of his investiga-
tion and in the absence of positive proof, h,.lj to his conclusions and adopted a policy
the wiadom of which was borne out by subsequent events. The long search for the
specific cause of the disease and its discovery; the experimental work patiently and
labouriously pursued year after year; the determination of siK:.cific serum methods
of diagnosis, by the aid of which tb.e disease was (v.Mtiially suppressed; and the
difficulties met with and over,., me both by the veterinary insi^ctors in charge of the
tield work and by the pathologists in the laboratory— furnish an exami)le of persistent
and unrelenting effort and success achieved in an important problem of veterinary
research and sanitary science.

This report includes a review and summary of papers and records published in the
Annual Reports of the Veterinary Director General for the years 1904 to 1919, a
general description of the disease, and a number of appendices.

It is presented by permission of Dr. F. Torrance who for the past seven years hag
directed the work of suppressing dourine and has brought it to success.

9340— U

i

CONTKNTS.

Paoc

Introduction *

History, Research mul Sup|ire»sioii, n review and Mimniur.v .1

Durine Statisfio' '■'

The Natural Disens.- W

Diagnosis. — Aptieiidix Nc. 1 l'>

The Complenwnt Fixation Test.— Appendix No. :.' I'l

Experimental Dourine. — Appendix No. ?. !15

Trypanosoma equipi'iilum. — Appendix No. 4 39

Kejtulations relating to Maladie du Coit (Dourine). Appendix No. :, 40

Iteferences. — Appeinlix No. t! 41

Kxplanntion of Plate-* 42

K.xplanation of Charts 411

Plates— I to IX.

History Chart*— I to HI.

Temperature Cl.arts-I. T... 11-, I.'; IT, 11... HI.; Ill; IV; IX and X.

HISTORY, RESEARCH AND SUPPRESSION.
Review and Sumnviry.

S
9

10

1.-.

It!
35
39
40
41
42
43

The (ligcnsp wiis tir«t obsorve«l in the provinm of AlU-rfn, in tho Lethbridgo
ilistrift, ill tiin i:irly -i'r>'i« »f ilx' .vi iir Ut<U. I'miii tli,. imniirics inii(l«« in tho
iifiKhbourhood it ;i|.|Mnri'.l prnlmhl<> that tho ilis.imc imd lii^n iiitroducoil s.>vpral
ypiira prcvi(iusly. 1 hoinjli thf oriiiin wax m vtr ixactiy lo.Mtcl tli<-rc is itoix] reason
for attributing it to aniniaU bnnitflit in from the Wi -tirn Knitoil State* in which,
iiiiionjr ranRo iiiiiniiii-'. th.' (Iis.>:i«f was itnowii to cxi^t from finu- to timo.

In March, 1!HI4. ln'*|«<-tor liurnrtt, Chipf V.t.riiiisry Officer of th.> Royal North
H.jst MouutiMl Poiic... report.',! iji,. <.:;iMf,nifH. of .jourin.' ( Mnhuli.- .lu Ooit) in a
shillum and srv.ral mar..«, lo.'ato,! n.-ar Ix-thbri.ljr.'. Alb.rta. Dr. Ituthorford. Vrtor-
inary Director Gcnoral, Dr. irarjrrnvo. of Medieino Hat. Dr. Davison, of tho United
States Bureau of Animal Industry, mid r.tli. • veterinarians examin.,! th,. atle.'fecl
atiinials and ajfree 1 upon tlie diaftnosis.

A «iuarantine station of 1,s(m» acres of fcn.^ed laii.i wi-.s thercuiKin establisbe<l
near Letlibridw., wlure all .*u>pe<ted animals were ,<athered in and held unlei-
observation. I'ndir the Animal ('onti.trious Diseases Ai-t, l!»i»,1, retrulations relatinir
to this disease were fornmhit.<d and bi murht into force (Apix-n.lix So. .'.>. Pro-
vision was also made for the slaujfhter of inf.-eto.l animals and tho payment of reason-
able compensation to the owners.

The (luaranfined animals were re-examined after an interval of several months
and when it was <xpeeted that acute or w.ll-markeil symptoms of the disease would
have developed. Dr. Hutherf..rd reported that instead of tindinff an njwravation
of symptoms aiul a progressive state of disease, a surpris.' was en.'ountered in that no
well-marke.l c;>s<. was presented, thi t many of the affected animals had improved in
a marked do>;roe, and that there was not nmonjf them any one ease sufficiently
pronounced to warr.mt an order for its destruction.

Donbts began to arise ns to whether the disease wa« actually dourine or .somo
other. M.iny examinations of blood and of the body fluids obtained from affected
animals were made in tlic field, and .specimens were sent to various laboratories, but
without revealing any trace of the presence of the parasite or any definite information
as to the nature of the disease. Dr. Rutherford drew attention to the fact that dourine
i-s an indigenous disease in Asia and in Africa had l>eeome more irregular and uncer-
tain m Its course and manifestations in Europe and in America, and. probably, still
further modified under tho i)eeuliar climatic conditions of tho northwest provinces of
Canada. The decision was made not to immediately adopt a policy of slauRhter, but
to hold the suspected animals for a further period in quarantine until more satisfaetoiy
evidence was obtainable, and also for the purpose of acquiring as much information
as posf ibie as to the behaviour of the disease in this country.

In May, 1905, the animals were again examined by Dr. Rutherford and accompany,
mg veterinary officers. In some eases the disease wps found to have made progress, in
others there was little change from conditions noted on previous occasions. One
hundred and sixteen horses were destroyed; a considerable number held over for
further observation. SiKjcial officers were detailed to deal with the disease and were
authorized to order the slaughter of any clearly marked castas coming under their
notice.

In September, 19(K"., it was decided to slaughter all the animals at the quarantine
station excepting a few which had been under observation for upwards of twelve
months and appeared to be healthy. It was then arranged to utilize the existing

5

quarantine station for experimental work in connection with dourine, and Dr. Hadwen,
an inspector at Nelson, B.C., was transferred to Lethbridge and placed in charge, wi*li
a number of condemned animals at his disposal. Three infected animals were sent to
the Biological Laboratory, Ottawa, for study and experimi^nts.

Investigations were continued in the field and in the laboratories during the
winter of 1905-06 and the following summer. The disease was found to exist in various
parts of southern Alberta, and bj October 31, 1906, 412 animals had been destr'ived.

At the Lethbridge quarantine station breeding experiments were conducted with
the object of ascertaining whctlier app.nrently recovered mares would bear healthy
offspring or would rem.iin sterile. I)i.«eased and healthy stallions were employed in
these experiments. Several slightly affected mares were spayed in the hope that such
cases could je spared destruction and usefully employed for work purposes only. The
search for the dourine parasite Trypanosoma cquiperdum was kept up continuously
both at Lethbridge and at Ottawa. Microscopical exami.ia'ions were made of great
numbers of specimen.^; dogs and other laborator>' animaia were inoculated with blood
and material obtained from diseased tissues, but all were negative in result and every
attempt to detect the parasite mot with failure.

At the Biological L.iboratory, Ott.^wa, Higgins and Watson had made a cytological
study of the blood of dourined horses comparatively with the blood of normal horses
and in certain diseases. It was found that the leneoeyto formula in dourine differed
from that in normal horses and in the other diseases '' lied, and was of somo
diagnostic value. A i)athoge:iio trypanosome, Tr. gamhiense, was maintained and
studied in a series of laboratory animals, mainly with the idea of acquiring a knowl-
edge of the habits ni.d eharaeteristics of a blood-haunting protozoan parasite elosel.v
allied to that of dourine. In October, 1906, Watson took over the investigation of
dourine and removed to Letiibridge. In November and 1 ' oember of that year he
discovered trypanosomes in tlie blood of native "cotton-tail ' rabbits and in a species
of northern deer-mouse caught on the quarantine grounds at Lethbridge. These
findings created much interest at the time, being the first record of trypanosomes found
in mammalian blood in Canada.

The important discovery of 'J'r. rqulpordutn was made in February, 1907, by
Watson, in a dourined mare in which tjie disease had been recognized by Inspector
Oallivan. The reproduction of the disease by inoculations of this parasite into healthy
horses, and the r'^i'ovcry of the parasites from the typical lesions produced, removed
all doubt as to wliethfr the dourine of America was a trypanosome disease or not, and
<'!early establiL^hed its identity with the dourine of Asia and Africa and of European
countries. However, in Africa and Asia, wliere the disease exists in its most severe
term, no great difficulty seems to have bi>en encountered in defecting the parasite or
in diagnosing the dix'ase by its rliiiical maiiifestiitions. In Europe, on the contrary,
the search for the tryiianosomc was for many year< un-uecessful, the course of the
disease was found to be irregular and uncertain, and diagnosis, as a rule, had to be
based np<in clinic .'. symptoms. \vlii<'h, as often a< not, w<'r( too vague and elusive to
permit of satisfactory conclusions. In .\merica it w:'s already evident that the discns<'
occurred in a still more insidious form and that fr<Hiuently in the early stages of
infection, which might coiitinne f<ir very lengthy periods, neither a characteristic
symptom iior a warning '^igii nf the ili-c'isc was presented. Further, and far from
bi'ing an invariably fnlal disease, many infected marcs retained or recovered apparent
health, though suspected of being capable of transmitting the disease to the covering
stallion. The bett<r bred ami espi'cially the pure-bred imported animals of hifrh
commercial value were usually tl'.c most severely nffeeled and the first to succumb.
Prairie-bred animals of the native pony and bronciio types were best able to resist or
tolerate dourine iMfi'<'tion and to lend tliemsclves as <'arriers of it, and in the absence
of any reliable means of recognizing them as such. cori..tituted a frequent and
danjrrrous suurrt- of ijii's.i-'.iiiii. Al this time large herds of hurscs lived ant! lifed e!:
the opiMi ranges where natural conditions all favoured the spread of the disease.

The horse industry wii:* thus faced with a serious menaoe and the Veterinary
Department with the formidiihlo task of overcoming it. The prospect of eradication
in face of the apparently insurmountable diflicultics in dingnosis did not look encour-
aging. It was decided to carry on research, to study the disease from its various
aspects and, if possibe, devise a specific or accurate method of diagnosis which would
enable the department to deal with dourinn in an efficient and satisfactory manner and
to finally suppress it in its entirety.

During the next five years, 1007-12, eases of duurine were discovered in a
number of the breeding districts in the provinces of Alberta and Saskatchewan and
reported on by Inspectors Gallivan and W. L. Ilawke. The procedure when an animal
was found atfpi^ted was to secure the fulhst possible history of tlio case, to trace all
animals which had been in contact by breeding and to quarantine sick animals
for an indefinite period, forbidding them to be bred, sold or removed without
a special license. Periodical examinations were made by tlie inspectors in charge, and
as soon as a clear case of disease had developed the animal was destroyed. But it
hap])ened quite eouinionly tliat no definite symptoms could be detected until well on
into the second year of the disease, and that in consequence horse-breeding among
such groups of animals, often several hundred in numbers, had to be prohibitied for
two and sometimes three years in succession. Even then there was no certainty that
some latent currier did not remain undetected. In fact, fvs became known later on,
in any large outbreak of dourine and espivially among prairie-bred animals, it is
practically certain that such carriers do remain and can only be detected by specific
methods of diivjnosis. Although no better measures of ei^ntrol could be devised under
the existing circunwt.Tnces, the prolonired iiuaraiitine for uliservation purposes, tlie
periodic examination b,v the veterinar.v ins))ectors and the maintenance of large
breeding establishments or herds of brood mares which must not i)e use<l for breeding
purposes, provefl costly, onerous and unsatisfactory. These five years f l'JOT-12), saw a
great amount of research and experimental work accomplished at the Quarantine and
Research Laboratory, T^'thbridge. Besides tlio original trypanosome strain several
others were oht^vincd from diiferent sources of dourine outbreaks. Parallel series of
experiments wi>re made with the different strains in a close study of the pathogenecity,
life-histor.v and variations in the virulence of I r. i-ii^iiprrtliiin. The strains were main-
tained in hor-es: the evoluticm of the disease in its various phages was followed from
.vear to year and many observaticms and ''Nperimeuls weie m.vle upon the survivors.

Mares wliic-h liad -iioutaneou^ly n vcreil, snuie frnm a natural and others from an

experimental iufeetion, were successfully bred and raised healthy offspring year after
year. It was found that these mares resisteii reinfection and that the serum of such
animals had immunizing properties. The ex|N'rimcMt-,il treatment of dourine was
iiivestigate<l both from a preventive and a cunktive point of view, immunizing sera
and sjx'cial drugs recoiumend<>d in other tryiiauo^iieie i!i<ease>, e.tr., ato.vyl and arse-
nophenylglycin, were employed and reported uimii. Tlie chief olijeetive, that of deter-
7uining a precise method of diagnosi-, was always kept in view, and to that end the
study of the serum proi>erties of dourined iniimal> wa-i a eoiitinuous one. Serum
reactions and serum methods of diatiiiosis were worked out and included a serum-
globulin test, a trypannsinne agglutination test and a precipitin test. Of these, the
agglutination test was the more satisfactory but <lid not nux't all requirements.

The winter of 11I11-12 was spent by Wat-nn in a study of trypmiosomc diseases in
Kuropean laboratories. A German strain of dourine t Mes<'hiiUeuche) and an African
strain were ohtained from the Imperial lleahh Institute. Herlin. and bro\ight to
f'amida for eomi)arnfive work. Good pro;ires< in the serum diagnosis of dourine was
made during the year 1!U2. Tlie Comiileuieut-fixation or ("omiilemcnt-deviation test.*
after several modifications and improvements in technique had been made, was found
to provide a sp(>cific, precise and uniform method of testing. As a means of diagnosis
it answered all requirements and came into general use during the year 1013. Towards

♦.See Aii|j.-nclix Nn, u. jimk.s Id. for cxiilanali.m and il.'Siripticm .>f tlu- ( ■iiiniilenii-iit-tixatain
tfst for ileui-iiu'-

8

the end of that year a recurrence of the disease was reported in the southern districts oi
Alberta. The field investigations showed it to be of very wide extent and to cover the
old centres of infection of the first known outbreaks occurring six to eight years pre-
viously. On one company's ranch 2,718 horses were involved, including a number oi
imported stallions and mares and several hundred animals of high grades and value,
Blood was collected from every animal and sent to the laboratory. As a result of the
first test over 400 animals were pronouneinl infected. Some scepticism was at first
evinced by the owners iis animab, apparently in perfect health, were condemned on the
strength of a positive serum reaction. But as many of these animals began to show
symptoms and when several had died, all opposition vanished and the owners were will-
ing enough to have the animals slaughtered and compensated for, and to fully
co-operate in stamping out the disease.

The Complement-fixation test has since been employed wherever the disease has
mode an appearance in this country. It has proved of inestimable value as the main
factor in the diagnosis and suppression of the disease, enabling an early and definite
decision to be made — just as soon as blood can be collected and sent to the laboratory.
Instead of holding suspected aniiniils in quarantine for indefinite periods extendinc
into years it has sufficed to apply the tost and, after a short interval and to eliminate
any doubtful reactors a retest. It was most fortunate tliat this test was available when
the recurrence and wide extent of the disease became known in the years 1913 to 1916.
Huring tJiis jK^riod every elTort was made to cover the whole field of the disease, and it
was then that the heaviest toll was paid in slaughter and in compensation. The large
Indian reservations situated within the infected area of southern Alberta were for long
suspected of harbouring dourine. It was rarely possible to detect a case among lihe
native ponies thcmselvos but cases were frequently found among the better grades of
animals of the neighbouring ranchers which from time to time strayed on to the
Indian ranges. A serum tost of these Indian herds indicated many cases of hidden
infection and of dourine carriers. This was not surprising as the constant inter-
mingling of herds, the Indian habit of letting young stallions go uncastrated until
three or four ycjirs of age, and promiscuous breeding had given every facility for the
scattering of infection. To drain out this remaining source the herds were gathered
up and tested repeatedly at intervals of six months to one year, and the animals whose
serum reacted jKtsitivoly in the complement fixation test were destroyed.

In connection with the serum-diagnosis of dourine over 40,000 Laboratory tests
were made during the period 1912-20.

Since the disease first came under departmental action and up to present date
(1904 to 1920), 1,933 horses have been destroyed.

The figures year by year are to be found in the table of dourine statistics given
below.

At the present day dourine appears to have been totally suppressed in Canada.
If further outbreaks should occur or the disease be reintroduced it should never have
an opportunity of gaining much headway now that there are the means of dete::ting
it either in its initial or active stages or in its latent and hidden forms, and thus
dealing with it promptly and efficiently.

Finally, the testing of all stallions imported into the country and stallions that
are travelled through the country for breeding purposes would prevent the reintroduc-
tion of the disease and afford an adequate protection to the horse industry.

DOt'illNK STATISTICS (1*04-20).

NoTB. — During the present year commencing April 1st. up to the time of going to press.
November, 1920, not a single case of dourine has been reported. The final test of the last
remaining herd under suspicion — about one thousand mares and ."itaHions running upon an Indian
(Reservation — has been concluded, all the reactions being negative.

DOURINE.
THE NATURAL DISEASE.

As It n,-piirs in Cnniula ilourino infect ion is dirodtly .•ommunicable from 1
disonsoii to the honltli.v animal by ritlicr the horse or tiie nss throuph the net of soxi
union an<l in no (.thor way. It is enused by a protozoan parasite named Trypanosoi
niuip,r,him and is tlic only known disease of trypanosomian oi-itcin that occurs
( aiiada. Xatural dourine is very rarely, if ever, obst>rved in an acute form. Sta
iiiff from an mteetive coitus, at s<ime point in the Kenitnl organs the disease tal
an irrepular and uncertain course but is essentially chronic and intermitte:
Determining factors in its evolution are (1) the di-pree of virulence of the ti
panosome strain. (2) the decree of resistance or of susceptibility of difTerent brec
of horses, and (3) physical and functional conditions and disturbances dependc
upon climate and seasonal .•banps. brcding, exhausting work, anil stabliiifr or livi
a natural lite on the open raiiKt^s or j.a.tures. As to viruleiKv. Laveran and Mesi
have well stated that "ihe trypanosome of dourine constitutes one of the b«
examples of variations in the virulence of the same siH'cies following its origin a
genealogy.' The higher bret^ds of horses show the greater susceptibility. In t
thoroughbred the disease is apt to make quick progress and manifest itself mainly
derangements of the central nervous system. In the heavier and sluggish breeds, su
as the "Clydesdale," the disease is more halting and is marked mainly by oedem
an, distiirl.anees of the circulatory system. Native pony brwds are the most rcsista
and irc^iuently serve as carriers and reservoirs of the virus for a duration of yea
l)oiik.ys are aUo well able to tolerate infection and to become dangerous carriei
t imatic changes and extremes of temperature may cause .>ither an arrest or i
.ulvanee in tlie progress of the disease, cohl retarding and heat favouring it. Thi
m Canada it often lies dormant through the winter months, becoming suddenly ai
inereaMiigly aetiv,^ during th.> first hot spell of weather in .Tunc or July, find
natural e .nditioiis on the open range or at pasture more resistance is shown to
than in stable-kept animals. Breeding, heavy work and physical exhaustion favoi
llie .lise,,-e. e-peeially in the stalii,.n. Man's that have lieeome impregnated at tl
time of the infective coitus occasionally abort, but more often tiiey carry their youi
to lull term. It ha- been noted again and again that pregnant infe<-tJd mares ha'
remained apparently healthy up to the time of parturition and that shortly after ar
about the time of the first oe,trum have changed to a state of obvious disease a.
rapid breakdown m which marked syni|ytonis of dourine were presented. In shoi
Willie all hor-^es are more or les.i susivptible to dourine, the disease itself, like mo
.■!ir..ine mleetioiis is susceptive to various modifications and interruptions.

/«.'T^'„,i.-^ Alihoiif.h infectiim is passed from the diseased to the healthy durii
llie act ot copnlaticn, and in that manner alone, it dm^s not follow that tran'smissic
i<- always etfcned at such time of coni.ict. A stallion, for example, may infect a
the mar.-... he eovirs dnriiig one week and none .iuring the next. The contact <
two mucous surfaces, the liiseased and the healthy, provided the dourine parasii
1- present at the tune, is the only ni'cessary condition for transmission. The tr\n)ani
som.- IS quite capable of jiassing through an intact and mucous membrane and no wouii
or abrasion is needed for t!>r> vir,;s f g.iin rtn enlrv to the tissues. But there ui
ponnds „, the course of the disease when the jiarasite is absent from the genit,

10

11

mucosa and lifs dormant in the tissues. These may he called " non-infioctive"
periods and may have a duration of weeks or of months; they arc most apt to oocur
in the laler staRcs and, consequently, animuls in the earlier stages of infection
are usually the most active i.ropaftatorM, while those which enjoy a temporary or
partial immunity become the carriers.

Period of Incubation. — The interval fri)m the time of the infective coitu? to the
first appearance of symptoms or of visible chnnRcs varies and averages between two
weeks and three months. It may be even longer and sometimes it happens that no
symptoms can be recognized until the firsit winter has passed and the second spring
or summer arrives. From an immunological viewpoint and as a period of sensitization,
the incubation interval is less than one month, for specific serum reactions can be
obtained within that time and usually in from ten to twenty days after infection.

Evolution of the Disease. — Commencing as a local infection dourine habitually
takes a chronic, intermittent and irregular course both in its local and general evolu-
tion. With trypanosome strains of low virulence and in animals of strong resistance
the infection may l)e arrested locally and held in check for an indeiiiiit<? period and,
in some cases appears to terminate tlierc. In otliiT cases, and such are in the
majority, inf(>ction is both IoimI and general, that is to say, it predominates locally,
in the genital organs, throughout the course of the disease but, from time to ti ic.
finds its way to distant organs and ti>^ues. In a third type of case the opposite h Ids
good, that is, the genital organs acijuire a partial or temporary immunity whih the
disease is progressing in other dire<>tion-, and especially in its attack upon the ci itral
nervous system.

In most accounts of dourine the <'vo!iition of the diseas'! is divided into three
wcll-defii;ed periods or stages to which certain symptoms are ascribed, thus: (1)
/'Wni'iry—oedema, tunicfaetions and changes in the genital organs; (2) Sicondani—
pla<iues and skin eruptions; (.",) T.-rii'iri/- paralyses, anaemia and cachexia. This
arrangement is convenient for de>criptive p\irpi)Ses but is arbitrary and rather apt
to mislead an inexperienced observer of the disease in his conclusions or diagnosis
based upon the clinical history and evidence. Actually, the disease operates in a
succession of attacks, each attack being followed by a period of tolerance or temporary
innnurity of very uncertain duration. One attack may follow another so closely
that symptoms overlap or are never altogether absent. On the other hand, the attacks
may be so far apart that in the long intervals between them there is no visible sign
of the disease. Unless such an interval has continued for at least two years without
liny relapse it would be hazardous to assume that the animal had permanen'lv
recovered. As for the grouping anil sequence of symptoms it should be remembered
that symptoms of all three groups nniy be coexistant; <>r, that symptoms of one group
may api>ear sinudtaneously with those of anothiT. and that "primary" symptoms.
or those categorized as such, may r.Miipear after the tirst, second, third or any later
attack, and, similarly, that those of the "secnndary" or "tertiary"' groups may recur
repeatedly and irrespectively of the order or sei]uence indii>ated.

In short, the disease is marked by stages of exacerbation, tolerance and relapse,
any of which may be of short or of long duration. They may occur but .ince or may
lie repeated over and over again l>efore the final stage either of recovery or of
decline The symptoms most frequently noted are fever, tumefactions and lo(>al
oedemas of the genital parts and mammary glands, oedcniatous eruptions of the
skin, knuckling of joints, and inco-ordinate movement, espt^cially in tlie hind limbs.
facial paralyses, ocular lesions, anaemia and emaciation. The one true pathognomic
symptom is the oedematous patch or " plaque " ; but. unfortunately, it is a rare
symi)tom and can only be observed in a comparatively few cases. In the earlier stages
of infection fugitive oedemas of tlie genital organs and paroxysms of fever are the
rule, thus: —

In a slalUon the tirst symptom is a slight oedema of the sheath, often accom-
panied by a slight swelling of the plans penis. The mucous membrane at the urethral

exit mny show n buljiinjr. infiltratpd npponrnixM' niul imiy ivou protnule from tl
op«>ninK. Those first oo.li miis tond to .limipiH-nr within n iVw days. Aftor nn intt>rvi
usually fiftpon to twonty diiys, tiio oitl.«mn returns nnd is apt to bn moro pronoun<T
It may involve u iwrtion of the sorotuni tind fl\ictiiato« ns a <-old and painless swollin
As a mil' tlicro are well marivcd stajfes of aiifnnentation and diminution with varyii
intervals between. The oe<K'nia advances in a sueeession of waves and durinir <Mn

iiTing
recess an inereasiuK extent of ixTUianently tliiekened and indurated tissues can 1
noted. Tlie penis nuiy he in a stat<' of piirapliyinosis, temporary or more or le
permanent. In stalii.M.s of the h.v.vy .-h.nKish hre.ds the oedema may extend over tl
whole ot the floor of tlie ahdoin.ii and reaeii alonjt tin- eliest wall
doniinatinj,' the elinieal pieture throughout tlie disease.

to tilt; liriski
. In the lifthter breeds, ai

esiioeially in the more aetive and hiKhly-struuK runninR liorscs, this svniiitom may 1
sliirhf and quite transient.

In a morr tlu- first symptom is a jmleh of sliprhtly swollen mueous niombrnnn
the vulva or viiirma nnd is elosely follow.,! by tumefaetioiw of the vulva. Sometin^
on..v one labmm i. involved, iriviiis a distorted appearaneo to the vulva. The oed.ii
vanishes ami returns, the intervals beeoniiiitr more and more irn'Rular. The ou'i
black skin of the \ iilva. nnd s.imetinics of the luius, may s!u,w irr(>^Milar shaped patch
of depisnnenfation. The patch.<s usually rvtend outwards from tlio inner borders i
the labia, at first :i!>pt>nrinff pink and then becomins,' white. Thev are of unecrtai
duration: m some eas.^s fh.'y luTsist or remain as permanent leucoderniic patches, i
otliers tliey return to a ncrinal app.'arance within six montlis to one year. At tb
stape the vapinal mucosa may sb.nv raises! and fbiekened. penii-transparent patches of
jjlassv or straw-coloured app< araiice or somewhat motfbil. Folds of swollen membrai
may protrude into the vulva opcninif: the clitoris is often swollen and crec
l'rt>iuently there are spasmodic attempts fo urinate, prolonged periods of oestrum ar
increased sexual di>sires. At such times there is an excess of vaginal mucous mixt
with a serous exudate and occasionally tinged with blood. In the later stages c
disease, and especially when nn infected pregnant mare has aborted or foaled, thei
may bo a chronic vesico-uterine di.scharge. In some mares the genital symptoms mn
be so sliglit as to escape observation. After infection has existed for six mouths (
longer, and when it may be impossible to dete<-t any abnormal condition of the vagiu
or vulva, it is not uncommon to find a slight oedema of the udder or of the skin '>ii
underlying tissues just anterior to flie udder. Less connuonly there may be noted a
oedema about the anus, or one or more patches between the vulva and the udder, (
upon the inner surfaces of the thighs. Such symptoms, slight and vague as they ma
seem to be, are important, for it is in these si>rosities that tiie trypanosomcs of dourii
are most likely to be found by patient searching.

The true -phuiuc." or "dollar spot," differs somewhat in appearance from th
oedmatous patches already m-ntinned. The "plague" has been aptly described as
th u disc of metal slipped under the skin. As such it may appear upon any part c
the body surface, but seems to f.avonr the sides covering the ribs. In Canada th:
eharactoristie symptom of dourine can rarely be observed. In many animals held und<
continuous veterinary observation, and in which this .symptom was especially watche
lor month after month, in winter and in summer, a plaque was never know'n to hnv
occurred. Others have exhibitt d plaques in the first, second and in the third years i
the disease. In the most of cases the plaque has occurred singly and at wide interval
of time; less often, two. or even three, plaques were observed at the one time; and, i
some rare cases, a rapid succession or " crop " of plaques during one brief period in tli
course of the disease The average duration of a single plaque is four to five day;
Rarely docs the eruption and absorption of the swelling occupy loss than three da.v
or more than ten days. The average size is one and a half to two inches in diamete:
and one-half inch in thickness or elevation. The site of a plaque, especially in animal
■with a smooili and glossy eoat, is often made conspicuous by the staring or bristlin
appearance of the hair at that point. During the eruptive stage of the swelling and i
the serous fluid tlie trypano-omc of dourine can always be found.

].]

Fever, distiimtly of the intfrmittftit or riniiltLUt type, may occur durinif any
stage of the disease. It i* more retfulnr and proii'juiici'd during the early stages anil
before any marked tolerance or immunity has hecn acquired. A typical paroxysm ha-
an average duration of five lo six di.ys, the maxiniuni temrierature, \isually 104 to IWi-
F., being reached about the third or fourth day. Tlie intermis?)ion ranges between
twenty-one and twenty-ei^ht days. These febrile paroxy-ms are closely associated with
trypanosome cycles of devcloimient. A renewal of K'nital symptoms, the eruption of
plaqui'S, or a recurrence of oedima on some part of the animal body can usually b.-
detected during or just preceding the rise in temperature. As the disease advances
the febrile conditions become more variable and incon.-tant. In the later stages a
subfebrile tem|)erature of about 102'' F. nuiy continue for weeks or montlis. Very
little depression or constitutional disturbance accompanies the fever, even during the
severer paroxysans, and the appetite is maintained throughout.

The nerrnus fysdm is subject to severe disorders and disturbance, indicated by
inco-ordinate locomotory action and paraplegic conditions atTectinR the hind limbs,
lips, nostrils, ear and throat. The animal shows a wabbling gait and sways from sid>'
to side. There is knuckling over at the fetlock joints, and, in unshod animals, the toes
of the hind hoofs wear down from dragging and shulHing the feet. The animal is prone
to trip over small obstacles and there is fre(iuently a spasmodic contraction and
relaxation of the muscles of the hind legs. Facial paralysis is nearly always unilateral.
The ear, upper eyelid, nostril and underlip of one side of the head may be aff(»cted at
the same time or only one of these appendages. The nervous symptoms are not as
fugitive in character as the symptoms of the other groups. They tend to persist or to
become aggravated independently of other symptoms, and, as a rule, are the more
pronounced in the absence of other symptoms.

Ocular symptoms are not constant and occur in only a small percentage of casi^s.
They comprise photophobia, lachrym.ation, keratitis, cornea! opacities and chanj^es in
the interior of the eye. Stallions seem more prone to ocular troubles than do mare-.
It is of interest to note that on more than one ocoa-ion a corneal opacity was the first
symptom observed in animals that came under suspicion of dourine infection.

The (hirali'iii of the difcase average? betiveen one and two years. Rarely is it
under si.\ months; sometimes it extends to three years and occasionally to five years.
As a rule, if an animal survives the third year of infection a permanent recovery of
health follows. In such cases specific infection and immunity reactions are obtained
with the complemcnt-fi.Nation test for an indefinite period and even up to 10 years.

Termination and MoriaUtp. — In cases advancing towards a fatal termination the
decline is usually slow and gradual. The appetite remains good almost to the end and
often appears excessive. There is a progressive anaemia and amaciation; the animal
has a most dejected appearance and continues a miserable existence until finally, from
exhaustion and paraplegia, it is down and unable to rise, and, unless destroyed, suffers
a lingering death.

In stallions the disease would appear to be fatal without excrptiun; but in mares
the evolution of the disease is slower an<l more irregular and micirtain in its result.
Certainly, a large number of mares would make a natural neovery. Tlie mortality
depends upon the fxetors of virulence of the pathogenic agent and upon the resistance
of the breed of animal infected. In the prairie-bred, native animal, the mortality is
low; in i)ure-bred, imported animals it is high. Among the naturally infected animals,
representative of different types and breeds, at the dourine research station, the
mortality has not exceeded 50 per cent.

Pathological Anatomy. — The end stages of dourine are usually marked by a
progressive emaciation and anaemia and in many cases, and more particularly in
stallioiis. by ;n-t!enuituus swcHings extending along tiio lower surface? of the body.
The oedema is sometimes of enormous extent and the swollen tissues in the region of
the scrotum and under the abdomen may be up to eight inches and more in thickness.

14

At the autopgy ono finds more or less gelatinous exudation under the skin. I
the stallion the scrotum, sheath and testicular coverings are thickened and infiltrated
and in seme coses the testicle* are embedded in a tough mass of new sclerotic tissu
in which they are scarcely recognizable. In the mare the vulva, vaginal mucosi
uterus, bladder and mammary glands may show thickening and gelatinous infiltrotioi
The lymphatic glands, particulnrly the abdominal, are hypertrophied and aoftene
and sometimes hsemorrhagic.

Further changes similar in character to those found in other chronic, wastin
disease and anaemia may be noted, viz., gelatinous infiltrntioii, serous tranaudatio
from the pleura, peritoneum and pericardium, and fibrinous deposits, adhesions am
new growth on the liver, spleen, diaphragm and thoracic organs. Cystitis, pyelitii
nephritis, and bone marrow changes may also be noted. The spleen is not enlnrgri
iind may be under weight. The liver may be engorged with blood.

The principal changes are to be found in the nervous system, and are mos
marked in the lumbar and sacral regions of the spinal cord. In animals in whicl
paralytic conditions in the hind limbs have been severe and of long duration thi
cord shows softened, pulpy and discoloured areas. Histological examination shows i
deseneration of the nerve fibres of the posterior columns, and a cellular infiltration
degeneration and atrophy in the extraspinal nerve trunks, particularly of the hint
Ijmbs.. In nocordance with these findings Marok names the disease Polyneurifu
mfeetiosa.

16

APPENDIX No. 1.

DIAGN'OSLS.

'"^mptomahc (liagnosu.—Thi! type of oedema affecting the genital organs, tlie
eruption of the characteristic "plaque" and the peculiar nervous symptoms, ami
inco-ordmato locomotion in connection with the hind limbs may suffice for a
diaprnos.a But in the early staijes of the disease and in latent cases it is diffifult
or impossible to detect any such symptoms or to recognize them with any c.rfaintv.
Ihe history of the case or cases suspected and the examination of the contact animals
IS important and not to be overlooke.1. Where it may be impossible to come to a
<'oncIusion in the case of a siniflo animal, the symptoms presented in a group of
animals may clear away all dout.t. If it is found that only stallions and brood mare,
are affected there aro but two coverii.fr diseases to consider, namely, dourine and
coital exanthema. The hitter is the disease most commonly confused with dourine
owins to a misconception of the relative importance of ulcers and depismente.l sjwts
in these diseases. In coital exanthema the ulcers, precedcl bv vesicles and followed
fro<iuently by depurmcnted spc.ts are cbaracteristic lesions and the bi.-is of a svmpto-
iiiatic diHpnosis; in dourine they are not and h.ive very little importance, 'it has
been said and repeated in many descriptions of these disea-^es that the .lepigmentation
in the former is transient and in the latter permanent. I have f„und the opposite
conditions to prevail. With regard to ulceration of the genital organs in horses it is
extremely doubtful if such lesions ever occur as a direct result of dourine or Any
other trypanosome infection. I have never oI>served an ulcer in any case of
experimental infection in horses. I'l.ers have occa^onally been noted iil naturally
infected mares, i:sujilly in aired nninial-, and in staKions, and also in normal mares
and stallions. In appearance the le-ions are quite similar in the dourined and in
the healthy animals. In mares they are usually situated near the wlges of the vulva
and in stallions on the inverted portion of the penile sheath. ilicroscopical
examination of deep scrapings frequently show spirochaetae infection.

Detection of the parasite— In the naturally infected animal the trvpanosnme
appears and multijilies in the serosities of hwal oedemas. It is futile to search f<.r
a in the blood. As soon as a plaque is signalled or an oedematous patch in the
region of the vulva, anus or mammary gland is noted, the search should commence.
The skin over the patch should be washed, shaved and dried. Two or three punctures
are then made with a fine needle aud if .1 clear or only faintly tinged serum exmles
on pressure wet cover-glass preparations and stained smears are examined. A very
long and thorough search should be made before pronouncing a negative result.
Sometimes there aro one or several trypanosomcs present in each microscopic field
but this is a rare finding. Sometimes only one or two organisms are to be found in
the whole of the specimen examined. The object in puncturing the swelling with a
fine needle is to avoid the blood vessels; the more the serous fluid becomes diluted
with blood the less the chance of detecting the trypanosome. As a rule the p^irasites
are present in the serous contents of the oedema only for a few days though the
swelling itself may persist for several days after parasites have disappeared from it.
It may accordingly be necessary to examine a number of oedemas before the para-ite
can be detected.

In searching for the parasite in the vaginal tract the most oedeniatoos l^ikin"-
patch of mucous membrane is slightly s<-arified and specimens of the exuding fluids
are prepared for examination. A previous irrigation of the vagina with warm salt
solution secures fresher specimens, richer in trypanosomes and facilitates the search.

Id

Tlie trypanosonio is very rarely to be found in the niassivo oetlrma that aprenda
nlong the under aurfuco of the obdoinen nor in the oedema of the acrotura.

In horses the parasites are always few in numbers and for long periods in tl.o
lUsoBse lie inactive so that detoction is unually a very difficult matter, and dia^osis
by niiorosoopioal examination is i>oji8ible only in rare cases.

Srruin diagnosi*.— The serum of dourine infected animals posswsea certain
gpocitic properties which can be determined by (1) the complement-fixation test, (2)
the nirRlutiniiticm test, ami (,1) the prtx-ipitin test. Of those, the first named is reoojf-
nized ns a delicate, safe and satisfactory method of diagnosis. The full technique
and description is given further on (Appendix No. 2) together with some observations
on the incubation period and longevity of dourine indicated by the scrum reaction
in the complement fixation test.

The agglutination and precipitin tests'.' are useful aids to diagnosis but less
satisfactory than the complement fixation method especially when large numbers of
animals are involved.

' Winkl.r * Wyscheltmiky. Berlin, Tterar:ll, Woch , Dec. 8/, eitl.
• WatBoii, I'ror. Amrr. Vrt. Vnl. A>.inr., mil.

APPENDIX No. 2.

DOURINE AXr> THK OOMFLEMENT-FIXATION TEST.

The full description and detailed technique of the complement-fixation test method
of diagnosis was published in 1915 in "Parasitology," and is reprinted in this appendix.

Among the animals experimentally and naturally infected with dourine are some
which have survived the whole period of these investigations, 1905 to 1920, some which
showed tolerance and relative immunity for many years, some which succumbed to
the disen^e awd some which died from other causes (charts I, 11 and III). The sera
of many of these animals were used as controls in the 40,000 serum tests made during
the period 1912 to 1919, the records of these re-actions furnishing some remarkable
data, summarized Wow in table No. I.

All the animals there listed (table No. I) excepting the last two, Nos. 180 and lh2,
were !nfccte<l five to seven years before the complement-fixation test was available, so
tliat the reactions for that jieriod cannot be given. However, Nos. 180 and 182 were
tosted at intervals of a few weeks from the first to the fourth and fifth years and
constantly pave positive reactiftns. Further, all other horses which succumbed to
ilourine in from one to five years, not includeil in this table but which were serum-
tcste<l at frequent intervals, gave constant positive reactions up to the time of death.

Normal horses and horses in different stages of swamp-fever infection, used year
after year as "negative controls,"' never gave a positive reaction.

It may therefore be concluded that in dourine a positive reaction to the
r.implenient-fi.'tatioii test is given in all cases up to the time of death from the disease,
and up to tlie fifth year after infection in cases that recover, and continued in some
rtieh eases up tu the seventh, ninth, eleventh and thiilecutk year.

In experimental dourine of the horse and donkey the incubation period is de-
termined by the complement-fixation test as ten to eleven days (table No. II).

17

TABLE Xn. I— Showinj the wrum resclion flo ilourine) in the Cotnplenient-Rution tent for a number

u( yesn m/t«r infection.

Hurao No.

2 il'hHrt Nil. I

10 ■

13 I

17 ( ■•

24
•M

7t I

2« I

48 I

42 I

W (

67 I

m I

70 (

71 I

75 I

82 1

180 '

182 I

I
I
1
II
II
III
HI
III
III
III
III
III
III
III
III
III
III

Complement-fliation Test.

I'onitive.

Otli and 10th year

5th to 11th year..
StIi ami 8th year.

7th to I3th year.

7th year.

j7th anil 8th yt-ar. .
9th anil lOlh year

lint to 4th year

'Ut ti) .Ith ytar

I

Negative.

0th yi<ar onward*.

8th year onwarild .

4th year onwardu
»th "
flth "
th "

:7th yiiar i>nwarils
i7th ••
|7th •■
17th ■•

T.VBI.E No. 11. -.•showing the inculmtion poriod of ilourine as detcniiiniHl by the Cmnplciiient-fuation

Test.

Horse No.

190 (Table No. Ill) Ut positive reaction, 11th day after in lion.

130 .. ■• No. Ill) " •• 10th •*

Donkey

183 (Table No. Ill) " " loth "

DOURIXE AND THE OOMIT.EMEXT-FIXATIOX TEST.
By E. A. W.tTsoN, V.S.,

PatlmlooM, Veterinary Research Lahoratory 'Dominion Dritartmeiti of AgricuUuK),

Lethhridge, Ctinada.

Introductiok.

This paper is written with tlio purpose of drawing further attention to the value
of the complement-fixation reaction ns a diasmostic test in dourino and of recommend-
in)? a method of procedure and technique arrived at with an experience of 15,000 t»st3
f<ir dourino made at the Veterinary Research Laboratory, Lethhridge.

In a previous paper I have briefly described tlie serum renetions in dourine.
Since that paper was published in 1912 {Proceedings of the Amer. Vet.-Med. Assoc.)
diagnostic tests for dourine have been carried along continuously at this laboratory,
together with exhaustive control and experimental tests and the searching out of
fvery possible source of error. By the number.* of hoiwe available for exneriment,
the prolonged trial of the test through every known pha^e of the disease and its
widest application in naturally occurring outbreaks, coupled with observations in
company with the veterinary officers in chiirge of the field work, the oomplemeat-

034O— 3

18

Hviiliiiii ri'iii'l lull liii> Ih I'll llioriiiitilil.v ■■•Inlilinliril im h Mirr, siifr uiul aiHi'itii' iiiitlii
• >!' ilinKim-iiiif iloiiriiii'. Tlii' ixpiric ih'i' (tlinw* lliiit tin- ii^l iiiri'lH I'vcr.v rcfiiiirciiu'i
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I'y llif 1 |iliiiiriil tixiilioii lixt it Iiii- lifi ii |uih><ili|i uml witlnnit ilittiiiilty

Ih'l ii|||\ to inilkl' II llTtlllll llillHIlliri- 111' lllf llliirt' op ll—« ••> lll|ltoMllllil' lil-l .. Illll. Ill

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^iml lalriil l'orni> ol' infi otioii.

• •lily liy n «yj»li iiiiitii' iipp! iiiil loii ot' llio li-t to iViry iiiiiiiiiil lApo-iil lo iiil'i.'lii

iiinl ill no oilur «n,\ known nt pn-inl ciin llii' lii.iltliy lookinir, ■Ki-rallnl inniiiii

I'liriiiT-. ol iloiiriiio Im' (lite olid. Will II it i- niiiiniliiTi'ii tliiit lior^ii iimy toli riitr

iloiiriiio inli'i'lion I'or piriml-i ol' ono to ilinr ymr- iiinl ri inalii lor tlmt linn- iioriii

ill (rriiiTiil liiallli ami iippiiirinuo iml oiipiili'.r at tiiiiiT^ of ti-aii>iiiitliii;f tin ili-oa-o. ll

nior..iity of an iiirly and di liinto diau'iio-i- i- rvidrnt. 'I'll, iiipli'liii'iil tixalion li-

lunii^jii- llii- and linn Im .•oiin- of irriai iinportaiii-i' a-i a lui-i- for tin' oonlrol ai
-iippn-^ioii o'' ijoiirlno, ll i- \»\t\n applird in iviTy known outliroak ol' ilonrino
Canada, and, a- a pri''aiilioniir> moa-nro, in Ih. \arioiii ,«liid. a. id to >tallion. -tamlii
for -. r\ l.'i- a tlir 'i-iri.'i- tlial liavi' ooini' I n-idrrod a- doiiriiii- info'tid ariM

Hill I {-.' I Ih'illl'llillll.

'I'lir L'llii'ral prill. iplo* and iiin-li ini-in of tlio roiniiioiiioni lixalioii riM.'lion a
now >.i widr'ly ki;own that it mtiiis iiiitiii-i>!iiiry for tin' purpoM'- of this papor
11 in at lluin in dilail. a fiw roiiiarks on tlii> sulijrrt Mirticiiifr to inako il cli'ar ai
iiitilliyiliii'.

\\ In 11 an anti^ri'ii i» inlrodiii'id into an aniinal citlur by way of imtnral iiifn'tii
or l.y artiti.ial adinini-tration a t;roiip of nai-tion prodiii'ts ari-i- in tin- aniiiia!
M-niiii known a-i antiliodii'V' liniriiiH: a ,-piMific ri'lation»!iip to tlio antiu'rii and ali
to ooinliinr with il lnlt^ill^• of tlic nniinal Imdy undrr i-rrtain rondition-. Micr
ort.'ani-iii'', loniirn Mood cills and M'ra, allminriis and niaiiy fonn< of protriii matt
Mo alilo to ai't M> autinrii-. 'I'luis an aniinal infiM'ti-d with doiiriiio produci'- ant
lio.lu- ri,-iiltin>f from and <pii'iti rally nlatfii t.p tlii> limiriiu' niiti>:i ii, na.nrlv Trfiim
• -HOI , iiiiiii, r'hiiii , tho actual raiisc of tlio ili«oasi-.

In a -imilar nianinr. an animal wliiidi has rci-rivnl injiTtion- of llio hlood i
aMotlar animal spo.-ios hrooinos po-<,-sod of antihodiis haviii)^ a >p.'citic atHnity f.
tho hlood of that partiriilar spci-iiw of iinimal. In otliiT words, the antihody nriti'
in rispon-o to tho t'X.'itiiiK' r.ntipn in the proi-it-s of intVrtion, siMi>ilization, .,r imiiii
liizatinn, has the sivcitii' fniii'tiou of aoiinr npon tin- aiiti>ri'ii to iiriitrali/o it or pr
parr i; for dcstrtn'tion.

Tho coiiip'.fmcnt-ti\ation ti-t applird in thr dia.irnosis of disra-r roii,i,ts .,f tw
mN ot antijrm and antihody, that i>, two di?*tinrt mid M'parable coiiibinimr jjroni
lia\ inir no rrhition-liip to oiir aiiothrr Imt in rarli of whirh ('omiili'iiunl- x\ ronstitnei
of normal srruni- is an i-M'iuial fai .r. It i- roiivcni.'iit to distin^ruisli these j,'roii|
hy rrtrrrin^.' to thr onr rompri-iiifr .airinolytir i-rriiin, rrd rrlls and rnmplrnirnt ;
tlio •• h:vnic.|ytio >y-trin " and to thr othrr— rlosrly rrlatrd to thr discasr — (.-omprisii
doiiniir, antihody rorri-iiondiiiK anliirrn, and romplrnirnt as thr " antihody-roinhinir
;.'rouii." Thus: —

llariiiolvtir Mrnm 1

Red cells [llai-molyiic siisl

'■"'"'""'"•" y""'' I Antihody

stem

19

lU't'ori- till' tot ciiii iicluiill.\ Im' ii|i|>liril till' cxiHt iliisHKi' of ilir ililTcritit flciiitiitu
in carh irroup iiiiwt l>t» wurkcl ri\it y i-iiri'fiii qiuuititiitivi' titnition'k -tlio rmmt
iiii|Hirtaiit Ktrp in tht' wlinlc proi'i'iilinw iiikI thi> npi-riitur inii't li>' iili«oliiti-l.v axxiiri'il
tliiit null Kroiip rt'ftctinii U uiuli r lii" pirfi-ct I'mitnil iitiil tliat tin- iciwt itlHtiirliinir
rurtiir will Im' known to liini. In lln iictuiil Iciit only <>n>' I'oniplrnuMit unit i« ctnploycil

illiK niiniliml uuioiint iu'('t**)iry for tl iiiplitiiti of tin' lim'ninjytii' »y^ttin) no that

only out- of the ri'iii'tion jtroiipn can niiii' into o|Mration; it i* ai riliii(f to wlirtlur

till' conipli'incnt unit i< attrartnl aiiil atKxid to the antibody. 'uniliininir (froiip or ti
ilic liarniolytii- »froiip that wo obtain a po-itivc or a lu'trativc rcai'tion. The fornii r
'vill nlwiiyg ho offi'i'tod uhon tlio antiiton and I'.ntihody i'orri').|«iMd, that ii. wlioii tho
-.nun to^lrd oi.ntaiii'* tin- -po.iH.' na.lion prodiiit-* ol' doiirino iiifcotion cvon tlioiiKh
in niinutost (iiiantity, -.o .i. 'i.ato i< tho naotion. Niitlur tho anti^on alono nor tho
-ornMi alono, whoii properly priparod. oan taki^ up tlu' I'oniphnicnt unit; to do «o, all
thri-o factor'^ ^lu^•t Im- hrout'lit into intiinati' oontiut, aii.t whon tho t<«t "iriiiii dooH not
loniin spcoitio doiirino aiitilxdios tho ouniphinont is imt tixr.l to thi" frroup, hut
loinaiii-* froo to join with and <'oinpiito the two fa.'tor- of the haoniolytio "y-toiii. •>.>
tliat tho rid iil^ undirtr.i haiinolvri^ ami a in'iriitivo riaction is in.Hcatod.

'I'll ll\!./l 1' (M) I'll." Km UK 1{K( i)M\U;M)K|i.

Ai'iMinilus rriiiilml. — Iliiivy (rla-> ti.ho without lip, ."i iiuho-. hy i of nn iiioti,
and racks to !'old twonty-t'our tulxs in a doiihlo row, oiio alio\i> tli.' othir. Small t.-it
tiiho-, 4 in... ' ) of .III inch, tor -iriiin iiiactivatioii. Finely jtriiduati d iiica.-iuriti«
]pipottos of 0 '. (t. ,"i O atid lit (> r.i'. .'apaoitics. (iraduatcd .ylindors of .M) and KXt
• •.(■. capacities. I'>lenineycr lla^ks of heavy i.'la>''. standard -iz. - up t.i ,"aK) i-a: r-apa.'ity.
I.ar«c centrifuKo cups iind small .•oiitrit'uj.'o tuhe . Ampoule* and vial-. ,\ hiuh power
contrifiiBo machine, larj-'e water hath, and iii<'uliat<'r riKun.

-Ml ^'lii--wari' i- -terili/.e.l hy dry Inat.

'Ill matter
nee- anti-
V Jriiiiiitt-

1

■ hIo,,d of

i

itHiiity f..r

i

dy ariti'iK

1
1

or imiiiii-

it or pre-

l)iliil!ii!i, iriisliiiiii iiiiil jiii mi riiiii /hiiil-i.

(1) Normal fult solutien- -O" .,, prr .f-nt pure f.tMliuin clilori.l,- in 1'
tUleil water. A larKi^ quantity sliir.ilil I).' ina.le up (TjiJuii c.v.)
lixo.1 In fl.isks liavinB a siplion attai)\nient.

(2) Citrate.l »alt Pollution:

Nonnal salt solution

Sodium citrate

(?,\ I'ris.rvinB flui.l for tr> paniii-i,niis :

Nonnal salt solution

Pure n.'Utral glycerine

Konnalin (Schi-i'lnKs)

(4) Preservinff llnid for seruni :

.'.lyrerlno

rhi'iiol

p-shly dis-
md Mteri-

ino'o

1-5

fiO'O

lO'O

0-1

9r.-ft

T. I'm i'MIMloN Id UKM.rNlS.

A. Tin- Il'iiiiKiliiHr Siifch III.

(a) Rid C.llx. — A quiet slipop may ho h\M in th.o -tandiiip p.i-ili.'ii. nthorwiso it
-hould be phu'otl upon its hack in a V--liap<d troujrh and held tliorc hy t!ie attendant,
an assistant shavinir the .leok- and propariiiff the site of operation. The oinrat.ir draws
troin tho jiipular vein, under aseptic conditions, ,"0 c.c. (more or less) of blood into a
il.-.sk ooiitaiiiiiitc ulass lioad- and in whicii tho Wood is dotibrinated. It i« then run
throtifth a double layer of tino. sterilized pauze info larpe oeiitrifupo eups. about 20 c.c.
of i.lood in each, addiiip three to fotir times the amount of salt -olutioti. Tho corpuscles

0.'t4(V-2A

20

are thrown down by centrifugal force, the upper fluid taken away and replaced with
fresh salt solution, and the mixture again centrifuged. Washing in this way is repeated
three times, when the red cells are carefully measured and suspended in an equal
amount of salt solution, this 50 per cent stock suspension being stored in the ice
chamber until needed.

(&) Haemolytic Serum. — ^Rabbits have a variable amount of natural haemolytic
amboceptor for sheep's corpuscles — 0-1 c.c. of fresh rabbit serum will usually haerao-
lyze a like amount of 5 per cent corpuscle suspension. For test purposes a serum with
a much higher haemolytic index is required and to obtain this rabbits are hyper-
sensitizcd or immunized by repeated injection of sheep's corpuscles until a serum is
given showing a haemolytic index of 00005.

Not leas than six largo healthy rabbits should be selected for the immunization,
for one or several are apt to die from shock during the process. The rabbits are
injected intraperitoneally with a first dose of 2-5 c.c. of the 50 per cent stock suspension
of sheep's corpuscles. I'>ery four to five days a further injection is given, each time
increasing the dose until, after five or six injections, it has reached 10 c.c. This dose
is repeated once or twice. After the sixth or seventh injection 5-0 c.c. of blood is
drawn from the heart of each rabbit, using a hypodermic syringe and a fine needle.
The operation can easily be performed and does no harm to the animal.

The serum of each rabbit is then heated for one-half hour at 56° C. and the
haemoljtio index established by titration (vide p. 22). It will be found, probably,
that in only two or three rabbits out of six can the haemolytic index be raised to +ho
desirefl degree, namely. 00005 or better. From such rabbits as much blood is drawn
from the heart as will not cndangor the life of the animal — about 25 c.c. The rabbits
are then kept in reserve and can easily be reimmunized as required.

Finally, the serum is separated from the corpuscles and storc<l in very small
ampoules — 0-3 c.c. in each ampoule for convenience and economy — in the ice chamber.

When the serum is not to be used immediately it requires neither inactivation nor
carlwlization, and is, in fact, better without, the index remaining constant or but
very sliijhtly lowertMl even after six montlis. But unless the serum has been collected
under aseptic "onditions, rather than risk it siwiling, 1-0 c.c. of the carbolized glycerine
preservative is added to 00 c.c. of scrum before measuring it into the ampoules.

The whole procedure of imnainizing rabbits, drawing hlood from the heart,
separating and bottling serum, can and should be carried out under aseptic conditions.

(c) Compliment. — INonnnl guinea-pig sennii in a fresh state furnishes ". rich
complement. Blowl may be drawn from the heart, if desired, but as guinea-pigs are
usually plentiful at a lal)oratory it is simpler to anaesthetize the animal in an ether
jar, remove and susi»end the guinea-pig over a centrifuge tube of 25 or 30 c.c. capa-
city, sever tlie arteries and veins on one side of the neck, and collect all the blood.

Centrifuge immediately, liefore coagulation takes pluce. The clear scrum is taken
off and placed in tlie ic^ i'hanil)er. Coniplenieiit is always l>ettf r used in the fresh state
80 the guinea-pig should not 1k' bled until just before complement is neede<l for a
titration or a diagnostic test.

1?. Dovrino (aiitihadui rnmhinhif! (iruup.

(.i") Anibiin. — A stix'k dourinc antigen i> obtained as the result of inoculating
a number of white rikts with Trypanosoma iquipcnium. coUecting the rat's blood when
teeming with trypannsonies, and separating tlie trypaiiosomes from blood cells and
serum by washing and ceiitrifuging.

The 1)1o(k1 (if a dourine infected rat is colleottxl in a vessel containing sufficient
salt solution to prevent coagulation. Not less than ten large white rats— twenty or
twenty-live rat*;, if a considerable amount of antigen is needed — are inoculated intra-
perltuiu-aily with ihi- diluted blood, injecting ,iii equal amouhl, about 0 3 O.c, into each
rat. This may l>e done very conveiiiv'ntly by taking a ^mall sharp-pointed pipette, with
rubber tubing and moiithpiwe attached, drawing the 1i1.«h1 up to a point marked by

?^^^^S?SS^SS

ifii;v*>^:;«i:v

21

a file or pencil, and cNpcllinjr it into the abdomen, repenting the process with the same
pipette for each rat. The object is to have all the rats come down together with a
heavy infection. In the ordinary course a white rat dies of dourine between the end
(f the third and the beginning of the -fifth day of infection. When twenty-ifiTe rats are
inoculated at the same time about fifteen of them show a heavy trypanosomo infection
at the end of the third day, the remainder within the next 12-34 hours. It is necessary
to make a rapid covcr-plass examination of the blood of each rat forty-ei^ht hours
or so after inoculation and to sort the animals according as thry show a light or a
heavy infection into two or more groups. The result of the fir.^t blood examination
will indicate approximately the time for a second examination and upon that the hour
for bleeding may be judged. The timing of this operation is important for in the last
six or eight hours of infection the trypanosnmes multiply enormously, and if the rats
are left until well on into this stage a very rich antigen will be furnisi.cd. Careful
timing, however, is nooossary, for it may easily happ«>n that eight <!i ten rats will
all die within one to two hours, if left too long. The blooding should be carried out
as rapidly as possible. The writer's method is simple and effective and may be worth
describing in detail :

A running noose is made out of a two-foot length of thin copper wire, doubled
over in the middle and twisted to the end-;, the ends Ix'ing passed through the ring
formed at the beginning of the twist to form the noose and attacliod to any convenient
fixture over a laborator.v wash basin, si.\ iiches above an operating board resting across
the basin. An ether jar, a flask of oitra •id salt solution, two sterile coveretl beakers
and a razor complete the outfit.

An ass'stant passes the rats one at a time into the ether jar and hands them over
as required. Tlie animal is held back downwards in the left hand of the o()erator
whoso i;nlex or middle fiiigi-r presses on tiie left front limb of the rat. The noose is
slipped over the head and iirrantrcd so that tlie pull stretches the left side of the neck
bending the head sliglitly to the opposite side, kickwards and downwards. A beaker
half filled with citrated salt solution is placwl in position under the neck, the arteries
;!nd veins on that side and cl<i-e to the shoulder then severed witli a sintrle sweep of
the razor. I'sually, the animals bleed belter if one avoids sev<Ting the traehea. In
this way ten rats may 1k' iiled in half-au-hour. The volume of i)lo()d and citrated salt
siilution should be about (^pial or a slight excess of the latter. The mixture is then
I>assed through a double layer of sterile irau/.e to remove any smiUl clots and fibrin into
narrow centrifuge tubes, 10 mm. diami tor and 10 c.c capacity (when wider tubes aro
used it is more dithcult to separate; the trypanosonies and the wastage is greater).
Centrifuge not longer than four to five minutes at I.-IOO revoluti.ms yxr minute so that
the bulk of the corpuscles are thrown down while the trypanos.imes remain in suspen-
sion. Draw otf the cloudy suspensicui lluid into fresh tubes, then the upp<.>r layer of
'•orpuseles — more or less mixe<l .vith trypanosonies — into another tidv, and the next
layer into a second tidm. adding citrated salt solution and again eeutrifuging for
^10 minutes. Draw otl' and discjird as much nf the upix-r fluid as apjiears clear and
I'ree from trypanosonuv. Then colhx-t from each tube into a siu.i-'le tube the upper
pure whit«; layer of trypanosonu's, in another tube the middle layers slightly soiled
with blnod, and in a third and fourth tube the lower layers in contjict with the blood
cells. Add normal salt solution now, rot citrate, shake up well and centrifuge again,
n'lK'jiting the washings until all the tr\-panosoiues are obtaiiu-d in a pure white mass.

Ten rat< bled at the right time will furnish ."lO <■,>■. of try|iani>soines. Twice the
volume of the glycerine-furnudiu preservative is added .I'ld the mixture stored in
ie:i!ed and)er ampoules, J-O c.r. in earli, in a block of i.'i-.

.")•(• r.e. (if trypanosonies will make lnO r.c. lA aiitigi'Ti, -ufiicicMit for luon
.'iOO diagiiostii' tests. The antigen will ke.-p iiulcfinitely it' -<jlidified by fr.
tor (!~S weeks or longer when .-tored in li(iuid form, in (healed ampoul

(.v) Anltbu:!:/. I;i ir.r (!ia;.-nnrTi.- ii.,t- the ,Uitli«jdy. ul euurje, is or is not
prese:it in the suspected scrum. Rut for purposes of control and titration and to
thoroughly understand the crmbii.ing action of dourine antigen and antibody it is

than
eeziiig. and
on ice.

V ^:'^s^aMm^'.'*'.:A'^:ita'i>tiV^^^--sa

■.57^>-^if,'r,S4iai5?--*~

22

absolutely net-essary to huvc oiip or more series o£ known positive or specific dourine
horse sera, of which the antibody content '• u he determi-ned. To obtain this a hor^o
is inoculated with Triipanosonui rquiperdum. Ten days later and at weekly intervals
thereafter, blood is drawn aseptically from the jugular vein, the serum collected and
tested for antibody content (vide p. ->8). A series of specific positive sera are thus
obtained, representative of different periods and stages of the disease. Stored in the
ice chamber the sera will retain their specific proi)ei'tie3 for many months, even years,
if collected sterile. If not absolutely sterile the serum may be preser\-ed by adding
1-0 c.c. of 5 per cent carbolizcd glycerine, or the same amount of iodized glyveriiio to
9-0 c.c. serum. At the same time one should collect and store a number of negative
control sera inider the fiime conditioiui.

II. Titration of Hevof.nts.

(1) Tiliation of llacmohitic !^rfiim (Amljorrptor).

Prepare the following stock dilutions of scrum and corpuscle suspension: —

1 Haemolytic ■•■■rum irahbit anti ahcopl 2 i ".'.'

Nor . :! salt solution i,0-85 per eontl ■><>

100 " . .1 : 100

2. C'ompli'inont ■ in"

Fresh Kuinea-piKs scrum ' ' .,

Normal salt solut inn "* "

200 •• 1 :20

3. t'orpusele suspensiyn: , ., a ..

Washeil slii'<-p's corpusHes (50 per cent stink suspension -U

.■Sail si>lut ion -' ' "

2,7-0 " .. .1 ;25

Further .lilutioiis of the hiiciunlytii' periiiii arc made as under: —

Sail Haemolytic

solution serum

TuliP No I ':tO V'o a : IIWI equals 1 : 400 (0 002.i serum in 1 0 c.c. )

'"' ..-^ ■ .', 5.,) 1.0 •• •• 1:000 tOOOUi • " )

•"{ T.n 1-0 " " I :800 (00012 " " )

4 (,.(, 10 •■ •• I : 11)00 (0001 •• '■ )

-, i).,-, lOUilOtW) " 1 ; 1500 (000066 " " )

,; ,.n 10 •• ■• 1 :2000 (0 0005 " " )

~ .>0 10 ■■ " 1 : 3000 lOtKKKiS V •' 1

K j.n ].() •• •■ 1:4000(0 00025 " " )

,j 4 0 10 •■ ■■ 1 :5000 lO 0002 " " )

In each tubt.- 1-0 c.c. only of the dilution is held batk, the excess amount being
discarded. 1-0 c.<'. each compb'nient and red cell ^usl.cnrion iuUUhI, which with i-O c.c.
snlt'solutiou make a tctal volume of 5-0 c.c. i;i 0M-\i tulx-.

The complete titration set is then:—

tscrum

dilutions ;

Salt

Comple- j !

mont I Red Cell

iasal«)ve) solution , 1:20 [suspension

Tutws 1-9
Tulie No. 10
•• No. 11
" No. 12.

10
10

c.c.

2 0
30

3 0

4 0

10

10

CO.

10
10
10
10

! Titration set

I Scrum control (1 : 100 dil.)

Complement contrt>l

Red cell control

Mix well and incubate for two hours at i!T" C. , , , •

rj-j^,. ,..~.,.trol set. tubes 10. 11. and IL', must not show any trace of haemolysis.
The tHre of the haemolytic M>rum is indi<iitcd by the amount present in the last
tube of the ecrle.s l-!t in which dissolution of all the red cells irt compMe, that is. the
leant amount iieces*iiry to dissolve a definite aniou-it of red rcUs.

wm^^Bm^^^:m^%ff^mm^s^^%

23

F<ir (.'Xiiinpic', if in tulxs Xos. l-t! Imciiiolvsis is complcti'. not niiitp foniiiloto in
tube Xo. T. and still lt'r« in Nos. 8 wul it, then tlio titre is the amount of serum in
tube Xo. 0. or 1-0 c.c. of a 1 :2fl<K> dilution, 1 unit being exiires^'d at 0.0005.

A MTUm with a unit value of U-tween 0-00()2-O.00O5 is quite satisfnetory, but
when the value of a si-.iKle unit ex( ee<ls the latter amount the results are not so good.

The relationshii) and eombined action .if liaeni(d,vtic amboeeptor and "omplement
should bo olearly understood. To do this a numtxr of experimental test*; should be
undertaken, usiiis in one series only one unit of nmboeeptor with fraetioaal amount.^
of eoniplement. in another series two units of i\nibo<eptor and lesser complement
fractions, frur units iiiul so forth, prosressively multiplying the -.lumber of ambo-
ceptor unit.- while further reducing the fractious of complement. It will be found,
for in<tan:-e. that two unit^ of aniboceptor re<iuirc a les.*er amount of complement
than one unit to completely haeumly^e a rtandard amou-it of red cells. The lesser
the amount of complement that can be safely emplny,>d in the i.ractical tests the more
delicate l«-conU'S the fixation reaction, the equilibrium of the hacmolytic system being
more easily ujiM't, even by a test .-erum naturally weak in antibody ontent and which,
if a relativelv large ec^mplena-.it unit was employed, might be insufficient to give a
complete reaction. On the other hand the r(«luction of complcmcii't must not 1*
(arried to ^uch an extreme i)oint that any slightly inhibitive property of one of the
ether reagents would tend to obscure it and give a fake fixation.

It i> e-sential that for all subsequent titrations a"id test.s a standard dose of
liaemolvti • amlniceptor l>e fixed and rigidly adhered to. For all practical purposes the
use of two units of amboceptor permit of a sufficiently fine gradation of complement,
while still allouing a margin of eafety. The dose is therefore fixed constantly at two
units, t.. which complement is always titrated as in the next procedure.

(2) TUralion of Complement.

Preiiare (1) a stock dilution of guinea-])ig eomplenn'iit and (2) a suspension of
sheep's corpuscles, as in the previous titration.

Al-o (.T), an hacmolytic serum (ambocci>tor) diluticui. so that 1-0 cy. of the
diluted serum contains two amboceptor nnits. For example, if the value of one unit
is (».(i4M>.->. then 0-(i01 will be that of two unit.*, the dilutio-i being accordingly 1:1000.

The titration of complement is of the utmost importance and requires the greatest
r.ccuracy. as alre«dy indicated. T'ntil one has become familiar with the technique
I'ud expert in reading the reactions the titration is best carried out in a double set,
the second having o-ie-balf of the amount of each reagent used in the first set. the
one serving as a check to the other.

Tli(> two set.s are arranged as follows: — ■

Titratwn of Complemenf.

First Sot

Soi'ond Sot

Tulio Ni

9
10
II

! Salt 1

{'oiiiplo-

HaoiiKit.

Salt

t'omplo-

Haoniol.

' solution 1

motit.

sorum !

solution ;

inont j

scrum

c^. 1

c.r.

c.c.

r.e.

c.c.

c.r.

; 2-0 !

o:m ;2oi

10

10

015(1:20

) 0-5

2-0 1

0-4

10

10

0-2

0-5

2-0 !

0 4,j

10

10

0 225

O-.i

2-0 j

OS

10

10

0-25

0-5

2-0

0 55

10

10

0-275

0 5

2-0

0 fi

10

10

03

0-5

20

Oll.">

10

10

0-325

0-.5

20

0 7

10

10

0-35

0-5

20

OS

10

10

0 4

0 5

20

10

0-5

10

0-5

0 2.'

, ..:, 3^0

—

10

—

—

1 3U

1

1 0

20

1

Add I CO. red roll suspension
to each tul>e.

Add 0-5 o.o. rod coll suspension
toeai-h tul)e.

'^mkfm^^i^'-'^'m^^vi'^i^ '^^'^-g^-

24

Mix wi'll (I'voiditiit undue frothing).

Incubate at 38-3U' C.

Agitate the mixtures again by shaking the racks after ten minutee incubation.

Rend the reactions one hour later.

Tube No. 10 controls the originiil hnomolytic titration, only one unit of ambo-
ceptor being used with an excess of complement. In this tube complete haemolysis
should occur.

Tube No. 1 will show only slight or partial haemolysis; as one descends tho
series the reaction is seen to be increased, until, usually between Noi. 4 and 7, a
tube is reached in which the reaction is absolutely complete. The first tube in the
series in which all the red cells are completely dissolved indicates the complement
titre. If this occurs in tube No. ,'j, for example, then 0-55 c.e. of a 1:20 dilution of
complement is the titrc. equivalent to 10 c.c. of a 2-75 per cent dilution.

For the antigen titration and final tests the complement is accordingly made
up so that 10 c.c. of the dilution contains the amount of complement indicated by
the above titration.

From now on it is optional whether one employs the relatively large amounts of
reagents as givcii in the first set of complement titration, or the one-half amounts
as in the second set. The latter is the more economical, especially when a large
number of tests are being performed, and is given personal preference to by the
writer as it seems to provide nn even more highly sensitive test reaction than when
the larger amounts are employed.

(3) Titraiion of Antigen.

Dilute 10 c.e. of stock trypanosome antigen with 190 c.c. of normal salt sulution.

Prepare tie ccimplement and haemolytie scrum according to their titration
values already determined.

Inactivate by heating for half-an-hour at 5S^ C. in a water bath, 20 c.c. of
known positive doiirino horse serum anJ. 20 c.c. of known negative or uomial horse
scrum.

The antigen is then titrated in a double set, the one being with the ])usltive
serum, the other with double tlie amount of negative serum. Thus:--

P<»sitive set

!

Kn<.)vvn '

1

positive

i

liorse

Comple-

.-'alt rol.

scrum

AntiRcn

ment

c

nut r

.1

C.c.

c.c.

c.c.

c.c.

■|i|l>e

Nil.

1

11)

01

0 02

O-.i

■»

10

0 1

0 0.')

0 .1

A . ...

10

01

101

0 o

4 . .

Id

01

0 1.5

O-.I

.1 ...

Id

01

01'

Oj

ti

10

01

0-25

0 .i

^

1-0

01

03

»■:■,

.s . ...

10

0-2

0 .'i

N

10

—

0 2,i

(1 ■ .i

1(1 ....

15

—

0-5 1

1

Ncgat

vo <et

' Known

1

1 ntgative

1 horse

1

Com|)l(>-

Suit sol.

1 serum

An(iKca

ment

c.c.

j c.c.

c c.

r.c.

1-0

i "■-

0 (I.')

O'.'i

1(1

1 0-2

0 1

()■,")

1 ()

0 ■ 2

0 2

(I-.")

1 (1

0 2

ti:\

()■.')

10

0 2

(14

Oo

10

0-2

( ) .">

()■.">

1()

; 02

(Id

0')

Id

10
2 0

Mix well and incubate for one hour and ten minutes at 3S-39' C.

ili.x together eipial (iiiaiititii's of haeniolyllc .-<-iMini (ambuceptur) and rid cell
siispi'ii^ioM, then add loe.e. of the nii.sture to each tnl)e.

Shake again and iiii'uliate for two hours longer.

It is usually possible to read the antigen titre in 1\ hovi's and proceed with tlie
fiiUi! ti>!^; iieVerthek-;, the tubes ^l:Oti'd be li-ft or rrplaeed iii t!-" ilioubfit-r f-r
the full two hours and then put on one side for further reference and to see ;f
any further a<'ti(in has taken phue.

'samcsssxEx^m^ss^^isss:^.

25

Tube No. 10 is tlio control for tlie haeniolytic systrm and must show completfl
haemolysis. Xo. 10 in the second sot contains only haemolytic amboceptor and red
cells and must not show the slightest degree of hicmolysis. No. 8 controls the horse
serum, No. 9 the antigen, the red cells being haemolysed in all.

The positive sot will show more or less complete fixation of complement — no
haemolysis, except perhaps in the first and second tul)cs, the negative set complete
haemolysis. When the antigen appears very strong there may bo some inhibition
ill the negative set in the tubes containing the larger amounts of antigen.

The amount of antigen to bo selected as the titre for the final tests is that which
gives complete fixation with the positive -irum while double the (juantity in the
corresponding tube nf the negative set does not prevent or inhibit haemolysis.

Til. The SKRr>r to hk Tested.

(I'.'i

O")

(I-.")

O-.')
U-."i
(!■.">
I)-.')
1 ')
O-.')

Collection of spruiii. — The chief point aimed at in collecting Wood from suspected
animals is sterility, especially when the specimens have to be transportcii over long
distances and mailed to the laboratury. Absolute sterility is not essential, nevertheless
as near as p^jssible aseptic conditions are to be strongly reeommcnded and the
avoidance of adding carbolic acid or any other antiseptic fluid to the sample specimen
as a preservative. The blootl clot should be well formed and the serum odourless and
clear or only slightly tinged with haemoglobin.

The condition of a sample of blond may vary greatly according to the size and
shape of the vial or tube containing it, the slowness or rapidity with which blood
is run into the vial, the partial or complete filliii;i of llie vial, the shaking of the
speeimcti before coagulation ha* occurred, and in otlur \\ays irrespective of asceptic
conditions and of abnormal properties of the blood itself. In square or rectangular
bottles and in specimen vials witiiout a neck the clot has a tendency to cling firmly to
the sides, the scrum being separated with more or less difficulty. In small round
bottle^ curved into a narrow neck ami mouth, for corks, filled with freely flowing
blood to within a margin of the narrowest diameter but not touching the cork,
and allowed to stand for at least half an liotir for coagulation, there is usually an
abuiulance of clear serum.

Such bottles, of one ounce capacity, one inch in diameter, three-eighths inch
neck and mouth, arc vt ry suitable for field work. They must be absolutely clean and
free from any trace of soap, alkali or acid. These bottles are distribiited from tb.is
laboratory after being sterilized in the hot air o\ou. corked, labelled and we'l wrapped
in sterile paper wrappers. Also, ..,rge bore needles attached to three inches of rubber
lulling with a small glass nozzle. s.>paratoly wrapped and sterilized. With this simple
apparatus and observing the u^ual iircrautions during operation it is an easy matter
to draw blood from tlie jugular vein ot a horse, aseptically.

Among the last G,(MMt samples ,,t' blood seciired in this manner le~s than twenty
have reached the lab.tratory in a condition unfit for testing and tlii>so few unfit
>l>ei-iinens have been ten days or more in transit.

On reaching the lab.iratnry the -peeinicns are briefly examined and where
ne,.es>ary the clots are detaclie<l from the sides of the bottles with a sterile wire. They
are th(>ii left to stand in a cool chamber overnight for the serum to clear. The Serum
i> then drawn off into >inall test tubes, uli.mt ■2-0 c.c. in each, and is ready for inactiva-

luartivation of scrum. — Before any specimen of liMr-c scrum can be used in
the complement fixation t.'.st it li.is first to be inactivated. All animal serum in u
Very iresh state contains completuent in a varying amount. This constituent is
readily destr .ycd by heating the scrum to o.'.-.'.O ' C. for one half hour. No eomple-
niciit other than that emphiyed in the haemolyt':; system may take part in the

a matt(>r of f;ict horse eotnpleni.-et v"ry rapi<lly !--.-oaies h::-n i-.-.-A

in specimens several days old is a practically negligible quantity. However, in

serum there arise several other factors whicli, uidess destroyed or

re.'n^t'oTi

^rmal horse

miomti^r^a^j^^^mitk- ':^&

^ESo¥^

..-•ja.-1-i

mwmisfk^s&m

30

renilcrcil inactivf. arc alilo to net uik)ii <'i)ni|iUin<t\t anil aiitipcii niiil iliaturb nn
hai'nii>l.vtic s.vstom. All untrpatod horso, (Imikcy and mule ?ora possess onzymofio
and profiHilytir properties, potentially nt lea^t, and beeoming active in sera a day
or two old. Tliey act upon most preparations of antiften, especially upon macerated
or»ian-i, such as the. liver and spleen, and arc all more or less anticomplementary,
iiHjre so in the presence of antijren than without it. Such action, of course, is non-
specific and must be elimInat<Hl, otherwise it woidd be difficult or impossililc to dis-
tilittui>li a sju'cific from a non-specific reaction. Fortunati ly it can be eliminated, and
the e(|uilibri\im of the scruin fixed, by a proper an.l sufficient inactivation. It is morr
rrsisf.'iiil In hrnl than ik romiihmcnt (iii.l it not whoUii ili'xini/rd at 50° V. Tiiis
is an important iwiint, and one that appears to have been overlooked. T cannot help
thinking' ilia; it is the explanation and tlie sounr of error of many of the ai)parcnt
failures or discrepancies, esix'cially that of non-specific fi.xation, which some scrojofrists
e.xiH'ricnce. A reference to the literature on complement-tixatiou methods shows n
remarkable lack of uniformity in respect to the deirrt>e of heat and the length of time
for the inactivation of suspected sera--fiftecu to thirty minutes at dejjrees varvlng
between ,",0 and 58" C.

A few experiments with sets of ten or twenty ditTi'rent horse, mule and donkey
sera, each set beinp heated for thirty minutes at different decrees between .lO and
Oi2 C and then tested in the haemolytic system, witli and withont antigen, will show
the im|5ortance and necessity of a very careful inactivation an<l the temperature
peijuircd {ridr p. 27).

Mithiiil of inactivation rcrommcndnl.- -X water bath, sufficiently lar^e to liold
200 -mall test tubes, is heated to GO' O. The tubes, eontainintr 20c.e. serum in
each (numbered for identification in waterproof in<lia ink, labels beiiiff apt to become
detached), are placed within the inner tank which is to contain .sufficient water to
mount to the level of the serum or to about half the heitrht of the tubes. The cover
of the tank should have two perforatims for thermometer tubes which are inserted
into control tubes within the tank, enabling the temperature to be read without
removintr the cover. Another thermometer pa.-ses directly into ti-e outer tank. For
the first few min>it(^ the tem])erature will rapidly fall; it is broufrht up to 59° ajiain
— takinjr about ten minutes— and maintained at that point for a full half hour, for
horse serum, and to 02° for one half hour for ilonkey cr mule serum.

There is no danger of destroying tl;e specific antitxidies of dourine sera by heat-
ing to the points given. Dourine sera can, in fact, be heateil up to 05°, or to the
point of coagulation, and still retain an active antibody content to prive the test
reaction, but the anticomplementary and non-specific factors in horse sera are wholly
destroycil at 5!t", and in donkey and mule sera at 02'.

To control tlie inactivation, with each batch of suspected sera several known
positive dourine sera as well as known ("anticomplementary and n.in--pecific) negativo
sera are included and all tested together in the final diagnostic tc-t.

-j.^.'^'if^^^si- ■.-.s^'. 'KJim^:s-^'<x^»i?m'^i \imnvv'iu i ,m^esssissmss9s^^iss^rss!Fm-

Eriii riiui nl slinwiiiij thi' ilrfiri'r of iiimtualinn itf suspcrleil ."I'rinn nrri'snarif

far .ipi'cilic rcacliDiis.

DoM> (if scrum. 0-2 <•.!•., ui\li(iiito<l imd lio;iti'il ;it iliffcnut iltjirtiv- «{ tiiii|>i'niture
and tfstod with trvriiiiDSDnu' iititisirn as in tlic (li:iiinii,tii' ti'*t.

Nuriiiul j
heultliy lior.-es j

1

rnhoa(<'(l
stTum

I SoruMi

lieatcd for onp lialf

! ''»

54

5H

Vo. 1

1

+ + + -^

+ + + +

4- 4- ■

-^

+ + + +

+ + + +

4- 4-

4-

" 'i ,

+ + + +

^ + + + +

4- 4-

4-

^■ + + +

+ ++■'-

-4- 4- -t-

4- 4-

+ + + +

1- + -t-

-t-

—

4- 4- + +

+ 4- -i- 4-

4- 4-

1 ■' ,

-+- + + +

1-4-4- +

'4-4-4-

4- 4-

T + + +

4-4-4-

4-

( ? )

" )t

+ + -!-+■

f f f +

4- 4-

1, ^ 1

" 10

.

+ + + +

4-4-4-

4- 4-

4-

m

ti2

Inhiltition. Non-spi'cific.

Hourino hitr.^cH

"

' ■ 1 1

1

No. Mst yi'iir i

f (liseast

4- + 4-4- i 4-4--f4-

4-

4- -t- -f 1

+ + + +

" 2 1

4-4-4-+ , + + + +

+

+ + +

+ + + 1-

•• 3 1

: + + + + ' + + + 4-

+

+ + +

+ + + -1-

■■ -i •

+ 4-++ + + + T- ;

+

+ + +

4- + + +

" 5 I

' + + + + 4- + 4- +

4-

+ + -^

4-4 4- +

" 7 (

+ + -I + + + -*-

4-

+ 4-4-

-h + + +

•■ 7 iJnil ••

i + + + + i + + + +

-1-

+ + 4-

-f + + +

•• X i.'iril ■■

: +4- + 4- + + -,- +

+

+ -t- -r

4- + -t- +

•• y i4tli ••

i + + + + +4-4-4-

+

+ 4-4-

+ + + +

•• 10 i.itli •■

+ +++ + + + + !

+

+ + +

+ + + +

Sp»'iilic coinpleEiient-fixation.

N'drinal

'i Cnhoatel ^ 5) | 54 1 5B i

5S ' (10 i

fl2 1 M-M

Mule N'o. 1

i

1 ■' + +

1 1 i

+'++++;++++■ +++

4

- i

2

+ +

+

4-'+ + 4-+.+ + + +;4-4-4-4-|

+

+ ' + i

— ! —

Donkey No. 1

! 4- 4-

+

+ .4- + + +!+ + + +'+ + 4

+

+ ' +

_ _

2

j4-4-4--|-,4-4- + 4-i-f4-4-4-: 4-4- ;

-

~ -

lnliil)ition N'on-f^p^' ii'ir.

4- 4- +

-1-

See pasje 28 for the ineiininz of the-;e reacti

m expressions.

Xoti . — In the non-epeeific iiiliihition reactions tiic rod cells are loosely sedimeiited.
In the siie<'ifi(> eoniplcment fixation reactions the red oelk are precipitated in a
nia?s or ag-^lutiiiatod it; lumps. When the sera are tested without antigen, as in the
serum controls, the douriiie sera, of <'ourse, {rive no spe<'ifio reactions, hut the
inhihitinu reactions are pivcn hy normal and dourine sera alike when in'suffieiently
inactivated, though to a lesser (lejrree than when antipen is f sent.

Cuiii-liision. — ■S\ispp«->ted horse .serum Must he heated to at least a"^' C. (59-C<+ for
safety) ;,nd mule or donkey serum to (>2" C, to eliminate non-siK-citic reactions.

Tile .XntHtodu content of Dourine Sera, ami the done of .■••lu^pectcd Serum )wccf>'arii

for a Biagriostic Test.

The maximum dose of horse serum used in a diajaio-tic fixation test is 0--2
CO. This amount is not exceeded for fear of any disturhance to the haeniolytic system
hy the non-specific reactions which larger doses are apt to cause. Douhle the amount

^■Jl^. '•:i^tXMt'tilifiii>!:i-:-<^r:.<iiti[S^-,SjMer;jfi:^-^: ■,^;l.L--h^--v?

.,>-■-! v. •RWaWftii^i

28

can actually bo used with perfect safety provided the serum is correctly inativatcd
But it is unnece»8nry to use more than 0-2 c.c, for that amount of ^crum fif a dourin<
horse will contain in the case of a scrum very weak in antibody content at leaBl
one unit, and in the case of a serum stronir in antibody ten, twenty, forty or mor(
units — and one unit of Antibody is sufiRcient to frive a positive reaction with th(
fixation test.

That this is i«o may be determined ^y taking a series of sera ciilKvted froir
animals in active and in latent i)hases of the disease and titrnting out each seruir
for antibody content.

The experiment is carried out jis follows: —

The sera are first inactivated by hcatinjr for one half hour at 5!)' C. Thrc(
stock tubes arc then taken for each scrum, (1) containing the pure serum, (2) c
dilution of 1:10, and Ci) a dilution of 1:100, these dilutions permiftinp of the
accurate nicastircmcnt of the smaller do^^s.

Twelve tubes arc now arrantred for epch scrum to \>o tested-- the first and last
to contain 0-2 c.c. of undiluted serum, the larfrest amount used in the tc^t, the last
tube heinfr the scrum control without antifren, the interveninp tubes to contair
gradually decreasing doses of serum. Enough salt solution is then added to make
up to 1-0 c.c. in each tube, then tiie antiscn and complement in amounts previously
determined by careful titration, and fiiuilly, afto- incubation for seventy minutes,
haemolytic serum and red cells — as in a diagnostic test.

An experiment of this kind is given l)clow, the titres of seven sera from ditfi'renl
horses in different iihascs of the disease being determined.

E.rp<'rimcnt for dilirmininff (lip Atii'lodii content of Vinivhu^ Srra Ini tlf
Complemrnl Fixation Method.

Dose of
iniicf ivatfMi ituurino sorum

('(jiiiplpinent (ixation reactions with ilourini' mtuiii

NO. .-•

.\o. 7

+ + + +

+ + + + moans complete fix"*ion of romplemnt -absolutely no tr:ii(.' of Imemol.i -is. Hed lella
morp or le.ss elumpeil. \ very stronK positi\f reiictitm.

-i — f- + is also u strong? positive reaction, witli just a f.iint trace or tinp;e of haemolysis.
-f + is a ratlier weak positive, indicating partial fixation -about one-half the red cells iiaemulysed,
+ , a very weak or faint positive— slight fixation, with more than oni^-half the re<l cells liaemolyaed.
— , a negative reaction. Complete haemolysis of red cells.

The smallest dose of serum which comhiiies witli antigen to cause complete
fixation ( +- + -f 4-) indicates the value of one antibody unit, iind from this may bo
calculated the number of units in the st.iiidard or maximum dose and the value of a
douriue scrum in antibody content.

rif^m^^^wtii^^i^^^^m^bs^

mw^m^p^s^^s^^^sm

30

Xo. 7

Such values are more relative than absolute, for the titre of a Jourine serum
may be «omowhat higher or lower aocordiiifr to the amount of dourine antigen present
and the fineness with which the haemolytic system has been adjusted — just as the titre
of the haemolytic serum itself is correlative to the amount of complement and red
cells (antigen).

The seven sera, Xos. 1-", used in this experiment are taken from dourine control
horsiB in the first, second, third, fourth, fifth, sixth and seventh year of the disease,
respectively. Xo. 1 from a maro showing clinical symptoms; Xo. 2 from a stallion
showing oc(>asional symptoms; Xos. .3 and 4 from mares very rarely showing symptoms
and progressing towards recovery; Xos. 5. C and 7 from mares that have not shown
any symptoms for three, four and five years respectively, and which have made
complete recovery, been bred to a he.^lthy stallion each year — without transmitting
infection — nnd raising healthy offspring.

In addition to the above, among our experimental horses that have recovered
from dourine, there are two mares that gi%o a iK)sitive (+ + + +) reaction with
(1-2 c.c. scrum after six years, and on<> manr a positive (-\ — i — \-) after seven years.
On the other hniul, there are thre<.' marcs that have entirely ceased to react, even with
two or three times the amount of serum, after six to seven years of recovery,
although they reacted positively up to the fifth year.

ConrhisSon.- — 0-2 c.c. of horse scrum from a dourine infected animal contains
up to forty units of specific nntil)<)dy. In the ca^c of horses that have completely
recovered from dourine and which are no longer able to transmit the disease, one or
several units of iintibody are present in the same amount of serum up to the fifth
year of recovery. After that period they may cease to react — indicating that not
only was an .absolute recovery made but that the immunity was lost in about five
years (proof of whi.'h has Iwen given by inoculation experiments with T. cquiperdum,
on recovered horses).

For diagnostic tests it is sufficient to use three doses of senmi, namely, 0-2,
015 and 0.1 c.c.

elis

Tho. first appearance of a positive srrum renction in dourine infectivns.

Having fixed upon a standard dosage of susi)ected serum, it is now necessary to
know the incubation period of dourine and when a first positive serum reaction may
be cx()ected, for otherwise a negative reaction would bo valueless or even misleading.

In this conne<-tion tiiere follow the records of some experiments: —

Ex peri merit for determining the length of time between doarine infection and the
first appearance of a positive svrv.ni reaction.

.\ healthy filly '2\ years old, was infected with dourine by smearing nver the
vrtginal mucosa a few drops of blood containing Triipanosomii equiperduni.

Serum was collected from this young lare before infection and daily up to the
fifteenth dny after infection, and tested by the comploment-tixation method, with
ti'y]ianos<iinc antisriii. The results were as follows: —

Doc lit Hefore

f-fruni infertion

Days after infectiun

1 )<> 10

r.c.
;(l :! .
II- Ij

■• 1 .

^! l).l..
(I III

0 iKi:i

+ + +
+ +

12

13

14

+ + + +
+ + + +

+ + + +
+ + + +

+ + + +
+ + + +

+ +

15

+ +

+ + ■

+ + + +
+ + + +
+ + + +
+ + + +

+ +
+

'smpf^rn'mfsmsmm

v^'ii^m^,?^^!sm^^mi^smi^^§

.■iO

Thu*. the first appearance of u iH)8itivo s<Tum rciiotidn whs i levin dny^ nt'tir
info'tion.

In three earlier exiierinu-nt* of this kind, hut in which Bcrinn wiis not cnlKi'tcil
for testing until the twentieth <lny after infection, the reaction in ciich .u-c wns
strongly positive.

The incuhation jH'riod of doiirine in tlic light of the coniplcinent-tixntion tc-<t i»
indicated, by the above experiments, as not less than eleven days and not over twenty
days. However, the strain of douriiie iis«'d in tht>so e.xix'rinients was of IukIi virulciici';
when horses l)eeonie infeeted with strains of low viruh'noe — an<l there is nunli viiriii-
tion in dourine strains — the incubation jx^riod is probably prolonged.

A negative reaction should not be taken as final or conclusive when the iniiiviil
between exp<j8ure to infection and the collection of test seniin is less than two ni.>iitlis.

IV. The Duoxosnc Tkst.

Two methods of prHC<>dure are here ri'<"oiiiniended: —

(1) When only one or several tests are to be made.

(2) For daily routine testing or when .JO, HXI, or more tests are t.i In- iiiadi' iit
one time.

In either case a necessary preliminary is the titration of coiiiplenient (riilr. p. Hit;).
This establishe<l, sufficient complement dilution is made up — O ,■, c.c. ,,( tlir (liiuti..n
to contilin the smallest amount indicati-*! by titration — to do for the tiliiilinn (,f
antigen and for as many sertim tests and controls as are to be maib*. It i- iidvisuble
to make up an exces.s of complement rather than have a deficit, so as x-< u<e one-
stock uniform dilution throughout and avoiil having to make up fresh stuck- during
the testing.

The trypanosome antigen is then titrat(>d against a known po-itive doiiriiic -num
and a known negative serum (riile, p. 167).

First nu'lhod of procedure — for one or srvmil ^,v^^•.

Four tubes and one pipette of 1-0 c.c. capacity, graduated 1 -1<H>, iire u ..il for
each serum to be tested. l-O c.c. salt .solution is measured into each tube. In
each set of four tubw 0'2, Ol.'i, 0.1 and 0-2 c.o. of the inactivate*! test siTiini is
added. Antigen in the amount already decided by titration is now added to the first
three tubes in each set, omitting it from the fourth tube which serves a- a serum
control. Complement, 0-5 e.c. of the dilution re<iuired, is then added to a'.l tubes.
Sets of positive and negative sera are included with the above, and. in addition,
<'ontrol.s for the various reagents. For the reagent contrids five tubes nre needeil ;
(1) .\ntigen control, omitting the test .-erum, (2) hacmolytic control, ( 'nittinar serum,
and antigen, (ii) hacmolytic serum control, omitting test serum, antigen iiml eoin-
plement. (4) complement control, omitting fo^t serum, liaemolyti" .sertnn and antiiri n.
(."i) red cells control, containing only red cells and .salt solution. The eoiitroU nr.'
made up to a uniform volume of 2-5 c.c. by adding salt solution as rniuired.

When the test scrum, antigen and comi)leni(>nt have been mixed toL'.-iiu r, tlie
tul)ef» are incubated at .18-30" ('. for 70 minutes.

Equal quantities of the hacmolytic serum dilution and the red cell >iis|„.iision
(A per cent) are mixed together and 1-0 c.c. of the mixture added to ,vcry tube
excepting the last two controls, Xos. 4 and .">, to which 0..') c.c red cells only are added.

The tubes are again shaken and incubated for another two hours wIumi the
reactions may be read, a second reading b<'ing made the followirii: niornini.'. abour
twelve hours later, the racks being left at a cool looni temperature nieaiiwlii'<\

Tin; above procciliire is ii.dieiited in ilie foiiowing tai>io:--

;«-_':^

T^sstwtBumsaa'ia^ses^k

m

^^5WtSi^f^

''''T^~

S?S^^

M

■ 1- + -f +•

,' +^'

t -*-+ -(■ +

I I I

1

ri^t^i *iri -1

a /.

c

' 5 ' £ .

t 2.2

i i -r . =5 . C

I

T. z ■/. :

!• wJ ?>^ s;/!-:?-:

:;i^9S9E£EiJSBi5%&]BIESiaQ^«;:£fi@SSIir%^-.

39

Second m>lhod of procedure— for daily roufinf tenting or iili.n :.<>, 100, or vwr^'

teits art to be made.

Thi« it only a ilight modificntioii of the first method of promluro to allow of
more rapid and loiw luboriou* work in tiwtinu lurKo nuniUrs of «iw|Kvtt'd »erM.

Two tc«t »«'ri<'* nro niudo, the first M'r'w*, in which only oiw tube for rnch serum
is uiied (instead of four tube* as before), oontaininK the nmximuni dose, fl-2 c.c, and
antigen, eliminating all ni-Kative gera and at the same tune indii-atinif the positive
»era. These latter are aRuin tested on the followinK 'lay, usinif the four tubes— the
three standard dos<» of scrum an.l fxrum ('ontrol— ii» in procMHiure \o. 1, ineludinR
them with the next lot of situ to underKo the first test in which the single tube
IS used.

If a iKWilive scrum dot>s not Rive a fixntior iclion with 02 v.c. serum it
certainly will not with the lessor di««'s, and as .i serui.i contr..! is ..nlv nee,led in the
case of a w rum which fix.s e.miiilement. the sinirle tuU' U obviouM.v all that it
roquir.nl to .ietrrmiiie a n.'Kiifive serum. Further, the sera with which complement-
fixation takes place in the one series serve avi additional controls when included with
and fully t.-stcd out in the sc.v.nd t.-.ri.w— -no day's work thus chc.'kinjr the oUier,
continuously.

in routine tesliuR nt this laboratory, when Inrfje numbers of sera arc beina d.'alt
with. It is the practice to make a repent test with each scrum nofjiitive at the first
test and to nrranse the work ami the dilTcrcnt scries so that each day's t.'sts include:
(a) a s<.ri.-s not before tc^tefl (one tube for each scrum) ; (h) the «Ta tc.mc<l the dav
before with ncRative result (one tube f,.r each serum); (c) the scni t.-sted the .' iv
before wMtli fixati,m reactions (f,,ur tubes for each serum); and, in addition, the usual
9ori.« of known positive d.>urinc sera, negative cdntro's and rcnircnt cntrols.

All siw,,ected sera arc thus test.-d twice over so that if any error or ..mi.s»ion in
the tcchni(|ue has been made it will surely bo indicated.

_ Int>rpr,'fa(ion of the nvir^on,- -Fixation of the complement, n..t in itself visible
in t. n tost tube, is indicated by the prcv..„t;.^., r' ' icnolvn, .,f the red cells and
constitutes a jwsitive reaction, on which a diaanosi^ of dourine \* (riven.

\Vlien no complement is fixcl the red ceils are complctelv lineiiiolvs,..] „nd the
reaction is then said to be mvative.

The i,revciition or inhibition of haemolysis may be comph'te, partial or slight—
aeeording to the richness of the serum in specific antibodies. However, with the
stanclar.l -loses of serum, in the ffrcat majority of nases. the reaction is either clearly
positive or clearly ncsrntive. Occasionally, complement fixation complete with 0-2 c c
serum, partial with O-l.', o.o. and slipht with 0 1 c.c. may be iri-cn. This is a ,K.«itive
reaction ami indical.s. that the scrum is weak in antibodies, onlv one unit being
prcsi nt in 0-2 c.c. serum. '

Partial ti.Nafion with O.o serum and complete haemolysis with 0-1 serum is. a rare
reactn.n and of a (iu.Ntionablo nature. Tn the serum controls, without antigen.
hacmolysi. should a ways be complete Very rarely in.lced it happens that haemolvsis
in the scrum controls is not compl.H.-. the mixture havimr a cloudv or opaque appear-
fine,^ and some of the r,^ cells remaining unhaemolysed. Thi.- mar be the result of
insum..i..nt ina.'tivation or of changes in the serum due to certain bacterial growths.
V\ hen su.^h questionable reactions are given a fresh specimen of serum is asked for
and a rctc-t made.

Oextcral Rkmahks.

"■.-■' "• i-"^--' ••' T.t,- -•.mfuLiia^'iil is.tation test ariHndi n.uuilv ui.on the

preparation and use of powerful reagents, their siwificitv and the accurate deter-
mination of their relative values, the fixing of s.tandard doses wherever possible, and
r. constant, uniform technique and method of procedure.

^^aumea.^sitiissMSiikL-i'v;

as

riy«., f>.rnili,.rily uill, tl... ....fivily .,f tlu- rrn^r,■uU i. ..«M..„iittl for th., l,.^t r.«ult«

M..,.k miKmlH ,i,„»l,l l„. |,r..|,„n..| ii, .|Ui.i.tili,.H ■•al.-ulHt.Kl to um-t hII r«,iiii«-
. w'lit, f.,r ... 1....^ .. tin.., I,* tho «,..ivif,y .,f the r.».^....t. tvumIuh ,,r..c.i..ally ....nHtant.

Ihu.: Hult.,M,.,.t I.,...,,.,.,,,..,. H,.ru... (nv ,ix .„„..tl,s' w„rk; antlK,.,, t,. sutli..,. for ono
M.n.ill., uork: fr.-l. r.l .vll mh|«i,m„„ an,.,, „ w.vk; In si. , ,„„,,|,.ni.-..t .J.Jv or on
,.lt.r.n.t.. .Imv,. or ... -m,..!.,!. |. . n.lvi-al.|„ to „,.■ tl... Moo.l of two .l..v|. fo'r scnHi-
M/.,..K r,.l,|„N ..M.l .„ ,.«. tl... r..| -..lis „f tl,., ..„n... ,1,,...,, f„r tl... lu,..n.ul,.|i.. HyHtom.
11... folluvMii^ |„„iit>, uf ..xn-.....,. imiH.rti.n...' will l.-.^r r..|...|iti..n :

ID Tl... ...„..„„t of r,, II- i„ .,„,...,.io„ „u.M 1,. v.rv a...-,.n,t,.lv .n.-.,-,.r,Kl nml

III.' st.iniliircl ;iiiinuiit iiiv.-r v^irii'l.

(!') Th,. U-r nf ll,,. l,.a,| |,.„-il,|,. ,„.,.,„Mt of r„„i|,|..|....„t u|,i,.l, with tWO UnitB
"I l.n..m..|,Vll,. ,-rniMI ..au-.- Cloplrl.- hM.li...|\,l, „f r,.,| ,.,.11.

(;i) 'Ih,- ,i-,. ..f i«i,... t|„. „i„..u.il .if aotir.ii whi.li with a .l,.iii.iii,. antibody unit

i. ■"■'•'-^•"•v In l.x ll„ „|,i,„u.|,t. |,ro,i,|,.,l tho ,a.iio a.ii..nnl of aofi^-,.ri ,.h,no ha-.

iio loliiljilo.-.v aiiioii.

( 1) la.-.-f..l .•.,„i|...l ,,f .!„. imutisatin,. of s..s|M.,.f,.,| ., n. l.v ki.own ,>o8itive and
l^iiowi. ii.'({at.M. s.ra.

(:.) Control of il„. .lianno.ti.. t..,t- hy a .,ri,.- of knou,, |,o-i,ivo s.ra, ...ui. having

all antiho.jy unit o| .lilTirml valiio, hi«h to low.

|)ls( CSSION.

Th,. r.-liahility of ,1„. ,.„n,|,l,.„,.„. lixation f,.,t a. a oortain an-l -iH.-itl.. nu.nns of

.lia;rno-u ha. h.-on ,„„.,,■ ,|, ,„„. [ ,|.ink. v.-ry ...ri.oi-k or o„ strh'tlv s,.io„tifi..

;4i-.oiml,s la,t innn. ,n r,.-|H.,.t to it, prac'tloal ap|.li,.atlon aial on a.. i.nwarrant.Hl sup-
|...Mt.on hat It K ,1,11 v..,y in,|,..,.f,.,.tlv „n,h.,.,too.l. iha. thr to,.|,ni,|n.- ami method
..t i,nH...,h„.,. „ ,o i,„ri,.a... ai.,1 Iah..rion,. that tho na.-tio,,- th,in,Plv..s are .ul>-
l-t to a,al hav,. to ho ;riian].-,l a;rain,t ,o n.any pos-ihl,- ,li,|,irhinK intlu.'no,^* that
""•■"'"'"'"" "' -"'I' 'I'"'! •>( 'Vuitiu.,.]^ is att..n.I..,l witi n,i,h.rahlo risk.

,, '''"',!".' ' ' '■; ''•";''■''■'" .;/././/■.'■ ■■■ -s without .louhl. an.| with as mi.<-h oaa.-

nl ...d ..nt in fo,. a laho,.ato,.y t..... : „, tho o,hor ,i„. roa^'.i.ts ar.. ,.ro,,an>d in the
I., o tory and „.|,t o„t tor a H,.ld ,o,t. |.-„rth..r. a. many n-t.^ts oan h.. Ina.l.^ hv the
;..ri„.„ tixal.on Muthod as ,|,.,i,.,.d. f„r no toxins or i.nn.nni.in.^ -nhstano... are
'".f"U->\ mo th,> s„-|..,.|,.d animal to mf.rfor,. with suhst-qm-nt ,iiaKno,ti.- t.Ms This
oM ,s no h.n^or a n, w .hparlor,. i,, ^,,,.,.,„ary .lia^no-ls; it is su<.....ssf„lly applied
. L-landers. oonta^nons al...rt,on and ,n o.hor sp,.,.;ti,. di-eases and is v.^arlv .-ntnin^
111!,, i,ior,. jri'iHTal lis,.. ■ "

-../.p/'-../. S,„.i, a MOW i, no, laid hv -..,.oh,^i,,. and ..,.n only ho n.lain.-d hv'tho*.
;;" '•'^••' ""• ll;-' .■IM-itumty of ol.-.ly ,-,u,lyinK the s,.hj..et and heoo.nin^.' familiar
;^ "1' tho t„, ., |,„„.,- „, „. A„v atiompt ,o apply iho to-t hy one who ha. not ,h.,rou.,hlv
-a- ,.,.,.d tho „.ohn„pio and r^nuHl oon.ph.lo oontn.l of t!,o n.a.',.nts wonhl. nf eoursp
;<■ .l,,i,i:,.,.ou<. I,„l tho ooinphnient Hxation r..arth,n fn,.„l-hr- tho most p<.rfe<'t. bio-

->.-al, d,a«„o,,,o ,o-t yo, .lovisod. one in whieh all advo,. r di-turhin^ faetors can

"■_ -lniH.ii.1..d and ,,, wl,,,-!, a ..h.ar knowlodK.. of the pro,«.,t!os and nio.le of action
■Mho HM^onts ha- ho,a, a...or,ainod.--_r.n. ,„„n. -o, i„ ,,.,. ,|u„ ,1 ,f a mallei,, or

: ' ■ iil.i, v..act,o„ w-„,.h ,n appl,oat,o„ ami interp.-ointion. is er„. in ecmpnrison.
I I." \oi.y delicacy of the hxation reaction and the sr.-iot lans 1 iu;,,„„ ...,..^^.,:^^

■■ H.ni to Ti,e r\-actn...s, value and reliability of the to,t. '' " ""

/^ Ihr trrln,!,,,.' I.,,, in/nn.lr „n,l l.,l..n-n,„.^^-^Sot more .„ ihan many other
'^■-..-ary and a-eept,.,l I.d.oralory metho,|.. a'a.l tlii,- i- ,.>sontiallv a lalmratorv test

irtiri^ar •j.'afxiy^***

;".» -.•■J9'5^t'#"jlS!Ka«£<<S:%SS5»S'a

34

/« it necessary to vse a pure siispmsinn of triip<inio:t<)nws as antigen?— <By the
'•mployment of a pure susppiision of ('ourino tryimiio-oinos an iintiKoii iion-ept^^ific
Hnd false or mis'.cadm? reactions are avoided. Many othiT ways of preparing antigen
for the doiirine test have Wen tried by ditferent iiivestisrators but, with one ex<'eplion.
with little success. Alohler and Eiclilinrn recominond a spleen iirejiaration of a rat
den-1 from surra. I have Ufeed the Bpliy'ns of rats dead from dourine in several thou-
sand tests and with very good results, but, on the whole, .-uch jiroparatio^is are inferior
to the trypnnosome suspension and possess a numU'r of disadvantapf*;. Sjileen prej)a-
rations are often troublesome on a<>count of a more or los.^ anticomplementary action
or owing to a weak-ness in specific antiiienie property. Tluy are very unstable and
of inconstant value and give rise to many borderline <>r questionable reactions whidi
can be eliminated or definitely decided by the trypanosome antigen. Ju comparative
titrations of dourine sera with the two foi'ms of a-.itij;cn I have found that approxi-
mately one-tenth of the amount of serum necessary for a jRwitive reaction with spleen
i-.ntigen suffices for a clear positive reaction with trypanosome antigen. Very weak
positive reactions with the former become clearly and strongly positive with the latter,
which, therefore, should always be given the preference. The trypanosome suspension
has also the great advantage of retaining a constant value for several weeks at least,
for six to eight weekfi if carefully prepared, and thus allows of the keeping of a
uniform stock antigen.

What is th« pcrcentn'ip of positive reactors in ihuriiic outbreaks?-- ilus of course
varies according to the length of time the disease has been in existence in a stud or
range herd before being checked by preventive mea-siires. In the most extensive out-
break that we have had to deal with 4,"ri positive reactors were found in a total of
2,000 animals tested; nearly S-T per cent. In an outbreak on an I-.ulian Reservation,
]27 animals gave positive reactions out of 1,4(!4 tested, or less than i) per cent. Usually
it is between 15 and 20 per cent. Our experience iudicatw that 100 per cent of
dourine infected animals, wliether in active cir latent stages of disease, give positive
«-erum reactions, provided that an interval of two to throe months luis been allowed
for an incubation period in the more or less resistant animals, less tha'i one month
being sufficient in most cases.

How does the value of the douriu-c test romparc with the Wassennann test for
typhilis? — The old name of horse syphilis still clinirs to dourine infections, esi)ecially
among stock owners and the general public, and comparisons have bwu made both in
regard to the nature of the disease and the diagnostic tests, tending to lead to mistake^i
conclusions.

Th« reaction in dourine by the nuthod recommemled in this paper is a specific
cne. A positive reaction in other diseases or with animals in which dourine i-ifoction
could be excludetl. remain.s unknown to us, while in every authi'ntic case of dourine
the reaction is invariably positive. In my wIkiIo experience there is only o-ic case in
dispute— a negative serum reaction being given where a .symptomatic diagnosis of
dourine was made. However, the symptnmatie dingno-is may have been at fault;
unfortunately, the animal was divtroyed before any tiro.)f or disproof of dourint
iTifection was forthcoming.

The very few cases on record where a negative <iouriiio reaction at a first test
was followed by a positive reaction at a second or later te5t can be aci'ounted for by
infection taking place only a few davs U^fore the s,.rnm was first collected, or by
continued axposure to infection between the fi'->- i later test.

In syphilis, on the other hand, negative rear are of more value for prognosis

than for diagnosis. A positive reaction may l>e; le negative after a short course of
treatment., vctt.rnir.g again to po,itlvc ii' a cure -.s ■. u eilecled. Further, it is

admitted that a negative reaction is fre<|uently giver , primary s.vphilis and again
at times in latent and tertiary syphilis. A source of error, operating in the negative

•HIKV^lWBI-a «?«»SS.W5»*''

iitfM

36

direction, is, as NokucIu hiit^ iniintcd out, in tliiit human scrum contains a variable
(.mount of nati.rol llnti-^ll('(•|) anilioof^ptor, wliioh in somo cuijes may be sufficient to
hide u positive reu.'tion. Ifnrsc fcruni dooe nut contain anti-sheep amboceptor, as I
nave found by maTiy experiments, so tluit the nnti-she«!p liaemolytic system can be
uced in hoi'se serum tests with perfect reliance.

A pcsitivc '''^sserniaMf re.iction may be ffiven in several diseafies in which
^vphilitic '^if ' "fi ciiu he isi-liuleil, in leprosy, scarlatina, certain forms of tuber-
culosis aii'i e.ir i!U'iii;i. I ! 'Vassermaii-n reaction is not specific. Owinf? to the great
(litficulfy < I lilaiiiiiii:' :< ii-ire syi.hilitic antisren, the extract of a eyphilitie liver was
first used . AV tvCiniaiHi's i .ipinal method. But, later on, it n-as found that non-spe-
cific extrac* -f normiil livei: and other oiRnns answered equally well, and such are now
commonly used. 1 Me ,,ii"tion in syphilis is not accordingly a true and specific
antinen-antihody combination and is depen<lent upon more or less (rro«s chaTigee in
tlie serum of syphilitic pati<'nts. It is not to he compared, therefore, and ie preatly
Inferior to our test method for dourinc; either in delicacy, specificity or trust-
worthiness.

In concliisio:i, I venture to express aKsohite confidence in the complement fixation
test for dourine as it is now iiresented. and to claim that apparent failures or diecrep-
;;ncies are due, not to the method itself, but to faulty tivhnique on the part of the
operators or of the collectors of the ti'<t serum.

APPENDIX No. 3.

KXI'KItlMK.NIWr, DOURINK.

Horses. — Tr. eqicipfrdujn is always pathopenic for the hor.'c. If introduced into
tiie vaginal tract of a healthy mare it rcproducf«: the disease in a similar fashion
to that which follows an infective coitus. A subcutaneous inoculation of trypanosomes,
obtained from a plaque, ocdematous swelling or vaginal secretions, is followed in
about 10 (lays time by a small swellinir at thi' siat of inoculation. In the serous fluid
of such a swellini' multiplying trypano^omes are to he found. Tli's prinu^ry lesion
disappears in a few days niul the diseise evolves as ir. a case of natural infection,
especially if the inoculation has be,Mi made via the genitjtl organs.

It is essential that the material inoculate<l contains the si>eeitic trypanosome of
dourine. Blood drawn from the general circulation of dourined horses rarely
eoiitains trypanosotiies aiul thus rarelv reproduces the disease when inoculated into
healthy subjects. Direct transfusions of blood or inooilations of large quantities
re[K>atcd at intervals over several weeks have failed to set up an "ifoction. Only
■luring a very active staire of the disease, as during a period of plaque eruption, is
.in inoculation of Llood from the general eirculatiim likely to be successful.

1"he virulence of a strain of dourine can be riiis( d to a high degree by a series
't rapid passages through young aiiimal>. In tliis wiiy the disease is rendered more
le'iite in foals or yearling horses, running it- course in about six weeks in males and
n about three months in females, a high and remittent fever being the dominant
feature throughout (ri>l,j Chart \o. II and conqvire with Charts Nos. I and III).

The trypanosome virulence for horses is raised to its niaxinuirn degree by
passage through laboratory animals, viz., white mice, rats and guineapigs. In
horses infected with trypanosome blood taken from a rat or guineapig the disease
makes rapid and continuous progress. There is scarcely any remission of symptoms.
Trypanosomcs soon apinar in the general circulation and are present there in varying
numbers up to the time of death, which occurs in from six weeks to three month?,

xr,:^if^''

afi

in adult horses. In tliis .■„inip,.ti,.n it is well .vorlli uotlui: tlmt 11 liors- and 1
donkey have been ex,«.rinun,nl!.v infe-.ted with tr,vj.M.ios,„„e. whi.ji had h...n parsed
through a rat or puineapijr, an.i that in all V> animal, tr.v,>anosom.s appeared in
the general .-.nMilation. On the other imnd, 24 hoi^e. have i„-en experimentally
infected with trypano.onie^ of seven or eipl't different <><|nine origin, and previous
to passage thruufrh lalmratory animals, and in none of fli.se H animals wer»
trypanosomes ever lo he seen in the general eireiilation. Simiiarlv, hundreds of
Wood_ examinations for trypan, .somes in cas.N of natural infection likewise proved
negative.

This exalted trypanosonie virulence and the aef|uiiv.l habit of the parasite to
live in the hloodstr.'am of horses after a passage throngb laboratory animals has
been maintained for several generations in horses.

LaboTatnn, animah.-W'vy irreat diflinilty is e.N,H.rieneed in transmitting eiiuino
dourine to laboratory animals. The white nunise .seems to be the least resistan'-
even so. it may be neeessary t.. in,)eulate series after s.tIos before obtaining a sing','
suoeessfnl infeetion. However, ,.nee this is aeeomplisl-ed. the trvi.anosonie ean be
pas.sed through rats, guineai-igs, rabbit-. doL's. eats, .'te.. with great ease, from an
animal of one speeies to tlijit of Mn,,tlior ,ii„l earrle,! on indetinitelv wilbont loss „(
virulenee.

The adaptation ,.f tiie ti-vpaii.i-,.n],. f..r l^il„.r;ii,.ry aiiim.ii- i- M..t l.-i 1,\ -nbs,.-
quent passage tlmnigh li,u-s,.., nt li'a-i m.t l',,r >ev,nil ir,'ii,Tati..iis.

M,W .w,I ml..- Tla. tra.,M,ii,.i„n ,,f Caiuulian d,.nrin,- ,., i.ib,.r;,l,.rv aniMi:,!- xva-

etfocted by a sueeesslul moeulation of a white ni.nis,. :,Uvr inindivds of „ns„ ..-fol

attempts had hern ma.h' with do-s, rabbits, gniiieaiii-s, ,.t,'. The ,,ri-in ,,f the str'iin
Nvas a natarally infeeted mare. During a perind of fonr w.vks in whiel, tliis'in-.r,.
presented fre.sh plaques almost o^ry ,lay, m wliite mi,v an,l L>4 rats were inoeulate,l
with fluid erlhvt.,1 from th.^ i.laques. rieh in irypai.osom.s. The first S;! iMoeulati,.ns
(..!) in mioe and L'4 rats^ failed: tlie eighty-fonrtl, (in the sixtieth nmuse)

succeeded. From this .nouse others were infeeted without anv .litli.^ulty and tli,.
strain was passed on thnnigh the usual laborat,,ry animals. In mie,. nu,\ rats ,,mIv
one trypan.xsom.. <-ye]e is rej.resented. The parasit.-s appear in the bb.od ,,n*tl,','
s(vond m-thir,: day after in.xailation and multiply with rapidity up t,. tiie tim,- ,,f
death, whieh in miee occurs usually in three to five days, ami in white rat- in f,>iir t.,
SIX days. Death fakes plaee snd.leniy and with s.-aroely any warning svmpt,,ms
When rats are inoculated in batches of l,", to l'.", „t on., time, as wla.n pr.'.parii.tr a
trypnnosome antiL'cn for eom|il,.ment-tixati,.n w,.rk, if may hapi«'n tliat 1.", ,.r in,,r,>
rats out of -JO will die within the space of two t,i three h,uirs ,,0 the f.,iirth or fifth ilav
Often thereare one ,.r two rats in every iT. that show in., re or l,ss resi-tan,',.. Tw.nlv-
three out of 2,") raf^; inoculated may die on the fourth t.. fifth day, tli,' twenlv-f..urtli
rat may liv.. fen to fifteen days ami the tv.cnty-hffh rat twenty t.. tiilrtv .b,ys not-
withstanding the fact that the trypanosomes present in the bj.io,! of tli,si> iw.. resistant
rats bwome even more numerous than in the rats whi.'h ,lie in tl„. n..rnial tim,- of
four to six days. It would appear that in a na.us.. or rat inf,.cti,.n wl„ n the en.l
of the first tryi.an..s„m,, ..y,.],. js r.^achci th,-ro is a smbbn ivb.a-,. ami ab-orjilioii
of toxin which cau-i's sudden .h^afii in tl„' L'rcat maj,.rify (.f .■as,.... \,,t m,,re fiiaii

5 to 10 per cent of inf. ,.t,.,l rats ,„rviv,. fh.' >li,.,.k. an.l th.-.. s„,.cunib f.. th,. - n.l

or third trypanosonie .yele.

nu!nr,ji>l,,s.— The duration of infe.tion averag, s b.'twecn -ix an.l -•i-lit ue.'k- a^
a rule. Ho^vever. with a highly virulent rat strain of dourin.. guin..,|,igs mav
succumb within two t.> four weeks. The appearance of trypanosonas in th,. blood
IS remittent or intermittent ami the parasites are never as num.T..us as in rat's

blouJ. Fe

sympfon,,- can be noted and ijeati

number of generat

easily restored by passing through a si

n guineapigs the strain is a[

1 occurs wi

th

pt t.. l,...s

PU.I.Iennes.
'c yiiul.nce

rie-

'f rats

.\ft..r
ut this

37

Rdhliils. Iiifi-cifil Fiil'liil- |iri -'lit a train nf niorc or Icr^s cliaractcri^ric >.vni[)tii

\ , I KM

Ifinatoiis ~ui-lliii^:^ nl' ihr iaI,

riia! (4:iiiilal ■.i';;aiii an

I nf till' cars, purulent

I (•■ junctivifis anil either e.-nlar le-iun-, riifai i- li~ii,n~. iu'ilti --ivc eiiiaeiati'tn.

r, eacliexia iiinl death in I'r.nii nn.' t.p lln-.e njenth-. '1 hi> para-ile- a|>|"ai- in the y-eneral

c'irculation "ilh reini-^ii>n> er iiitc rnii--i'.iis ami are rarily inuner.ais.

I)(i;ls. I'rnnnuneeil aihl t\|'ieal -\ni|itnni^ lit' lry|pan"-'>inia~l- are i.n-intiij

f tVver. iieclenias n( the -Ixin ami ;;e!iiial .iruMii- aiai vi'ry ?e\i r iilar Iri'nMr-.; ilio ey(-

1 hall l>rotrilcle~ anil has an an\iiMi-. -taring; e.\|iii ~-i,in 1 1 Ni'i.hl hahiiia ', jikI there is a

I keratitis ami sta|ih.\ h'liia. Ihire is 1 inMtar> ili-tni hani-i aial a nr. at feelili ni>-

I aial a teinleney to ~lee|i alino-t e, it iniii ai-ly, Kinaeialion anil |iara!ytie eonilitions

I pre le ileatli.

I As alreaily -laleil. in tin- lir-t ilireel iransniis-i.in ef ilnnrine to l.iiioratory animals

1 all atfeni|ils t,, inlVet il, ,■;■-. ral'l'il-. ami (.MiimaiiiiJ-s faihil, anil -m e,>, was onlv

i? aellieveil lliripnf:h a uliile uion~e. Tlio 1 ry|iaiio-oiiie wa- Hun pa -i li tlnnni;!! rai-

I anil no further iliU'eiilt.v \va- eN|ierieiii-i-.l in inl'ietinLT ntln r -|Kei( - ..f -mall aninials.

I Fnrthir, uiaai a Imr-e wa- infieii.l with a (hiiiri ■ -train mainlaini-il in ial'oralory

I animal-, the try|iano-iine- re.-M\,!'eil in the hi 1 .f thi- hor-e ui|-i aluays infeetive

I aliil lli;:hl>' virnhait I'or doL's am! all other .-|ioeie- .,f laliorator\ animal-. So easy

J liad ti'ari-mi--ion tlieli lueMine that ilotrs were iiii'eeio.l hy inji-linn. liv eatinir the

■f 111 -h of horses uhii'h had -ueennihiil to Iryiiaie-onie inl'r.-f i, ,n.

J \ hiase \va- de-lroyod hi irlrruii-. .md a ini-l-mortom examinatii.i was made

f i 'diati ly after d.atli. 'riyiMiio-ona - wiro iouiid |ire-ent in the hi 1 ,.f all the

I organs and mns,|e- examined. .\ eii.o ..f ile-h «as fed to two doi;s witli little ox|iee-

1 latioll lliat tlle> would leeoleo ilil'.rtid. !|oMr\er. tliro,. w.il,.- af'il' tlie feiilinf.' the

I dof.'- aiipeared to he ea-ily fati^'md, duii ami -oiiiowhal d pro-sid. On ilie twenty-

I ninth day a earrlul -eareli of h! 1 i.ikiii from th,. ,.,-ir reMMJed -,. \er..- l"ew trypano-

-1 -onies. On iho ihiiiyliftii il.i\- tin para-iti - wore !',iii>I\ iiumh i' -le . .-iiid the do'j-

I appearid to ho \er.\ ill. 'I'lio i.\.- Imd a loiltjinir. -ion\ ai»iiear.inee. Tho faei^ was

rather pntfy and e\pr.--i-id.— . l-'or - m ;■ 1 day- lliero had in-en attaeks of vomitiiitr
:ii:d diarrliiea. l'"or -I.ori inlei\,il- 'li,' animals wore \-ery re-tlo-s. hut most of tlip
time were Very depres-rd .Hid -'i pi i'..i- iiian.v li mrs a,t a -tretep.

.\n intra\enou- injeoti.iu ..( n.j yr.inimi- of Sal\ar-.-iii ei;oi;'i jn iniie.e. of
alkaline s,,]nlion wa- ;;i\en ..i.d \'. i'hin f .rt\-oij-Ml hour- i\!l trv]iaiio-onies had dis-
apjieareil from the hlood -li. am. I'll,- eiiai.Lio and improM ment :\oro a-tonislnnfr.
.\t the linn- of iho injeeikin ih. animals wero -o w.ak tliat ihoy had to ho prop|)ed
no and -upimrled. 'riiioo dav- later th.y v.ero runniiii;- .ainoit. harkinir and idayinfr.
Ilowover. on the lifiy ninlh da.v in o,,,., :i|,,| ,,,, ;]„. -I My-t'. airtii da\- in the oflaa'. tin n?

o.-eurred a rel.ap-e. S\i,,piMni- i imm iieod and the pai-.i-i:,- i'oap|.o.arod in the

I'lood. .\ soo.aid injeotiMO ..t' S:il\ar-a!i, Ol'.'i "rammi- in the ..no and n:: j.-ranunes
Ml the otln-r. was itIm n. Thi- tinio ihi-ro wa ■ -oaroi i\ .my i,oiJo,.mM.. oli.inp-. 'I'lii^
par.asites rem.ain,,! in Ih,- oinnlat 'on. iko -xniptoui- pr,.uri --ed .ind diaili i oeiirred in
(iLdity-oiio dav- in ike o,,,. ,..i-o .and in nine!>-li\o dav- ii! ■ ho o'k, r.

M'm.&mm

tiS

a
1
I

s

'2

I
S

s

n

s

a
■?

^^

1
I

is
o

i-l

J

JTlift^liCSt*

39

APPENDIX No. 4.

TRYPANOSOMA EQUIPERDr.U.

i

s

The microscopical appoaruncc of the trypnno.-ome of d'liirine is very similar to
hat of other pathogenic trypaiio^onu-; of the Hrticei type. In morpholoj^y, vitality
and general biological features, there is little to distiiiKiiish it from the trypanosomos
of surra, nap:aiia, mal de eaderas and sleei-'.iir sickness of man. Very coinn- i features
of the dourine trypanosome are (1) the .presence uf a vacuole borderi- ^' upon the
centrosome and (2) the blunted, ciit-otf nr ti-^h-niouth appearance of the posterior
extremity.

Tr. cqiiiperdum do(« not require any insect intermediary carrier or host and
completes its life cycle in the body of its natural host — the horse — and is directly
c(jmmunicable from one horse to another.

Tr. cquip'rJum is the only known patlmgenic try[)anosome of tropical or sub-
tropical oriprin that is maintained by natural means in the temperate zones and in
countries far removed from tropical conditions.

In its natural host, naturally infected, Tr. rijitipordnm does not live as a true
blood parasite but rather as a tissue parasite, selectinf? special organs, mucous mem-
branes and the skin for its development and life cycle. In these rcfpects the tr.v-
panosome of dourine differs fro"i the trypanosomcs of surra, luigaini and the other
trypanosome diseases.

The appearance, development, multiplication, confrlomeratic.u or afrglutination,
phagocytosis and intracellular dijrestion of Tr. c(HiiiierJum. takes place iti a singls
oederaatous patch or plaque, and the whole phenomenon can be traced within, and
usually occupies, 3 days. The following is an example: —

In a young mare, experimentally infected with a trypanosnme strain i liffore pas.<i-
ngp through laboratory anini'ih), three- to four-day periods of fever recurred at
twenty-four to twenty-eight day intervals and, at the same time or i)recediiig the rise
in temperature by one to two days, an oedematous swelling of the labium i)udeud..
Kxaminations of the Jluid contents of the patch wen; made on the first sign of tume-
faction or oedema and continued at intervals of a few hours throughout the stages
of eruption and absorption. The procedure at each examination was to make a fine
needle puncture and with the drop of serous Huid exuding make two dry smears and
one wet coverglass-celled preparation. Tlu! latter was examined immediately, the
former being stained by the Giemsa method. In the first i^pccimens thus obtained the
trypanosome could be located only with great difficulty being so few in number. In
the subsequent specimens increasing numbers and multiplication forms were noted
up to the thirty-sixth hour. At the 4(ltli hour conglomerations of trypanosomee and
attacliMient to the phagocytes were noted. At the forty-fourth hour nearly all t;u!
trypanoji incs were injested by the macrophage cells and in various stages of digestion
and disintegration. At the forty-eighth hour no trypanosomcs could be identified
as such, though in some of the macrophage cells fragmenting parasitic nuclei could
be made out. On the tliird day the swelling diminished in size, was further reduced
on the fourth and was quite absorbed by the end of the fifth day. Xo trypanosomcs
could be seen in thr specimens taken on these days.

Similar observations were made on a number of occasions and in different horses.
In one case in which depigmentation of the skin of the labia pudendi was taking
place, it was noted that simultaneously with the macrophagic injestion of the parasites
numbers of these phagocytic cells were cn^wded witli blackish granular pigment. In
smear preparations many of these swollen cells ruptured and let escape masses of
black granules.

■':'Sinp«Br..v»'

40

.^ftrluliiiiili"ii mill phim'i'X l"-i> "f tlir tiAp:

lio-nlili - .-riPh

•ii«ii^ illlil. 111

fiU'f,

■ iil.v 111 till' iiHii'in

tv. 'I'lir hilrr lii-l.iiv cf l!ii

il 111' iili-irviil In ;:il
;i-i < -linw^ iIkiI

lln> inri'i'linii w;is w.'.l ti .li'iMti'il mill llii' iurrrriiri' i-i tluil ,il;:^1iiI liiiili'iM mul [iliimi
I'vti-ii' i* mi iiiilii'iitioii of iiirrr:i-iiiLr r.- -imirr ;in'l "I tin' pr."'!- "I iniiiiiiiiitv.

I.i

till' is kimwii 111' ihi- iKiliin ami pr"|icrl;i- m| tr.\ |i.iii.i-"itu- tnxiius.

in III.

iilr. i;i'ii' ru.l/ril iiilri-l H'lu

rlr

il' ~iii:ill lal'i'iMt.'vy aiiiiiuiU. iihil in uliicli ■■iilv uiir
trypmiiisoiiii' i'>rlr i- nprrsnitiil. ili .iili ;iii|.:irriilly n-uli- I'r. m ,lii- -mliliii nlia-r iiii i
riil>iil alisor]itii'ii of tnxiii at lln' tiii;r if niaNiiiijI lry|iaii"-iiiiii niultiiiji'alii'ii ami
I'liiniiii'iii'iiiu: liiiinti'irriiinii. In ''ir I'lirMi.n', iuli iiiiiltriil i11-;im-i> in tin' ],.'-.i f
anininls i-dnniii'iiciiiir in a I.m-iI iiifrii' n, iIh' ir.s paii'i-oiiir rijiiaW it- ryrlv airaiii aiil
ilirain mul tlio toxir arti"n uii tin' iiri^i ll- -\-lrni apinai-. to In- i>r.iy:ri -^-ivi' ami
.•uniuliitivc.

Attriin>t> til I'liltivalr 'Jr. • 'iniin nlinii mi avtitii'ial riilturr imilia iiml tn i-inlal'- a
tiixin have nut >i n-il il in Ihr liami- nl' ll:i' Mi-itrr,

APPENDIX No. 5.

UKOI'LATIOXS IMM.AllNt; To MALAlUi: DT ((Ml'.

/\ Or.lr in I'nmfil 'hilrJ .';.,./ Juhi. V<l!. /';• 'i'//' -/ l''f J ,11, mil Cioilihildus

I)is. „.., V .1, /, l;.>.f.. I'.Kfi;,

I. N'n animal whirli is atl'i rtiii. .t .-ii-|.. .-i.-il nf i'liiiL' alf.'.-t.il witli Malailii- <lii

(•'.It sliall Ic pcrniitti'il t.i run at lar^'.' "i- I im- in i"n!a.'l uitli any animal uliirli

i- m.t jii atlirtoil. anil im .-m'li aiiim:i! ~li;il!. in :iny 'H-i'. I'l' n-'il f'"' lirri'ilini; pin-
tiiiM'>.

L'. Any insiii-rt"r may ili'-lari' I" In- an inlVi-tril iila.'O witliin fhc mcaninc; of
•■ 'I'lir Animal ('"ntairinu- l>i-ra-i'- A.-i," aiiv r'.mni.'ii. tii'lii. -talili-. or otliiT plaoc or
prrmi-i's win rv anin .il> avr fniinil wliii'li aii- all'irliil nr .-u-pi-'i'ti il "I liriiii; afli'i-ti'J
v.itli Malailii- -111 <'oit.

;;. Ni. I'.'.r-i'. a-- "1- III ill' sliall I'l- Villi "Mil .'lit nf any plan' -" iloi'liirrd to be rm
■■it'i i-ti'il I'lai-i' witli.int a Tn'i n-i' siLinnl ly an in-|.i'i-t"i-.

-1. Till- \'i-;i 1 inary I tin rt'.r (;iiiii:il may fviii liiii'- t" tim -diT tin- -lauplit. i-.

ra-tratii'ii "T i'iIht ili-p"si!i,,n "f animal- all'i''-l.'l witli Ma'aijii' i!n ('"il.

.".. ]ii-|"it"rs ai-i' Iniiliy antlmrizi il t" iii-iurt any animal- atl'i 'ti-.j uilli Malaill
.hi C'.II. IT -n-iii-.'ti'il i.f I'l-iiii: .-'. atfirt.'.l "i- wliirl. lia\.' l.'.n in .-'.iita''! witli animal
■;.. all'.-.'t.'il, .'!■ sii-p.fti-.l .'f L.-im: .-. atl'i'.'!' '1 "i- wlii'-li liav.' I..', u in any way wiiai-
i'\ii' ixpi'-. .1 to till' infi 'tii.ii "f Malailii' lin ("11, ai,.l iit,i.\- "Vilir any -m-li aniiiial-
1,1 1.1- I'.'ll, I't.-.l. il.'tainiil. i-'.'al."!. .-a-IiMl.-'l, "V "ili.'r'A ;-■ iLall with a- may t.. tliri!;
api'i-ar a.lvisal.li'.

'i. 'I'lir I'Xpin-i'S I.f. ami iii.'iili nial t.. ili.- ■-.'!!. .■•i.'ii "f. i-..la! li.ii. M-i/iin-. ra-tra
li'.n. '.!• nliiiru i-o ili-aliiii: «iiii aninia'- f'.r ll.i- piirp..-.-. ..I' tlji-.- r.-;;iilat i.'ii-. -liall
1.1' l.nrnr- l.y tlir ..wihts ..f tin' animal-, aii.l m. imliiiiiiii.. -hail \n- all..«iil t.. tlh' i.wm i'
in .-a-' "l' 'lam.;:..- ari-im;- "Ui "f '.r r.-ir; ii.ir fr"in -11. 'ii arti'Mi-. rxi'rpt as luTriii-
afli'i' i.r'.\ iik-il.

7. Xi. iiitirc li"i-i', as- m' niii'i' ii'i' any riilelim.' m"ri' than ..n.' yiar "lil -hall hi'
).rnnili.il t" I'liii at larjji' mi nnfini'.'il lan.l- in li.i' ninv im-i' nf -Ml., ria ..r in that p..i'-
ti'.n I.f till' pr.".vim'i"' i.f Saskati'licwaii lyini; u,-t "I tlm tiiin! pi'iii'i|',il iiu fiilian.

>. .\ny I'litii'ii lii.f-i'. ass m' mnli' i.r any riili^liiiir ni"i.- than ..m' y.ai' nal Imiml
ruiiiiinir at lar;.'.' wltliin tin- nri'.i ililiiail ah"\. may 1'.' -ri/i il ami lii'lil mi llic nnlir

w?^m^^9i4

^^^^^ixM^:jwiw^^rjTj^z7777~SL^:x^^^r^n>^.

41

vt Mii,\ i|ui,\ aillh.ii'i/fil vit. i-ii]iii'\ i'i-|ii . I'.r mT iIi, 1 1. |imii m, i,t >.l A'.

t'.iMll 111'.', \\ i

ill' "V\ llrr •■!' llir -.11. 1 .lliilrl

li

■ M -1 1 ,' I ! r' •

-li;i!l I'llliw llli will iii'Vi r |iii--il,l,- riMiil'v t

■lll'l ''"■ >ili'i Mllilrilll, if ||r,t ri.li.i,,-,! V, illiilt ll,i|I\ .j.iv- ,,|' -il.-ll -, i/ll!. , lll;l\ !.,■ .M-

iratiil. mill ii.i iiiilciniiily -liiill !..■ ull.n.nl i.. iIh- ..«ii,i- Im .■,,-,■ ,,r ,l;;iii..'j,. iii-.iiiu ,,ih

"f ..!• I'l >iiIiiiiL: tV.'iii >;iiil (M-lniiiMii, -il/ur • .|i;. ni ;..ii.

'■K Aliiiiiiil- :illri-|..| uiili .\|:,l,i,li,^ <lii C.ii, ,1,,,., ,,,, ,,,, ,,|,|.,. -i;;!ii.| l.\ ,, Jul'.

■1| l.i.Mltrll \r!i I'iluil-V ill-|iirlnr .l.iiiiL llli.l. r -|'ii-i:.l ir-lril.-|il)- (l.^lll ll- \'. I rri l,.i . .

Jtii-iTtor (iriii-r.il. Ill- f.irlliuidi -hnii^iiKr,-.!. ,|.,,| tl„ ■-.a-r.,... ,ii-|„.., ,| ,.f ,, i,, ,;„.]
.nil r iiri.Uili.l. iind r..iii|ii-ii-;il ii.ii iii;i,\ l.i^ |.;iiil |.. il,, ■•.■ii.'i-- -f -iirli jm'mi:.!- i i' .i;,,;
wlii-ii till' Ai-l -.1 provide-.

'"• lii'l"!''- an ol-ijiT i- iiiiiili' I',.,- ill,. |..,\ii!, HI ..f .•..iii|i,.|.-,.i i..;i ill n,\ ..) ih
."i-i- .ifof, -;,;,! ili,.f,. i,in-t l„. ,,r,.ilnr, ,| i,, |li,. Mmi-tii- .,|' Auiii-n'iiin ;i -.r i-l;,,-i,.r>

n'|l'.l-t. lil'lllT fii-- -l;llll;llti r. liliil .•.■rtilir;lli,,|l of V:l'll.Mi..Tl .ill.l - i I 1 1 _• | ! 1 1 i'. .'!! -i:;:i.i! '.-,

.■Ill iii-|)i'i'l.ir.

APPENDIX No. 6.

i;i;ki:k'i:.\(I-:s

Ki rill iii.m:i.. .1, (i. Ann. K,].. \'ii. Dir. (;,ii.. Ciin;..!:,. I'.miI ]'.<\-j

III" i^v C. 11.. ami \V\T,-ii\. I-:. A -S,„.-i;i^ i;,i...n ,,., l),,|i,-ini
I'rlH. .\-l-ii-.. Ciliihlii. .\,,\. I'.inT.

I"!:''^"-' l:. I' Ann. \[:\.. \', I. Ilir. (..n.. ( iin.nli,. l'.> I-'.- !'.' I-

''■I i:M.TI. .1. !•' I.„H

\V\i-ii\ !•:. .\. ■■ pi,,., pip-,

•■I'"nrin.- j- I'i iiirni - ".. -X,,!.. ..,, i],,. jj.-,. j,;.i,,,.\ ,,,■ y,
';"','"•■■■'■ • l''i''.i; ■ .\:i l''.,\|'i r'niniiw! ,'^iii.i;, . n' 1 io,iriiii- '.
■ <)ii ill,. |li:ii!no-i- of Moiiriiir ■.. I'.ilii; ■ Th. !;iti v In-o.r^ .,
■_'il -,irvi\.i|. ,.f !»,, ill-ill,.'. p.i|_'; • I-:, ;,| ln\, -.iij- itinn-
Di'iirini' '.. ■'rill- ■rraii-iiii--l,,!i of lloin-ii;, i. I. !'..,i-iit,,r
.\niinn:-'.. I'.ilo: • I'lio S,i-iiiii Tr-t for I ).qiriiio '. I'.ilt.)

■ I *'inriiii-. it- piillio-,.n,-ri!\ mni...:! ).-■ of •!-, , t'., .,.\ of ,!;-.|.

ti-i'iilliii'iil . . , '. .loin-, ( omp. r:it|i. ,v 'I'Iht.. 1!i1 1.
i'lir SiTiini );,■:, oiioii- nil, I .^.-nini |li:,i.:ii
.\nii r. \', :. .\1,.|. .\--,„-.. An-.. I'.M:',.

■ I'oiiriii.- iiMii tlio Coinploiiiiiii l-iN.itioii T, -r r;ii',i-;|,.l,„.s \',,i

Vlll. i;ii.-.. p. i.m;.

.in.l llMiupN S.. ■ Tivp;ino-oiii, - f,,ii|.,| ,,, {■;,,|,i,liiiii .\l.i,mn.il-
I'alMsit,, '..:;>, \',il. \". PHl'.

IKum;. \\'. I.

v.. Ill (

■ f lloii 11 .■ ■ I'l-..,.

■;. :. \- '! 1 ;

.\ii (lutl.rciik of jl
.Vii. :.'n.

ni'iiio . .\

in li.p

\Mi«;,. r.'i I .\

pp. n.lN

i^js^»!3e« fc>;^^.»i«iftiffiwwysgigiSfe?_wiiT»iiifi 'i*[r«'«Ma5w 'aBr-isaeasmiamr^

KXn.ANAl ION <»1' n.ATKS

I'hil.s I. II 'II,. I III

N'iitiir;il iiifiM'tioii ..1 Scilliou N,.. :;:.. -li..wii.K '>'• li':*'"!!-. llii. -111:11 iiii: I'Mii:i.

|ir"i.'n'--ivi' I'liiMriiilpi'M :incl tinul -tM;;i- ..f tin' disfii*!'.

riiit, i\

Stiilli..!,. \.. :;:;, ikiiiiim11.\ Mil.-.'tr,! «itli Itntiriin' i 1!mi:.-1!>ii7 •

/■/„/, r

KxpiTiiiK-iiUil iiilVctior; ..f iiiMi-.' N.I. ilii. -li..\Miii: .i riiiji->liiip.il "i- lioliow-ci'iitr.'

•■plniiuc." the uiiilli I" ii|ipi;ir. in llic t.-ntli iii..iilh iiicM-iilnl ion. Kiiinrc 1.
l-'i.i:. u'. Niilnriil iiilV,-ti..n ..f marc N... Ii'. -Ip.wiii!.' fa^inl piiriil.\>i-.
I'ii;. ■!. l':iiMpli'i;ia in :t iiatiiriil'i.v inl'i'i'li.l inari .

I'I'itr 17

l-'iu'-. 1. ■-' an. I :•'. Kxp.riiriinlal mfVi'ti.'ii in voiinn tillio. -hmvinn typical ocdi'iu
jitou. piih'luv- ..f the aim- (tin. 1). .if tlic lal.ia i>iiil.'ii.li (fit'. •_>>. ami of tho -kin
!r.-tw(^'ii till' iMitt.x'k-. I I'iK'. :'.).
Kit'-. I anil ."i. Natural iiificti.ni ..f marc N... l.V.'. -licwinjr .Icpitmicntal skin ol
till' vulva anil ami- ami tin- transient cliaractcr of such dcpipniciitation, Vip
.•, -hows the ccindilioii ten months later than fip. 4.

At ihc lime the-e photograph- wen- taken ( tip-, iro. ii-.vpan..-..mc- ucre pre-ent
in (he ...■.Icimiloii- lliiiil-. in each cas.-

I'hil, III

Tiiiii,iiii,s„i„„ •././</<. r,/'i(m III the ..eilianal.iU- lliii.l ..f a -u.nlen lahiuin pinlemli.
.li..\\in:; the (h'Velopmcni. iiiulti!ili.-ati..n. appliitiim'ion, phatrf»\vtosis ami
.lici-iii.n ..f ilie pari-it.- all tal.iiii; p!a.-.- within th.- -pac- ..f 4^ hours

ri.it. \ III

I'niiuniiistnii.i . ./.'(/I. iiliiiii.

J-I.il. I.\

Triiii'iiii.^'iiii'i -/»<;.. I. /»!» iiic liaj^.ilal. .1 i:ncr.i-..r«anisMi .■au-iiic l>..iiriiic. a-
-cen in llic vauiiiai iiiiic, i- Ihii.i I ti--. 1 ^1 .in.l in the ..e.l«-ll\atoiis tluid fr..m
-w.ill.'ii pal.-h.- ..f tlie v.iyinal ni.Miihran.- (ii--. lii-1>) ; taken from a tiaturall.v

inf.'.'l' i in;.!'.' '■ animal Vo. :'.i; i

r.^BET^smrs

tm^mi}jr:iW'-:Ky!f^''r^,

KXI'l.ANA I l(»N «»l- ( IIAUiS

iiisrouv ( iiAiris \..v i. ii. and hi

I lii-sc I'ilarts 1 iiitdiiiizi' the liistury nt' I'lirty-tw i ciinr.-. cit' douriin' iii lior>*cr<. twi nf,
lu.i liciiip iialiinilly iiifiM'tiil mid twenty i-xiieriirifiitiilly iiiocnlafi'il

The rh'wf points* of intcriNt ami I'onclusidns arc:-

The fatality in Htallions.

Till! toleranoi' anil recovery in nuiro and tin- nlatiM' minuinity olplaimii i.
N'infectiou by naturul means.

Tolornnco and n<'quirpd immunity tu inoculations of a natural strain of douriii.-
' liorsc) is broken by inoi-niations of a laboratory strain (rats and (fiiineapitfs) and
death follows.

In some of thcso mares '.ha [leriod of observation is fifteen years, in others twi he .
ten, and a lesser number of years. Marw spontaneously cured of the disease havi
-howii their usefulness for work puri>o8(^ and for brcedinK. No case of natural trans
mission to the offspring has bef>n ol>served. The four healthy stallions kept for brii>i|
iijr these naturally cured marns escaped infection.

SiXH'itic. serum proiHTties, as shown by the <Mimplenunt-tixation test, are retained
in some i.'ases for many years after recovery and cure; in others these properties are
i"«t in aliout five years (st« Appendi.x 2).

History ('hart No. II shows the orijriii aiul Kenialojrv of a strain of douriiie in
:i'>rses. and the exalted vinileiic-e obtaini'd by serial passap* through foals, and i-
-uppleniiiited bv : Ten'peratiire Chart* N'os. I, la. Ih. I.-; II. Ila, Mb; HI; IV: IX
tnd X

fiRl<Iii

r7.jj^-(c^Bs:^.-ii^tma^>'i»hmiVivir«?ftm-^<&r::sp'^?f..v'

Vi^SB?WB*f«9Rr

~'^' arg*^ jjB^M:.^mpjiw^^^^^gBiF— g'^ai'^M^g^im— ^^^^^^a^gq^^^^^n iT^g f^ . tw •MMiwr'-jpiwite^^^^iJ^ ■'* ra^w f^^MW ~ r~ JTrilM-".f~.WL ^i ^Vli

Platb I.

\

5S^S5^?^5^5^?S5E^^^SS!?^S5^^ES^J*!

• >JW..J«Jta!JBS«JaBBg!!ag.g" .lywLiai!

Plati I.

7i'«-;7>,

Ai-^S.IJBKTv'^Sff

1

Plati II.

p., in IIL

&:-i*^£:^.it:jfl

-I

.'I

,1

•trasT?-?"?

Plat»! V.

'^S^^Sr.-Rf

e®llK»■'«^^^■';i^V^t^^«.^ 'S-*'

TrvpanOBomn equipenlntn.

I'l.ATl VII.

h-i

■.mm^- n^MJui m..

.'me:d'<

^S^^^'^^E^'

at UlTTlltai ■.■KLS! II

i^ /-^

/

i^.

V

1

•V.

•^^••1

N

•*o *u r

3 1073 OOOSMOIb S

I

HISTOEY-CHAiT B

Hkalth ok Aximm^ »RANc„._VKT«...NMn Kksea,

law

1MI7

l»l»l

IMIt

1910

I»1J

Stttti4nt (.1/)
.SVoH«,n (,i.)i

Stall ivn r.'l

Von •(;

.Vote /-.I)

I/or. r.

.l/flr# r.'))

l/n/r ;.;

.t/»rr ;•;

Mir,

1/ur. ,.',.<

• "'uturat
iQfi>ctu>n.

■Vaturitl

N'liturul
inffction.

NuriirnI
infi^rtion

N'u'tir.-i!
infi'i tion

infirtuin

Ntittiral

1 l>HMtniy(il.|

* *«*<lf ma.
)iniu>mia.
•>iiift<'Uili im,
IHTiiy r<i..

,.l.-lilli
•July 2i)

'»<*«i<'mii.
Ciwiwxiu,
ilratli

T"ipriinl Normal

< "ViTi>.lliy,(„v,.r(.<l |,v
■"'l ■11. .stl 7.. •

. Inlimnt. I .Viinual

.'I "'viTinm .1 iTovcrine.
I>y Stl„, I,,.

:llan,l;l.l .Sir72

.T.iliTiint, Xiirmal

l>v

N'ormal.

Hrwl V,
lionlthv
»t«llion

F.wl,..)

•Niirmul

.N'ormal

1 .Vormal

J.'nipl .

>.vivl a. „ K

"rk marc!

rottJoii.

I

Nortiiat.

fniirubiUMl

with try-

piimtHtmieN

from riiari'

I ft.)

CI

•il unci .{;

Niilur.il
infiTiion

\ Situ flit

iiiff«ii,,ii

Niiturui
infftMinn,

.Nullum I
tnft-ci ion

7 (vtviTintc-*

I*'l<'r:int
'>y Sti ;ll.

rolcriitii.

Si I. ::.

IniM-. of

trypan-*-

■*oiin'M from

iiian' ,W.

Norriiril.
Inm-. of
Tr\ pitno-
M'lm-s from
marc '[ti.

■Normat.
Mn-.l to
(i-:tllh.v
■'itillion.

(Km pit
.Vtirnijil,

Kerurrinn

oudoma of

viuEinal

'X-il ;i^ fdst: ilrivinx li

■N'Ttiml. Normal
Inor. rtf
Iryptino-
*jincs froin
r<ml (if.

-Vorinal.

ir .s, anW ;

Th 111-

in'x'iilurril.

'■ov.Tin« trviMn.H

. 'V -«)nnv from

Ml .,.{, man.^!(

■in-l .':*,

Xnrmal,
i'oal.'.l.

Normal.

I (l>«uth
from
rolir.)

■ (no.- „f 'Aperii«|„f
tru-:ino- ' i„^f, f,.^.^.^

*>mt'3 from ;jt.T
FfHtl l-'f. in<M-ulunon

' If for ij >..

N'Tmal.

V(vi.||.nt-
■iliy huri,
I>c»tn)yed

N'-rmal. Normal.
iKinpl.tw- .1 ;,,^ .1 wr>rk

l.ro.

mart

iniM'ulated,

tr\punr^.

■^HUf-. from

r-Ml.^ If. 7f.

Normal.
»;mplo>*..|

Norriutl. Niirn

.»-. a work iiianv ■

al.

'lof.-rant

< >van.-,-
ri-mo\iH|.

"rmal I, ,.akl, .,,,,1 ,,. „,liti„„ ,'„,„i„„|

' Vjipan.nll vr.... ,v,..v.i \ ,.rv ... .1 i

-Niitunil
infection.

Nalurul
ir..'..<-(i..ii

ToliTint .\,.rTii:,l

r.-.,v,.rinK,- l..i,l,.,|
l>.\ .'^tl-

•liKlit

■L.TN,.

N'..rmiil V.,rn,al

I'.mi...!

I.ji.l,.«l.

IniK- ..f (• 1'

(r\|«ino- .\o ?

^'iiii-... from i.Kij..

man. IH.V MarrI

n,.,|,.|n:i l(,K^.,i

In.N- of (■ (.■ .

irypiuin. Imx-

-"""'" from trvpa

mart' Iti.i -oriii'jt
.Vori-ai'tion inf r;
lJ.aI

< ,|-

Matf /.I

Nutiiral
infi'Cliuii.

\:ucinul

•liwharK*'.
< inai-iafu.ii
P(ira(.!<'ni:i

S|.,«

iMipnu..

iitfiit

Inr...

onlin.ition

I iirth.-r

iiiiprri\. .

inroHjr.ImatioooflMn.ll,mh. r.
cnronir f-yiii piotii

'"i. thoiiuli

( ' r. -

trypan

•om,-s fi
:t rat
Di'atI

M>m,*f from foal iif.

II

t'vatii

' I <Ja>

HAET No. 1.

i Kesearch Laboratorv, Lkti.uuiwjk, Aijiu.ta.

l»l-> 191.1

lOU |,,|.,

MMU |i,|7

1"^ "" i

•,«i..,.. i J •^•^' '^ <7 . ■

.- .-'.._ Walt),

ith try. < v,|,.,„„. I h . .

uw.mw. irypum.- r„il,n,r li .,

(.'nriipli'iiicn • hiijitum

i("«>nipl<'nn-nf Knutinn

P»»iitivi-

'•• - I r. -

I'l :ia a l.r.«,l jT.arf f„r ( y,-ar»

fuulinff.

'■'iM,i.||.n,.nl. rn,ii„,n

inal. C.y, -. :- . (• |, , _
' work i„ar,- , l.„,,|„,

lal.

<■ I .

'"^'i.-'i r...,k.,i

1„:,),.,| p.>..lliv.

' ■'ii.iilc'nii nt Kivilii.n

^"!<ymp- |n,,r .
l.mi.frnin trypun..-
Marrli u, huh,,., (r„
Diwiiil,..,-, inf. rut
D.ath

"f (■ I', . . .
'""■ IniKv lit
Ir.ini trypiiii.i-
l«.i ..,n„.» (r,,ri
turn inf. rat
l>i-alli.

hoalisl.

I.. a I,., I.

K.^ I

I-..ali..l

<**"i,pIcii,fnT Kn;iti«,n

I '■'' . ■• Itll In.H- ,,i
trvpaniMinipH fn,ii, ;,n
mr,.('tf(l r.at
Ma.\. Ill Mil 1),.„||,.

Mii\ .', In,M' ,,f riyp;,n,,.
^^iiitf, fnm, an inf,H-t.,|
rat

Aia: . 1 I'll.! I),,,th,
< "ii,pli-,ri,nt liiatii.n

tHi-,n\ '■

I •■ii.pl,-i,..nl h„at,.,n
n.-y ,ii\,-

I' r. ■- ^ i-
IntM' of
Irypuno-

oKleM frnn,
a rat .
IMitli.

•■•■riipi „„.|ii lixat,..n -

May. ,-, I'lll i„,„.
trypiiniw-.,i„,.s fruij, ■
infiHti'il rat
,>'1>t . J'J I'Ji.i ll,.all,.

•■"•'wl J..n.Taei.,iii,n.||ia>vi„.

"I 7i-|,;«iiu,„„„.,,,„,,„^,(^

'"1 >yii/;i»ii 1,1,-1 \,itui:,i

in/tftion

-»'' "»" <■''■• S:.U,r.,l ,„

"™™ - - "^^s....^^;^'^

•'•■.I.rn,,.

anai'iiiiu
t'niHf-iuti iin
IMtniplfKJ ji,

IV.it I,

""■'''•""i viiKin ill ,11 m
111 «>ir piiriipl.-(u,

7 jPliniui*?i

''■"•"'>»'» |lV„ll,

mi-.'

:ir.l F„o( i.». l:<p..r„„..„,,,|
imiruliMion. trvpjinii-
»*»nii',- fridu nj.ir.- ;!ti

:ir.l, \lc„,l\,, l:,|„.ri„i,.„,„|'
in«M-uiati<>n. tr\iiani>.
^«*^nllM fruiii iiiiiri>;tt>

•ifl. t\.«l J'., l:>p..ri„„.„,,,|
iniH-ulaiH.n. tr.viuim*
^uniiv fntiii man- Iin

«1. f.N. .m l:,p,.ri„„.„,;,|
llKM'iilaMiin. Irjpani. ■
■umii". frijii, i,,„| _..,

K.Mtl,

iltlil

^auinal

Ml III

I'li"^ I .Voriilal, ; .\,,ri,i,,l

Nuniiiil

>liKhl

Ml h, i',"l ^■■'■'""1

'"' '■■•■'' '■'; l:v|».ri„„.«„| '

vjiiiiv- (nun i,,.,l "I ,., '' I'lpx- Mlur,.

< ti,iri, I. 1;,, I), I,.

'»• il.-.im .,( Normal

lah jia puili'iiili

.Nnrinal

.Nnrnial.

"■•.I.^riia. :

fi' viT par(iT\<. iiiv, , .VIiM^ncf

'"■ ulur and piir apli-ti.- "'■"■"v.-rine ,,(

^.\ nipt. nil., ^.Miiptiiin-

'"' ^'-'' -v. l:<|«.rNi„.„,;,|

llM«-ulaUi.ii, Iripam, Si- T.'in

I'lni-, It. nil f,„i| .M|

< '■■<"- II. Il„.||li

■ill.. f,ml .tr l:„».r,„.,.ntal

in'H-ubta.n Tl^uaIH.. ^ r
■■«ini,., (ri.Mi i„r,| .-,f li'M'p.- raliir.. (In, rtlll

'''h >■■■»' li; l:.,«Tl,n..n,„|

■"'in,- fn.n, |..,;i II 1 ini|». r.-,tiin- (|,,, rll\

•"' 'W|7/. i:,p..„ni,.„,al
in-»-ulali,in. Irvpaii.,-
.■"iini',» Iroiii /.lul i;i

Mil. AW ,M-; ,,:^,.,,, „,^_|

iniH-ul.iii.in. if.pani.-
"<'llii'~ Iroin (.,al 71

-"'■■ ^W 11, l:M..n„„.„,,,|:

iniN-ulatinii. trwvin.. <. r

>•>•;.: I,„„.!..!,l^r ■"■ '■■'"l'"'-a'ur,. n,,, rl|.\

"'"'• >Wll-;.' l:xp,.n„„.„tal

X'".l-» f...,n 1u:'mi" "' ''■""- ■■"""'■ < t... '1 .\

f^l V. r i)iirfi\\H 111-.
Pi' rrtplt-Kir ,„^ .niptnin^

I

U.H-iirrinn

iMn.i\>iiis i'iiiM'r.\.

(itii-urrin ^ immI,.
Ilia. r™ii,t..„, |,,,.,„|,

Kiiur rins ".'.li.- v.

"la. re imrti.nt . '""'" '"'

''•>'-r. urti,.„rn,

\ppjiri'tit riH^>vi'r\

afliT .1

nji' rtiiin.v of

ar^,.n<ipli(. n> lulyi-iii

K..X
l)..«
41

IT
111
• lav

-

IV
I)..

.il

MT

nil
.la%

rii.atli
1 ila^s.

Y-CHAHT No. 2.

V.IH

mr,

I'm:

I'lii

lUUI

tr>p;in..«,in,-.. (mm .uinca

PIK

.\..v..,.il..r 4. HI12 Dfatl,
■<i'i- TuIjI,. |\ ,

*K.Hl..,r '"'•'"''■•^■'■nt Kii:il,„„. -\v,.,,k
jhwitivi' rotictitin:*.

'■■.n.pl,.ni,n> I i,.,t,„„, „,

, "lei.. U. 19l:i I„„r ,|„ur,„,,
; 'rypaiK^inie, fr,„„ „n ,„

•Jur... I.'. 1,.|.V In,,.. ,,„u,j„,.
tr>pan.woinM fr„m ;,„ i„.
It'ctttl eutneapiif

Jr>pM„»,„„., ,,„„, ,,„ .^
leotetl i;uin<>]ipiK

(-.•..ri.pl.-i.ii.nt Hwtwj, -ni-ju-

HISTORY-CHi

'"'"1 xljillinn ;i:i ' ""'

V"v :■! ,„ 1^,. .

",'?/"' 1'!'""'"-'"" "'•>'»'"•','
>i>l :'i. ii«i7

""'■ «..;.| ,r ..1,1, I,.,,,.,,

Piin.«.,n„^ fr,„„ ,„.,„„„,
in.UI nn.l fr,.i„ (,„| ;( "
V|.l -M. I»ll> \|„> .., |,„,.,

""" ■«■' .1 yr ..1,1, I,.,,,.,,

"■'^' "■■■'i .l,r ..l.r I ,.,,..„

'"'"•«'"«•» lr..,„,„„r,.»,
>UK IT. I'llls

""'• ■"' >2 vr ..1,1 , .

""nt"l in.,ul;.li,.„."„V'"rT'

l«.n.»..,,„., ,,,, >

''"'H' I '.'I 1, oil;

"•"■• '•" M yr .,1,1, |,„„r,

■;' ''I'""! 'I..1.I ii,,„,. ..,

».ltt J1I fin*

"• .«' ,-1 yr .,l,r I „,.,,
""•nl.il m,.„n,t„.n, ,,,,,,; .J

nnnT'""" '""""'I inf.-..

""" ;»l I.-, vr ..1,1, s„„..,|

"'"• •" •: SI .,1.1 s,,,„

inf''<'h..n

>' "I'l \:,.,|,,,|

I/....- ,/*,l ,.-, yr „l.|,. v.,„„.,|
]•';»■'«'•> mil:,, ,,y ,,X;„

*';T"'""' ' i'|"'i.i.-

■l.-linii.. ViiK .", \, ,,

-MMrii..iii, rn,w,n.,

H.,J|1,.,
.|.-|.-,-.,..|

'nl.-iinirrit
l':.r:i|ll,..,.. \„,,„;,| ^ ,

'' l,«,l,,l

V,.fi.j.,l

II

ii
iillu

IV.|,.„|,, „|
'-il' liu.l.n.li
- p.l.1,,,1.- h.lu.Ml., M.,, . ,

'l>-.l.,lii.-,l,,.
-«i-ll.n«. .\,.,,„,,|

tin.l.-r
i.l.,|,..i„.„

"[.■.■.jriin „,..,, ^, ^

"' l^il.i.i |.ii,|,,n.li. ,,,.,„ ' ,

■'' in ti \ii .iii,.^,,,,,...

•"■'i in ii\ iiiin,,^.!,,,..

\, .11111,1

' "■•!> I.,:,l,.,|

,l.-l,n,„. ^" •■■'••■I

->iii|ir..iii>

l.«,l,-,|

"■'•!•■. ^■-■1 .,„|,|

l„:,l..,l

I,.mI,.,|

■|,.l.l;,nT \,,r,„.

-I'l'i" In. ; ,"""■'

».,i...„., ,,, '" " '" ln,«- .,r

.•iil.l,.l I|>|B„„, l,vi»,n..

""'I '( i..',i 1 v

>>_..i.iu.l \„ri„„|

I- I.,> ,•.!

'I -

I,'-' .Irmrii: „|.| ,,. ,„ .

■r,,l.;i:,nl \,„„,..,|

>lilthl
'"■'''•"'I' •"■'I l.h.i,!,,,,. ,| ,
~>lil|.t.>ll,.

I'M I.- III.,,.. /,r III ,,,

''■'"■'- .( v.winai \,,r„,,,|

li"l.-,.„, „„ .| „, i„|, .,,.,'"'■''

I"i'l-n.li. ri.-h in ''" ^"iii.,.l I, ,-„lil,
I'viKin,. ^,11,,.,

H.-.UII

'in,l.-i :,l,.l ,,„„.„ IrM, „„,„'„„„„ '.•.-v.-r,

"•■■•I'l -nlly ,|,.t,H. ,,.,(

^■'"11.1 ll.-.ltl,

"■'F"-'^;MV"v,y-x^

in'.v.i,„ riV, '•■""'=''

I'lii'iu,- :in

l''>|«in

pri>

)RY-CHAET No. 3.

I'MI

I I

(■ (■

'■•"'-' >■«'•■'> i;-i,.,,
i III i„.„i,h

Mi.'d

lltlli

•■"nipkiii.-nl (■iiu.iio,, „.„,.

1 1.

Illllll
fliii-nxii

(• ^ ...

Ill Ix'lllth
riirulv!.!-

I><'»rli

' III S-pl

1' 1

1 .«ll,..l

< ■ 1 -
l'.«,l...l

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Expertaental Station IHealth of Anlr'uls)I.')<-'''Jrl-!(,o,Alta, v <<»

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Exi..; .' '.T -•..r.'. ,-a Do%! r'rc .

November

December J^'^ •"""•''' '■'»">

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Chart Lb. Co»„nu^^.,909 (E. A. *it.on. )

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Trypanoi

98 1 I 1 : i I

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105
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