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CORNELL UNIVERSITY

THE
Flower Veterinary Library
FOUNDED BY
ROSWELL P. FLOWER
for the use of the

N. Y. STATE VETERINARY COLLEGE
1897

This Volume is the Gift of

356

Cornell University Library
SF 779.P8A25

The veterinary bacteriological laborator

HN i lM

Cornell University

Library

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924000278485

THE LieHt HON. (eNERAL Louris BoTHa, P.C., F, B. SMITH, Exy.,
Minister of Agriculture, Director of Agriculture,

Dr, ARNOLD THEILER, (.M.G,,
Government Veterinary Bucterialagist,

THE

VETERINARY BACTERIOLOGICAL

LABORATORIES.

ISSUED IN COMMEMORATION OF THE OPENING OF THE NEW LABORATORIES
AT ONDERSTEPOORT, PRETORIA, OCTOBER, 1908.

PRETORIA :

THE GOVERNMENT PRINTING AND STATIONERY OFFICE.

1909,
4198—8/7/09—1,200.
TG. 35.—’09,

y Surgeon.

Principal Veterinary

RAY. Esq..

(

E.

IDER, C.M.G., Government Veterinary Bacteriolog ist.

Dr. ARNOLD THE

CONTENTS.

History of the Lahoratories

Description of the Laboratory Buildings ...

Imununity in Tropical and Sub-Tropical Diseases ...

The Diagnosis of Bacillary Piroplasmosis of Bovines in the Transvaal
Haemolysis in Practical Veterinary Science

The Anatomy of Stilesia Centripunctata ...

Notes on the Pathological Anatomy of Pleuro-pnewmonia

torsion.

The Staff of the Veterinary Bacteriological D

TRANSVAAL DEPARTMENT OF AGRICULTURE.

Minister for Agriculture :-—The Right Hon. General Lours Borna, P.C.
Director of Ayriculiure --—F. B. Surru, Esq.

THE VETERINARY BacrErIoLogicaL Dtviston.
Government Veterinary Bacteriologist :-—

ARNOLD THEILER, (.M.G., Schweiz. Tierarz. Staats-diploma; Dr. Med. Vet.
University, Berne; Hon, Associate R.C.V.8.. London: Associé étranger de
la Société de Pathologie Exotique, Paris; Membre Corresp. de la Société
Centrale de Med. Vet., Paris: F.R.S., South Africa.

Assistant Government Veterinary Bacteriologists :-—
JAMES WaLKER, M.R.C.V.S.
WattEeR Fret, Schweiz. Tierarz. Staats-diploma; Dr. Med. Vet. University,

Zurich.
Zoologrst :—
Lewis Henry Goucu, Phil. Dr., Basle.
Pathologist :—
Karu Frieprich Meyer, Schweiz. Tierarz. Staats-diploma;: Dr. Med. Vet.

University, Zurich.

Superintendent :—
E. B. H. Parkes, B.A., Cantab.

Clerical Staff :—
H. W. R. Kine.
C. F. Hryps.
F. T. Maucute.
J. C. H. v. p. Heever.
C. AnvILL (Messenger).

Lay Assistants :-—

Tueo. MEYER.

R. J. WuHiIte.

W. F. AVERRE.

J. F. Scuurtz.

T. J. M. DEvERs.

F. Cocatus

Miss L. Basson.
Farm Foreman: W. B. Breton.
Farner: S. B. Tre.
Storekeeper: J. B. GiupEa.
Caretaker: F, W. PFISTER.
Yard Foreman: R. J. VARLEY.
Enginecr > 8. SoLoMon.
Assistant Engineer: W. R. Parmer.

“uoIsIagd dapuitaja A ay? f9 JfPIS FUT

= Ss
Z
ae

Tue Verertnary Drvision (for Contagious Diseases).

Principal Veterinary Surgeon :—
C. E. Gray, M.R.C.V.S.

Assistant Principal Veterinary Surgeon :—

J. M. Curisty, M.R.C.V.S.

District Government Veterinary Surgeons :-—

J. Cuaumers, M.R.C.V.S., Government Veterinary Surgeon for Witwatersrand.

G. May, M.R.C.V.8., Government Veterinary Surgeon tor Waterberg.

J. I. Epear, M.R.C.V.S., Government Veterinary Surgeon for Zoutpansberg.

8. I. Jounston, M.R.C.V.S., Government Vetermary Surgeon for Wakkerstroom.

H. M. Wexz, M.R.C.V.S., Government Veterinary Surgeon for Lydenburg.

W. G. Evans. M.R.C.V.8., Government Veterinary Surgeon for Lichtenburg and
Marico.

J. Donaupson, M.R.C.V.S., Government Veiermary Surgecn for Evmele and
Carolina.

H. X. Turnputt, M.R.C.V.S., Governinent Veterinary Surgeon for Barberton.

J. M. Tatz, M.R.C.V.S., Government Veterinary Surgeon for Heidelberg and
Standerton.

J. G. Buso, M.R.C.V.8., Government Veterinary Surgeon for Krugersdorp.

R. 8. Garraway, M.R.C.V.S., Government Veterinary Surgeon for Pretoria.

F. Linpsay, M.R.C.V.S., Government Veterinary Surgeon for Middelburg.

G. W. Lez, M.R.C.V.S., Government Veterinary Surgeon for Rustenburg.

T. H. Dats, M.R.C.V.S., Government Veterinary Surgeon for Potchefstroom,
Wolmaransstad, and Bloemhof.

F. J. Dunnine, M.R.C.V.S., Relieving Officer.

D. T. Mircuett, M.R.C.V.S., Relieving Officer.

P. Conacuer, M.R.C.V.S., Seconded for Service with Portuguese East Africa
Administration.

History of the Laboratories.

Tue Veterinary Research Laboratories of the Transvaal Departineat
of Agriculture comprise a large block of buildings on the farm Ondersty-
poort, situated eight miles to the north of Pretoria.

The site was chosen first, for its central position, being in the vicinity
of Pretoria, the capital of the Transvaal; the laboratory is in close touch
with the headquarters of the Department of Agriculture and the Govern-
ment of the Colony. It is also well served by the railway, Pretoria being
a junction for the eastern line to Delagoa Bay, the northern line to
Pietersburg, and the western line to Rustenburg, and on the south by
the lme via Germiston, it is connected up with the south-eastern line to
Natal, the south-western line via Johannesburg to Potchefstroom, and
the Cape border, and by the direct southern line to Orange River Colony
border at the Vaal River.

A railway siding at the laboratory makes it possible for animals from
infected areas to be sent through in quarantine with the least possible
delay, and a good service of local trains runs between the siding and
Pretoria. The laboratory is also connected by telephone with the Pretoria
Central Exchange, and in this way is in direct telephone communication
with Johannesburg and the whole of the Rand, as well as the towns of
Pietersburg, Klerksdorp, Potchefstroom, and Zeerust.

A site to the south of Pretoria would in some ways have been pre-
ferable had other considerations been as easily satisfied, but climatic
conditions had to he considered, and the high veld would not have offered
the same facilities of studying the progress of the diseases of the low veld
as the situation that was decided upon. Being practically on the verge
of the bushveld, it has a climate perceptibly warmer than Pretoria, but
at the same time perfectly healthy for human beings; it is consequently
not only geographically but also climatically centrally situated.

A further consideration in the selection of a site was the necessity
of there being attached to the laboratory a farm of sufficient area to
afford. grazing ground for the live stock in addition to some arable land
on which to raise crops for feeding the stabled animals under experiment,
and so it would not have been convenient for the laboratory to have been
situated in the town itself.

The portion of the farm Onderstepoort that was purchased meets all
these requirements. In area it is 512 acres, and is NOW divided up into
paddocks. Sixty acres are under cultivation, irrigated from a furrow
taken from the Aapies River, which flows along the whole of the eastern

2
boundary of the farm, and to the north a further portion of the same
farm, in extent 1770 acres, is held under lease, and on this portion there
are 60 more acres of irrigated land.

The selection of a site was a difficult matter, and it was some time
before a suitable one could be found, so that it was not before the end
of November, 1906, that the Colonial Government appointed a committee
of gentlemen to report and advise as to the purchase. The farm was the
freehold of the late Mr. Cornelius Erasmus, but was held under a
ninety-nine years’ lease by Mr. E. P. A. Meintjes, but these two gentle-
men met the Government in a public spirited manner and readily agreed
to transfer their interests in the farm at the price it had been valued at.
It was accordingly purchased and was taken over by the Vetermary
Bacteriological Division immediately.

In the month of July preceding the Colonial Government had voted
£1,500 as a first instalment towards the founding of a new laboratory,
so that on the purchase of the farm plans for the new building were at
once taken in hand.

Mr. Charles Murray, the Secretary of the Public Works Department>
placed the work in the hands of Mr. Eagle, the Chief Architect of his
Department, to whose care and to the untiring efforts of whose staff, it is
due that the fine edifice which now stands as a landmark in the surround-
ing country is so complete in every detail needed to make a perfectly
equipped modern laboratory.

In the month of March, 1907, the administration of the Colony by
the Crown was superseded by a Responsible Colonial Government, and
the Right Hon. Louis Botha, being the first Prime Minister of the Trans-
vaal as well as Minister of Agriculture, gave his whole-hearted support
to the project, so that in the first session of the new Parliament, a further
sum of £40,000 was voted for the completion of the buildings. From
that time the work was rapidly pushed forward, and by the Ist October,
1908, the buildings were ready for occupation.

Although the new laboratories are built on scale that will compare
with similar institutions in Europe, and are fitted up with every requisite
for research into tropical diseases, it was not always under such auspicious
circumstances that the work of the Division was carried on, and it was
in very humble surroundings that scientific veterinary research in the
Transvaal was first started.

When in the year 1896 rinderpest devastated South Africa, and caught
the Transvaal farmer almost by surprise so that thousands of cattle died,
the preventive inoculation then introduced first showed to the farming
community of this country the value of scientific treatment

After the pest had finally been successfully dealt with, the Govern-
ment of the late South African Republic decided to continue to keep up
a small laboratory for scientific research into other diseases of the

*kLOJDLOQDT ay} JO MalA JUOLYT YW

»
2)

country, and accordingly a small sum of money was voted to fit up a
temporary laboratory on the town lands of Pretoria.

At that time there was at Daspoort (a suburb of Pretoria) a wood
and iron shed which had been erected as an outcome of a resolution of
the Inter-Colonial Rinderpest Congress held at Vryburg in the year 1896.
Delegates from the various States and Colonies of South Africa met at
this congress to discuss the position with regard to rinderpest, and the
best measures to be taken to combat the disease in future. One of the
resolutions passed was to the effect that the exportation of hides should
be permitted only if they were first subjected to proper disinfection, and
it was for this purpose that the building in question had been erected.
It had, however, never been used, and the suggestion was now made to
utilise it with such alterations and additions as might be necessary as a
veterinary research laboratory. The scheme received the hearty support
of the landdrost, Mr. C. E. Schutte, and eventually a new three-room
building of wood and iron, lined with brick, was erected for a laboratory,
the existing building being turned into a stable, and after a few small
additions made with material and fittings taken from the temporary
Rinderpest Field Station, the buildings were ready for occupation by
the middle of 1898; the equipment, however, was sadly deficient as for
the present the best had to be made of such as was available from the
temporary Rinderpest Laboratory which was now no longer needed.

The first undertaking im the new laboratory was the preparation of
calf vaccine lymph. A serious outbreak of small-pox had occurred in
the Transvaal, and the necessity of providing a large supply of lymph
for vaccination of Kaffirs prevented much useful research work being
undertaken, and the war which broke out in the later part of the year
1899 temporarily stopped further work.

After the occupation of Pretoria by the British troops in 1900 the
laboratory premises were made use of by the military authorities as a
stable for the horses of the Transvaal Constabulary, and it was not until
the following year when rinderpest had again broken out in Basutoland
and the Orange River Colony that thoughts were again turned to a
veterinary laboratory.

Though the outbreak of rinderpest had not occurred in the Trans-
vaal, it was feared that due to the movement of troops in the field, there
was very little doubt that it would soon spread into this Colony, and on
the recommendation of Dr. George Turner, at that time Medical Officer
of Health for the Transvaal, it was decided to start a rinderpest station
at Daspoort, so that the laboratory could be utilised in connection with
the preparation of serum. Dr. Turner took charge of this station, at
which considerable additions had to be made. A yard 200 ft. square
enclosed by a galvanised iron fence was made, in which the cattle could
be tethered to poles; and on two sides open sheds were erected, an

4

incinerator for the disposal of the carcases was built, and outside the yard
a brick building to serve as further laboratory accommodation.

From this time on as necessity arose small additions were made,
first a building for the preparation of vaccine lymph, then some quarters
for the staff engaged in the rinderpest work, later some stables and more
quarters; but always the additions were of a temporary nature and
chiefly constructed from such old wood and iron as was obtainable from
buildings pulled down during the war, so by the end of the year 1905 the
station had already grown to some size, though it was merely a hetero-
geneous collection of old wood and iron buildings; most of them of a
very unsuitable nature for scientific work.

But an important change had in the meantime taken place in the
administrative control of the laboratory. Until the conclusion of the
war in the year 1902, both the laboratory and the rinderpest station were
a subordinate branch of the Public Health Department, but with the
establishment of a Department of Agriculture under the direction of
Mr. F. B. Smith, they were both transferred to his Department, and the
laboratory which had hitherto received but half-hearted support as a merely
temporary institution, now became organised as the Veterinary Bacterio-
logical Division of the Department of Agriculture, and from this time
onward a steady policy of progress has been pursued.

The preparation of rinderpest serum was continued until in 1903
the country was considered free from the disease; it was then thought
advisable to close down the serum depot, as the keeping of infected animals
at a place where the facilities for segregation were by no means perfect
created a quite unnecessary risk of starting a new outbreak in the
immediate neighbourhood of the laboratory.

Meanwhile at the close of the war, importations of stock on a large
scale had begun, not only from neighbouring Colonies and States in
South Africa, but also from oversea; and due to these unrestricted
importations, both during the war and after, many diseases before not
known in the Transvaal, were introduced, and amongst them a new
disease of cattle, which at one time seemed as if it would rival rinderpest
in destruction and sweep off the last head of cattle that rinderpest and
the war had left. The history of the introduction of East Coast fever,
and the methods taken to combat it are now a matter of history, but the
Transvaal farmer has need to congratulate the Department of Agricul-
ture on its foresight in establishing a laboratory which was ready at once
to take up research into the nature of the disease and so arrive at the
best means of attacking it and preventing its spread.

From the first inception of the laboratory horse-sickness, which is
so peculiarly a disease of South Africa, and which annually takes so heavy
a toll of the horses and mules of this country, was naturally studied, but
at first the facilities were scanty and money lacking. As, however, with a

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more progressive policy the laboratory increased in size and funds were
more liberally supplied, more attention was paid to this disease, and it
was possible to enter upon a systematic course of experiments, which
eventually culminated in the introduction in the autumn of 1905 of the
preventive serum inoculation of mules which is now practised with such
successful results.

It was a matter of good fortune no doubt that the Division in its
eatly days should have had the opportunity of bringing such practical
evidence of the value of scientific work before the people of the Colony,
so that the laboratory has gradually become to be looked upon not only
as a necessity for the prevention of the introduction of diseases from other
countries, but also as the farmer’s best friend and adviser.

As more and more work was thrust upon the Division the time came
when it was impossible any longer to attempt to meet the demands in
the temporary and inconvenient quarters of Daspoort. Moreover, the
situation, low lying at the farther end of the town and in close proximity
of the Kaffir and coolie locations, was extremely unhealthy ; used as a
depositing site before, and as a burying ground for horses during, the war,
it seemed as if the ground itself were infected. From 1902 to 1906 every
year saw the occurrence of typhoid fever amongst one or more members
of the staff, and that of a very virulent nature, which ended fatally in
several cases; it became, therefore, a matter of necessity that a move
should be made to a more healthy situation.

The enteric outbreak of the year 1906 made it urgent that the future
policy of the Government with regard to the laboratory should be decided
upon at once; whether appropriate buildings should be erected and a
Veterinary Research Laboratory should be established as a permanent
institution in South Africa, or a move should be made to other temporary
quarters on a new site. Happily the former policy was decided upon
without hesitation, and South Africa is now able to take a place amongst
the older countries of Europe as a leader in veterinary scientific research.

PUBLICATIONS OF WORK DONE IN THE LABORATORIES.

By Dr. Apnotp Tuuiter, C.M.G.
1. Das Wiedererscheinen der Rinderpest und die Erfolge der Schutzimpfung in Sued
Afrika. (Published in Monatshefte fuer Praktische Tierheilkunde, Band. 13.)
2. The Danger of the Simultaneous Immunisation with Serum and Virulent Blood
for Rinderpest in Cattle not Immune against Redwater. (Annual Report,
1903-04.
3. ee ace (Results from Former Experiments and Serum Treatment applied
to Horse-sickness). (Annual Report, 1903-04.)
. Notes on Haemolysis. (Annual Report, 1903-04.)
. Horse-sickness Experiments. (Annual Report, 1904-05.)
Further Experiments with Immunisation of Mules against Horse-sickness. (Annual
Report, 1905-06.)
7. Transmission of Horse-sickness into Dogs. (Annual Report, 1905-06.)

oe

6

8. The Immunity in Horse-sickness. (Annual Report, 1905-06.)
9. Horse-sickness. The results of Inoculation in Practice during 1905-06. (Annual

10.

11.

12.
13.

Report, same year.)

Horse-sickness. The Results of Inoculation in Practice during 1906-07. (Annual
Report, same year.)

Horse-sickness. The Results of Inoculation in Practice during 1907-08. (Annual
Report, same year.)

Further Notes on Immunity in Horse-sickness. (Annual Report. 1906-07.)

The Immunisation of Mules with Inadequate and Adequate Serum and Virus,
and the Immunity obtained therefrom. (Annual Report, 1906-07.)

. The Inoculation of Mules with Polyvalent Virus and Serum. (Annual Report,

1906-07.)

. The Inoculation of Mules with Polyvalent Virus. (Annual Report, 1907-08.)
. On the Variability of a Certain Strain of Horse-sickness Virus. (Annual Report,

1907-08.)

. Fever Reactions Simulating Horse-sickness. (Annual Report, 1907-08.)
. The Rhodesian Tick Fever. (Transvaal Agricultural Journal, 1903.)
. The Rhodesian Tick Fever. (Report of the South African Association for the

Advancement of Science, 1904.)

. East Coast Fever. (Journal of the Royal Army Medical Corps, 1904, and in the

Annual Report, 1903-04, also partially in Fortschritte der Veterinaer-Hygiene,
1903.)

. Further Transmission Experiments with East Coast Fever. (Annual Report,

1906-07.)

. The Influence of Cold on Ticks and Piroplasma Parvum. (Annual Report,

1907-08.)

3. Equine Malaria. Journal of Comparative Pathology and Therapeutics, 1902.)
. Equine Malaria and its Sequelae. (As above.)
. Notes on Piroplasmosis of the Horse, Mule, and Donkey. (Annual Report,

1903-04, and Zeitschrift fuer Thiermedicin, 1904.)

. Further Notes on Piroplasmosis of the Horse, Mule. and Donkey. (Annual

Report, 1904-05, and Journal of Comparative Pathology and Therapeutics,
1905 )

. Piroplasma Equi as a Complication of Horse-sickness. (Annual Report, 1904-05.)
. Inoculation against Equine Piroplasmosis. (Annual Report, 1905-06.)
. Transmission of Equine Piroplasmosis by Ticks in South Africa. (Annual Report,

1905-06, and Journal of Comparative Pathology and Therapeutics, 1906.)

. Piroplasmosis in Horses due to Hyperimmunisation. (Annual Report, 1905-06.)
. Continuation of Experiments for Inoculation against Equine Piroplasmosis.

(Annual Report, 1906-07.)

2. Further Inoculation Experiments against Biliary Fever of Equines. (Annual

Report, 1907-08.)

. The Piroplasma Bigeminum of the Immune Ox. (Annual Report, 1903-04, and

Journal of the Royal Army Medical Corps, 1904.)

. Piroplasma Mutans (Nova Species) of South African Cattle. (Annual Report,

1905-06, and Journal of Comparative Pathology and Therapeutics, 1906.)

. Further Notes on Piroplasma Mutans. (Annual Report, 1906-07, and Journal

of Comparative Pathology and Therapeutics, 1907.)

. The Immunity of Cattle Inoculated against Piroplasma Mutans. (Annual Report,

1907-08.)

. Experiments with English and South African Redwater. (Annual Report,

1906-07.)

- Immunity in Tropical and Sub-tropical Diseases. (Commemoration Publi-

cation, 1909.)

The Office of the Government Veterinary Bacteriologist.

The Technical Records Office.

By

By

re
(

39. Trypanosomiasis in Camels. (Annual Report, 1904-05.)

40. A New Trypanosoma. (Journal of Comparative Pathology and Therapeutics,
1903.)

41. Spirillosis of Cattle. (Journal of Comparative Pathology and Therapeutics
1904.)

42. Transmission and Inoculability of Spirillosis Thieleri (Laveran.) (Proceedings
of the Royal Society, 1905.)

43. Transmission and Inoculability of Spirillosis in Cattle. (Annual Report, 1904-05.)

44. Notes on the Immunity of the Piroplasmosis of the Dog. (Annual Report, 1903-04,
and Centralblatt fuer Bakteriologie, 1904.)

45. Heartwater in Cattle. (Annual Report, 1903-04.)

46. Immunisation against Heartwater. (Annual Report, 1904-05.)

47. Blue-Tongue in Sheep. (Annual Report, 1904-05.)

48. The Inoculation of Sheep against Blue-Tongue, and the Results in Practice.
Annual Report, 1906-07.)

2

Dr. A. THEmLeR and Srewarr Srockxmay, M.R.C.V.S. (late Principal Veterinary
Surgeon, Transvaal, 1902 to 1903).

1. Experiments to Show how long an Area which was at one time Infected (with East
Coast Fever) will remain Infected. (Annual Report, 1903-04, and Journal of
Comparative Pathology and Therapeutics, 1904.)

2. Inoculation Experiments according to the Methods of Professor Koch. (As above.)

3. Dipping Experiments. (As above.)

4. Possible Influence of the Different Seasons on the Outbreak of East Coast Fever.
(As above.)

5. Further Experiments to note how long an Area will remain Infected with East
Coast Fever. (Annual Report, 1904-05, and Journal of Comparative Pathology
and Therapeutics, 1905.)

6. Do Salted Cattle contain Piroplasma Parvum in their Blood? (As above.)

7. Experiments with Serum against East Coast Fever. (Annual Report, 1905-06,
and Journal of Tropical and Veterinary Science, 1906.)

Sypney Dopp, M.R.C.V.8. (late Assistant Government Veterinary Bacteriologist,
1906-07).
1. A Disease of a Pig due to a Spirochaete. (Journal of Comparative Pathology and
Therapeutics, 1906.)
2. A Preliminary Note on the Identity of the Spirochaete found in the Horse, Ox,
and Sheep. (Journal of Comparative Pathology and Therapeutics, December,
1906.)

James Watker, M.R.C.V.S., Assistant Government Veterinary Bacteriologist
(appointed July, 1908).
1. The Diagnosis of Bacillary Piroplasmosis of Bovines in the Transvaal. (Com-
memoration Publication, 1909.)

Dr. Watrer Fret, Assistant Government Veterinary Bacteriologist (appointed
November, 1906).

1. Viscosity of Blood. (Transvaal Medical Journal, April, 1908.)

2. Surface Tension of Serum. (Transvaal Medical Journal, August, 1908.)

3. Physical Chemistry in Veterinary Science. (South African Association for the
Advancement of Science, Grahamstown, 1908.)

4. Remarks on some Experiments with Snake Poison. (Royal Society of South
Africa Proceedings, 1909.)

8

5. Vergleigende Physikalische Chemische Blut und Serum_ Untersuchungen, mit
besonderer Berucksichtigung der Pferdesterbe. (Zeitsch. fur Infektions-
krankheiten, 1909.) ; ie

6. Physikalische Chemische Untersuchungen uber Piroplasmosis. (Zeitsch. fur Infek-
tionskrankheiten, 1909.)

7. Physical Chemical Investigations into South African Diseases. (Annual Report
Government Veterinary Bacteriologist, 1907-08.)

8. Haemolvsis in Practical Veterinary Science. (Commemoration Publication, 1909.)

By Dr. Lewis Henry Govueu, Zoologist (appointed July, 1908). ;
1, On a Coenurus in the Duiker. (Proceedings Royal Society of South Africa.)
2. The Anatomy of Stilisia Centripunctata. (Commemoration Publication, 1909.)

By Dr. Kart FRrepricnh Meyrr, Pathologist (appointed October, 1908). _ :
Notes on the Pathological Anatomy of Pleuro-pneumonia. (Commemoration Publica-
tion, 1909.)

Description of the Laboratory Buildings.

The laboratory building, which is in the Colonial Dutch style of
architecture, is of brick, faced with cement. It is 303 ft. in length « 51
ft. wide and from the centre a wing runs back another 80 ft., making the
whole a T-shaped building.

A second storey extends for 77 ft. over the central part, and this
is surmounted by a clock turret, from where a magnificient view of the
surrounding country 1s obtained.

The building les east and west with the main entrance on the south
side. A corridor runs from end to end, and beneath the floor of the
corridor, with branches into all the rooms, is a conduit covered with
removable granolithic slabs; in this conduit run the pipes for hot and
cold water, steam, gas, and waste.

The rooms on the ground floor are 13 ft. high, and the walls are dis-
tempered above and painted with a chocolate coloured dado below.
There are steel ceilings throughout the building. The floors, except in
the offices, are of granolithic and are finished off at the sides of the rooms
rising in a curve to meet the skirting so as to avoid corners that might
collect dust, and the laboratories have been designed throughout with a
view to absolute cleanliness and freedom from dust. The windows in
all the laboratories are 2 ft. 6 in. from the ground and on a level with the
slate slab tables used for microscope work, and the lower part of the
windows are fitted with panes of plate glass 36 in. x 2 ft. so that there
shall be no obstruction to the hght on the tables. The whole building
has been constructed to be perfectly insect proof, wire screens being fitted
to all the windows and doors.

The several laboratories are fitted on a uniform plan as far as such
will conform to the particular requirements of each, and hot and cold

The General Office.

g

water, gas, steam, and electric light are laid on throughout the building.
Down the centre of each laboratory runs a teak table 3 ft. 3 in. high,
having a row of drawers on each side and semi-circular sinks fitted to the
ends: gas nozzles are provided on each side at suitable distances down
the whole length of the tables: they are let into hollows scooped out of
the tables so as to be flush with the table tops. Hot and cold water taps
are provided at all the sinks.

In front of the windows are tables for microscope work made of slate
slabs 2 ft. wide, fitted on iron brackets fixed in the wall; between the
windows and at each end of these tables are small sinks.

Fume closets are provided in all the laboratories 6 ft. long x 2 ft.
wide, tapering to the top, which is connected by a flue with the chimney ;
glass doors shding upwards open the closets to a height of 2 ft. 4 in., and
in each hot and cold water, gas and electric power are laid on. A sink is
also provided, and the floor of the closets are covered with sheet lead.
These fume closets are of great value ina hot climate asa place for keeping
the gas stoves as well as for carrying on the smaller sterilizing operations.
Brackets 2 ft. 6 in. long x 11 in. wide, each fitted below with two plate
glass shelves 6 in. wide are fixed to the walls over all the microscope tables :
the brackets are used for carrying the larger bottles of disinfectants,
and the shelves for reagents.

Smallincubators, heated by gas, are in those laboratories where they are
required. Vacuum pumps as well as small centrifugal machines driven by
electric motors are also provided where necessary, and there are large
cupboards with sliding glass doors in all laboratories.

The sterilizing rooms, which are three in number, in addition to tables
similar to those provided in the laboratories, have, in each, large slate
tables fitted against the wall, which is lined above them to a height of 18 in.
with glazed white tiles. These tables are for sterilizing operations and are
in two sections—one 2 ft. 8 in. high for smaller apparatus. and one 2 ft.
high for carrying the large autoclaves, which are heated by steam from
the central supplv. Beneath the former a smaller slab is fixed to be
used as a shelf, and over the whole is a hood of sheet iron connected at
the top with the chimney to carry away the hot air. There are also two
large sinks with draining racks, bottle racks, and other necessary
accessories ; hot and cold water, gas, and electric light are provided as
in the laboratories as well as glass-blowing apparatus and vacuum pumps
in each.

A general idea of the arrangement of the laboratory can best be
obtained from the plan, from which it will be seen that on entering
at the door on the south side and passing through a small vesti-
bule, the main corridor runs right and left. This corridor taking a turn
at each end leads to two doors at the back of the building, that on the

10

east side opening out into the quadrangle yard, and that on the west side
into the yard in which the power house is situated. Facing the front
door is the staircase which leads to the upper floor, and going direct from
the door and passing the staircase a passage leads through a glass panelled
door to the back wing of the building. Into these two corridors all the
rooms on the ground floor open, excepting the serum store and the packing
room, which open on the back. The four front rooms in the centre of
the building are the offices, the two to the right are, first the office in which
all the technical records are kept, and next to it, with a connecting door,
that of the Government Veterinary Bacteriologist ; those to the left are
first the office of the Superintendent, under whose control is the organisa-
tion and all the business management of the station, and next, opening
out of it, the general clerical office.

Adjoining the office of the Government Veterinary Bacteriologist
and connected by a door is his private laboratory, which opens out at the
farther end also into a sterilizing room. This laboratory is at present
chiefly used in connection with the study of Protozoa and their relation
to diseases of live stock.

The last room on this side of the building is the Pathological
Laboratory, which also opens by another door into the same sterilizing
room. This laboratory is 25 ft. long x 16 ft. wide, and lighted by two
large windows facing the south, and one facing east, and is very com-
pletely fitted up for histological and bacteriological research. Here also
the examination of blood and other smears ix carried on, as well as of
numbers of pathological specimens that daily come in from the country.
The extent of this branch of the work will be realised when it is stated
that during the past year 1,362 specimens were examined, and the number
is on a steady increase. The principal object of this laboratory is the
study of histological pathology of tropical diseases of the country, and
more especially of horse-sickness, Hast Coast fever, and the various
piroplasms.

Next to the Pathological Laboratory, with two windows looking on
to the east, is the Zoological Laboratory. Though not directly connected
it is conveniently situated for using the same sterilizing room mentioned
before. This room is 18 ft. x 18 ft. 6 in., and is now chiefly devoted to
the study of intestinal parasites, and more especially to those found in
sheep, a matter of most vital importance to the sheep farmer of this
Colony. Here also the breeding of mosquitos is carried on and experiments
on their relation to the transmission of disease.

Passing from the corridor through the door at the back of the building,
one comes out on to a wide verandah which faces the north and runs half
the length of the building. This was designed to protect from the sun’s
rays the windows of the rooms facing the north, and so to preserve a
more equable temperature in the laboratory. On the left of the door is

The Centrifugal Room.

The Still Room.

la

a small lavatory, and on the right, fitted with shelves for the cages, is the
room. in which small animals under experiment are kept; this room has
a table in the middle and is fitted with slate slabs and a sink by the window.

Returning through the door into the laboratory building, the first
room on the north side is a room used exclusively for bacteriological work,
and mainly for the preparation of mallein, tuberculin, pleuro-pneumonia
cultures, and quarter-evil vaccine, and connected with it by a double
door is an incubation room 8 ft. square and & ft. high insulated with
asbestos and heated by a gas stove which is automatically regulated by
the Roux system. Next to this room is a small room fitted as a pharmacy,
which is also used as a chemical and drug store; against the walls on
each side are large cupboards with sliding glass doors, and a table with
a sink is in the centre.

Adjoining is another small room used for making distilled water,
for which purpose a large Murrle distilling apparatus, fed by steam from
the main supply, has been provided, and is so arranged that it can also
be used with gas; connected with it is a drving oven, the temperature
of which can be raised if necessary by super-heating the steam by means
of a gas jet. A Buchner press, mounted on a concrete stand is placed in
this room, and there is a small fume closet containing the serum
coagulating apparatus.

Adjoining is the cold storage room, previded with double doors,
the walls and ceiling of which are completely insulated with asbestos and
silicate cotton. In the centre is a large ice chest in which compartments
are provided for keeving pathological specimens. The walls are lined
with shelves filled chiefly with bottles of horse-sickness virus.

Returning again to the other side of the building next te the general
office is a room at present used as a workroom for the lay assistants ;
adjoining this is the room set aside for the large centrifugal machines ;
these, two in number, are by Lautenschlager, of Berlin: the larger, driven
by a 5 horse-power electric motor and making 3,000 revolutions a minute,
is provided with four receptacles each of a capacity of one litre; the
smaller, driven by a 24 horse-power motor, makes 4,000 revolutions per
minute, and is capable of centrifugalising one litre of liquid. A smaller
machine, driven by 4 horse-power motor, is fixed against the wall, and the
room is fitted wp in other respects as a laboratory. Opposite the last two
rooms are two storerooms for laboratory apparatus and glassware, and on
the same side is a strongroom for the records.

The corner room on the front of the building is the room used for
photography, in size similar to the Pathological Laboratory, situated on
the opposite corner of the building, with two large windows facing the
south and a window on the west. A working bench extends the length
of the room under the windows, with asink at each end, and the walls are
fitted with glass panelled cupboards. In this room there is a Zeiss

12

micro-photographic ‘apparatus and appliances for ultra microscope work.
The illumination for these is provided by a thirty ampere arc lamp, or
by means of a heliostat placed on a brick pedestal outside the window.
Heavy curtains are hung at the windows so that the room can be com-
pletely darkened when required. A well fitted up dark room adjoms and
opens out of this room.

The three next rooms, all facing west, are used for the preparation
of rahies vaccine, according to the Pasteur method. The centre room
is a small laboratory. An inner room opens out from it which is fitted
with double doors and windows, and here the temperature is regulated
by a similar stove to the one in the incubation room. This room is
exclusively used for drying the Rabies cords. On the other side of the
laboratory is a room for keeping the small animals and fitted similarly
to the small animal room on the eastern side of the building.

In the back wing the rooms devoted to the preparation of horse-
sickness serum and blue-tongue vaccine are situated, and the last two
rooms to the north are the operating theatre and the post-mortem room.
Passing from the main corridor through the glass panelled door mentioned
before, on right and left are doors opening out on the verandah facing
the quadrangle yard, and on to a verandah running north and south on the
west side of the back wing; in front is a corridor with a door at the end
opening into the operating theatre. There are two rooms on the right—
first, a small laboratory used for testing the serum for its haemolitic effect
and sterility; second, the serum preparation room, a large room with two
windows facing east and a hatch opening into the operating theatre, so
that the bottles filled after tapping can be expeditiously passed through
from the latter. In this room the preparation of horse-sickness serum
for the immunisation of mules is carried on. The laboratory annually
sends out more than 1,000 litres of this serum to all parts of South Africa.
Blue-tongue vaccine is also prepared here, and of this in the past year
nearly 200,000 doses were issued.

Facing this room on the left of the corridor is a sterilizing room which
is also provided with a hatch opening into the operating theatre, to
facilitate the easy passing through of sterilized bottles; and there is a
second hatch opening into the post-mortem room, which adjoins the
operating theatre. Next to the sterilizing room, and connected with it
by a door, is a small room fitted with shelves for sterilized bottles, and
opposite the Serum Laboratory is the tick and insect room, where ticks
used in connection with the experiments in the transmission of the various
protozoa and diseases of the country are bred and kept during their
moulting stages.

To the left of the door opening on to the west verandah is a door leading
into the serum storeroom, where the serum, blue-tongue vaccine, and
other preparations, after having been bottled, sealed, and labelled are

The Serum Store.

The Small Animals Room.

13

kept ready to be sent out as required ; adjoining and connected by a door
is the packing room, which also opens by a door into the yard. In this
room the bottles of serum and other preparations are packed in boxes
for despatch by train into the country districts.

The operating theatre and the post-mortem room are situated at the
end of the back wing and are connected by sliding doors; the former is
38 ft. x 28 ft., and the latter 29 ft. x 21 ft. The flooring of both rooms
1S oo and they are well lighted by windows placed high up on the
walls.

The post-mortem room has in addition two large windows on the west,
beneath which are slate slabs similar to those provided inthe laboratories for
microscope tables. Tram lines are laid on the floor which, passing through
the double doors on the north side, circle all round the back of the stables
and branch off to the destructor. Carcases are thus easily taken from the
stables on a truck and brought into the post-mortem room, and, after
autopsy is completed, can be quickly removed to the destructor. The
truck is so arranged that the four sides fall down to form a table on which
the post-mortem examination is made, and they can afterwards be replaced
in position for the removal of the carcase. There is a desk for entering
in the register at the time the record of each post-mortem, and all con-
veniences for the curing and preserving of pathological specimens are
provided.

The operating theatre, in addition to windows on the north and east
side, has a large skylight in the roof with windows on all four sides ; there
is therefore an excellent light in all parts of the hall. Tethering rails are
fixed around the walls, and there are two padded boxes for retaining
horses under operation, and, in addition to every necessary convenience,
a small centrifugal machine with haematocrytes 1s fixed on the wall, which
facilitates the measuring of the blood volume of the living animal on the
spot. Double doors open out on the north side towards the stables, and
there are two doors on the east side for bringing horses into the boxes,
and a small door at the south-east corner leading into the quadrangle
yard.

On the first floor there are six rooms; the centre room on the south
side is a lecture room and library, arranged with benches in tiers facing
a table fitted with a sink and provided with water and gas so that laboratory
demonstrations can be made; behind are the blackboards. In this room
there is a large projection lantern illuminated by a seven ampere arc lamp,
which is fitted with a projection microscope, and is arranged for direct
projection of diapositives by transmitted hght or for projection of solid
objects by reflected light.

On the north side there are only two rooms, each 15 ft. x 32 ft. One
is fitted up as a students’ laboratory with all the necessary facilities for
bacteriological work and having four working tables arranged down the

I+

centre fitted with sinks, hot and cold water, and gas; they are also pro-
vided with small cupboards and drawers below, and are modelled after
the pattern of the students’ tables at the bacteriological laboratory at
Berne. The other room is devoted to physical chemical research, and
has also complete laboratory equipment and it is proposed will be used
later for teaching physiology to students.

Opposite the students’ laboratory is a sterilizig room, fitted up
similarly to those in the other parts of the building.

The remaining room on this floor is the pathological anatomical
museum. Here already there is a fine collection of specimens, and the
large glazed cases on the walls are filled with a verv representative collec-
tion, amongst which are some interesting specimens of tropical diseases.
Tn the centre of the room is a chest fitted with drawers for specimens, and
under the window is a table so that work connected with the museum
can be conveniently carried on.

All the principal rooms are connected with a telephone installation
provided for communication within the building, a matter of great con-
venience in a large laboratory where a numerous staff is employed.

To the west of the laboratory, completely detached, is a separate
building designed for the preparation of calf vaccine lymph. This
building complete in itself has four rooms; a large room well lighted by
two windows facing the south and fitted with a table is for the vaccinating
operations ; a door leads from here into a stable with three loose boxes
for the calves, to which also there is an outer door for attendance. In
front, opening on to a verandah, as well as connected by a door with the
operating room, is the laboratory for preparing the vaccine and filling
the tubes ; there is also a small room at the back, opening into the stable,
used as a storeroom for forage. The preparation of vaccine lymph is an
important branch of the laboratory work, over three-quarters of a
million tubes having been sent out into all parts of South Africa during
the last two years, and the work is now carried out under the most modern
hygienic conditions.

The situations of the other buildings are seen on the block plan.

The stables are all built of brick and are lofty and well ventilated ;
the mangers are of iron and the stall partitions are made with iron pillars
and iron frames covered with boarding; the floors are paved with blue
bricks set in cement, and are drained by open channels, which pass through
a pipe into an open gulley that runs the whole way along the outside of
the stables and discharges through traps at intervals into the main
drainage system. The windows are placed high up and are covered, as
also are all ventilators, with wire gauze to keep out insects, a matter of
some importance for the prevention of natural infection of horse-sickness,

Hii’
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The Post-Mortem Hall.

fe

15

The stables used for the animals under experiment are situated at
the back and.form, with the east and north wings of the laboratory, a
quadrangle. In these stables, accommodation is provided for 100 horses
and mules, 50 cattle, and 100 sheep and goats. There are twelve loose
boxes as well as six isolation boxes, which latter are arranged for slinging
horses if required. Doors open into the quadrangle yard, and there are
also doors at the back, and passages for feeding with doors at each end
run between each stable. Jn this way in the hottest of weather there is
abundance of ventilation, and all cleaning and feeding operations are
carried out through the doors at the back, the decors opening into the
quadrangle yard being only used for bringing out animals in connection
with the laboratory work. A tram line runs the whole length of the
stables at the back to facilitate feeding and cleaning, and dead animals
are removed to the post-mortem room by this means.

Behind the stables are the forage stores and feed mixing room, with
chafi-cutter and corn-grinding machine, the buggy house, harness and
tool sheds, a carpenter’s shop, a saddler’s shop, and the wagon and imple-
ment shed. There is also a farrier’s shop and a large platform scale.

The dog kennels are in a building which contains 20 pens, with a
passage dewn the middle of the building; each opens into a yard paved
with granolithic and enclosed by iron railings on dwarf walls.

The piggeries are built on the same plan, but the yards are enclosed
bv walls 3 ft. high, and are fitted with cast iron feeding troughs and gates.

There is also a segregation stable capable of bolding 30 horses, to
receive horses and mules when first admitted to the station, in which they
can be malleined, and a room is fitted up at one end of the stable for
facilitating this.

An isolation stable more especially for suspected cases of glanders
is apart from the other buildings, and has twelve loose boxes with a
feeding passage running down the centre, but the latter has no communica-
tion with boxes except by a sliding trap door through which the food can
be passed ; a tap supplies water to a trough im each box operated by a
cock in the passage.

There is also a house for breeding smal! animals, rabbits, and guinea
pigs; this is fitted with removable partitions so that a number of small
boxes or larger runs can be arranged as desired, and the whole can be
taken down and thoroughly cleansed and disinfected when necessary.
The design for this house was copied from the plans of a house for the
same purpose in the Berne Laboratory.

There are eight wood and iron stables, 50 it. long x 15 ft. wide,
opening into yards 50 ft. x 50 ft., fenced with corrugated iron 8 ft. high.
These are used for horses kept for serum and for animals not under
experiment and reserve animals. Water is laid on to troughs in the yards.

16

The cremator is situated at a little distance from the other buildings
and was supplied by Messrs. Manlove, Alliott & Co., of Nottingham.
It consists of two separate destructors placed end to end and working
through one chimney, and is capable of cremating five or six horses a day.

The electric installation, the work on which was carried out under
the direction of Mr. F. Stephens, the Electrical Engineer to the Public
Works Department, consists of an engine and dynamo supplying current
at 250 volts pressure to about 200 lamps throughout the laboratory
building and to a number of points whence current can be conveniently
drawn for working small motors for various purposes. Electricity 1s
also supplied for driving the centrifugal machines by means of two motors, one
of 5 horse-power, and another of 2} horse-power, the motors being on raised
platforms to economise space. The system of distribution for power purposes
is kept entirely separate from the lighting circuits. Current up to 40
amperes 1s also available for running the large arc lamp used for photo-
micrography and ultra microscope work, and for the projection lantern
in the lecture room.

A pump driven by a small electric motor is provided for filling a tank
in the roof. This pump is automatically stopped when the tank is full
by a float operating an electrical device, so that the pump requires no
attention when once started.

The electric light plant is located in a detached building on the
north-west side of the laboratory. It consists of a steam-driven high
speed, direct-coupled Crompton-Belliss set, running at 400 revolutions
per minute, and capable of developing 80 amperes at 250 volts pressure.
The current from the dynamo is taken direct to a main switchboard located
in the engine house, whence the cables radiate to the main building and
to the pump house. Several spare circuits are provided on the board for
future extensions. The boiler is of the Robey semi-portable type. It
is fixed under the same roof as the engine, but in a separate room, and
is capable of supplying all the steam required in connection with the supply
of electricity, and for sterilising purposes, as well as for making distilled
water.

The auxiliary plant im connection with the boiler consists of a feed-
water heater worked by exhaust steam from the engine and a hot water
cyclinder for the supply of hot water for all purposes throughout the
laboratories. This water is also heated by means of exhaust steam, use
being thus made of heat which would otherwise be wasted. These heaters
are situated in a chamber below the level of the boiler house floor.

Installed in a recess in the boiler house is the retort for making gas
from petroleum, a Mansfield generator being used. As the gas is pro-
duced it flows from the generator to the gasometer about thirty yards
away; this gasometer has a capacity of 1,000 cubic ft. and holds sufficient
for about two days’ supply. Gas is of course largely used in the

The Pathological-Anatomical Museum.

jasieiesn

The Lecture Hall.

17

laboratories, and fittings for incandescent and ordinary gas lamps are
provided through the building, so that it can be used also as an auxiliary
to the electric hght in the event of light being required at any time when
the electric plant may not be running.

An ample supply of water for all purposes in the stables and
laboratories is drawn from a borehole situated about 200 yards to the
east of the main building. A Rees Roturbo high efficiency pump is
installed in the pump house. This is driven by a 5 horse-power vertical motor
direct coupled to the pump spindle, and is capable of delivering about
3,000 gallons per hour when running at a speed of 2,500 revolutions per
minute. The water pumped from the borehole is delivered to a tank
situated near the gasometer, this tank being raised to a height of 40 ft.
above ground level in order to produce sufficient pressure for the water
to reach the taps in the upstairs rooms of the laboratory. The tank has
a capacity of 30,000 gallons which suffices for a two days’ supply. A
system of mains distributes the water to the various laboratories, stables,
and the dwelling-houses where the staff reside.

The whole of the above-mentioned plant is under the charge of an
electrician resident on the spot, an assistant being employed to run the
engine for lighting purposes at night.

In every way most complete both in buildings and all accessories and
fitted up with the best and most modern equipment, the new Laboratories
have a wide field of work before them in research into the many and
hitherto little studied tropical and sub-tropical diseases of live stock of
South Africa.

The Old Laboratories at Daspoort.

‘piv ajsuvispongd) aul

“PAD ajsupspongd) ay} Uloly &40}DL0QDT ay} JO yoDYG ayy

A Cattle Byre.

Some Loose Boxes.

One of the Stables.

The Piggeries.

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In the Quadrangle Yard. Some Sussex Cattle imported for Experiment,

‘2]9QDIS Bulaiaday ay]

“S]DD4Y 2]330D pup assozy ay

Immunity in Tropical and Sub-tropical

Diseases.

By Dr. ARNOLD THEILER, C.M.G.

UDA ETD)

The Laboratory of the Government Veterinary Bacteriologist.

Immunity in Tropical and Sub-tropical Diseases.

THE term “tropical” or “sub-tropical” disease is applied in this paper
to such maladies of stock as are principally encountered in Africa, and
more particularly in South Africa.

Those of any economic importance are, so far without exception:
either due to protozoa (Piroplasmoses, Trypanosomiases, Swpirochaetoses
theileria) or to a specific group of ultravisible organisms (Chlamydozoa)
which, in the same way as the protozoa, are dependent for their propa-
gation on a host in the form of a tick or an insect. (Heartwater,
horse-sickness, blue-tongue.)

In South Africa other infectious diseases also exist, such as anthrax,
quarter-evil, glanders, strangles, etc., caused by specific bacteria, and not.
sO many years ago epizooties such as pleuro-pneumonia, foot and mouth
disease, and rinderpest have played considerable havoc amongst stock.
These latter two are also due to ultravisible organisms, but are spread by
direct contact with the sick animals and their morbid products.
These and the mentioned diseases caused by bacteria do not require
hosts as propagators, and in this fact lies the important difference between
those designated as “ tropical” or “ sub-tropical.’ They are also encoun-
tered in colder regions and have been known for a considerable length of
time, whereas the tropical ones are more or less new to science.

Ever since I have undertaken the studv of the diseases of South Africa,
I have laid particular stress on the question of immunity. A possible
application of any immunity for the purpose of protecting the greatest
number of animals exposed to infection must bear immediate results, and
for this reason, wherever possible, it forms the best weapon under present
conditions for successfully combatting these maladies.

It is my object in this paper to compile the facts known concerning
the immunity in tropical diseases and to compare them with those known
in other diseases, selecting for examples such as are known or have been
observed in this sub-continent.

Immunity is spoken of as general or specific. In the first instance
is usually meant the absolute insusceptibility of a certain animal or a
class of animals to a disease. When we apply this to some of the tropical
diseases, it would, for instance, express that cattle and sheep are immune
against horse-sickness ; cattle, sheep, and dogs are immune against the
piroplasmosis of horses ; sheep and dogs immune against the piroplasmosis
of the ox; cattle immune against blue-tongue of sheep; horses immune
against heartwater, etc.

22

The reverse of immunity is,susceptibility, and in speaking generally
about certain diseases having no influence on particular animals, we most
frequently use the term non-susceptibility ; thus indicating that the word
immunity has a more specific sense. It is more especially applied in such
instances where animals or certain classes of animals which are usually
susceptible to a certain disease have, by the process of recovering from
that disease, acquired such new qualities as to render them insusceptible
against the further attacks of the same disease.

For the purpose of this article, the term will be used in its restricted
sense ; for instance, a horse which has recovered from horse-sickness or
from piroplasmosis will be called immune against these diseases. For
the present the question will be left open as to the nature of such immunity ;
this will follow as a corollary from the deliberations.

CoMPLETE IMMUNITY.

An immunity is complete when the cause of the disease has no influence
on the recovered animal under any conditions whatsoever ; that is to say
whether the test is applied naturally in the form of an exposure, or
artificially by introducing the specific germs, and irrespective of the quan-
tity, manner of injection, and lapse of time succeeding the recovery. We
know of one disease where this has been observed to be the case, viz., in
rinderpest. In South Africa two epidemics of this zoonose have been
observed—the first during the years 1896-97, and the other during
1901-03. Animals which recovered in the first outbreak were refractory
to the second one, and it is admitted that in rinderpest immunity lasts
for life.

After the fact was established that the serum of immune animals
had protective properties, use was made of this fact and immune oxen were
hyperimmunised to a great extent bv injecting several litres of virulent
blood. In no instance under my observation did such an injection lead
to a breakdown of immunity. We may thus safely accept that immunity
in rinderpest acquired by recovery cannot be broken, neither naturally
nor artificially.

TEMPORARY IMMUNITY.

The immunity of pleuro-pneumonia in cattle has been made use of
in South Africa probably. ever since that disease has been introduced.
It has been noted that an ox which has recovered from the disease is
immune. The expression pleuro-pneumonia indicates that the pathological
lesions are found on the lungs and the pleura. Although the primary
seat is in the lungs, yet the disease is by no means specific for this organ.
It is rather specific for the lymphatic system, starting in the interstitial
tissue of the lung and involving the lung parenchym proper secondarily.
Therefore typical lesions can after artificial introduction of virus develop

23

in any region of the body; they correspond with those of the interstitium
of the lungs. Recovery from these lesions are succeeded by immunity,
that is to say, a subsequent inoculation within a reasonable short time
no longer produces the typical lesions, neither does an animal so treated
contract lung-sickness within that time. When at a later period, a year
or more, the same animal is again injected, a reaction can take place,
or when exposed to natural infection, it may contract pleuro-pneumonia.

We thus stand here before the fact that an acquired immunity
diminishes in the course of time. This, of course, varies with the animal,
and in some occurs sooner than infothers. Immunity acquired by recovery
from the disease naturally acquired lasts, according to Nocard and
Leclainche,* three to four years.

IMMUNITY TO DIFFERENT QUANTITIES OF VIRUS.

Quarter-evil in cattle is due to the introduction of a specific bacterium
into the subcutaneous tissue of young cattle. Recovery from the lesions
thus produced is succeeded by immunity. This immunity can be brought
about in various ways; one of them is the introduction of the virus in
small quantities, and here the observation is made that the immunity
stands in direct relation to the quantity of virus injected. Thus, if the
dose of virus in increased, the immunity previously obtained does not
protect, and breakdowns occur (Kitt). This fact is generally observed in
diseases caused by bacteria; an immunity obtained through the introduc-
tion of the minimum lethal doses will protect against this dose, or perhaps
a small multiple of it, but not so against a larger one. ,

Immunity To DIFFERENT VIRULENCY.

Another contingency which occurs in experiments with bacteria is
the different virulency of one and the same bacterium. Artificial
immunity against anthrax, for instance, is obtained by a double
injection of a first and second vaccine, differing in virulency. This
differentiation in virulency can be so exalted that the immunity thus
obtained can be broken by such a virus. This exaltation can be obtained
by passing the virus through a series of susceptible animals of the same
or of different species. It is noted and generally accepted that the more
virulent the virus, the more solid becomes the resulting immunity.

Toe Immune ANIMAL AS A RESERVOIR OF VIRUS.

An important fact to be considered in connection with the immunity
obtained, either naturally or by inoculation in the above-mentioned
diseases, with the exception of pleuro-pneumonia, is the observation that
these animals no longer propagate the disease. The principle holds also

*Nocard and Leclainche, ‘‘ Les Maladies Microbiennes les Animaux,? 1903.

24

for pleuro-pneumonia, inasmuch as a completely recovered animal is
harmless; only such animals in which the sequesters have been found
in the lungs carry the contagion with them as long as this sequester has
access to a bronchus.

IMMUNE SERUM.

The recovery from an infection is accompanied with the acquisition
of certain qualities of the blood, more particularly of its serum; the most
prominent one of which is the preventive effect such a serum exerts on the
virus to which it corresponds, when injected previously, simultaneously,
or subsequently to the virus or when mixed with the virus, either totally
or partially prohibiting the effect of such virus. In addition to this, in
certain cases direct influences are noted in vitro on the virus known as
precipitation, agglutination and cytolvsis (Bacteriolysis). These qualities
become specially pronounced in hyperimmune animals, which process is
carried out by subcutaneous or intrajugular injections of large amounts
of virus. In rinderpest the injection of an appropriate dose of serum
some hours previous to virus prevents the latter from developing; when
injected simultaneously, a reaction ensues which, in the majority of cases,
ends with recovery. The same can be noticed when the serum is injected
not too long after the virus; the action of the virus is completely
destroyed when it is mixed in vitro and then injected. In pleuro-
pneumonia, according to Nocard, the inoculation of an immune serum
(40 c.c.) protects against a subsequent inoculation of virus (1 c.c.). In a
mixture of serum and virus in equal quantities injected subcutaneously,
the virus does not develop. In quarter-evil, according to Kitt, an
immune serum (40 ¢.c.) injected some days previously to virus protects
against this virus. In a mixture of serum and virus injected sub-
cutaneously, the virus becomes inert. The serum has agglutinating
properties. Jn anthrax an immune serum protects against a subsequent
virus injection. A simultaneous inoculation of serum and virus produces
active immunity (Sobernheim).

The résumé concerning immunity in non-tropical diseases may be
classified as follows :—

(1) Immunity may be complete, both regarding quantity of virus

and length of time it lasts.

Immunity lasts only for a limited time.

Immunity varies with the quantity of injected virus.

Immunity varies with the virulency of the virus used.

Immunity renders an animal unfit for the propagation of the

contagion.

(6) Immunity and the process of hyperimmunisation gives the
serum preventive qualities.

2
3

(
(
(4
(5

25
I— DISEASES DUE TO ULTRAVISIBLE VIRA.

A.—Horsk-sicKness, BLuE-ToNGUE, HEARTWATER.

Horse-sickness is due to a filtrable ultravisible organism, which in
vitro preserves its virulency for several years. Blood of a sick animal
acts as a virus. A horse or a mule which has recovered from an attack of
this disease is known to have acquired immunity (salted) ; it is generally
understood that immune animals do suffer from relapses (aanmanings),
from which as a rule they recover.

The following notes will demonstrate the nature of the immunity
acquired, and the cause of the relapses. It will be advisable to record
the observation in chronological order, as this will best explain the develop-
ment of our knowledge concerning the immunity in this disease.

Immunity 1n Muues.*—The method of immunising mules consists in
the simultaneous injection of virus (2 ¢.c.) and serum (average dose, 300
c.c.). The serum is obtained from horses or mules which have recovered
and which have been hyperimmunised. This process consists in the
intrajugular transfusion of ‘blood from the sick into the immune animal,
averaging in amounts to 10 litres, transfused in two to four operations in
intervals of 6-18-24 hours. About three to four weeks after infusion,
the serum of the infused animal is fit for use. The virus with which the
experiments were started was collected from a horse in Pretoria, which
had contracted the disease spontaneously. As nearly all our initial experi-
ments were carried out with this virus, and later it became necessary
to distinguish it from vira collected from other animals, it was called the
“Ordinary Virus.” Once the fact had become established that through
the simultaneous injection of virus and a corresponding dose of serum
immunity could be obtained, it had to be decided whether this immunity
was complete, both concerning quantity of virus and time it lasts.
The former could naturally easily be settled, whereas the latter has not
yet: found a definite solution.

The best proof for immunity was the process of hyperimmunisation.
The statistics at our disposal show that up to January, 1907, the total
number of 295 mules were tested with the Ordinary Virus. Amongst
this number were 41 mules which were hyperimmunised twice, 13 mules
three times, 4 mules four times, and 2 mules five times. It must be
remembered here that the infusion for one hyperimmunisation averages
8 to 10 litres. The observation made was, that in no instance did the
infused animal show any reaction typical for horse-sickness; indeed
usually not even a rise of temperature due to the huge infusion of sick
febrile blood was noticed. The animals-used for hyperimmunisation had

* Annual Report, Gov. Vet. Bac., ‘Transvaal, 1903-04.
f Annual Report, Gov, Vet. Bac., Transvaal, 1907-08,

26

passed through immunisation within eight months previous to the infusion;
those hvperimmunised a second or third time were done so after an interval
of three months to a year.* The longest period between immunisation
and hyperimmunisation was two years without breakdowns. The longest
period between immunisation and test with the same virus was six years.

ConcLuston.—The immunity obtained in mules by the simultaneous
injection of serum and Ordinary Virus is complete concerning
quantity of Ordinary Virus with which it is tested ; concerning time,
at 1s so at least for six years.

Immunrry 1n Horses.—No method of immunising horses has as yet been
introduced into practice. The horses referred to here have recovered
in experiments carried out for the purpose of finding such a method.
With a few exceptions the immunity obtained was due to the injection
of serum and virus in various combinations, but principally by simultaneous
injection. As this article deals with the immunity, the details of
immunisation does not enter within its scope. , All the recovered horses
were used for hyperimmunisation in a similar way as indicated in connec-
tion with the mules. Thus were hyperimmunised or otherwise tested with
Ordinary Virus 104 horses, amongst which were 10 hyperimmunised twice.
The amount infused for hyperimmunisation averages 8 to 10 litres. Five
horses were hyperimmunised three times. The intervals between hyper-
immunisation was usually short, averaging two to three months. The longest
interval between two hyperimmunisations was two years. The observation
was, that out of this number no horses showed a typical reaction due
to this test of hyperimmunisation.

Norre.—There was one horse which reacted with symptoms of dikkop.
At that time this occurrence was not understood. Soon after the test
injection, the horse was grazed at Onderstepoort during the horse-
sickness season, and a new infection must have been contracted
naturally, a fact which will be explained later.

ConcLusion.—The immunity of horses obtained through the recovery from
an injection of Ordinary Virus is complete concerning quantity of
Ordinary Virus. Concerning time it is for at least two years.

The virus used for hyperimmunisation and test did not correspond
in generation to that”used for immunisation. The vira used principally
for immunisation were the fifth and the thirty-seventh generations,
counting every subsequent animal through which the ordinary strain had
passed as a generation. These animals were indiscriminately taken
amongst horses and mules, and did not follow each other with any
regularity. Only after the sixty-fifth generation the virus was continued
through horses exclusively. The greatest interval between the generation

* Annual Report, G.V.B., Transvaal, 1906-07—Imniunisation of Mules with Ordinary Virus,

Ww

7

used for immunisation and hyperimmunisation in mules was forty-three
generations ; in horses, seventy generations.

It is usually observed in other diseases that the passage of virus
from animal to animal of the same species increases the virulency, and
it may be expected that such virulency can break the immunity obtained
by a lower generation. However, the virus of the Ordinary strain of a
high generation, and even the hyperimmunisation with such had no
effect on the animal immunised with a lower generation.

ConcLusion.—The immunity of mules and horses obtained by the
recovery from the injection of Ordinary Virus protected completely
against the infusion of large quantities of virus of a higher generation.

(The reservation should be made here, that in the passage of virus
through the different generations, mules and horses were used, and it is
possible that this may account for the stability of the virus.)

EXPERIENCES IN PRactTicE.—A.—TzaNgeEN ViRUS.—Once it had been
settled that the immunity in mules could not be broken by huge quantities
of virus, it was expected that such an immunity would stand natural
exposure, and accordingly the method was introduced into practice.

During the horse-sickness season, 1905-06,* a total number of 3,235
mules had been inoculated, and were exposed to natural infection. At
the end of the season, of this number 21 were reported to have died of
horse-sickness, and 45 had shown symptoms of relapses (aanmanings).
There was but little doubt that death was due to horse-sickness. Post-
mortem reports in several instances were obtained from the District
Veterinary Surgeon. At the same time some horses which had recovered
from experiments, and had also been hyperimmunised, were exposed to
be tested. One of these died in Tzaneen, the Government estate in the
Letaba Low Veld, a notorious country for horse-sickness. Blood from
the dead animal was obtained, and named “ Tzaneen Virus.’ It was then
used in the following experiments. A number of mules, totalling 139, all
immune against Ordinary Virus, were tested with Tzaneen Virus, with the
result that 12 showed typical horse-sickness fever reactions, 4 with lesions
of dikkop, and 1 died of horse-sickness. A number of horses, totalling 17,
all immune against Ordinary Virus were tested with Tzaneen Virus, with
the result that 5 showed reactions and recovered, 3 had reactions with
symptoms of dikkop, 1 died from the pulmonary and | from the dikkop
form of horse-sickness. There were only 7 animals out of the 17 which
did not react. Some of the tests were made within a few months after
recovery from Ordinary Virus, and on animals (horses) which had been
previously hyperimmunised once, twice, aid three times with Ordinary
Virus.

* Annyal Report, G.V.B., Transvaal, 1906-07,

28

Conctuston.—The immunity obtained by the Ordinary Virus did not
protect all the mules “and horses against the test with Tzaneen Verus,
even when this test” was carried out soon after immunisation, and
even in horses which had been hyperimmunised.

B.—Bvrawayo Virus AND ORDINARY IMMUNITY.

This virus was obtained from a mule which had been immunised in
Bulawayo during the season 1905-06 with Ordinary Virus. When
exposed, it showed a relapse, from which it recovered.

A number of mules, totalling 36, all immunised with Ordinary Virus,
were tested with this Bulawayo Virus, with the result that 7 mules
showed fever reactions typical for horse-sickness, 6 animals showed
the symptoms of dikkop and recovered, 1 mule had a doubtful reaction,
and 6 died of horge-sickness.' Of 9 horses immune to Ordinary Virus,
which were tested with the Bulawayo Virus, 3 showed reactions with
dikkop, and 4 died of..horse-sickness. There were 2 animals out of
the 9 which did not react. Some of these tests were made within a few
weeks after immunisation.

Conctusion.—The immunity obtained by the Ordinary Virus did not
protect all the mules and horses against the test with Bulawayo Virus,
even when this test was carried out soon after immunisation.

C.—ImMMUNITY FROM TZANEEN VIRUS AND TESTS WITH ORDINARY VIRUS.

Shortly after it had been noticed that Tzaneen Virus could break
the immunity of the Ordinary Virus, it was concluded that the former
would be stronger or, better, more virulent, and therefore probably
better suitable for the practice. It was at the same time ascertained
that the serum of animals hvperimmunised with the Ordinary Virus
could advantageously be used with the Tzaneen Virus, the mortality
from inoculation averaging about the same percentage.

In the first half of the season 1906-07, the Tzaneen Virus was intro-
duced, but in the latter half withdrawn, as it was found to have changed
spontaneously in virulency. Subsequent to this, for the purpose of

ascertaining the immunity obtained from the Tzaneen Virus, tests were
made.

(1) Tests with Ordinary Virus.—A number of mules, immune against
Tzaneen Virus, totalling 59, were tested with Ordinary Virus a com-
paratively short time after immunisation. The result was that 47 animals
showed fever reactions more or less typical for horse-sickness, 2 animals
showed the symptoms of dikkop and recovered, and 5 animals died of
horse-sickness. There were tested 19 horses immune to Tzaneen Virus,
with Ordinary Virus, of which 7 showed fever reactions typical for horse-
sickness, 5 had reactions with symptoms of dikkop, 2 died of horse-
sickness, and 5 had no reactions,

29

Concrusion.—The immunity obtained by the Tzaneen Virus did not
protect all the mules and horses against the Ordinary Virus.

(2) Tests with Bulawayo Virus.—There were tested 13 mules immune
to Tzaneen Virus, with Bulawayo Virus, also a comparatively short time
after immunisation, with the result that 8 showed horse-sickness reactions
and 1 died of horse-sickness. Of 4 horses immune to Tzaneen Virus and
tested with Bulawayo Virus, 2 showed horse-sickness reactions, and 1 a
reaction with the symptoms of dikkop.

ConcLusion.—The immunity obtained by the Tzaneen Virus did not
protect all the mules and horses against the Bulawayo Virus.

D.—ImMMuNITY FROM BuLAWAYO VIRUS AND TESTS WITH ORDINARY VIRUS
AND TZANEEN VIRUS.

(a) Three mules immune against Bulawayo Virus and tested with
Ordinary Virus all showed reactions; one immune horse reacted to the
virus.

(6) Of 5 mules immune against Bulawayo Virus and tested with
Tzaneen Virus, 3 gave reactions. Of 2 horses, 1 died from the test.

Conchusion.—The immunity obtained from the Bulawayo Virus did
not protect all the mules and horses against a subsequent test of
either Ordinary or Tzaneen Virus.

E.—Tests oF MULES IMMUNE TO TZANEEN VIRUS WITH TZANEEN VIRUS
or A HIGHER G=NERATION.

The experience of a rather large number of animals had shown that
the immunity obtained by the Ordinary Virus could not be broken by
the same virus, by whatever quantity of virus and whatever generation,
and after even a lapse of two years. This proved not to be the case with
the Tzaneen Virus; it is particularly typically pronounced in horses.
Of 81 horses which were immunised with the Tzaneen Virus of a lower
generation, and within a comparatively short time tested with the same
strain of a higher generation, 16 showed reaction typical for horse-
sickness, 6 showed reactions with dikkop, 9 showed reactions and died
of pulmonary form, and 3 showed reactions and. died of dikkop. A
similar observation was made with horses injected twice with the Tzaneen
strain of two different generations, first of a lower one and later with a
higher one. When tested with the virus still of a higher generation,
breakdowns of immunity were noted. Of 40 horses thus treated, 4 showed
simple horse-sickness reactions, 3 showed reactions of dikkop, 9 died of
pulmonary form, and 3 died of dikkop. The same observation was
repeated to a smaller extent even in horses which had received three
subsequent injections of Tzaneen Virus of different succeeding generations,
as the following statistic shows: Of 15 horses thus treated, 3 showed
simple reactions, and 2 died of pulmonary horse-sickness.

30

Conctusion.—The immunity obtained from Tzaneen Virus of a lower
generation did not completely protect against the test of a higher
generation; even the immunity obtained from a Virus two or three
generations below that of the test was not complete.

IMMUNE ANIMALS DO Not Act AS RESERVOIRS.

It has experimentally been proved that the blood of an animal
suffering from horse-sickness is virulent during the febrile reaction and
only for a limited time afterwards. This applies of course only to
tests made by inoculation with blood from recovered animals, and
these have in all instances proved to be negative if carried out almost
at any time after recovery up to a number of years, but the reservation
must be made here that under natural conditions, that is to say, through
the host, a transmission might occur from immune to susceptible
animals, a contingency which so far has not any support by analogy
with another disease. We are therefore entitled to conclude for the
present that the immune animal does not act as a reservoir.

SERUM OF RECOVERED ANIMALS.—In speaking of serum and virus in
horse-sickness, one has in the first instance to consider whether they are
adequate to each other, in other words, whether the serum used corre-
sponds to the same strain of virus with which the animal is injected.

The serum of recovered animals has but little preventive value, but
acquires this when injected or infused in short or longer intervals as
already described.

The observations refer to a virus which in the dose of 1 c.c.
subcutaneously injected is invariably fatal, and mav be summarised as
as follows :—*

(1) Adequate serum and virus mixture of equal quantities and
injected in multiples of the minimum dose, subcutaneously or intra-
jugularly, is, as a rule, harmless, but no immunity results. (2) Inadequate
serum and virus mixtures of equal quantities, mixed and injected
im multiples of the minimum dose are usually harmless. (3) The
inoculation of adequate serum previous to the subcutaneous injection of
virus, generally speaking, prevents the development of the disease ; the
same is sometimes observed when virus is injected intrajugularly ; no
immunity results. It is also sometimes noticed with inadequate serum
and virus. (4) The simultaneous inoculation of adequate serum and virus
does not prevent development of the disease in mules, and when the
doses of serum are properly adjusted, 98 per cent. of animals recover.
Under similar conditions recoveries in horses are less (about 60 per
cent.) ; when the dose of serum is increased all development may be
stopped. This refers also to some inadequate sera. The recovered
animals are immune. (5) Adequate virus injected previously to serum

* Annual Report, G.V.B., Transvaal, 1904-05,

31

is generally not influenced by the latter; the longer the interval between
the two, the surer a reaction will result. This applies also to in-
adequate virus.

RrsumMé.—Our present knowledge concerning immunity in horse-
sickness can be résuméd as follows :—

(1) Recovery from an attack of horse-sickness causes immunity ;
this immunity is complete to a virus adequate to the one which
caused the disease (Ordinary strain).

(2) Immunity may vary according to the virulency of a strain
which can be influenced by passage from animal to animal.
(This applies to the Tzaneen strain.)

(3) Immunity obtained by one strain of virus does not protect all
the animals against a different strain of virus.

(4) Serum of immune animals, hyperimmunised by either strain,
has protective qualities against virus of the same and of
different strains.

(5) Recovered animals do not retain the infection in their blood.

Biue-TonGcuE IN SHEEP.

This disease is due to a filtrable ultravisible micro-organism, which
in vitro preserves its virulency for several] years. Blood of a sick animal
acts as virus. A sheep which has recovered from an attack of blue-
tongue is considered immune: there are no definite data at our disposal
to show how long the natural acquired immunity lasts, but it is generally
admitted that “salted ’’ sheep contract the disease in a lesser degree.

IMMUNISATION.—There exists two methods of immunisation, viz., the
first introduced by Spruell,* consisting of an injection of adequate serum and
virus in appropriate quantities either simultaneously or mixed at the time
of injection ; the second one is the vaccination introduced by the writer of
an attenuated virus obtained by passing it through a number of
generations.

Sheep which recover as a result of either inoculation are immune.

The following data refer principally to my own observations, where
not otherwise noted :—Mortality from vaccination is almost nil. The
sheep show visible symptoms of blue-tongue in a slight degree; the
intensity varies in various flocks and under adverse conditions (rain, for
instance).

CHARACTER OF ImmuNITY.—The following note explains the nature
of the immunity :—

A sheep which recovered from a 20 ¢.c. injection of virus reacted to
a subsequent injection of 30 c.c. virulent blood injected 33 days later

aoe Cape Colony Agricultural Journal,”

32

of the same strain, and again an injection of 100 c.c. another 33 days
later caused a typical reaction. A lamb which had contracted blue-tongue
and recovered from an injection of virulent blood showed typical lesions
of blue-tongue when injected 71 days later with 60 c.c. virulent blood of
the same strain. A number of sheep injected with increasing doses of
virulent blood for the purpose of hyperimmunisation generally show
fever reactions after the first and second injection. In later injections
these reactions are no longer noticed. The fact is noteworthy that when
the hyperimmunisation is carried out soon after immunisation no deaths
result from this increasing amount of blood. The reaction becomes the
more pronounced the later the injection of virus is carried out after
immunisation.

Of 40 sheep tested with 20 c.c. virus four and seven months after
vaccination, severe fever reactions were noticed m 15 sheep and slights
reactions in 14 sheep; in some of these typical symptoms of the disease
were noticed, and 1 actually died as a result of it.

ConcLusion.—Immunity against blue-tongue conferred by virulent or
attenuated virus is not complete. It may be broken with a larger
quantity of the same strain of virus, and this breakdown 1s more
certain to occur the longer the period which elapses between wm-
munisation and test.

The breakdowns of immunity in the majority of cases do not end with
death.

EXPERIENCE IN Practice.—The vaccination of sheep was introduced
into practice in 1907, and during the time when blue-tongue was rampant.

The mortality within the first fourteen days after inoculation may
principally be accounted for by natural infection. In the 5,875 sheep,
of which records were kept, the disease stopped after this period; the
mortality amounted to 7 animals, equal to 0°4 per cent. Of 16,218
susceptible non-treated sheep running under the same conditions as above,
1,817 died of blue-tongue, or 11:per cent.

ConcLusion.—The vaccination of sheep during an epidemic of blue-
tongue stops the disease after fourteen days, and the sheep remain
immune for the rest of the season.

The post-mortem examinations of seven sheep mentioned in the
foregoing notes out of the vaccinated number could not be controlled ;
thus some doubt as to correct diagnosis does exist. However, in the
same season amongst the sheep belonging to the laboratory exposed on
the notorious farm Onderstepoort, some breakdowns were noted, of which
the particulars are as follows :—

One sheep contracted spontaneous blue-tongue ten months, a second
one eight months, a third one fourteen months, and a fourth one fifteen
months after immunisation.

30

In the year 1907-08 about 200,000 sheep were vaccinated, the season
was a good one; the only breakdown which occurred was in the Orange
River Colony affecting a ewe. .

In the year 1908-09 about 85,000 were inoculated, and about 80
breakdowns were noted in three different farms of three different districts
(Lydenburg, Ermelo, Piet Retief).

ConcLusion.—A certain number of breakdowns under natural conditions
are observed, even soon after vaccination ; these breakdowns vary in
thew extent according to certain localities.

THe Bioop or Immune Animats.—A number of sheep immunised six
to eighteen months ago were tapped, and the blood was injected in the
doses of 5 c.c. into susceptible sheep. In no instance were the lesions
of blue-tongue noted.

_ When immediately afterwards tested on their immunity, with the
minimum doses of virus, these injected sheep proved to be susceptible to
blue-tongue.

ConcLuston.—Immune animals do not retain the infection in the blood.
‘The same reservation as in horse-sickness has to be made here.

Serum oF Immune AnIMALS.—Serum of hyperimmunised sheep was
tested on its preventive value, with the following results :—

(1) Simultaneous subcutaneous injections of serum and virus,
with the excess of the former, prevented all development of
disease ; intrajugularly injection was followed by reaction and
death.

(2) Serum injected twenty-four hours previous to virus prevented
the development of the disease.

Conciusion.—The serum has preventive qualities.

RESUME.

(1) Immunity in blue-tongue of sheep is never complete; it can be
broken either by virus of the same strain if applied in larger doses or by
a virus of a different strain.

(2) The serum has preventive qualities, and immune animals do not act
as reservoir for virus.

HEARTWATER.

Heartwater is a specific disease of cattle, sheep, and goats, due to a
filtrable micro-organism present in the blood, which preserves its virulency
for not longer than forty-eight hours. Fresh blood of sick animals must,
therefore, be used as virus. Any animal belonging to the genera men-
tioned above acquires immunity through the recovery from the disease.

34

There does not yet exist any practical method of immunisation, and
the observations made on the immunity refer to animals recovered from a
disease transmitted to them by injection of virulent blood.

CHARACTER oF ImmMuUNITY.—Oxen, sheep, and goats which have
recovered from an attack of heartwater can be injected with large
quantities of virulent blood adequate to the virus which produced immunity.

Five cattle were hyperimmunised to the extent of 1,000 to 3,000 c.c.
virulent blood in one operation, and eight sheep from 50 c.c. to 400 c.c.
in one injection were hyperimmunised in this way.

This hyperimmunisation was carried out in various intervals. No
breakdowns occurred. The longest interval between two hyperimmuni-
sations was in cattle, seventeen months, and in sheep, eighteen months.

Conciusion.-—Immunity against heartwater when tests were carried out
with the adequate virus was complete concerning the quantity of virus,
and, concerning time, at least for ecghteen months.

EXPERIENCE WITH INADEQUATE Virus.—KEight sheep, all immune and
hyperimmunised to a particular strain of virus called the Sjamboks Kraal
Virus, which were injected with virus of a different strain obtained from
Komatipoort, showed the typical heartwater reaction, from which | died.
Spreull* injected 3 sheep and 2 goats previously immunised by virulent
blood injection and then exposed to natural infection with 5 c.c. blood
of a sick animal into the jugular vein; 1 sheep sickened and recovered,
1 goat sickened and recovered, and 1 died.

To judge from Spreull’s statement, the virus must have been inade-
quate to the one with which immunity was produced. The experiment
of the same man proved that immunity obtained by the inoculation of a
virulent blood did not protect against natural infection (tick infection).
These facts are probably also due to a virus of a different strain.

ConcLusion.—The immunity obtained from ene particular strain of
virus can be broken by an inadequate strain. (Strain of a
different locality.)

EXPERIENCE IN Pracrice.—This is only limited and refers specially
to a lot of cattle which were the recoveries from the experiments and
had been hyperimmunised to various extents; they were exposed as
controls in connection with an experiment of a different nature two
years after immunisation.

There were altogether exposed eight animals immune against heart-
water. In no case could with certainty a breakdown due to the tick
infection be noticed, although the control animals contracted this disease.
Dixon,t in his experiments with goats which had been immunised by

** Cape Agricultural Journal,” Vol. XXIV, No. 4, 1904.
+ Cape Agricultural Journal,” Vol. XV, No, 12, 1899,

35

subcutaneous inoculation of virulent blood, found that they were not
protected when exposed to natural infection. His results after five months’
exposure were: Death from heartwater in 307per cent. of Boer goats ;
50 per cent. of Cape sheep; 40 per cent of Angora goats; 40 per cent.
of merino sheep, and lambs 25 per cent.

Conciusion.—Immunity acquired by inoculation and increased by
hyperimmunisation protected erght cattle against natural infection of
heartwater. The immunity conferred on sheep and goats by inocula-
tion of virulent blood broke to a great percentage.

Bioop or Immune AnimaLs.—The blood of an ox which had recovered
from an attack of heartwater was injected into two susceptible sheep
without any result.* The same results were obtained by Lounsbury}, who
injected blood of recovered goats into susceptible goats. Further, his
experiments with ticks reared on goats which recovered from heartwater
demonstrated that these were unable to transmit the disease.

Conc.Lusion.—Animals which have recovered from an attack of heart-
water do not retain the infection in the blood.

PREVENTIVE VALUE or SERUM.—The serum of an immune animal
which has been hyperimmunised has acquired preventive qualities which
may be summarised as follows :—(1) Mixture of serum and virus, with
the excess of the former, injected subcutaneously, did not cause the
disease. (2) Serum injected subcutaneously in excess and virus into
the jugular vein or subcutaneously prevented a reaction. (3) Serum
injected twenty-four hours previously to the virus prevented a reaction.
(4) Serum injected twenty-four hours after virus did not prevent
development of reaction.

ConcLusion.—The serum of hyperimmunised animals have preventive
qualities.

RESUME.

(1) Immunity in heartwater is not complete; it can be broken by
virus of a different strain.

(2) Immune cattle seem to have a better protection than immune goats
of sheep against virus of different localities.

(3) Serum of hyperimmunised animals has protective properties.

(4) Immune animals do not act as reservoirs.

II.—PIROPLASMOSES.
Redwater of cattle (bovine piroplasmosis). Biliary fever of equines
(equine piroplasmosis). Biliary fever of dogs (canine piroplasmosis).
Infection due to Piroplasma mutans.

*“ Cape Agricultural Journal,” Vol. XXJ, No. 4.
+ Annual Report, Government Entomologist, 1901.

36

REDWATER oR TEXAS FEVER.

This disease is due to the introduction of Piroplasma bigeminum into
a susceptible beast naturally by the blue ticks, artificially by inoculation
of blood containing this parasite. The blood kept in vitro retains its
infectivity only for a limited number of days (fourteen days). Animals
which have recovered from an attack of this disease are known to be
immune. An immune Africander ox injected with various amounts of blood
from cattle actually suffermg from redwater, in intervals and varying in
quantity to the total extent of 8 litres, showed no breakdowns of immunity.

In connection with the immunisation of oxen against rinderpest,
country-bred oxen were injected with large doses of blood, which
undoubtedly contained Piroplasma bigeminum—no accurate records were
kept about the reaction, but judging from results, no breakdowns occurred.

EneuisH Cattite.—Of 10 English heifers, which were all immunised
against redwater and had shown Piroplasma bigenanum in the blood, 6
showed reactions and rare Piroplasma bigeminum when injected with blood
containing this parasite. Of 6 English heifers recovered from an attack of
redwater brought on bv injection of blood, when exposed to natural
infection, 2 died. Smith and Kilborne* also, in their experiments came
to the conclusion that one attack of Texas fever does not completely
protect against a succeeding exposure to new infection.

ConcLusion.—The immunity conferred by an injection of virulent blood
can be broken by inoculation, which is, as a rule, not accompanied by
severe symptoms or death. I mmunity conferred by unjection of virulent
blood does not protect completely against natural infection. The
ummunity thus conveyed varies im the various breeds of animals ;
u is better and almost complete in South African born and bred cattle;
at is not so good in imported cattle.

THE Bioop oF ImmMuNE AnimMALS.—Animals recovered from redwater
retain the infection in the blood and such blood remains infective when
injected into susceptible animals. The progeny of the blue ticks feeding
on such immune animals likewise transmit the disease. The blood of an
immune animal exposed to tick infection remains so during the whole
life of such an animal and probably so in an animal not exposed to tick
infection. There is an observation recorded from America,t where the
blood of such an immune animal proved to be infective after twelve
years. It has been observed that the injection of immune blood causes
in the majority of animals only a slight reaction during which Piroplasma
bigeminum is found in the blood, and this fact has been made use of as
a preventive inoculation. The immunity obtained in this way protects
the greatest number of animals against heavy infection and deaths from
redwater when exposed to redwater veld. (Vide foregoing conclusion.)

*U.S.A. Bureau Animal Industry, 8th and 9th Annual Reports.
7 Schroeder and Cotton, 22nd Annual Report, Bureau of Animal Industry, 1905.

Or
of

OBSERVATION IN Practice.—The history of the importation of cattle
from various Texas fever countries into the Transvaal after the conclusion of
the late war gives us a demonstration of the immunity under the condition of
natural exposure. Importations from Madagascar numbered 10,000 oxen.
These oxen are immune to redwater, as experience and experimental proof
have shown. The general experience was that these animais did not contract
redwater, and ever since cattie were imported from that country this fact
is generally admitted. Contrary to this, an observation of mine shows
that freshly imported Madagascar cattle can suffer from redwater. Eight
Madagascar oxen brought from Natal, where they had been running for
a short time, were brought to the laboratory and directly after arrival
two succumbed to acute redwater.

Texas cattle were also imported to the number of 10,000. Speaking
generally, these cattle proved to be immune to the South African redwater,
but also here were exceptions to the general ruie. Some cattle which
had been running in Natal were brought to the Transvaal to be
used in connection with the Hast Coast fever experiment, and four
died within eight days after arrival from acute redwater. They un-
doubtedly must have contracted the disease previous to departure.
From Queensland 500 head were imported. In this case the experience
amounts to experimental value. The 500 head were inoculated with the
blood of an immune South African ox, with the result that some of the
cattle, few in number, contracted acute redwater and died.

Speaking from general observations, all cattle born and bred on the
veld of the Transvaal must have gone through an attack of redwater and,
therefore, must be immune. The immunity is a generally accepted fact.
Yet during this summer outbreaks of redwater in all parts of the country
were very numerous, and in many cases in cattle which were born and
bred on the farm, or had been there at least for some years. In some
instances the redwater took the form of a real epizootic.

It must be stated here that the climatical condition of the current
year was exceptionally favourable for the breeding of ticks.

ConcLusion.—bservations im practice prove that ummunity against
redwater naturally acquired gives a great protection against
subsequent exposure, but i ws by no means complete. The
breakdowns may be either due to infection with a dijferent strain
or to the over infection by means of ticks.

REDWATER NOTICED AS COMPLICATION OF OTHER D1sEASES.—When
East Coast fever was first observed, it was noticed that in sick animals in
conjunction with small piroplasms, the typical Piroplasma bigeminum was
found. When later the small piroplasms were recognised as a species of
their own-—Piroplasma parvum—the fact of the simultaneous presence of both
piroplasms in redwater immune animals found its interpretation in accept-
ing that, under the influence of the acute East Coast fever, the Puroplasma

38

bigeminum undergoes a further multiplication process and leads to the
appearance of redwater complicating the former disease. Statistics for the
year 1903-04 show that of blood smears sent to the laboratory for diagnostic
purposes :—
311 were pure East Coast fever infections ; and 23 complicated with
redwater.
Statistics for the year 1904-05 show :—
334 pure East Coast fever infections; and 16 complicated with
redwater.
Statistics for the year 1905-06 show :—
152 pure East Coast fever infections; and 5 complicated with
redwater.
Statistics for the year 1906-07 show :—
133 pure East Coast fever infections; and 5 complicated with
redwater.
Statistics for the year 1907-08 show :—
239 pure East Coast fever infections; and 6 complicated with
redwater.

ConcLusion.—Immunity against redwater, can be broken when the
immune animal is suffering from some other febrile disease. (It is
possible that the cases of breakdowns noted by me in Texan

and Madagascar cattle, shortly after a railway journey, were due
to this.)

EouIneE PIROPLASMOSIS.

This disease is due to the introduction of Piroplasma equi into sus-
ceptible equines, either naturally by the bite of the red tick (Phipicephalus
everlsi) or artificially by inoculation of blood containing this parasite.
The blood retains its infectivity for a limited time, about three weeks in
our experiments. Animals which recover from the natural or artificially
contracted disease may show more than one reaction.* ‘The recovery
means immunity. Horses born and bred in the Transvaal veld become
immune by the natural tick infection. Foals suffer but little from the
disease.

Bioop oF Immune ANIMALS.—Experiments undertaken to this effect
have demonstrated that the blood of immune horses is infective for
horses, mules, and donkeys, and vice versa. The blood of a zebra
caught in the bushveld proved to be infective for horses.

An immune animal retains the infection probably for a lifetime when
exposed to ticks, and for a considerable time when not exposed.

In our experiments for the purpose of hyperimmunisation in horse-
sickness by transfusion of blood from sick to healthy horses, we noted

*Annual Report, G.V.B., Transvaal, 1903-04.

39

equine piroplasms in 38 cases out of 402 horses treated, and in 3 mules
se of 282, due to the infused blood, which kept the infection in a latent
‘orm.

A donkey foal kept for eighteen months out of the infection proved
to be virulent in the inoculation of 100 mules. It has been observed that,
similar to redwater, the injection of blood of immune foals is not so virulent
and that the majority of injected animals recover. This has been made
use of as a method of immunisation (blood of donkey foals).

In this way were treated with horse foal blood : 34 horses, 27 donkeys,
and 135 mules. Three mules died of piroplasmosis.

With donkey foal blood were injected: 80 horses, 81 mules, 15
donkeys. There were no deaths.

Conctusion.—The blood of an equine which has recovered from an
attack of piroplasmosis remains infective.

Test oF Immuniry By Invection or Bioop.—A horse* which had
recovered from the disease in July, 1904, was tested by the injection of
2,500 c.c. blood of a horse which at that time was suffering from piro-
plasmosis, and containing numerous piroplasms. No reaction was noticed
in this horse.

Of 35 horses immunised against piroplasmosis by injection of immune
blood, 3 horses died of piroplasmosis when hyperimmunised against horse-
sickness.

Conciusion.—Immunity against Prroplasma equi may be broken
through infusion of large quantity of immune blood. (Horse-sickness
virus.) (Probably different strain.’

EQUINE PIROPLASMOSIS AS COMPLICATION OF OTHER DISEASES.--
In our experiments with horse-sickness, we frequently met animals
suffering from biliary fever whilst undergoing a horse-sickness reaction.
All possibilities of an artificial infection simultaneously with the injection
of serum or virus had to be excluded, since virus and serum were of old
standing. Similarly to what has been described in redwater is the case
here, a breakdown of immunity due to a concurrent fever.

During the year 1905-06 these breakdowns were noticed during the
immunisation of 3,195 mules; 26 showed piroplasmosis complicated with
horse-sickness (0-8 per cent.), of which number 11 died (0-3 per cent.).

Of 402 horses immunised against horse-sickness and passing through
a horse-sickness reaction, 12 showed complications with Piroplasma equi.

Conciusion.—Immunity against equine piroplasmosis can be broken
when the immune animal is suffering from some other febrile disease.

* Annual Report, G.V.B., Transvaal, 1904-05, page 104.

40)
PIROPLASMA MUTANS INFECTION.

A disease of cattle, due to the presence of Prroplasma mutans
either naturally contracted by ticks or artificially by the moculation of
blood containing this parasite has, as a rule, a chronic course, micro-
scopicaliy pronounced as an anemia. Recovery is usual, and recovered
animals are known to be immune.

THE Bioop or Immune Antmats.—Blood of an animal which has
shown the presence of Piroplasma mutans at one time, either due to artificial
or natural infection, is infective for susceptible cattle when inoculated.

The experiments never failed when animals were used of a country
known to be free of Piroplasma mutans infection and the blood used for
inoculation was derived from an animal in whose blood Piroplasma mutans
have been traced; whilst animals immune against redwater as a rule do
not show the presence of Prroplasma bigeminum in microscopical examina-
tions; this is the rule with Prroplasma mutans, which for a long time
can be traced microscopically.

IMMUNITY UNDER INFLUENCE OF INTERCURRENT FEvER.—The number
of these parasites undergo variations and, similar to redwater, the
existence of another febrile disease can evoke an increase of parasites.

Two head of cattle used in a heartwater experiment developed a
typical heartwater ieaction, due to the virus injection, and during this
reaction Piroplasma mutans increased considerably. A similar observa-
tion was made in an ox which contracted heartwater naturally.
Usually the presence of Piroplasma mutans is accompanied with that
ot Piroplasma bigeminum, which can easily be understood, since every
animal born on the Transvaal veld is immune against redwater. We
succeeded, however, in finding an animal which was infected with Poyro-
plasma mutans alone, and, in the course of the various experiments, twenty-
six animals were injected with blood of recovered animals and all showed
pure Piroplasma mutans reactions.

ConcLusion.—The blood of an immune animal which recovered from
mutans infection remains infective. Similar to Piroplasma bigeminum
and equi, intercurrent fevers can cause an increase of Piroplasma
mutans, which can be considered as a decrease in the existing
emmunity.

PIROPLASMOSIS OF THE Doc.

Canine piroplasmosis is due to the infection with Piroplasma canis,
introduced into susceptible dogs either naturally by means of ticks
(Haemaphysalis leachi) or artificially with blood which contains the para-
site. The disease has a relapsing character. -

The final recovery renders a dog immune. This immunity in an
experiment of ours by inoculation with virulent blood of one and the
same strain could not be broken.

4]

_ Tue Boop or Immune Dogs is infective and, contrary to the other
pizoplasmosis, such blood is as virulent both for young and old animals
as is that of sick animals. The blood of an immune dog retained its
infectivity in our experiments during one year.

Robertson* quotes an instance of thirteen months and another of
two full years. Robertson was able to infect this latter dog again by
mjecting virulent blood.

Conciusion.—Immunity against canine piroplasmosis is present as
long as the dog retains the infection in the blood ; when this wears off
then it again becomes susceptible. Under natural conditions, this
will hardly be noticed, as the presence of ticks is the permanent cause
of re-infection.

SERUM oF IMMUNE ANIMALS.—The knowledge concerning the serum

of hyperimmunised animals can be summarised as follows} :—

(1) The mixture of serum and virulent blood injected into suscep-
tible dogs was harmless ; no immunity followed.

(2) Injection of serum twenty-four hours before virus injection
prevented development of disease; no immunity followed.

(3) Injection of serum twenty-four hours after virus prevented
development of disease; no immunity followed.

(4) The serum was active in the way indicated both against a virus
of a different animal (Heterologous), as against the virus
obtained from the serum supplying dog (Homologous).

ConcLusion.—The serum of a dog hyperimmunised with virulent blood

has preventive qualities.

Immunity IN INocULABLE PrkovLAsMosis.—Recovery from a piro-
plasmosis causes immunity. This immunity is not complete and can be
broken by virus of a different strain. The immune animals retain the
infection in the blood for a considerable length of time, and during this
time intercurrent maladies can break the immunity. The degree of
immunity varies somewhat in the various breeds of animals (redwater) ; it
seems to last as long as the blood remains infective (canine piroplasmosis).
The serum of hyperimmune dogs has protective properties. (Not tested
in redwater and equine piroplasmosis.)

IIL.--SPIROCHAETOSIS IN CATTLE.

SPIRILLUM THEILERI causes in horses, cattle, sheep, and goats, a febrile
reaction of short duration, sometimes of a relapsing character and the
microscopical lesions of anemia. All animals suffermg from a pure
infection recover. The infection is naturally transmitted by the progeny
of infected blue ticks, or artificially by the inoculation of blood containing

the parasites.

* Jour. Comp. Path. and Therap., Vol. XIX, Part 2.
} Annual Report, G.V.B., Transvaal, 1903-04,

42

The presence of Spirillum theileri had been seen in animais suffering
from other infections as, for instance, Piroplasma mutans, Piroplasma
higeminum, Trypanosome theilert. In the former two cases it is sometimes
difficult to say which would be the primary infection, especially when the
fact is considered that Spirillum theileri remains in the blood of immune
animals. The appearance of the spirillum, together with trypanosomes,
must be considered as a vreakdown of immunity of spirillosis.

Conciuston.—The immunity obtained through the recovery from a
Spirochaetosis infection is not complete ; the blood remains infective
and intercurrent fevers cause an increase of the parasites.

IV.—EAST COAST FEVER (Thevlerva parva).

East Coast fever is a disease in cattle due to the presence of Thealeria
parva, a parasite of the red corpuscles, which can so far only be introduced
into the system in the natural way by means of ticks. Thus the disease
is not inoculable, and all attempts to transmit it with blood by infusion
or injection of juice of internal organs have failed. In one of our experi-
ments, a young ox was infused during fifteen minutes with East Coast
fever blood; there was no reaction due to this. The disease leads to a
mortality of 95 per cent. The recovered animals are known to be immune.
A number of eight oxen which had recovered in August, 1902, at
Komatipoort from East Coast fever, were exposed three and a half years
later at Sjamboks Kraal to natural infection ; none of these animals con-
tracted the disease.

ConcLusion.--The immunity is complete concerning natural mfection

and concerning time.

THE Bioop oF Immune OxEen.—Immune animals do not retain the
infection in the blood. This can be observed in practice where immune
cattle have survived the outbreak of East Coast fever, where subsequently
young, or new, fresh imported stock were running together on tick-infected
farms without ever showing an infection. Oxen which recovered in the
year 1902 from East Coast fever have been running since then with
susceptible cattle in tick-infected areas without transmitting the disease
to the susceptible ones.

Experiments* to prove this were also undertaken by feeding brown
tick nymphae on oxen which had two years previously recovered from
East Coast fever, and placing the moulted adults on susceptible cattle.
In no instance did such ticks transmit the disease, hence it can safely be
concluded that the blood of East Coast fever immune cattle harbours no
longer the parasite of East Coast fever.

ConcLusion.—The blood of an ox recovered from East Coast: fever does
not retain its infectivity.

* Annual Report, G.V,.B., Transvaal, 1903-04, pages 93 and 94,

43

SERUM or Immune AnimaLs.—Professor Koch had stated that the
serum of cattle which were hyperimmunised with the blood of cattle
suffering from East Coast fever acted, when injected into sick oxen,
directly on the parasite, causing them to disappear; unfortunately such
serum had also haemolytic effect, so that it could not be applied with
safety. Having overcome the difficulty of haemolysis in horses by
infusion of the virulent blood into the jugular vein, this method was
also used in connection with East Coast fever and some oxen were
hyperimmunised to the extent of a minimum of 20 and a maximum of 40
litres.

This serum was not haemolytic. It had no lytic action on the parasite,
and when applied on an infected herd it had no preventive action.

ConcLusion.—Serum of East Coast fever immune oxen hyperimmunised
by infusion of sick blood has no preveniive action whatsoever.

RESUME.

The immunity in East Coast fever is complete concerning natural
infection and time.

The immune animals do not retain the infection in the blood.

The serum has no preventive properties.

V.—TRYPANOSOMIASES.

There exist many different species of these parasites in Africa, which
from our point of view have not yet been studied. A certain number of
records are registered in the literature, which will give us an idea as to
the existence of immunity or otherwise. [ shall only refer to these
trypanosomiases.

The specific trypanosomiasis of cattle in South Africa is due to the
presence of Trypanosoma theileri. Generally speaking, this parasite has
but little pathogenic action; we meet it occasionally in bloodsmears
sent to us from cattle reported as suffering from gall-sickness, invariably
from the bushveld. This trypanosomiasis is only inoculable into cattle.
Recovery from such an attack seems to create immunity.

The inoculation of large quantities of blood containing the
trypanosomes into animals which had recovered from trypanosome infec-
tion gave in the majority of cases negative results, and in the positive
results there were only a small number of parasites.

Conciusion.—Immunity caused by the recovery from a Trypanosoma
theileri infection. can be broken by subsequent inoculation.

Of more importance are the trypanosomes of Trypanosoma evansi,
the type to which belong, in addition to the one mentioned, Trypanosoma
bruce, of Zululand, of Togo in West Africa, and the Trypanosoma

+4

sudanense. Morphologically speaking, these various parasites could not
with absolute certainty be separated from each other, although for epidemio-
logical and geographical reasons a difference between Trypanosoma
evansi and brucei suggested itself. French scientists introduced a method
of differentiation, based on the observation, that animals which recovered
from a trypanosome infection are immune against that particular strain of
virus. The duality of Trypanosoma evansi and brucei was decided in the
following wavy: *

Laveran and Mesnil made use of two goats, which had recovered
from mal de caderas (Trypanosoma equinum) whose blood had proved to
be sterile by tests on susceptible animals. They were then injected with
Trypanosoma brucei, developed the disease and recovered; thus proving
that recovery from mal de caderas does not cause immunity against
nagana.

One of the recovered goats was re-injected with the Trypanosoma
brucei, and since no development followed it had to be concluded that
immunity was established. The goats were now injected with the
trypanosome of surra; they both developed the disease and the presence
of parasites could be demonstrated by sub-inoculation into mice.

ConcLusion.—(1) Recovery of goats from nagana gave immunity
against the strain by which the immunity was caused.

(2) Recovery from mal de caderas did not gwe immunity against
nagana or surra.

(3) Recovery from nagana did not cause immunity against surra.

French scientists worked with surra of various origins, viz., a strain
obtained from Mauritius, another from India, and a third one from
Nha-trang, China, and based on the immunity re-action declared the
trypanosomes found in the disease “mbori’’ of North Africa to be identical
with Trypanosoma evans.

Vallee and Panissett inoculated two head ot cattle with surra of
Mauritius. After the course of a year they no longer showed trypano-
somes in their blood, and a subsequent inoculation with the same virus
proved their immunity. They then were twice in succession inoculated
with the trypanosome of mbori and proved refractory. Later they were
inoculated with surra from India and again proved refractory.

Laveran experimented in the reverse order. A male goat which
had recovered from mbori, and whose blood had proved to be sterile was
first. tested with the same virus and proved to be refractory. Subse-
quently it was inoculated with surra of Mauritius and no infection took
place. From this, Laveran supported the views of Vallee and Panisset

* Compt. Rend. des Seances de l’Acad. de Science, t, CNX XVI, p. 1529 ; A, Laveran et F. Mesnil,
+ Comptes Rendus, 27/3/05,

45

of the identity of the trypanosome of Mauritius and mbori, but is inclined
to consider trypanosome of mbori as a variety of Trypanosoma evanst.
Similar experiments were undertaken by Laveran and Mesnil* to prove
that the trypanosomiasis of Nha-trang in Annam was identical with surra,
after it was proved that they did not differ either in morphology or in
their pathological action. A male goat which was immune against both
mbori and surra of Mauritius, and whose blood inoculated into susceptible
animals had proved to be sterile, was inoculated with surra from
Nha-trang. It contracted the disease and died.

A goat which had recovered from an infection of a Mauritius
strain of surra and had proved to be immune to the test with the virus, and
after sub-inoculations into smaller animals had proved that the blood
had become sterile, was inoculated with virus of Nha-trang. The animal
contracted the disease afd died. Here the immunity of surra and mbori
did not protect against a third strain of surra virus of Nha-trang. Laveran
and Mesnil’s interpretation of these facts is of a close relationship of the
two strains, but of different variety; but they do not consider the
Nha-trang strain to be an entity of its own.

CoNCLUSION.

(1) Cattle which recovered from surra of Mauritius proved immune
to the test when reinjected with the same virus.

(2) Goats which recovered from mbori proved immune to the test when
injected with the same virus.

(3) Cattle which were immune to surra of Mauritius proved to be
immune to mbori and to surra of India.

(4) Goats which were immune to mbori proved to be immune against
surra of Mauritius.

(5) Goats immune to surra of Mauritius and mbori, or to surra of
Mauritius alone, proved not immune to surra of Nha-trang.

(6) The blood of animals whose immunity was tested proved to be
sterile.

Although there are pathogenic differences described by Laveran,t
Trypanosoma sudanense, which morphologically cannot be distinguished
from Trypanosoma evansi, had to be considered as a specie of its own,
owing to the interpretation of the immunity test. Laveran used a goat
which had recovered from an infection with Trypanosoma sudanense.
After the blood had become sterile and had proved to be immune
against a subsequent test it was injected with Trypanosoma evansi of
the mbori disease with positive results.

In the reverse experiment, a goat recovered from mbori, and after
the blood had become sterile proved to be immune against this strain,
was successfully infected with Trypanosoma sudanense. One virus did

* Recherches Experimentales sur la Trypanosomiase des Chevaux de l’Annam.
+ Tryp. du Haut Niger Ann, de l’Ins. Past. t. 21, No. 5.

46

not protect against the other, hence the conclusion of the duality of the
two trypanosomes.

CoNCLUSION.

(1) Recovery of a goat from Trypanosoma sudanense gave immunity
against this strain.

(2) The blood of the immune animal was sterile.

(3) The immunity did not protect against mbori strain, nor did mbore
ummunity protect against Sudanense strain.

Morphologically identical with the Trypanosoma brucei of Zululand
were also found to be two strains of trypanosomes found in Togo. Their
identity or otherwise formed the subject of investigations by Lavern
and Mesnil.*

They experimented with a Nagana from Togo obtained from Prof.
Schilling, which they injected into a goat (this goat had recovered from
an infection due to the Zululand strain and its blood proved to be sterile)
together with a control; thev hoth contracted the disease and died
respectively on the 34th and’ 35th days. Here the immunity of the
Zululand Nagana did not protect against Schilling’s Togo strain; hence
the conclusion that this latter one is not Nagana.

CoNcLUSION.
(1) Immunity of Trypanosoma brucei of Nagana strain of Zululand
did not protect against Schilling’s Nagana strain of Togo.
(2) The blood of the goat which had recovered from a Nagana infection
proved to be sterile.

Observations recorded by Kocht concerning a Togo strain of
Trypanosoma brucei are of utmost interest to us.

Two horses, a mare and a stallion, were imported from the Hinterland
of Togo to Berlin. En route they had to pass a tsetse belt. They con-
tracted a trypanosomiasis (called the Togo-Martini strain of Nagana).
The stallion died about four months after infection, and its blood proved
to be highly virulent for any experimental animals which were injected.
The mare remained in good condition; microscopical examination did
not reveal the presence of trypanosomes, which were only demonstrated
after large doses of blood had been injected into dogs. Contrary to the
experience with the stallion, the mare’s trypanosomes were but slightly
virulent for smaller animals and these recovered to a great extent. Finally
the mare, which remained over a year in perfect health, was inoculated
with the trypanosome originating from the stallion, and now she developed
an acute tsetse disease from which she died. Koch interpretes this to a
different virulency of the two strains, although the horses had become
infected in all likelihood at the same time and at the same place.

* Comptes Rendus, 25/6/06.
+ Deutsche. Med., Woch., 1904.

47

Martini, who carried out these experiments, was able to increase the
virulency of the mare’s strain to a great extent. Dogs, injected with
virus of the first generation, only died after 100 days; but dogs injected
with virus from later generations died in 10-15 days. It was also noticed
that dogs which had recovered from an infection with the earlier genera-
tions died from the infection when, injected with a later generation. Here
no immunity was observed. _
CONCLUSION.

(1) The two strains of the stallion and the mare had for morphological
and other reasons to be considered identical.

(2) They were of different virulency.

(3) The virulency could by passage be so increased that the immunity
obtained from one virus, low in the scale of generations, did not
protect against the same virus higher in the scale.

Koch* has made some further observations which are useful from
our point of view: Two head of cattle which had been inoculated with
an attenuated strain of trypanosomes, apparently recovered, inasmuch
as in their blood microscopically trypanosomes could no longer be seen.
They were tested, together with some control animals with a virulent
strain of trypanosomes. The controls showed the disease in the usual
way; the two vaccinated animals showed trypanosomes only for a few
days and remained healthy. Apparently by the first inoculation they
acquired some immunity against the second strain. One of these animals
remained for about six years under observation and was tested during
this time on its immunity, again with positive results. After six years
the blood of this animal which on microscopical examination proved to
be sterile was tested on dogs, when the presence of trypanosomes was
promptly proved. Thus in this instance we have a proof that cattle,
although resisting to repeated inoculation of virulent blood and therefore
apparently immune, can retain the trvpanosome in the blood stream.

Bruce,t who studied the Nagana in Zululand in order to trace the
connection between game and Nagana, inoculated the blood of game into
dogs, which were kept outside of the infected area (on the Lebombo
mountains). Thus the blood of 8 buffaloes proved to be infective
once; of 13 wildebeeste three times; of 4 koedoes three times; and
once the blood of a bushbuck and a hyena. This fact demonstrates the
connection between game, fly, and disease in stock.

CoNncLUSION. .
(1) An animal proved to be immune against the inoculation of
Nagana, but its blood proved to be virulent for susceptible cattle.
(2) Game, which are considered immune against Nagana, may carry
the infection in their blood.

* Loco cit. 5
+ Further Report on the [setse Fly Disease or Nagana of Zululand.

48

The differentiation between the Trypanosoma pecaudi, the cause of
a North African disease called baleri, and the Trypanosoma dimorphon,
which in its smaller not free flagellated forms resemble each other to a
certain extent, was also demonstrated by the immunity test.

In Laveran’s experiments,* a sheep after it had recovered from a
Trypanosoma pecaudi infection, and its blood was proved to be sterile,
was tested with its own strain of virus, which in the meantime had passed
some guinea pigs. It was found to be refractory to this injection. It
was then inoculated with Trypanosoma dimorphon and developed the
infection in typical time, thus proving that the immunity obtained from
the former infection did not protect against the latter one.

The long flagellated forms of Trypanosoma pecaudi resemble Trypano-
soma evanst. It was therefore possible that the original animal in which
Trypanosoma pecaudi was first found contained a mixed infection of two
different trypanosomes, a flagellated and a non-flagellated one, and the
former might be Trypanosoma evansi of mbori disease. Laveran inocu-
lated a goat, immune against mbori (Trypanosoma evansi) with Trypano-
soma pecaudi; the goat developed the infection and the two forms of
Trypanosoma pecaudi appeared again, thus pointing to the true species
of Trypanosoma pecaudt.

CoNCLUSION.

(1) Recovery from Trypanosoma pecaudi gave immunity which pro-
tected against this particular virus, and the blood of recovered
animals was sterile.

(2) Immunity thus obtained did not protect against an infection with
Trypanosoma dimorphon.

(3) Immunity obtained through recovery from Trypanosoma evansi
(mbort) infection did not protect against Trypanosoma pecaudi.

Trypanosoma dimorphon was first described as a horse disease by
Dutton and Todd. Experiments proved that all stock can be infected
with it, and suksequently it has also been found in various species of
domesticated animals. The disease runs usually a chronic course, and
several animals have been observed to recover.

Concerning immunity, some observations have been made by Martint
ina goat andasheep. These animals had recovered from a natural attack,
and their blood injected into dogs proved to be sterile; when reinocu-
lated, they again showed the infection.

ConcLusion.—Recovery from an attack of Trypanosoma dimorphon did
not producermmunity. The blood of the recovered animal proved to be
sterile.

* Comptes Rendus, 1907.
7 Les Tryp. de la Guinea francaise : Aun. de l'Inst. Past., . XXI, No. 5,

49

_ Somewhat related to Trypanosoma dimorphon, but more uniform in

size, 18 the Trypanosoma congolense ; according to the immunity test it
must be differentiated from the former. Two goats which recovered
from an injection were used in Laveran’s experiments.* Sub-inoculation
into smaller animals proved the sterility of the recovered goats’ blood.
The animals were reinjected with the same strain; again they both
showed a slight infection. Further inoculation however did not cause
any more infection; the animals had acquired immunity. The two
recovered goats were now inoculated with Trypanosoma dimorphon.
Both animals contracted a typical infection from which one died and
the other was still alive on the date of publication (November, 1908).

ConcLusion.—Recovery from a first attack of Trypanosoma congolense
did not convey a completeimmunity. A second infection with the same
strain proved successful. Subsequent recovery increased this immunity.
The blood of immune animals was sterile. Immunity against
Trypanosoma congolense did not protect against Trypanosoma
dimorphon.

SERUM EXPERIMENTS.—The experiments of Laveran,t Mesnil,t Martin,§$
Kleine,|| and Mollers,|| and of others with trypanosomes can be résuméd
as followst :-—

(1) Serum of animals suffermg or recovered from an advanced
trypanosomiasis has distinct preventive qualities. When homologous
serum and virus were mixed before injection no infection took place ;
independently injected the results varied. In experiments of hetero-
logous mixtures of serum and virus no preventive actions were noticed.

(2) The serum of an animal suffering from advanced trypanosomiasis
may be active against the trypanosomes of the same blood from which
the serum was derived (Kleine and Moller).

(3) Animals which supply a serum with preventive properties may
retain the infection in the blood, and the serum of such blood has no
preventive action on the corresponding trypanosomes, even if such
trypanosomes have passed through a succession of animals (Mesnil and
Brimont).

SUMMARY.

Animals may recover from trypanosomiasis and then prove to be
immune against the strain with which they have been infected ; it is an
exception that the same strain again causes an infection, but the different
strains may cause reinfection.

_ Animals may be immune and their blood remain infective or they
may be immune and the blood becomes sterile.

Serum of infected or recovered animals has slight protective pro-
perties.

* Contrib. 4 ’Etude de Tryp. congolense: Aun, de l'Inst. Past., t. XXIT, Nov., 1908.
+ Comptes Rendus, t. CX LIT, p. 1482.
t Ann. de I'Inst. Past., t. X.

§ Zeitsch fur Hyg. t. L., 4/4/06.
|| Zeitsch fur Hyg. t. LIL, 1906.

50
COMPLETE SUMMARY OF CONCLUSIONS.

I.

Two different types of immunity may be distinguished :—

(1) Immunity whereby the recovered animal no longer acts as a
reservoir of virus. Immunitas sterilisans (East Coast fever and the
pe diseases due to ultravisible organisms ; trypanosomiasis partially.)

(2) Immunity whereby the recovered animal acts as a reservoir for
virus. This immunity may cease by intercurrent fevers; breakdowns.
Immunitas non sterilisans. (All inoculable piroplasmoses and spiro-
chaetoses ; trypanosomiasis partially.)

Il.

In speaking of immunity of a recovered animal, distinction must be
made between immunity generally without reference to any particular
strain of virus and immunity in particular with reference to definite
strains of virus. (For instance, immunity against horse-sickness can be
generally spoken of, or in particular the immunity obtained from the
Ordinary strain, or Tzaneen, or any other.)

We can then conclude as follows: With the exception of East Coast
fever in South Africa no other tropical disease produces complete immu-
nity. The cause of the other diseases is not uniform; there exist many
different strains or varieties of the species of organisms which al! form
the cause of the disease.

Concerning a particular strain, the following may be concluded :

(1) An immunity against a particular strain may be complete.
(Ordinary virus of horse-sickness; some trypanosomiases ; heartwater.)

(2) An immunity against a particular strain may be broken

(a) by the injection of large doses of the same strain of virus
(blue-tongue) ; or

(b) after a certain time has elapsed (blue-tongue) ; or

(c) by a more virulent virus of the same strain (Tzaneen virus in
horse-sickness) ; or

(d) by a different strain of virus (horse-sickness, heartwater, red-
water, and surra).

(3) Immunity can vary with the species and with the breed of animals
susceptible to the disease (redwater, heartwater).

II.

The facts noted in South Africa concerning variations of immunity
may be explained as follows :-—

Hast Coast fever is a freshly imported disease, and the outbreak
throughout South Africa may all be due to one and the same source,
whereas, for instance, horse-sickness, blue-tongue, etc., are diseases
established since ages, and are localised; the micro- organisms have
undergone and are still undergoing certain variations, the result of which
is the different immunity.

IV.

From a practical point of view, immunity against a tropical disease
can only be spoken of as sufficient when it protects against the majority
of various strains of the organisms forming the cause of the disease found
in such region, for which it is intended to make use of for inoculation
purposes.

V.

The immunity test for identifying two morphologically similar
organisms can only allow of a definite conclusion when the test is positive,
but not so when it is negative.

XI.

The serum of animals which have recovered from a disease and have
been hyperimmunised to a certain extent acquires preventive properties.
Kast Coast fever is an exception, but the injection of blood from
an animal suffering from East Coast fever into susceptible ones does
not communicate the disease, and this may explain the fact. The
preventive action of a serum is principally pronounced against a
homologous strain, either of the same animal (virus derived from the
same immune animal which supplied the serum ; in some trypanosomiasis
and in canine piroplasmosis) or of different animals and of different strain
(horse-sickness) ; it may be deficient against heterologous strains (strains
of different origin; some trypanosomiases, surra, and horse-sickness) or
even against a homologous strain derived from the animal which supplied
the serum (some trypanosomiases).

VI.

Concerning the immunity itself, there are no essential differences
between that caused by bacteria and that caused by protozoa. The fact
that an apparently recovered animal acts as a reservoir has its analogy
in pleuro-pneumonia of cattle, although the two cases are not quite
identical. In the latter, morbid lesions are still present, in which the
infective cause is retained; in the former, the cause remains in an
apparently healthy animal.

The Diagnosis of Bacillary Piroplasmosis

of Bovines in the Transvaal.

By JAMES WALKER, MLR.GV.S.

The Serum Room.

TY il
Hina

y |
ill

Sterilizing Room—Bacteriology.

The Diagnosis of Bacillary Piroplasmosis of Bovines
in the Transvaal.

THE term “ piroplasmosis’’ is applied to diseases caused by endo-
globular parasites belonging to the group protozoa and called piroplasmata.
Of the bovine piroplasmoses known in the Transvaal, two interest us
more particularly, viz., the two caused by bacilliform piroplasms and
which hitherto have. been known under the name Péroplasma parvum
and Piroplasma mutans; both from a diagnostic point of view are
interesting ; the former from an economic point of view is of extraordinary
importance, and at one time threatened to destroy the cattle of South
Africa. These two microscopical organisms are morphologically insepar-
able; this forms the reason for a difficulty in the microscopical diagnosis.
An early decision with regard to a diagnosis of East Coast fever is always
a matter of urgency, consequently the practical utility of observations
which would allow of a differential diagnosis would be very considerable.

Piroplasma parvum is the cause of the disease popularly known as
Hast Coast fever. A specific name for the disease caused by Piroplasma
mutans does not exist, but the term “ gall-sickness ’’ probably includes
that disease as well. The main characteristic of Piroplasma parvum is
its non-inocubility which separates it absolutely from Piroplasma mutans,
which is inoculable even with small quantities of blood.

At the Veterinary Conference held in Budapest * in 1905, the
question of piroplasmosis received attention. Dschunkowsky stated the
disease described first by him under the name Tropical Piroplasmosis,
and caused by small endoglobular parasites in the shape of rings, rods,
and flagellated forms, in Transcaucasia was inoculable; this statement
reversed his former one, and by this differentiated it from the disease
East Coast fever. Bitter communicated that he had observed a non-
inoculable piroplasm in Egypt, likewise due to small piroplasms, and
Ducloux, of Tunis, reported his experience of a similar parasite as the cause
of a disease in that country. In view of these new discoveries the
piroplasmata at that time were grouped as follows, viz. :—

‘“Typr.—Piroplasma begiminum, Piroplasma bovis (Babes) found
in European Haemoglobinuria of cattle. Piroplasma bige-
minum (Smith and Kilborne) of Texas fever.”’

“ Typr.—Piroplasma parvum (a) Inoculable piroplasmoses, tropical
piroplasmosis of Transcaucasia, Puroplasma annulatum
(Dschunkowsky) ;_ (6) Piroplasmoses (not. inoculable) Puro
plasma parvum (Theiler) of Hast Coast fever, piroplasmosis of
the North African disease (Bitter and Ducloux).”

* Proceedings of the 8th International Veterinary Congress, Budapest, Sept., 1905,

56

Since then new inoculable piroplasmata of the type Piroplasma
parvum had been described by Theiler and called Piroplasma mutans *
and by Miyajima and Shibayama in Japan,t which led Bettencourt to
suggest the following classification, viz. :—

1st Group.—Piroplasmoses produced by parasites presenting in one
of their evolution phases the form of a pear and disposing
themselves in pairs in the same corpuscle.
Babesia bovis (Babes).
Babesia bigeminum (Smith and Kilborne).
Geographical distribution, Europe, America, Africa.
2nd Growp.—Piroplasmosis produced by parasites presenting always
at one phase of their evolution the form hacillary—Prro-
plasmoses bacilleformes.
(a) Piroplasmoses bacilliformes, imoculable.
Babesia annulata (Piroplasma annulatum) Dschunkowsky.
Babesia mutans (Piroplasma mutans) Theiler.
Babesia bacilliforms found amongst the cattle of Japan
(Mivajima and Shibayama).

Geographical distribution.—Transcaucasia, Japan, South
Africa, to which may be added the following, viz. :—Piro-
plasmosis, described by Stephens and Christopher, in Madras :
Shein in Annam;f Bettencourt in Portugal;§ Boeret on the
Gold Coast of Africa;|| Does in the Dutch Indies;{] Martini
in Peycheles, China;** Lichtenheld in German Hast Africa ;}T}
Balfour in the Soudan;t{ Broden and Rodhain, Congo (Stanley
Pool).8§

(b) Pwroplasmoses bacilliformes, non-inoculable.

Babesia para|||| (Piroplasma parvum) Theiler.

Babesia of Tunis and Egypt.

Geographical distribution.—South Africa (Theiler), Egypt
(Bitter), Tunis (Ducloux).

*Theiler: Piroplasma Mutans.—Annual Report Transvaal Department of Agriculture, 1905-06 ;
Report of the Government Veterinary Bacteriologist, Transvaal Department of Avriculture, 1906-07.
+ Bacilliformis: Zietsch. }. Hyg., t. LIV, Sept.. 1906. Miyajima and Shibayama, pp. 189-200.
{ Shein in Annam Ann. Institut Pasteur, t. X XT, 25/8/07, pp. 656, 657, 669.
§ Bettencourt, extract Archives Institute Royal of Bacteriology, t. 1, Fax II, Lisboa, 1907.
|| Boeret, Gold Coast, Bulletin Soc. Path. Exot t. I, No. 4, 1908, p. 234. “
¥ Docs. Dutch Indies. Mededeelingen uit het Geneeskundig Lab. te Weltevreden, 2de Serie, B.
No. 4. p. 185, 1905.
** Martini in Peycheles, China, Arch f. Sch. u. Trop. Hygene, t. XI, 1907, pp. 507-511; 718-719,
August and November, 1907,
+7 Lichtenbeld in German East Africa. Zietschrift f. Hygene u. Infektious Krankheiten, bl. 61,
1908, heft 2. p. 261. :
tt Balfour in Sudan, 8rd Report Wellcome Res. Lab. at the Gordon Memorial College, Khartoum.
8§ Broden and Rodhain, Congo, Stanley Pool. Bulletin Soc. Path. Exot t. II, seance 10/3/09,
pp. 12 124.
||| Pheiler: Babesia Parva, Piroplasma Parvum.—Annual Report of the Transvaal Department of
Acriculture, 1903-04 ; Annual Report of the Transvaal Department of Agriculture, 1904-05; Annual
Report of the Transvaal Department of Agriculture, 1905-06; Report of the Governmeut. Veterinary
Bacteriologist. Transvaal Department of Agriculture. 1906-07,

a7

Bettencourt suggests creating a new genus which he proposes to call
“ Theileria,” including all the parasites which present the bacillary form
and which divide themselves in originating forms of a cross. In this
manner the name Babesia would be reserved solely for parasites of the
type Babesia bigemina.

From our present knowledge of Piroplasma parvum, principally its
non-inocubility and the presence of certain intracellular bodies found in
the internal organs of an affected animal which will be referred to later,
and from the fact that an immune animal does not retain the infection
in the blood, the genus—Theileria—has to be completely separated from
the rest of the piroplasms and to be considered as a genus of its own.
For the same reason it follows that the suggestion of Bettencourt to include
the bacillary forms in one genus cannot be accepted, and the inoculable
bacillary piroplasms either represent a genus of their own or should belong
to the group of Babesia.

The following grouping may represent the present knowledge :—

Inoculable (Babesioses).
L

(a) Babesia type, B. Bigeminum.

II.

(b) Babesia bacilliformes, type Babesia nvtans.

Babesia of Madras (Christopher and Stephens).
‘ Annam (Shein).
~ Gold Coast of Africa (Boeret).
- Dutch Indies (Does).
a Peycheles, China (Martini).
* German East Africa (Lichtenheld).
i Soudan (Balfour).
5 Congo (Stanley Pool) (A. Broden and

J. Rodhain.

Il.
Non-Inoculable.
(a) Theileria. Type: Theileria parva.

East Coast fever is a new disease for South Africa. In the year 1897,
Professor Koch when investigating diseases in German East Africa
described an endoglobular micro-organism of bacillary form, and which
at that time was regarded by him to be young forms in the development
of Piroplasma bigeminum, the cause of redwater (Texas fever), and accord-
ingly designated the disease by that name. From 1898 to 1901 it attracted
no special attention. About the latter end of 1901, an outbreak of a
disease corresponding to that observed by Koch in German Hast Africa
was found amongst a mob of recently imported cattle at Beira ; a number

58

died, the remainder were removed to Umtali, Rhodesia, where they con-
tinued to die. It appears that the cause of death at Beira was redwater
(Texas fever), and that the lot which were removed to Rhodesia developed
the usual symptoms of the East African disease as a result of being grazed
on ground infected by cattle introduced from German East Africa; the
disease now attracted more attention, although still associated with
redwater (Texas fever) by the first workers, East Coast fever was sub-
sequently introduced into the Transvaal in May, 1902, apparently from
Lourenco Marques via the Delagoa Bay Railway, and first appeared at
Komatipoort and Nelspruit, and thence eventually spread to other
districts and Swaziland. After its introduction into Rhodesia and the
Transvaal, and when its study was systematically undertaken, it was
found not to be identical with redwater (Texas fever), but to be a disease
of its own.

In the experiments with ordinary redwater inoculations it was noticed
that the blood of animals which were injected with blood of redwater
immune animals, showed on microscopical examination the typical
Babeswa bigemina. When, however, the examination of the blood was
continued endoglobular parasites similar to those of Thieleria parva were
also observed. This phenomenon led some observers to conclude that
these organisms are a stage in the life-cycle of Babesia bigemina. Others,
however, held that the small endoglobular organisms found in the blood

of animals had to be connected with East Coast fever. At the Veterinary
Conference held in Bloemfontein in 1903,* Professor Koch stated that
he had found the parasites of East Coast fever in smears of blood collected
from East London cattle, and concluded that that disease existed on
the south-eastern coast of Africa, basing his opinion on the presence of
the bacillary piroplasms. Theiler, who had carried out some investiga-
tions in connection with the appearance of small endoglobular parasites,
before East Coast fever was known to the Transvaal, in cattle out of
countries known to be free from that disease, considered the piroplasm
referred to by Koch was probably a species of its own and not connected
with Hast Coast fever. In the light of our present knowledge it is
evident that the parasites observed by Koch were those which were
formerly associated with the immune redwater ox, but which are now
known to be a distinct species, viz., Babesia mutans. Since Babesia mutans
exists in the Transvaal wherever redwater is found, and it is the exception
to find the one unassociated with the other (this association of Babesia
bigeminum and Babesia mutans appears to exist in other countries, e.g.
China, Japan, Annam, Madras), and since it is known that Madagascar
cattle are immune to both redwater (Texas fever) and mutans, its
introduction may be traced back to the importation of cattle from that
country.

* Report of the Proceedings of the Couference on Diseases amongst Cattle and other Animals in
South Africa; Bloemfontein, 1903.

59

Host oF THE PIROPLASM.

Numerous experiments (Theiler) (Lounsbury) have established the
fact that the transmission of the parasites which invade the erythrocytes
in East Coast fever takes place by the agency of ticks. From the most
recent experiments it has been shown that Rhipicephalus appendiculatus
is the principal transmittor; the affection being carried by nymphae
and adults, that Rt. everts:, Rt. capensis, Rh. simus, and Rh. nitens are
hosts of Theileria parva. Although the present knowledge with regard
to the means of transmission of Babesia mutans is perhaps still incomplete,
recent observations (Theiler *) point to Rh. appendiculatus and Rh. evertsi
as being hosts.

Course, Symptoms, and Pathological Lesions.

The neubatiwe period of Hast Coast fever averages twelve days,
variations being ten days shortest and twenty days longest period ;
following this a sudden rise of temperature occurs in the majority of cases,
reaching 106-107 and remaining high throughout. In a few cases the
temperature rises gradually day by dav, until the maximum is reached.

The fever period averages about thirteen days, variations being
six days minimum and twenty days maximum. Consequently the length
of the disease from date of infection averages twenty-five days.

The symptoms during life are not always characteristic; they are
subordinate to the seat of lesions. In some instances the animal may
appear in normal health up till the time of death, and in such is usually
found dead with a discharge of foam from the nostrils, resulting from an
oedematous condition of the lungs. In other cases in which the oedema
of the lung, although present, is not so pronounced as to cause immediate
death pulmonary symptoms, such as coughing and discharge from
nostrils, may be noticed. Where bowel lesions are present diarrhcea or
a haemorrhagic discharge may be present; depending on their severity,
swelling of the lymphatic glands of the head and throat may be detected
in a number of cases. Frequently the first symptoms observed will be
a discharge from nose and eyes and increased flow of saliva from the
mouth, or simply a loss of condition. Haemoglobinurea is not a symptom
of the disease, but is the expression of the complication with ordinary
redwater (Texas fever). Where the disease is associated with ordinary
redwater, as frequently happens in a country in which that disease is
known to exist, symptoms of redwater overmask those of Hast Coast
fever.

The post-mortem lesions vary. The organs found most constantly
affected are the spleen, liver, kidney, and heart. Lung lesions are
present in from 30-35 per cent. of cases (Gray).

* Transvaal Biological Society Proceedings, 29/3/09.
+ East Coast Fever—A Historical Review: Annual Report of the S.A. Association for the Advancement
of Science, Grahamstown meeting, 1908.

60

The following post-mortem appearances were recorded in a case of
pure infection with Theileria parva -—

Post-mortem examination of Heifer No. 683, 21st March, 1909.
Age: Two years.
Interim: Five hours after death.
Condition : Fair.
Blood : Not coagulated.
Subcutaneous tissue: Yellow infiltration.
Fat: Yellowish im colour.
Mediastimum : A few small hemorrhages.
Pericardium : Empty.
Lungs: In inspirium ; pleura whitish ; both anterior lobes and lower
half of middle lobe pneumonia, Stad. 11.
Bronchi: White mucus.
Pharynx: Slight hyperaemia; some pus in right tonsillae.
Heart : Left endocard yellowish, left ventricle contains blood coagu-
lated.
Right endocard yellowish, right ventricle contains blood coagu-
lated.
Myocard soft.
Epicard yellowish.

Liver: Enlarged, 45 « 35 cm., swollen, brown yellowish discolora-
tion on section; oedema; infarcts. Bile thick, dark green.
Spleen: 42 x 16 cm. In hgamentum gastro lienale a haematome
surrounded by fibrous tissue.

Stomach : Abomasum liquid contents, folds swollen. Mucosae slight
oedema and hyperaemia.

Small intestines : Jeywnum black discoloration, cross striped. Ilewm
whitish mucus and a few hyperaemic patches.

Large intestines : Caecum contracted, folded, slate colour. Valvula
ileo caecalis discoloured.

Colon : Folded and black discoloration.

Kidneys: A few small infracts the size of a pea; capsula easily
detached and oedematous infiltration.

Internal lymphatic glands : Swollen and hyperaemic ; white spots in
sinus.

Skull: Brain shght injection of pia.

Marrow of bones : Femur epiphysis soft, oedema and hyperaemic spots.

Ribs : Red, bone marrow, soft.

In the majority of cases lesions resembling infarcts are found in the
kidneys and sometimes in the liver. They are specific for Kast Coast
fever. In the kidney they may be detected on inspection of the surface
of this organ as regular shaped areas or spots, their colour varying from
red to white. If recent usually projecting above the surface and

61

surrounded by a hyperaemic zone, a yellowish zone may be seen within
the red zone.

Collaud,* as a result of the examination of sections from kidneys of
thirteen bovines sent by Theiler, noted an inflammatory process which
he divides into the following stages, viz. :—

(1) Hemorrhage from the capillaries (through their walls) into the
kidney tissue.

(2) Cellular infiltration of the hemorrhagic areas with leucocytes,
lymphocytes and fibroblasts and destruction of the red
corpuscles ; the leucocytes eventually disappearing, young
connective tissue cells and lymphocytes finally remaining ; the
epithelium canaliculaire is also destroyed.

(3) The connective tissue consolidates and invades not only the
hemorrhagic areas but the surrounding tissue as well. These
different stages are observed in the same kidney.

According to the author, the Theileria parvum secretes a toxine which
acts on the endothelium of the vessel walls and on the cells of the renal
epithelium. (Hzemorrhages are found in all the organism.) The author
proposes the denomination of Nephritis h:emorrhagica piroplasmatica. It
would thus seem that the process is an inflammatory one leading to
regeneration.

In a case of Babesia mutans infection, the wncubative period varies
from twenty to forty-five days. The fever period is not pronounced, but
there is usually a continuous rise of temperature lasting for some weeks,
ending in recovery. The main symptoms are as follows, viz. :—An animal
with Babesia mutans may show no outward symptoms at all; in other cases
a loss of condition is appreciable, and occasionally the symptoms of an
anaemia are pronounced. Death due to pure mutans infection has as
yet not been diagnosed. Post-mortem lesions.—in cases where piroplasms
were found to be present it was usually complicated with other diseases,
hence it is difficult to state where the lesions of mutans begin and where
they end.

The post-mortem of an animal which was killed at the height of the
infection was as follows :—

Post-mortem examination of Ox 660, 21st November, 1908. Destroyed.

Condition : Fair.

Pericard : Contains a little straw-coloured fluid.

Lungs: Normal. Foam at bifurcation of trachea.

Heart: “eft ventricle empty, endocard ecchymosis.

Right ventricle. A small blood coagulum, endocard a few
petechiae size of a pin’s head.

*L. Collaud : Beitrage zur Pathologischen Histulogie der Nicre bei, Rhodesian Redwater der Rinder
in Siid Afrika (Piroplasmosis) Inaug. dissert., University Zurich, 1906 ; Bulletin l'Institut Pasteur, t. V,
No. 6, page 252, 30/3/07.

62

Liver: On section a glazed reddish tint, hard, gall-bladder normal,
contents yellowish green in colour.

Spleen: Slightly enlarged; pulpae soft.

Stomachs : Abomasum—Mucous membrane patchy, hyperaemia.
Rumen—Full of pulpy ingesta, mucosa normal.
Reticulum—Contents pulpy, ingesta, mucosa normal.

Small intestines: Jejunum; mm. hyperaemia in streaks,
Ileum; mm. hyperaemia slight, in streaks.

Large intestines: Caecum normal.
Colon; mm. hyperaemia.
Kidneys : Right, normal.
Leit—A whitish coloured body, size of a pea, resembling an
infarct.

Bladder : Normal, contents normal.

MicroscopicAL EXAMINATION OF BLOOD SMEARS.

When the blood of an animal sick with East Coast fever is micro-
scopically examined after colouration by the method of Giemsa, or other
Romanowsky modifications, parasites will be found to make their appear-
ance in the globules a few days after a rise of temperature occurs; it will
be observed that the parasites assume various shapes, and that a visible
portion of each (nucleus) takes the chromatin stain (red), the remainder
(protoplasm) taking the basic stain. The shapes observed will be as
follows, viz. :—

Bacillary ring, ovoid (there appears to be a transitional form between
the bacillary and ovoid forms). The average measurement of a number
of the bacillary forms was found to be 1°5, in length. They present the
form of a baton with the chromatin concentrated at one extremity. In
some cases the chromatin occupies about one-half of the total length of
the baton. The cytoplasm may be of the same thickness throughout
and straight—clove forms, or it may taper to a pomt at the extremity
opposite the karyosome and be curved—comma form. The ring forms
average about 1, in diameter; they havea clear central portion
(vacuole), and the disposition of the chromatin varies. The ovoid
forms have likewise a central clear portion, their length being 1:°5.
and their thickness 0°75z.

I have noted that the preparation of the smear during life exercises
an influence on the size of the piroplasm, viz., when the blood corpuscles
are much contracted the piroplasms are also found reduced in size. The
cause of this phenomenon is in all probability the increase of osmotic
pressure in the periglobular liquid due to a certain evaporation which
takes place in making the smear.

63

A similar phenomenon is also noticed with Babesia bigeminum after
death. In a case of East Coast fever, as already stated, parasites make
their appearance a few days after a rise of temperature is recorded. At
the beginning of the disease, no corpuscles are found to be affected. As
the disease progresses an invasion of the corpuscles takes place. The
increase may be slow and never reaches a high percentage ; it may be
continuous and reach a high percentage, or it may be almost sudden,
invading all the corpuscles.

In the initial or middle stages when the percentage of cells affected
in a blood smear is yet small, it resembles that of a smear from B. mutans
infection. It is here that a definite diagnosis becomes embarrassing. In
such a case the only course would be to continue daily the microscopic
examinations when an increase of parasites usually takes place. In a
case of Babesia mutans no notable increase, or only a slow one, is observed.
Blood changes, viz., lesions of anaemia will, however, in most cases
be recognised with the appearance of this piroplasm. These take the
form of anisocytosis, poikilocytosis, basophile, and polychromatic cells
(nucleated red cells being rarely found). This is specific for mutans and
not for parvum infection. In those cases of East Coast fever in which
lesions of anaemia are noted, it is due to the complication with Babesia
bigeminum, ordinary redwater (Texas fever), and other blood infection.

Other elements found in smears from spleen, kidneys, lymphatic
glands, and in rare cases in blood smears of cattle affected with East
Coast fever are the so-called Koch’s granule. They were first described
by Professor Koch. They are contained within a cell-shaped body and
vary in size, shape, and numbers in the same smear; two varieties may
be distinguished, viz. :—Ist. Those in which the cell-shaped body stains
a deep blue tint and with granules varying in size; the cell-shaped body
varies in size from 1:5-9, in diameter. 2nd. Here the granules vary in
size from 0°5-0°75,, and the granular matter does not appear as compact
as in the former variety.

It will be interesting to review here the result of investigations of
Martin Mayer,* Assistant in the Institute for Tropical Diseases, Hamburg,
in connection with the study of spirochaeta Duttoni. In an end note
he points out that he found bodies containing granules corresponding to
Koch’s granules in various numbers in the endothelium of normal kidneys
of cattle, mice, and guinea pigs, and of different other animals which had
passed through a certain infection or intoxication.

He also points out that these globules are produced by contraction
of a part of the plasma of the cells, and that phagocytes transport these
to other cells, and that it seems a new proof that the endothelial cells
of the kidneys have a very great importance for destroying parasites and

* Archiv fiir Schiff und Tropen Hygiene, Bd. XII, No. 22, 1908.

64

chemical noxious products. It thus appears that Koch’s granules have
been found in normal kidney smears. This is not in accordance with the
result of the examination of smears from normal kidneys of cattle carried
out by the writer for the purpose of ascertaining whether the bodies
referred to by Koch were present in normal kidney and spleen smears
of cattle. A number of each were examined; in all cases the results were
negative. On the contrary the examination of a considerable number of
smears of spleens of East Coast fever cattle sent in from different
districts of the Transvaal always gave positive results. The writer has
observed during the course of a microscopical examination of a number
of normal smears from cattle minute reddish coloured granular bodies
which are found scattered throughout the smear in some cases; in other,
arranged in groups. In some instances they appear to be contained
within a cell wall, in which case they resemble somewhat the appearance
presented by blood plates in normal blood. They differ from Koch's
granules in size and also staining reaction.

The constant presence of Koch’s granules in the lymphatic glands,
in the kidneys and spleen of animals affected with East Coast fever affords
a means of basing a diagnosis in those cases, in which, as already pointed
out, a microscopical examination does not permit of this. The presence of
these granules in the lymphatic glands, kidneys, and spleen is specific
for East Coast fever. The Bacteriological Laboratory make use of the
fact for the definite diagnosis of all doubtful cases of infection with small
piroplasms.

Haemolysis in Practical Veterinary Science.

By Dr. WALTER FREI.

pe (11 TPR RO

HL TAFV ITERATED

The Physical Chemical Laboratory.

si
7 !

The Photographic Room.

Haemolysis in Practical Veterinary Science.

IL—INTRODUCTION.

In producing and using anti-sera for curative and preventive purposes,
it must be kept in mind that the injection of virulent matter gives
rise, not only to specific anti-bodies against the microbes, but also
against every proteid substance which accompanies them. Since the
virulent matter in horse-sickness, for example, is contained in the
blood, and the anti-serum is produced by the injection of such blood.
it must be expected that anti-bodies are formed, not only against the
micro-organism, but also against the constituents of such blood; the
prevalent anti-body is, however, that corresponding to the horse-
sickness organism.

The following table may elucidate the theoretical possibilities in
passive and active immunisation :—

A. BB. C.
Result of injection of serum contain-
Antigen, Gives rise to anti-bodies. ing the anti-bodies mentioned
under B.

1. Pure living culture of | Bactericidines, lysines, _ Bacteriolysis and agglutina-
microbes. agglutinines (anti- tion.

toxines and precipi- ,

tines), opsonines.

2. Dead culture or fil- | Anti-toxines, precipitines. | Neutralisation of the toxins,
trate. precipitation of the bac-
terial products.

3. Blood or serum con- | Anti-bodies as under 1 Action asunderland 2. In

taining the virus and 2. In addition addition: precipitation

which is generally haemolysines, hae- of serum constituents,

ultra-visible. magglutinines,serum Haemolysis.
precipitines.

In the Transvaal the following methods of immunisation are
actually in practice :—
A.—Against Bacterial diseases.

1. Black Quarter :--Active immunisation by means of a vaccine,
that is to say, with an artificially attenuated virus (heated
and dried muscle substance)—two injections.

2. Pleuro-pneumonia:—Active immunisation by one injection
of a pure culture (in bouillon Martin).

6S

B.—Against Protozoic diseases.
3. Piroplasmoses of equines:—Active immunisation with the
blood of immune animals.

4. Piroplasmoses of cattle (redwater):—Active immunisation
with the blood of immune animals.

C.—Against diseases caused by ultra-risible organisms.

5. Blue-tongue in sheep :—Active immunisation by vaccination.

6. Rinderpest :—
(a) Simultaneous injection of virus and serum of hyper-

immunised oxen.

(b) Injection of bile (vaccination).

7. Horse-sickness:—Simultaneous injection of virus and serum
of hyperimmunised animals (as yet only applied to mules).

One injection of a small quantity either of

(1) fully virulent matter in order to infect an animal and to obtain
Virus ; Or

(2) specific anti-bodies leads to the production of the required
anti-bodies and only of a negligible amount of accompanying
anti-bodies.

Only the specific rinderpest anti-bodies were observed, even at the
hyperimmunisation of oxen, with great quantities of virulent rinder-
pest blood in order to obtain a strong immune serum, recognised in
vivo by the successful treatment of animals; in no instance were
clinical or anatomical phenomena due to haemolysis or precipitation
recorded. We are therefore confronted by the remarkable fact that
cattle do not produce isolysines in their blood, i.e. substances with
the property of dissolving cattle blood in vivo or in vitro, even after
the injection of enormous quantities of blood.

Goats are able to react with the production of isolysines on in-
traperitoneal infusion of considerable quantities (S00 and 900 c.c.)
of dissolved goat blood corpuscles. But these anti-bodies do not appear
in all goats treated in the same manner, nor does an isolytic goat-
serum dissolve the corpuscles of all goats.*

As in goats, isolysines arise in the blood of horse, mule, or donkey
after subcutaneous or intravenous introduction of blood, serum, or
peritoneal liquid of one of these three. The serum of animals treated
in this way is able to produce haemolysis in an emulsion of equine
blood in vitro, and also, as recorded by Theiler,f in the living
animal.

Theiler hyperimmunised horses, mules, and donkeys already im-
mune (or salted) against the South African horse-sickness, that is to
say, he injected them in different manners with large quantities of

* Ehrlich and Morgenroth, Berl. Klin. Wochenschrift No. 1, 1899.
+ Annual Report of the Government Veterinary Bacteriologist, 1903, Transvaal.

t In the following paper the conception of isolysines is rather wide, and it would be better,
perhaps, to call the active substances in donkey serum which dissolve horse blood—heterolysines.

69

virulent blood (serum or peritoneal liquid) taken from sick animals.
The horse-sickness anti-serum obtained from hyperimmunised animals
proved to have effective anti-bodies against horse-sickness virus, but
accidentally also isolysines which became active in some injected
animals, and killed them by haemolysis.

Hence it was necessary

(1) to recognise a haemolytic serum amongst the immune sera
by experiments in a test tube;

(2) to find out the haemolytic limit below which a slightly
haemolytic serum could be used without any danger for
injection. For this purpose it was advisable to express the
haemolytic power by figures which he called the haemolytic
‘index ;

(3) to find a method of hyperimmunisation which gives only
horse-sickness anti-bodies and no isolysines, or as little as
possible.

The experiments were carried out with the object of solving these
problems in a practical manner. Dr. Theiler kindly permitted me to
use his experimental records. I am here publishing a collection of
them arranged from the theoretical standpoint* of haemolysis, which
is somewhat different from the abovementioned guiding ideas. It so
happened that several experiments which, from my point of view,
would have been useful in completing a series of deductions, were not
carried out.

METHODOLOGY.

In a series of tubes, containing each 2 cc. of the sterile serum
which had to be tested, were added 0°5 cc. of defibrinated sterile
blood of different animals, horses, or mules. The mixtures were kept
at 37° for two hours, then placed in an ice chest over night. The
change which took place was recorded as follows :—

Marks.

1. Trace—indicates a slight red discolouration of the lowest layer
of the serum just above the deposit.

2. Slight—indicates the haemaglobin diffused up to the top of
the liquid.

3. Distinct—indicates a deep red discolouration of the entire
serum, but the deposit still dark.

4. Complete—indicates the deposit colourless, and the haema-

globin completely diffused out of the corpuscles.
ew

The Haemolytic Index.
Example: 10 tubes, containing all the same serum, but blood from
10 different horses.
(a) Number of tubes showing haemolysis=5 (i.e. the blood of
5 horses is dissolved by this serum).

* Compare: W. Frei, Zur Theorie der Haemolyse, etc. Zeitschrift fur Infektionskrankhciten,
parasit. Krankheiten, Hygiene der Haustiere, 2, 158 and 360, 1907.

70

(6) Number of marks of all 10 tubes together=8 (total of haemo-
lysis degrees).
(a) and (b) multiplied=5 x 8=40.
This figure divided by the number of horses whose blood was
used (in this example 10) gives the degree of haemolysis

for 10 horses="*" = 4.

Again divided by the number of horses whose blood was
used, gives the average degree of haemolysis for 1

howe = =0" 4.

O-+ is the haemolytic index for the serum in question. In
this way it was possible to compare the results of different
haemolysis tests.

I may mention that for the purpose of drawing conclusions,
only those tests in which the blood of at least 10 different

horses were tested with one serum, have been utilised.

TII.—ISOLYSINES IN EQUINES.
A.—HYPERIMMUNISATION BY SUBCUTANEOUS INJECTION OF THE VIRUS.

(a) Hyperimmnisation with virulent defibrinated blood.
1. 11 horses hyperimmunised with virulent defibrinated horse

blood.
2. 3 horses a ; mule blood.
3. 3 mules x i horse blood.
+. + mules Ae i mule blood.
d. 1 donkey 4 Ge horse blood.

(b) Hyperimmunisation with virulent serum.
1. 3 mules hyperimmunised with virulent horse serum.

2. 4 mules re ss mule serun.
3. 2 donkeys Fe = horse serum.
4. 3 donkeys wy 3 mule serum.

) Hyperinunrarisation with virulent borillon.
(From peritoneal cavity.)
1. 2 mules hyperimmunised with virulent horse bouillon.
2. + mules i “ mule bouillon.

B.—HYPERIMMUNISATION BY INTRAJUGULAR INFUSION OF THE VIRUS.

(d) Hypertmiuaiaisation with not defibrinated wvwirnlent blood by
tnunediate transfusion into the jugular vein,

1. Hovses intused with virulent horse blood.
2. Mules "i i mule blood.

It must be kept in mind that the immune serum derived from the
hyperimmunised animals was never injected alone, but as the experi-
ments were in connection with horse-sickness, and not haemolysis, a
previous simultaneous or subsequent injection of virus took place,

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Discussion.

Horse SH 1.—The haemolytic index was higher for mules than for
horses. In no instance was an injection of this serum followed by
haemolysis. Horse serum with a haemolytic index of 0°3 for horses did
not seem to have any harmful effect on horse blood in vivo.

Horse 147.—The serum was in one instance more haemolytic for
mules than for horses; in another test vice versa. When injected it
killed tour horses out of five.

Horse 160.—This serum seems to have been move haemolytic for
mule than for horse blood. With an index of 13 it did not prove to
be haemolytic in vivo, (AIL animals being injected subcutaneously.)
With regard to the rise and disappearance of the isolysines, it may be
observed that from the 29th September, 1903, to 24th April, 1904 (seven
months), its haemolytic index for horses fell from 1:3 to 0284, although
two injections of blood, totalling 2,500 c.c., had been made. We must
consider, therefore, that within the three months which passed from
the date of haemolysis test, 29th September, 1903, to the tenth injection,
12th January, 1904, a large amount of the isolysines—perhaps all—had
disappeared. I am not able to say that the loss of 32,880 ¢.c. of blood
is the cause of the loss of haemolvsines; the haemolytic index for ales
had, however, increased between 25th April to 26th September, 1904,
from 0°} to O84, notwithstanding the withdrawal of 35,000 ¢.c. blood
from the horse, and that no further hyperimmunisation had taken
place.

Horse 172.—The haemolytic index was higher for mules than for
horses.

Horse 611.—This serum also had a stronger haemolytic power for
mule than for horse blood. The haemolytic indices were somewhat in-
creasing from the third to the sixth injection ; injections of 4,500 cc.
within three months did not give rise to haemolysines for horses.

Horse 612.—This serum was also more haemolvtic for mules than for
horses. The activity for horses—adversely to h. 611—decreased, although
three more injections were made. Both horses 61] and 612 were injected
with equal quantities of blood of the same origin at the same time, and
the haemolysis tests were made on the same days; but the quantities
of haemolysines which they developed in their blood were different.
For instance, the haemolytic index for mule blood 611=0°2 for 612=0:125
at the same date. The only difference between the horses at this date
is that 612 had been bled to the extent of 6,000 c.c. more than 611. (Com-
pare h. 160.)

Whilst the haemolytic index in 611 increased during three months,
it decreased in 612. The complete disappearance of haemolysines in 612
within two months is demonstrated by the haemolytic index (for horses)
of 0°167 on the 29th November, 1904, and nil on the 2nd February,
1905. These horses clearly show by their parallel treatment the indi-
vidual variations and differences in the appearance, disappearance, and
absolute efficacy of haemolysines,

74

Horses 615 and 624—Both gave a serum with higher haemolytic
activity for mules than for horses. In 615 the haemolytic index for
mules increased. The haemolytic index of 615 was four times higher
than that of 624, whilst the amounts of injected blood were equal ; from
horse 615, however, five times more blood was taken than from 624.

In 627, as in 612, haemolysines disappeared within two months.
Comparative Table.
HORSES INJECTED WITH VIRULENT DEFIBRINATED HORSE BLOOD.

No. of pret es Haemolytic Index.
No. of 3 Auimals from Total crepes Total
os No. of re tween first
Horse. ee which the Amount 7 Amount
Injections. injected blood} Injected endl es Bled :
ie = tale J , Hyperim- , Horse. Mule.
ae EDS munisation.
C.c. Gc.
SH 1 11 11 13,020 123 m. 43,900 0-3 O-4
|
147 — — | 10,050 — — 0-3 0-
160 R 11 oe 12,010 74m. 32,880 0-284 0-5
1724R 9 9 ‘ 9,150 15m. 15,000 0-148 0-563
473 2 1 | 6,000 2 days _ = Ol
545 3 2 | 8,500 4 days 6,000 0-02 0-02
611 NoR 6 2 9,000 3m. 35,500 0-005 0-2
612 NoR 6 2 9,000 3m. 29,000 0-02 0-125
615 NoR rm 2 | 8,000 2m. 35,000 0-125 0.3
627 NoR 3 1 | 4,000 3 days _ -- 0-045
624 3 2 8,000 3 days 7,000 0-045 0-075
CONCLUSIONS.

1. The most remarkable fact is that from eleven horses hyperim-
munised with defibrinated horse blood, the serum of
8 (= 72°77 %) is undoubtedly more haemolytic for mules than
for horses. In one instance the haemolytic indices were
equal, and in the other two cases the haemolytic tests are not
satisfactory enough to make a definite statement.

It appears, therefore, that the organism of horses “avoids”
or is incapable of reacting on the introduction of horse blood
by the production of anti-bodies exclusively specific for
horse blood. We cannot tell whether the isolysines arising
are the same for horses and mules, as the enumerated
experiments do not settle this point. It would have to be
carried out by specific absorption (according to Ehrlich and
Morgenroth),

75

2. The horses SH 1, 147, 160, and 172, showing the highest
haemolytic indices for horse and mule blood, had also been
most frequently injected with the greatest quantities of blood,
but a strict proportionality between the amount of blood
injected and the value of the haemolytic index cannot be
demonstrated. The haemolytic index seems rather to a great
extent to be independent of the amount of blood injected
and to differ individually; for instance, in horse 611, after
having received 4,500 c.c. the serum had an index of 0°166 for
mules. Horse 612, treated in the same way, even with the
blood of the same horses, showed an index of 0°204; the
haemolytic index of serum 624 was only 0:075, though the
quantity injected into this horse amounted to nearly the
double of that injected into horses 611 and 612. On the other
hand, although horses 615 and 624 had both received 8,000 c.c.,
the haemolytic power of serum 615 is three and four times
greater respectively than that of serum 624.

3. The haemolytic index varied considerably in the same horse.
(a) The haemolytic index increased when further injections
were made (horse 611 for mules and horses, horse 612 for
horses, horse 615 for mules and horses).

(0) The haemolytic index decreased, notwithstanding the higher
hyperimmunisation (horse 160 for horses, and horse 612
for mules).

(c) The haemolytic index increased without further injection of
blood (horse 160 for mules).

4. A serum of which the haemolytic index was 13 or lower
could safely be injected into horses; it did not produce
haemolysis in vivo.

; OLXOor OLXOL : ‘ j iz Bai nes
91-0 400-0 ext Bnet colelsz wT 000% colelez 0¢0'F Folzt/oe—"9
gXg gXg ;
8-1 O-l aor or cof 1/s% skBp y 000'¢ <o/ 110% 090'F r0/ztloe—9 868
6X 6 . ‘ cS .
161-0 — ae = Fol I1/9% wy 000'8 r0/6/tz 000'¢ colale—t
ILX IL
6F0-0 _ wee _ FO/LU/TL “WY | 000'8 Folsl1z 000"¢ col@l7—t
; 9x9 9X9 ; = 4 _ 7 i =
688-0 860-0 yee cy +0/6/oz 000'¢ colle—t
: LUX AI Ga X EE -_ = —_ i afin
GF8-0 IZ1-0 BLE ET axe r0lelz 000'E e0/z1/os—e
exe gX¢ ‘ 5 .
O-1 O-T EXE axe | +0/ tog EF ee = 000° eo/z1fos—e
OLXOl OLXOL | , ; :
a is Se | 316%—¢ = — — ; OfG/FL—%
€-% 0 CARAT 3x¢ | €0/6/6¢-9% 00¢'T eol6ff1—e 10¢
6X6
LIT-0 _— ee a Fol LI/9% Wg on0'¢ rolefot o00's colel@—t
IIXI1
€LL-0 — eae aa —- TOLL TIT “Wg 000s tofefot H00'¢ colzle—F
<o LX€
~ 9X9 9X9! . Es ; : ‘ = 3
BL6+() a anaes oe = tof 6/0z wg Folelyt 000'¢ cofzle—t
ae FLXFL 6LX6I re = = = rf ' ne
B0F-0 bFE-0 oer || Fades Folelz 000 0/11
gX¢ exX¢
0 0 = ‘ eufau/1t _— = — oou's eo/LI/1I—'
x x0
LG 8e0 Ty me eofuf9e-6z — -- — 00eT col/FI—z #92
; =a a
“poor “poorq “pooyd “pooyq _ 1804
an as1OH] any 98.10 H *4soy | giana’ siskiouiae *payoalur ‘uorgoaluy 4svT
sis {jour Ey “sul Sisal H Al H JUNO att} Jo :
0 97Rq Onde paid otOJOq TROL ayVp pur coqaun yz eon
*xopuy “yuartgon?) 4 iquNOUTe [VIOJ, | Surpospq yse'T : . # jo
| raquny

‘OMY A UL 4804 SIs<[oWOR]

‘sduLpoo[g

‘uorqustintatwuidod 4}

‘COOTY WIA AALVNIYAIMAC INATAMIA HLIM GaSINOWNIYAdAY SASUOH] JO WAYAY HLIM LSAT, SISAIOWHVY
6 IOL
‘poog any yz sasuoyy fo uoypvsnuuiunriadhyA °F

~I
ba) |

Discussion.

Horse 264.—Throughout all the tests the haemolytic index was
higher for mules than for horses. It decreased for mules in four months
in spite of intercedent injection, increased however for horses, although
the horse was not bled. There still remained a haemolytic power for
mules in the serum ten months after the last injection.

Horse 301.—This serum was also more haemolytic for mules than for
horses, similarly to that of horses 612 and 264. The haemolytic index of
serum 301 for mules and horses decreased in four months notwithstand-
ing 3,000 c.c. more blood had been injected and the horse had not been
bled. The serum still proved to be haemolytic for mule blood ten
months after the last hyperimmunisation. (Compare h. 264.)

- Serum of horse 398 was more haemolytic for mules than for horses.

Comparative Table.

Number of Haemolytic Index.
No. of Number Animals Total Time of Total
He . ~ of from which Amount Hyperimmu-| Amount \
Cre Injections. the blood Injected. nisation, Bled. ase: Mule.
was taken.
264 R 2 — 1,500 1 month. 0 0-28 0-56
3 _ 3,000 3 months. 0 0-349 0-408
301 2 2 1,500 2 months. 0 0-4 2-3
NoR 3 3 3,000 5 months. 0 0-121 0-854
398 6 2 4,050 1 month. 23,000 0-005 0-15
CONCLUSIONS.

1. Three horses hyperimmunised with defibrinated virulent mule
blood gave a serum with stronger haemolytic properties for
mule than for horse blood.

2. In two cases the serum preserved its haemolytic power for
ten months.

3. The haemolytic index (a) decreased in spite of further blood
injections for mules and horses (301), (6) increased. with
regard to horses, decreased with regard to mules within four
months, in which time one injection took place (264).

4. The haemolytic index is again not dependent on the quantity
of blood used for hyperimmunisation and the number of
injections.

os

. OLXOI | OIXol 2 4 F
0-1 O-F €o/6/9% — — — Oslé eole/zi—'s 10
GIXL | OF XO1 ole! ,
6X6 |
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OLXF | yu!
IIXI0
LE1-0 = —— = OfIT
ea iiail|
BIT et | OL olalvg
ea ‘9X9 = = = — golsiai—'s 661
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| ZLXg
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8 ae | (6)
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sis{[outoe Ey “SULIT EA ea aaL aIOZoq i Wei07, ayy jo ‘om
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Jaquiny
‘OAQLA UL 489} SISATOUTOR YY *sSULpaalq “torjyestanumMad APT

‘doOTg ASUOH GULVNIUMIG( LNATOYIA HLIM CUSINANWIYGdAH SHIA JO WOYAS HLIM LSA, SISATONAV EL
€ OUD
“pool” as“oxy ypim sappy fo vorpsriaiesadhzy °e

79

Discussion.

Mule 150.—The serum was more haemolytic for mules than for
horses. The haemolytic index for mules decreased in 134 months to }
of the original value ; or perhaps the haemolysines disappeared and arose
again after a further injection. They increased even within 14 months
although no injection took place.

Mule 199.—The serum was more haemolytic tor horse blood. The
index for mules decreased first and then increased fifteen months after
injection.

~Mule 201—The serum of this mule dissolved blood corpuscles of
horses much more energetically than of mules.

Comparative Table.

Number of Haemolytic Index.
No. of Nov uf Animals Total Time of Total
Mul tnzestions from which Amount Hyper- Amount
He mY : the blood Injected. |immunisation. Bled.
was taken. Horse. Mule.
150 8 8 7,550 9 months 0 1-7 2-07
— 9 9 9,550 11 a 6,000 _— 0-264
199 8 8 9,000 3 is Oo | 1-3 1-12
201 8 8 9,150 — O | 40 1-05
CONCLUSIONS.

1. Of three mules treated with horse blood two gave aserum with
stronger haemolytic properties for horse than for mule blood,
the serum of one of them, however, dissolved mule blood
better than horse blood.

2. The haemolytic properties of the serum of one mule were still
present fifteen months after the last immunisation, they
seemed to vary in strength during this time.

80

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x
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FLX #1] 91 X 91 ; ;
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x
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by RD, i ‘
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x
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0@ X 02 ; ‘
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8 X 8 | ¢L xX &r ‘ at
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y
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SIsATOUTOV PT uLing d aa ayy jo P
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TIC UINN
OIYLA UL 48aq sIsA[ouav “SBULpooTg, “uorpestmumtutiad 4 A

|

‘dOO1g WINN GALVNIUMIGGG INATAUIA HLIM GHSINOWWIUGdAY SHIN JO WOUTY HLIM LSA, SISATONATV EY
F 299L
‘poog any yun sang fo woynsiunwurwadhy “$

81

Discussion.

Mule 320.—The blood dissolving properties of this serum were
stronger for mule than for horse blood. Notwithstanding two further
injections, the haemolytic index fell within five months. Serum 539
was already haemolytic before the hyperimmunisations were started.
After eight injections the serum was.more destructive to horse than to
mule erythrocytes.

Sera 552 and 573 also dissolved mule blood cells before the immuni-
sation, afterwards the isolysines increased, acting more energetically
on mule than horse blood.

Comparative Table.

Number of . 7
N ¢ | Number of [Animals from Total Time of Total Haenply te Inaex:
Mole T oy a Be which the Amount Hyper- Amount

i BISCHONS: blood was Injected. | immunisation. Bled. ; Mul
taken. Horse. ule.

320 2 2 1,500 1 month. 0 0-72 0-84
a ¢ + 5,000 ~ 5 months. a0) 0372 0-015
539 8- 2 12,350 24 months. 31,000 0-234 ~ 0-092
552. 9 2 10,150 24 months. 25,000 0-188 0-196
573 7 2 10,500 23 months. 20,000 0-597 0:75

539, 552, and 573 were injected with the blood of the same mules and on the same dates.
CONCLUSIONS.

1. Three of the mules injected with mule blood hada serum more
haemolytic for mule than for horse blood.

2. The sera of three mules dissolved mule blood before hyper-
immunisation. These isolysines were probably provoked by
the previous serum and virus injection.

82

ay} eaoge paseerour ATEyeIpeuIMT ynq ‘yIUOTT oO SuTMp A[qQuiepIsu0D pesvetoep comod oyATOUIORy eT,

‘MoMelUl poo[qd LeyAouR Jee oN[VA TVULSTIO-

‘soynu Jo ueyqyL

sasIOy JO Seposndaxod poorq ey} 40F coSu0rs APUoTedde ‘wmnaes orATowovy ATqvyaeutor B avd gQE AeyYUog—uorssnosigy

x 7 .
= 688 °% ma = : FOlT 1/92 UH eT 000'8 eolatis 0g8'é solotle—sa
Il X It ‘ ;
GLB i ee ee = FOlIT/ IT “Wl §T 000'8 eo/at/e 0g8'Z eolotle—"a
P OL X OL | OL X O1
wer “Tt |x or | er x or, — 80le/9e
9X9
== ] . — 7
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; az X 2% |_1s X 1%
9ET°S 886-3 [Oa x ae | eX Iz] eoleler
. o . ‘oO OO
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Taq WAN
‘OTYLA UL 4809 sIsAToMOey ‘sduIpsoTg “moestunututtiied 4 Fy

‘doolg aSuoy
CULVNIVAIMAG INAIOULA HLIM GASINONWIYAdAH ATNNOG BNO AO WAUAY HALIM LSA], SISAIOWHVYL

°G 89OL
‘poojg asuoH yun fayuog v fo uoynsiunuuradheT *¢

83

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0 91-0 2 ~ 2 2 . 7 Fols/¢z — — ~ OOU'ST r0l2/[91—'s
862-0 199-0 a 7 F * i polt/6z = — — oor'st | F0/zl91—s GLE
69-0 eee = : : * : Fol6/oz "WZ 0006 s0l6/s
. to = : : : volsiee sae = — 000'81 r0l9lF1—2 oF
6IF-0 | 929-0 it x _ a x a r0/9/08 = = -
68-0 0 ; z : z ; 2 B0l6/0z "mg 000'8 +0/6/8
0 0-0 : * : : ~ ; rolslee a a = - OOFFL rolz/cI—"6 elF
x
szt-0 | g00-0 4 . A = - Fo/L/6z — = = |
‘pooig | “pooq | “poorq | -poorq hes =
an asloFT any PSIOH |-4904 sishouoeTy sence | 2 ore “4804 coogi -poyootur pias 48P]
“xopuy “quaryong oe . raetan ete Suypooyq ysory [MNOUN TOL d7eP PUB TOUT NT ee
Toquinyy
‘OLJLA UL 4804 stsA[ouOR “sBUIPEoT ‘uorestunuuttrod «7

‘NOYES ASYOH LNAINUIA HLIM CASINOWNIYAdAH SHTAW JO WOUAY HLIM LSM], SISATIOWAVY
"9 49OL
‘WINNS AS4OFT YPN sony, fo uoynsiunuumreadhzT *T

‘WOH INATOWIA HLIM NOILYSINQWWIYddAH (q)

84

qx qo | ox | ‘ =
SF-0 #0-0 | F0ls/¢e — — — OOF ST FOls/L—"9 Z8r
9x¢|txt isl :
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0 LX I
80-0 20-9 |—t - us Bs * TL | yols/ee — a _ 00081 Folsi9—¢ ¥9F
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g¢ xX g @¢x¢ ‘ ete
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0 seo-o | “ i “ I) so/e/at zs = = o00'oT rolgle—s 89g
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rm ; “4894 ,
aT OSOTT OTN OSLOH 1 P iss) et eae *poqoolut mop So aT FEM
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L MOL

‘WNL anh, yun saynyy fo uoynsrunwuunwadh *Z

Comparative Table to Table 6.

[
No. of Number Number Total cu Total | peal ate.
Mule of of Amount Hyperimmn- Amount |
Injection. Animals. Injected. Hisations Bled. Horse: Mule.
i |
413 9 7 14,400 2 months 0 | 0-008 0-128
415 7 6 12,000 ye 0 | 0-576 0-419
479 8 7 18,100 22 ny 0 | 0+561 0-298

Hyperimmunisation of mules with horse serum still produced
haemolytic serum, which in two instances was more active on horse

than on mule blood.

Comparative Table to Table 7.

? Haemolytic Index.
No. of Number of | Total Amount ee of Total Amount.
ane - yper-
Mule. Injection. Injected. ftimmuniaatio Bled.
: | Horse. Mule.
368 8 10,000 5 months 0 0-033 0
4145 7 13,800 ' | 3 months — 0 | (8-0) (1:08)
—_ 7 13,800 3 months —_— -- 0-798
464 5 12,000 3 months 0 0-074 0-033
482 \ 6 15,400 5 months 0 “(0-04) (0°48)

Mules treated subcutaneously with mule serum produced
haemolytic serum, which in two cases dissolved horse blood better

than mule blood.

86

3. Hyperimmunisation of Donkeys with Horse Serum.

Table 8.

HAEMOLYSIS TEST WITH SERUM OF DONKEYS HYPERIMMUNISED
WITH VIRULENT HORSE SERUM.

Hyperimmunisation. Bleedings. Haemolysis test in Vitro.
Nun-
ber Tact Total
of | Number and! Total bleedi amount Date of Quotient. Index.
Ani- | date of the | amount eee bled up to| Dur- | Hae-
mal, last injected. Haemolysis| ing. | molysis
Injection Haenie- test test Hi Mul H Mul
jection. lysis test. : est. Lorse. ule. orse.| Mule.
cc. c.c.
381 | 7.—14/7J04 | 7,900 | — = ee Sa ee
6 xX 6 7X7
mle | 7900) — es: 2. | peta ee eT ys See
4x 4 5 x 5
7—14/7/04 | 7,900 | — = ee (Tinian
11 x 11
7.—14/7/04 | 7,900 | — = — |2efiijos| = — Sx P| | gage9
9x9
6 x 10 5 xX 7
8.—14/7/04 8,700 — a = —/7/04 1-667 | 0-714
514 {7 (0S ag ee
8.—14/7/04 | 3,700 | — = = | opigoa|_ = 8 | 8 By gy. | ong
4x4 5x 5
Comparative Table.
Number Total Period of Total Haemolytic Index.
No. of No. of :
of : Amount Hyperim- Amount
Dalkey Injection nas: Injected nisation. Bled
J : a ‘ Horse. Mule.
381 7 7 7,900 23} months 0 (3:0) (3°571)
514 8 6 8,700 Dk gy 0 (1'667) (0°714)
CONCLUSION.

Two donkeys injected with horse serum developed isolysines in
their blood for horses and mules.

mules in 381 decreased to the half of its value within fifteen

days,

The haemolytic index for

87

[
9 Xx x
0 0 2 és ae “ : FOl6/0z “mt g 000'9 Fol6ls
¢xX¢ | xX #F
: €90-0 z = es a
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€L_X €1} 3E X ZI
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; qx ¢ | tx F = _ =!
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rs
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el X €t | Zt X sr
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pul qu9T4 qunoure Tez07,| BUPA ase

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6 AIL

HLIM GHSINOWWIYRdA SAGUNOG fO WOYRS HIM LSA. SISA TONEY

“UNAS aN YqIN Shayuog fo UounsIUnNUWUIadhA ‘F

88

Discussion.

515 and 516 were injected at the same time and with the same
serum of the same mules (516 had’ one injection more). The haemolytic
index in 516, however, appeared to be much lower.

CONCLUSION.

The sera of all three donkeys injected with mule serum were
haemolytic for horse and mule blood.

89

q¢X¢ | ¢xe :
0 0 A 7 o/s/2z = — = 000'6 0/8/6—"F 609 9
gxX¢ gq xX g ,
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Tequin jy
‘OITA Ul 989} sIsAJouLNB ET ‘sd UIpaa[q “woryestunmuttiedé

‘(GLVaGOXG IVENOLIBag) ZTAW
WOU (9) ‘ASHOH WOUd NOTIINOG (Y) HLIM CHSINOWWIYTdIAY SHTAW a0 WOW HLIM SES], SISAIONAY

‘OT F79OL

‘NOTTINOG INGINUIA HLIM NOLLVSINOQWWIUGdAH (2)

9()
CONCLUSION.

One mule treated with peritoneal exudate of horses and another
injected with peritoneal exudate of mules showed haemoly-
sines in their serum, thus proving that it is possible
to produce isolysines by subcutaneous injection of peritoneal
liquid.

SUMMARY OF RESULTS FROM SUBCUTANEOUS INJECTION OF BLOOD,
SERUM, AND PERITONEAL EXUDATE OF HORSES AND MULES INTO
EQUINES.

The above experiments demonstrated that in every instance isoly-
sines for horses and mules arose.

(1) In horses after injections of horse and mule blood.

(2) In mules after injections of horse and mule blood and serum
(except 368 for mules).

(3) In donkeys after injections of horse blood and serum and
mule serum.

It was also possible to obtain isolysines for horse and mule blood in
mules by subcutaneous injections of horse and mule peritoneal exudate.

It stands to reason that horses would also produce isolysines for
horses and mules after injections of horse and mule serum and probably
of peritoneal exudate as well.

These experiments give no indication how soon the isolysines arise,
but they allow of a conclusion regarding the length of time a serum
preserves its dissolving properties. Generally they were present up to
six months after the last hyperimmunisation. In three instances
(horses 611, 612, and 627) the serum was no longer haemolytic after two
and two anda half months respectively, but in two cases (horses 264 and
301) it dissolved mule blood ten months, and in one case even fifteen and
a half months after the last injection (mule 199).

In the majority of cases after the injection of mules with horse
blood, the isolytic serum was more haemolytic for horse blood.

In the majority of cases after the injection of mules with horse
serum the isolytic serum was more haemolytic for horse blood.

In 50 per cent. of cases after the injection of mules with mule serum
the isolytic serum was more haemolytic for horse blood.

After the injection of a donkey with horse blood the isolytic serum
was more haemolytic for horse blood.

In the majority of cases after the injection of horses with horse
blood the isolytic serum was more haemolytic for mule blood.

In all cases after the injection of horses with mule blood the isolytic
serum was more haemolytic for mule blood. :

Of

It appears, therefore, that the injected animal avoids producing
isolysines against the blood corpuscles of the variety to which it
belongs. The same phenomenon could be observed after the infusion
of mules with mule blood, but it did not take place after the infusion of
horses with horse blood. The best isolytic serum, for instance, for horse
blood was therefore obtained by injecting donkeys or mules with horse
blood. The tables support this idea, for the haemolytic indices were
indeed highest of the sera of one donkey, and of mules treated with
horse blood it was also seen that injection of defibrinated blood provoked
a more intensive production of isolysines than serum alone. The blood
corpuscles are of course the best carriers of haemolytic, i.e. isolytic,
antigenes.

The haemolytic power of a serum is toa great extent not dependent
on the quantity of blood or serum injected, nor on the number of
animals the antigenes were derived from; the number of injections
and the total time of hyperimmunisation make no difference. Bleed-
ings have apparently no influence on the haemolytic index.

For the rise of isolysines in an animal it is not necessary that it
reacts with hyperthermia on hyperimmunisation.

ANIMALS HYPERIMMUNISED BY INFUSION.

Hyperimmunisation by infusion was done by connecting the
jugular vein of the animal which is suffering from horse-sickness with
the corresponding vein of the immune animal which has to be hyper-
immunised. This method is simple and expedient, and it was usually
continued for six minutes. Measured through a trocar and the pipes
in use, about half a litre of blood passed through in a minute. There
were usually three infusions made into one animal on two or three
succeeding days.

92

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The various haemolytic tests of the serum from the infused animals
Were made on the corpuscles of tive horses and five mules. Whilst
from the horses. mules, and donkeys. treated subcutaneously with blood
and serum, all gave rise to isolysines in their serum, there were
amongst the infused horses only 37°5 per cent., amongst the infused
mules 43°35 per cent., with isolytic serum. The transfusion proved to be
the best method of hyperimmunisation against horse-sickness, hence it
was chosen for the future.

The majority of the sera of the various horses infused with horse
blood, and mules with mule blood, was more haemolytic for horses than
for mules.

There were a greater number of mule sera more haemolytic for
mule than tor horse blood corpuscles.

The question arises whether the greater haemolytic index of the
majority of horse and mule sera for horse than for mule blood is due to
a prominent sensibility of the horse erythrocytes or whether there are
in an isolytic serum two different isolysines for horse and mule blood.
If this latter should be the case the isolysines for horse blood would be
in @XCess.

The quantities of isolvsines in the various horses and mules after
immunising by subcutaneous injection or infusion are quite different.
There again seems to be no relation between the number of injections,
the quantity injected or the number and amount of bleedings and the
haemolytic potence of the serum.

One would think that the individual properties of the blood used
for injection or infusion have an influence on the quantity of the
produced haemolysines : but it was not possible to find any quantitative
sinilarly in the haemolytic action of the sera of those animals which
were injected with the same blood. We must therefore consider that
the main factor ruling the quantity (and quality) of the isolysines is the
individuality of the injected animal.

Temperature reaction of an animal after hyperimmunisation would
be an expression of its sensibility; it has to be taken into considera-
tion that the degree of hyperthermia might be an indicator for the
production of antibodies, in our case isolysines. Indeed, it was found
that the majority of animals showing a temperature reaction after in-
fasion and the minority of those not reacting contained isolysines in
their blood : but at the same time a considerable number of sera derived
from horses or mules showing thermo reaction was not haemolytic,
whilst some of those without rise in temperature was haemolytic.

These facts proved that fever reaction after hyperimmunisation is
not always a sign of the production of antibodies and that immune
substances also arise, though no alteration of temperature followed the
injections.

o

EXPERIMENTS WITH SERUM MIXTURES.

Since we know that the immune substances are all colloid systems,
it is a priori not to be expected that the final haemolytic effect of a
mixture of various isolytic sera should correspond with the calculated
haemolytic index; or with other words: the final effect of a combina-
tion of several haemolytic sera must not be simply the sum of the
individual effects of the components, because the xera—though being
colloids of the same electric character and belonging to the same
species of animals—influence each other. They form colloid com-
pounds with new peculiarities.

Cernovodeann and Henri* found that the haemolytic effect of a
mixture of two different sera is more than double the calculated sum
of the individual effects of the components. The experiments I
wish to communicate here were all made in the same manner,
namely: To 2 «cc. of the mixture 1 cc. of defibrinated blood was
added. The haemolysis tests of the components and the respective
mixtures were made under the same conditions, the same day, and
with the same kinds of blood.

Thus it is possible to compare the haemolytic index of the mixture
as it is given by experiment with the calculated haemolytic mixture
index which is the quotient :

Sum of Haemolytic Indices of Components.

Number of Components,

As the mixtures were made with equal quantities of serum, the
calculated index gives the mathematical sum of the haemolytic effects
of the components.

Example.—Three sera with the indices 0-2, 0-4 and 0-6, mixed
together (2 c.c. each) would give a calculated haemolysis 1-2 with a given
quantity of blood. Since only 2 c.c. of the mixture are added to a
constant quantity of blood, the actual quantity of each serum is only

2 of 2cc. and the calculated haemolytic index would be 2-0-4.

* Comptes Rend. Soc. Biol., No. 11. 1905.

100

Table 13.
EXPERIMENTS WITH SERUM MIXTURES.
Haamolvtis: ladex of No. Percentage of animals
No. Haemolytic Index. esd ean of animals suffering from
ee injected. Haemolysis.
—_ 1
Horses. Mules. Horses. Mules. Horses} Mules.| Horses. Mules.
Horses. Ist Mixture.
SH 1 U+137 0-391
160 0-537 0-5 — —_ —_— 20 — 60
172 0-148 0-563 (died)
Calculated
Mixture | Index 0-274 0-458
Horses. 2nd Mix ture.
160 1-333 0-555 Lon .
172 O-111 Odd ua pias
C.M.1. 0-722 0-499
0-555 0.444 J 1 0 100
C.M.I. 0-562 0-701 (haemo-
264 0-77 0-972 -66 1996 ae
301 0-028 0-833 os Less eee)
C.M.I. 0-402 0-902
Horses. 8rd Mir'ture.
+73 — 0-102 = 1-12
615 — 0.061 —
C.M.I. _— 0-08
Horses, 4th Miature.
611 0 0-166
612 0-167 0-204 0-049 0-54 1 1 0 1)
627 0-056 0-045 he a
CMI. 0-074 0-138
Horses. 5th MVirlture.
611 0-005 Q-2
612 0-02 0-125
615 0-125 0-3 , :
624 0-045 0-075 ell! le = ‘ == C
545 0-02 | 0-02
398 0-005 O15
CMLL. « 0-037 0-145
Mules. 6th Mirlture.
B68 0-028 i) )
413 0 : 0-25 2
415 1-333 0-694 j pe = ] 1 0 0
464 Q-111 0-111
C.M.I. 0-368 24
Mules. ith Mix ture.
462 — | 0-036 )
463 — ()- 123 - — 0-061 oa 19 <= 0
468 ae aor7 |) =
C.M.TI. = 0-079
Mules. sth Mirture.
448 _ 0-056
462 0-25 0-110 | ae ;
463 0-03 0-110 x pas | Ter | Q 0
468 0-08 oto |) |
CoM.I. >v 0-097 i
Mules. Oth Vie ture. ;
448 — 0-056
462 O25 0-110
463 0-03 0-110
468 0-03 0-110 95 ‘ <
Horses. ee babies 9 0 0
611 ) 0-166
612 0-167 0-204
627 0-056 0-045
C.M.I. — 0-114

101
Table 14.

TABLE OF REAL AND CALCULATED HAEMOLYTIC INDICES
OF SERUM MIXTURES.

Haemolytic Indices of Mixtures.
No. of Mixture. a
Real 1. Calculated I.

2 0°25 < 0°722 Horse.
Orde < 0-499 Mule.
0°666 > 0°402 Horse.
1:333 > 0°902 Mule.
0°555 < 0°562 Horse,
O44 < 0-701 Mule.

3 112 > 0°08 Mule.

+ 0-049 < 0-074 Horse.
O54 > 0-138 Mule.

5 0°02 < 0-037 Horse.
O14 < 0145 Mule.

6 O111 < 0-368 Horse.
0°25 > O24 Mule.

7 0-061 = 0-079 Mule.

8 Oy: < >0O Horse.
0-008 < O97 Mule.

9 O11 < 3114 Mule.

The haemolysis produced by a mixture of sera is in about two-
thirds of the cases smaller than the average of the individual effects, in
contrast to the results obtained by Cernovodeann and Henri.

We have to admit that the isolysines produced by various animals of
the same species are different colloids and that they influence each other.
It is a well-known fact that colloid combinations have an optimum. of
efficiency only if mixed in certain proportions, characteristic for the
nature of the components. It is possible that isolytic sera would also
show maximal effects when mixed in optimal quantities, the proportion
of which evidently is not 1:1 asin the abovementioned experiments.
But as the sera were mixed in this proportion, very likely not being the
optimal one the maximal effect could not be obtained from these mix-
tures. This might explain the variations in the differences between
real and calculated haemolytic index and the absence of a constant
mathematical relation between both.

102

On the other hand, it must not be forgotten that quantitatively a
reduction of a haemolytic substance does not mean a direct proportional
decrease of its blood dissolving effect, as experiments of Cernovodeann
and Henvi* with serum and myself* with saponine have proved.

The diminution of haemolysis is at first very rapid and becomes
slower and slower as the haemolytically active substance decreases.
Therefore an equal quantitative reduction (for instance to the half) of
two sera of different haemolytic strength involves not a proportional
reduction (to one-half) of the effect of each on the same quantity of red
blood corpuscles, and the haemolytic result of a mixture of the two
halves of these sera need not be the mathematical average of the
individual effects.

Tam not able to say which of the above given possible explanations
holds good, but certainly they both might be applicable. It is, of course,
not permissible to draw conclusions from the calculated mixture index
with regard to whether a serum mixture will produce haemolysis in
Vivo.

(Compare indices and results of injections of mixtures 1, 2, and 4.)

II.—CLINICAL SYMPTOMS OF HAEMOLYSIS.

The chief symptoms can be forecasted if it is kept in mind
(1) what is the function of the living erythrocytes
(2) what happens with them, when they are dead
We record, therefore,
(@) symptoms due to the deficiency of red blood cells ;
(b) symptons due to the elimination of the destzoyed erythrocytes.

Naturally the symptoms mentioned under (@) appear first ; not
immediately following the injunction of the destructive serum, but
after a certain incubation. This is sometimes, after intrajugular injec-
tion, very short—in a few cases only about five minutes, in others afew
hours; it is longer, according to the resorption of the serum, after
subcutaneous injection, varying from 1-3 days. After this time the
decrease of the number of erythrocytes and icterus are the most striking
symptoms, whilst the predominant phenomena which follow immedi-
ately an intravenous serum injection are mostly troubles of respiration
and heart action. Under normal conditions the organism requires a
certain number of erythrocytes which are the carriers of a defined
cunount of oxygen from the lungs to the tissues which is wanted by the
latter. As the frequency of pulse and respiration largely depends on
the extent or intensity of oxygen metabolism in the cells, a numerical
decrease of red blood corpuscles, as it happens after haemolysis, con-
sequently emphasises itself by acceleration of respiration and heart
action, the haemaglobine dissolved in the plasma being unable to
transmit oxygen. Both symptoms sometimes take place very soon after
intrajugular injection of isolytic serum. In such instances the horse

* Loco cit.

108

falls, breathing rapidly with wide open nostrils, the head being stretched
straight forwards. The pulse is weak and very quick, tremor of muscles
may be noted, probably due to the sudden alteration of oxygen
metabolism.

I observed quite similar svmptoms after infusion of distilled water
into the jugular vein, whereby haemolysis by sub-normal osmotic
pressure (hvpotony) is caused.

Acceleration of pulse and respiration are well-known symptoms
in piroplasmosis of horse and dog, and human medical science records
inclinations to forced vespiration of patients suffering from anemia.*
Haemolysis is, of course, the principle of both diseases.

Corresponding with the progress of artificial haemolysis and
anemia the animals lose in condition from day to day and become
weak, as a result of which they frequently lie down. The mucous
membranes are pale, on account of oligocythaemia and yellow on
account of icterus ; the same is the case in piroplasmosisx. Sometimes
it was observed that a horse suddenly, during or immediately after
intrajugular injection, fell down, arose after a few minutes and could
walk round ; the next day, however, was found lying on the ground
unable to rise, and so on, corresponding with the periodical appearance
and disappearance of icterus and red urine.

I want to draw attention to the fact that paralysis and loss of
control of the hind quarters was found in some cases of haemolysis due
to isolysines, sometimes in piroplasmosis of dogs and in haemoglobin-
aemia (lumbago) of horses and cattle. This analogy points to a unitary
cause in all three instances—haemolLysis.

Rise in temperature during an attack of haemolysis was recorded
for horses. Fever was also observed in human beings after transfusion.

Blood examinations were made by means of three methods, which
gave indications on:

(1) Number and shape of red blood corpuscles.
(2) Viscosity or internal friction of the blood.

The nuiniber of erythrocytes (obtained first by counting with Zeiss-
Abbe apparatus) shows intensity and rapidity of the action of the
isolvsines. The destruction commences on the first day, or sooner or
later according to the haemolytic strength of the serum. It is not
always continual but shows periods of increase and decrease. The
number of erythrocytes gives no prognostic indication as to exitus, for
an animal might die in spite of a but very slight decrease of the quantity
of the red blood corpuscles ; on the other hand, an animal can recover
though its erythrocytes had diminished in number to 3. The eventual
exitus lethalis does not always occur when the number of blood cells is
ata minimum: in some instances it followed after the number had
increased again. (Compare examples given later.) Hence there must
still be some other factors as cause of death.

* Kraus, Lubarsch-Ostertag, Ergebu. 3, 416, 1896.

104

Lately, instead of counting the red blood corpuscles their volume
percentage was used (by means of haematocrit and centrifugalisation) to
find the number of erythrocytes, which, as numerous comparative

a - Vol
experiments have proved is 73

As the wiscosity of blood is chiefly due to the amount of blood
corpuscles contained in it, the measurement of the internal friction
also gives an idea of the degree of destruction, and series of figures
obtained from blood examinations on horses suffering from isolytic
haemolysis, piroplasmosis, or artificial anaemia demonstrate that the
viscosity of blood is subnormal and goes parallel with the volume
of red blood corpuscles.

There is no doubt that physico-chemical researches on the serum
of animals showing haemolysis from the injection of haemolytic serum
would have brought forward results similar to those we observed in
piroplasmosis in horses, namely. alterations of specific gravity, electric
conductivity, depression of freezing point, internal friction, capillarity,
etc., because the products of the destruction of erythrocytes circulating
in the plasma for a certain time influence the osmotic concentration and
the structure of the colloids of the serum.

The main factor producing the second group of clinical systems is
the haemoglobin.

Haemolysis means not only a diffusion of haemoglobin but also ot
salts out of the stroma through the membrane of the red blood cor-
puscles. These three residues, stroma, haemoglobin, and salts have to be
eliminated out of the blood. The organs which come in consideration
for this function ave: 1, liver; 2, kidneys; 3, spleen ; 4, bone marrow.

Erythrocytes which have been killed by any haemolytic substance
are phagocytosed, and those still containing haemoglobin are deposited
in the liver. The haemoglobin dissolved in the plasma is also kept
back and carried by the circulation. The haemoglobin is then decom-
posed: one part of the products which contains the iron (haemosiderine
iron-albumine) is either taken off by phagocytes and deposited in liver.
spleen, and bone marrow (Biondi*) or discharged in the urine. The
part free of iron is the basis of the bile pigments (bilirubine and bili-
verdin) which are sent partially into the duodenum, whilst the other
part after a further chemical process (bilirubine-urobiline) goes through
the kidneys into the urine. Such is the case already under normal
circumstances. (Several authors found in cases of haemolysis an in-
crease ef bile pigments.t)

When the accumulation of haemoglobin and the production of bile
pigments has reached a certain supernormal degree, a greater or smaller
amount of bile passes over—by a mechanism, unknown at present—
into the blood (cholaemia). These (colloidal) pigments are absorbed by
the tissues and thus give rise to the symptom called icterus (conjunc-
tiva, mucosae). The presence of bile pigments in the blood is soon

* Ziegler. Beitrage 18th Jau., 1895; cit. Schmidt, Lubarsch-Ostertag, Ergebu. 3, 542, 1897.
Tt Oswald, Chem. Pathalog. Leipzig, 1907, 127.

105

followed by excretion of a part of them through the kidneys. (Choluria,
bilirubinuria.)

If the destruction of blood corpuscles proceeds or increases the
liver is unable to decompose the entire quantity of haemoglobin which
it receives, and so the surplus of this latter goes, as it is, into the bile
and is mixed with the faeces. Then a further increase of the haemolysis
emphasises itself by haemoglobinuria followed by renal symptoms.

In cases of (haemolytic) icterus, simultaneously with the bile
pigments, salts of the bile acids—especially sodium glycocholicum and
tauro-cholicum are also in the blood.

As the latter salt itself is able to destroy blood corpuscles, icterts asa
symptom is at the same time a pathogenic factor of haemolysis, under
the supposition that the concentration of these salts in the blood is high
enough, which is perhaps seldom the case. More important, however,
is the influence of taurocholic salts on the heart and on the kidnevs,
a protraction of frequency being one of the effects on the former.
After all, the icterus influences the liver itself, and haemolytic sera are
also able to affect liver cells.* As the “internal” function of this organ is
enormously important, specially for the nitrogen and hydrocarbonate
metabolism and the destruction of poisons, interruption or disturbance
of the normal liver action influences the health of the entire organism.

Haemoglobin which is not retained in the liver, bile pigments and
salts of bile acids circulating in the blood, have to be eliminated by the
kidneys and thus give rise to a complex or symptoms due to affections
of the kidneys: (1) haemoglobinuria, (2) albuminuria, (3) choluria,
(4) haematuria. 4

(1) Albuminuria was found in slight cases of haemolvsis as a
consequence of the increased blood destruction. The most striking
and common symptom of haemolysis (besides jaundice) is the appearance
of haemoglobin in the urine. Subsequently the latter becomes stained
from a slight to a deep dark red colour. :

If the inflammation of the kidneys is acute, epithelial cells may be
found in the urine, and a more intense destruction of them (1 siti is
followed by the escape of blood cells into the tubuli (haematuria). In
such cases the quantity of urine is very small (anuria, uraemia), and
death is the general exitus. (Compare the following examples.)

Clinical symptoms resulting from the affection of spleen and bone
marrow have not yet been recorded.

The leading idea for the above classification of the symptoms of
haemolysis is the physiological course of life, function, and death of a
red blood corpuscle. It enables us to derive the following symptoms
of intensity and chronological succession of the clinical symptoms of
haemolysis, which are in accordance with the recorded facts :—

1. Very slight destruction of erythrocytes. Very small decrease
of their number and volume is the onlysymptom. (Eventually
slight albuminuria).

* Kretz, Lubarsch-Ostertag, Ergebu. 8, UI, 495, 1902.

106

i)

. Considerable haemolysis and decrease of the number of
ervthrocytes. Super-production of bile. Transmission of
bile pigments into the blood, icterus.

. Strong haemolysis (when more than ¢y of the haemoglobin of
the ‘body i is dissolved in the plasma) elimination of haemo-
globin through the kidneys: Red wrine.

Deaths very frequent.

4. Enormous haemolysis, kidneys strongly affected ; red blood

cells in the urine ; haematuria, oliguria, anuria, uraenia,

eritus lethalis.

os

EXAMPLES.*

Mule 356.—Injected on the 13th November, 1903, with a mixture of
different sera.

14/11/03, number of blood corpuscles, g; 100,000.

15/11/08, e 2.640, 000 Red Urine.
16/11/03, 2 <i 2.560, 000 as
17/11/08, ” . 3,366,000 i
18/11/03, s e 4,560,000

19/11/03, ss si 4,860,000

20/11/03, za - 4,246,000

21/11/08, ri 100,000

Mule 356 died on the 21st November, 1908. This animal suddenly
showed an alarming dyspnoe, accompanied by a whistling and
trumpeting sound. It died within an hour after these symptons had
started.

Horse 348.—Injected on the 20th November, 1908, with a mixture of
different sera. The count of red blood corpuscles was as follows :—

19/11/08, blood count, 6,020,000 per m.m.

20/11/03. : 6,332,000
TLIO, ; 4,706,000
22/11/03, ; 3,820,000

There was a distinct jaundiced condition of the mucous membrane
of the eves, on which several blood spots were also noticed. Red urine
was voided.

23/11/08, blood count, 8,006,000 per m.m.
24/11/03, i 3,906,000
25/11/03, 7 3,966,000
26/11/03, ss 3,892,000
27/11/03, is 4,632,000

Urine was again clear; Esbach’s albumen test gave a strong pre-
cipitation.
28/11/03, blood count, 4,126,000
29/11/03, be 4,612,000
Albumen was less in the urine than on 27/11/03.
30/11/03, blood count, 5,552,000
1/12/03, “i 4,320,000

* Copied from Ann. Rep. Govt. Vet. Bact., 1903-04,

107

: The urine was again red and contained a large amount of albumen.
The horse had symptoms of colic; it lay down at intervals.
2/12/08, blood count, 3,906,000 per m.m.*

3/12/03, : 4,846,000
4/12/03, : 5,066,000
5/12/03, : 5,152,000

Urine was again clear from the 3rd December, 1903.
6/12/03, blood count, 5,050,000
This animal recovered.

Horse 360.—Injected on the 20th November, 19038, with a mixture
of different sera.

19/11/03, count of red corpuscles, 7,650,000 per m.m.*

20/11/03, , . 7,416,000
21/11/03, : : 7,320,000
22/11/03, " 2 6,826,000
23/11/03, ' 6,612,000

A slight jaundiced condition of the mucous membrane of the eyes
was noticeable. ©
24/11/08, count of red corpuscles, 6,494,000
25/11/08, a + 6,850,000
Red urine was noticed since the day previous.
26/11/03, count of red corpuscles, 7,080,000
Urine had cleared up since previous evening, but still contained
albumen.
27/11/03, count of red corpuscles, 5,646,000
Urine still clear.
28/11/03, count of red corpuscles, 5,906,000
The urine of the previous evening was red ; of this date (morning)
it was dark brown, containing much albumen.
29/11/03, count of red corpuscles, 7,060,000
The urine of the previous evening was again red.
30/11/08, count of red corpuscles, 7,080,000
The urine was clear and contained but little albumen.
1/12/03, count of red corpuscles, 6,852,000

3/12/03. i : 6,494,000
4/12/03, : ‘ 6,180,000
5/12/03. : 5,086,000

The horse died on the 5th December, 1903.

IV.—PATHOLOGICAL ANATOMICAL LESIONS OF HAEMOLYSIS.

According to the clinical symptoms, we distinguish among the
pathological anatomical lesions of haemolysis :
1. Alterations of the blood.
2. Alterations of spleen, bone marrow.
3. Alterations of the liver.
4, Alterations of the kidneys.

108

1. The Blood.

After every serious attack of haemolysis the blood is watery and
thin, thus showing the loss of corpuscular elements. Sometimes it is
found well coagulated and in other cases not. In the former instances
the serum is red stained by the haemoglobin dissolved in it, and so is
the plasma clot when the corpuscles are deposited before coagulation.
In cases of cholaemia (and general jaundice) plasma clot and serum have
wu brownish hue.

Obturations of blood vessels by residues of erythrocytes or con-
glomerations of agglutinated blood corpuscles are in great probability
the cause of the infarcts which are met with in lungs, kidneys, and
spleen. It is stated by several authors* that in cases of poisoning with
specific blood poisons the blood coagulates easier, and even in the living
animal coagulations take place and in this way blood vessels can be
blocked and infarcts and thrombi due to stasis produced.t

6

2. Spleen.

The spleen generally is found congested. Spleen tumour has been
observed not only in cases of isolysine-haemolysis but also in piroplas-
motic infections, malaria, and infections with bacillus typhi, spirochaete
recurrentis, streptococi, and staphylococci, that is to say in all diseases
accompanied by destruction of red blood corpuscles and also after
experimental poisoning with the verv haemolytic potassium and sodium
chloricum? and toluylendiamin.

There is now ample evidence that the destruction of erythrocytes
alone. without infection is sufficient to produce splenitis and spleen
tumor; but the question remains, which of the products of the
decomposition of blood corpuscles is the real cause of the splenitis.
Heinz§ says that the morphological residues of the destroyed blood
corpuscles are chiefly deposited in spleen and liver, that the spleen
cells take the altered blood cells, dissolve them, keep the pigment back
whereby the spleen swells up. According to Biondi (1.c.) the pigment
containing the iron which is a derivative of haemoglobin is made in the
liver and carried into the spleen by leucocytes, and after being taken
by the spleen cells it might act on them as stimulus for hyperplastic and
hypertrophic processes.

3. Liver.

The liver is, after the blood glands, the first affected gland, because
the chiet decomposition of haemoglobin takes place in it. This work
being very much too great for the liver in cases of pathologically
extended destruction of ervthrocytes leads to degenerative and regenera-
tive processes in this organ. Therefore it is found in almost every case

* Cit. Kionka, Lubarsch-Ostertag, Ergebu. 7, 515, 1900-1901.

+ Several authors go as far as to take the pathological anatomical phenomenon of obturation of vessels
or infarcts as one of the specific criteria and even a diagnosticum for blood poisons (sulphites, argentum
colloidale, CO, saturnism, bismut phosphoric, salicylic acid, Kionka ; glycerin, carbolic acid, pyrogallic acid,
anilin and derivates, Si/hermun; arsen, Ba Cl,. phosphorus, Heilborn; arsenic, Heinz ; and others)
(Compare Kionka, lc.) Affections of the endothelia of the vessels however have to be taken into
consideration.

t Jawein, Virchows Archiy, 161, 3, 1900.

§ Cit. Kionka, Lubarsch-Ostertag, Erg. 7, 406, 1901,

109

of haemolysis congested, very often soft, friable, enlarged, in the majority
of instances jaundiced, brownish or greenish discoloured with an
increased quantity of bile.

4. Kidneys.

Haemoglobin is noxious to the kidneys. Parenchymatous degenera-
tions of these organs were observed after haemolysis due to the injection
of distilled water (Own experiment), in haemoglobinuria paroxysmalis
hominis in the haemoglobinuria paralytica of horses (lumbago), in all
diseases which occur with haemolysis, and finally in cayes of haemolysis
after injection of isolytic serum. In connection with this “serum
disease ~ the following lesions were recorded :—Adherent capsula,
congestion and dark appearance of the paremchym, abnormal size and
weight, oedema and friability, dark radial stripes.

Examples*: Horse 297.—Injected on 6th July, 1903, with serum
of horse 147. This animal showed similar symptoms as those described
in horse 298. It was better the next day (7/7/03), but would not eat.
On 8th July, 1903, haemoglobinuria was noticed for the first time. The
mucous membranes were yellow. There was loss of condition. The
animal laid down almost the whole of the 9th July, 1903.

Horse 297 died on 9th July, 1903. Post-mortem was made one hour
and a half after death. The condition of the animal was good. Rigor
mortis was present. There was a general jaundiced condition of the
flesh and the serous membranes. The blood was coagulated and
separated into black and white clots; the plasmatic clot had a some-
what brownish hue. The heartbag was filled with dark vellow liquid.
The heart muscle was pale. There were no echymosae on the endocard.
The liver was dark green in colour. The spleen was enormously
enlarged, its weight being 12 pounds; the pulpa was soft. The kidneys
were uniformly dark red, with haemorrhagic infarcts in the cortex.
The urine was black. The mucous membrane of the stomach was
covered with blackish mucous (coagulated blood). The intestines were
normal.

Horse 298.—Injected on 6th July, 1903, with serum horse 147. The
injection was made into the lower part of the jugular vein. Soon after
injection the horse lost control of the hind quarters, staggered for a few
minutes, and dropped suddenly, neighing repeatedly. The pulse was
rapid, the respiration was accelerated. Atter a lapse of a few minutes
the horse rose again: it now kept the head stretched out ; it breathed
quickly with wide open nostrils. In the afternoon of the same day the
horse went down again. It showed tremor of the muscles ; it moaned
and had a rapid respiration. The next day (7/7/03) horse 298 seemed to
be better. On the 8th July, 1903, horse 298 lay down again. The
mucous membranes had a yellowish colour. The horse tried to stale
repeatedly. Red urine was voided. The animal lost condition from
day to day until death.

Horse 298 died at 1 o’clock p.m. on LOth July, 1903. Post-mortem
was made one hour later. The condition of the cadaver was fair

* Copied from Ann- Rep. Govt. Vet. Bact., 1903-04.

110

There was a general jaundiced condition of the flesh, the serous
membranes, and all organs. The lungs were oedematous and a few
haemorrhagic infarcts were present. The blood of the heart ventricles
Was not completely coagulated. A few haemorrhagic spots were
noticed on the endocard of the left ventricle. The heart muscle was
pale. There was but little liquid in the heartbag. The spleen was
slightly enlarged, there were several haematoms in the tissue of the
spleen. The pulpa was black. A white thrombus was found in the
splenic vein. The liver was enormously enlarged. The tissue was
friable, of a green-vellow-reddish colour; it was studded with white
spots. which on close examination proved to be small abscesses ; minute
haemorrhagic spots were also present. The kidneys were enormously
enlarged, almost spherical. The capsula was infiltrated. The tissue of
the kidneys was black, oedematous, and friable. The mucosa of the
stomach was uniformly reddened and swollen; that of the duodenum
was also thickened and red stripes across the mucosa were present. The
mucous membrane of the coscum and colon were slate-coloured. The
bladder was empty. The lymphatic glands were not enlarged.

Microscopical examination of smears of the different organs proved
the absence of endoglobular parasites. Tle small abscesses in the liver
contained a bipolar bacterium.

The above-described complex of clinical symptoms and pathological
anatomical lesions, which are met in cases of haemolysis due to the
injection of isolytic serum, has to be expected more or less complete in
all diseases where destruction of erythrocytes takes place, namely in :

1. Various poisonings with saponine, digitoxine, cyclamine,
colloidal metals and metal hydvo-oxyvdes; acids, Hg Cl,, K ClO,,
Na ClO,, Ba Cl, As, As,O,, P, and many others.

After influence of very low and very high temperatures.

After infections with microbes secerning haemolytic toxines :
strepto—and staphylo-cocei, b. anthracis, b. typhi, tetani, bact.
cholorae a.o.

ft. After poisonings with snake venoms.

5. After infections with piroplasms; they are all endoglobular

parasites and are specially important for South Africa.

In cattle: Piroplasma bigeminum (Texas fever, ordinary redwater).

Piroplasma mutans (gall sickness).
Piroplasma parvum (East coast fever, Rhodesian 1red-
water), haemolysis clinically seldom observed.

In equines: Piroplasma equi (biliary fever).

(Horses, mules, donkeys, zebras.)

In dogs . Piroplasma canis (biliary fever).

ool

The clinical pathological anatomical appearance of these diseases is
of course not always the same as it is seen in cases of serum-haemolysis,
but mixed with other local symptoms of intestinal, nervous, or cardiac
nature, This applies specially to diseases due to microbes which secrete
other toxines besides haemolysines (tetanus, typhus, anthrax, ete.)

The Anatomy of Stilesia Centripunctata.

ee

By Dr. LEWIS H. GOUGH.

Zoological Laboratory.

The Anatomy of Stilesia Centripunctata (Rivolta).

By Lewis Henry Goucu, Pu.D.

Stilesia centripunctata (Rivolta) is a parasite of the jejunum of sheep
and, according to Von Linstow (Compendum der Helmenthologie, 1889),
of cattle, but there is internal evidence that this statement is due to an
error, as he does not quote it as a parasite of sheep. Linstow, however,
is there citing Rivolta, who found it in sheep.

Its geographical distribution appears to be somewhat limited, as,
apart from South Africa, I have only seen records from Italy (Rivolta,
Mattozai) and Algeria (Neumann, Tempére).

In South Africa (Transvaal) it is absolutely the commonest cestode
found in the intestine of sheep; several specimens are usually found
together—it is, however, rarer than the liver tape-worm, Stilesia hepatica,

Wolth.

The parasite is almost entirely restricted to the continent of Africa
and to Southern Hurope—to the Mediterranean and Aethiopean regions,
but especially to the latter.

The worm was first described as Taenia centripunctata by Rivolta in
1874 (Sopra aleune Specie di Tenie della Pecora, Pisa), it was again men-
tioned by Perroncito in 1882 (I Parassiti dell’ Uomo e degli Animali Utili,
Milano), and again in 1886 (Trattato teorico-pratico sulle malattie piu
comuni degli Animali domestici dal punto di vista agricolo, commerciale
ed igienico, Torino). In the same year (1886) Railliet refers to it in the
first edition of Elements de Zoologie Medicale et Agricole, Paris. In 1888
it was again referred to by Neumann in the first edition of Traité des
Maladies parasitaires non microbiennes des Animaux domestiques, Paris.

These records or references all appear to have been based on the same
material, or to have been quoted from one paper.

In 1891 Neumann obtained fresh material, and was able to supplement
the details given by the earlier authors. (Observations sur les Ténias du
Monton: C.R. Soc. Hist. Nat., 1891, 18 Mars.)

The revised diagnosis is found also in the second edition of the Traité
des Maladies parasitaires 1892, and in Railliet’s second edition of Traité
de Zoologie Médicale et Agricole, 1893. In the same year Stiles gives a
description, partly original, from Neumann’s material, partly abstracted, in
“A Revision of the Adult Cestodes of Cattle, Sheep, and Allied Animals,”
being the best description of the worm published hitherto.

114

Since Stiles I only find one further reference (using the Zoological
Record), by Tempére, in the Micrographe Préparateur, 1904, p. 27, which,
however, is evidently only an abstract from Neumann, the illustration
given being simply a copy of Neumann’s figure of the Scolex. Most of
the older literature is inaccessible to me (working in South Africa), and
has been quoted from Stiles.

GENERAL APPEARANCE.

Stilesia centripunctata (Rivolta) is macroscopically instantly recognis-
able among the parasites of sheep by the following details. It differs
from the species of Monezia and Thysanosoma by its small breadth as
compared to its length, the extreme breadth not frequently exceeding
3 mm.; its greatest breadth is usually near the scolex, the posterior
portion of the strobila being almost round in section.

It differs macroscopically from the other species of Stilesia by its
greater length, and also in the following particular. The strobila from
10 cm. distance from the scolex onwards appears to consist of five longi-
tudinal bands, a moderately transparent one laterally on each side, within

this a very transparent band on each side, enclosing medially an opaque
band.

The moderately transparent portions are the lateral fields; the very
transparent bands are caused by the extremely wide ventral longitudinal
canals; the opaqueness of the median band is caused by the female genitalia
and their supporting tissues.

In life the worm has a gelatinous appearance (except along the median
line, which is somewhat opaque) ; it is slightly yellowish or greyish.

DIMENSIONS.

Perroncito (quoted from Stiles’ “ Revision *’) gives the following
dimensions :—

Total length 2-75—2-84 m.
Width 10 cm. from scolex 2—4 mm., 50 cm. from head 2—3 mm.
150 cm. from head 1.55 mm., terminally 1 mm.

Neumann, Traité, second edition, gives approximately the same
dimensions. Stiles states the total length to be nearly 3 m., at 10 cm.
from head the segments are 2—3 mm. wide and grow narrower from here
onwards, posterior segments being scarcely 1 mm. wide. Tempére (1904)
evidently quotes the length from Neumann or Perroncito, giving it at
2-75 m.—2-85 m., segments 10 cm. from scolex being 2—4 mm., at 150
cm. from scolex 1 mm. He adds that this worm differs from other
Taeniadae in that its segments are widest near the head,

115

A specimen taken 4th June, 1909, measures 202 cm. (after fixing in
sublimate), it contains mature oncosphaeres. The total length must,
therefore, vary from 2—3 m.

As to the breadth, this varies very considerably according to the
state of contraction; the greatest breadth is usually, but not always,
attained close to the scolex. One specimen measured 2-8 mm. at 10 cm.
from scolex, 3-2 mm. at 20 cm. from scolex. Another measured 2-5 mm.
directly behind the scolex (normal value), but at 2 cm. from scolex fell
off to 0°5 mm.

The posterior portion is always very narrow and thick, almost round
in section, measuring at the most 1 mm. broad. Anteriorly the segments
are always much broader than thick. All segments are much broader
than long, the ratio of breadth to length varying very considerably accord-
ing to contraction; at normal contraction, sections passing vertically to
the long axis in the posterior half of the stobila will almost always pass
through genitalia belonging to more than one segment, the anterior and
posterior surfaces of each maturing segment not being flat, but having
projections caused by the bulky female organs, or indentations caused by
those of the preceding or following segments. The scolex measures,
according to Perroncito, 2 mm., Neumann gives the measurements as
1-5—2 mm. ; there are some discrepancies in the literature concerning the
scolex. ,

Neumann figures it with large cylindrical suckers directed, parallel to
each other, anteriorly; Tempére follows Neumann and copies lis figure, as
does Railliet.

Stiles states that the scolex in his possession has its suckers situated
at its four corners and that they are directed diagonally forwards.

I have had the opportunity of examining several scolices, over twenty,
and in every case they agree with Stiles’ description (Fig. 1). Sections
also show the suckers to be directed outwards and forwards.

The size of the scolex also varies fairly considerably : five specimens
were measured :—

Length. Breadth.
a. 1-5 mm. 2-3 mm.
b. 3-1 mm. 2-8 mm.
ce 1-8 mm. 2-4 mm.
d. 1-5 mm. 2-0 mm.
e. 1-8 mm. 1-5 mm.

Specimen 6 was the largest, e one of the smallest I have seen.

The segments are hardly distinguishable by the naked eye ; in several
specimens in which segmentation was apparently visible magnification
proved that what appeared at first sight to be segments was in reality
caused by the grouping together of four, five, or more segments, due to
contraction,

116

The genital openings are not prominent, and are very slightly
developed, as compared with those of other Anoplocephalids. The genital
openings are irregularly alternate.

ANATOMY.
The Cuticula.—

The cuticula on scolex and strobila everywhere presents a similar
appearance, and is composed of three distinct layers, differing from each
other in thickness, structure and in their behaviour towards staining
reagents.

For the study of the cuticula, specimens fixed with silver-nitrate and
developed with hydroquinone give on the whole the best results. (Method :
Ramonv Cajal for nervous tissue.)

In a specimen fixed in this manner and stained with haematoxvlin
counterstained with orange g, the cuticula at 15 cm. from the scolex is
about 4:5 thick; of this the outer layer accounts for. about 1-5 4, the
middle for 3 #, the innermost layer is extremely thin. The outermost
laver stains in this specimen darker than the middle, and has a bluish
tinge due to the haematoxylin. Excepting for a single row of minute
black granules on its external surface, it is apparently structureless. The
middle layer stains only with the orange; it is not homogenous, as it
contains in its entire thickness very numerous, minute, black granules.
There is no definite arrangement for these granules, and they are
equally abundant in all parts of the layer. The imnermost laver is, as
already stated, extremely thin, and stains black according to this method.
T consider it to be structureless.

Material fixed in Zenker’s solution, but stained in the same way, does
not show as much detail. The outer layer is dark blue, taking almost
the same colour as the chromatin of the nuclei. It does not appear
homogenous, however, but seems to be traversed bv fine pore-canals.

In the middle layer the granules are not differentiated, the entire
thickness of the layer being almost homogenous; very careful examination
shows, however, lighter spots corresponding to the granules, but as these
are very minute, they are extremely difficult to make out. The innermost
layer does not differentiate, or appears as a lighter orange line between
the middle layer and the muscles running parallel to the cuticula. Material
fixed in formaline (4 per cent.), but stained in the same way, gives the same
results. The cuticula of S. centripunctata is smooth, unlike that of
S. hepatica, which is villose.

The Subcuticula.
The subcuticula is best studied on not too contracted pieces fixed
with Zenker’s fluid and stained with haematoxylin and orange g; accord-

ing to the state of contraction or extension it, offers rather different
pictures. (Fig. 2.)

117

In places where the cells are separated owing to expansion of the
worm, the subcuticula is seen to consist of elongated cells, rich in proto-
plasm, standing at right angles to the cuticula ; “these cells are in contact
with the innermost cuticula layer by three, four, or more processes, which
are again often branched before reaching the cuticula. These processes
appear occasionally to form anastomoses with processes from the same or
other cells, before inserting in the cuticula. The proximal end of the
cell terminates in a single elongate process, which is continued into a long
fibrilla; these fibrillae can sometimes be traced continuously through the
ring-muscle into the middle layer of the worm and from thence to the
cuticula of the opposite side, sometimes interrupted on their course by
the spindle-shaped cells belonging to the transverse-muscle system.

The fibrillae of several adjacent cells appear to close together into
definite bundles before penetrating the longitudinal muscles; this is best
seen on more contracted portions.

The subcuticula cells appear to have traces of a membrane, are very
distinctly outlined, and their plasma appears to be slightly denser along
the walls of the cells.

The nuclei of the subcuticula cells consist of small vesicles; the
membrane surrounding the nucleus stains quite distinctly ; each nucleus
contains several round chromatine bodies, often five or six in addition to
the nucleolus, which is about twice the size of the chromatine bodies.

The measurement of a few subcuticular cells is (Zenker’s solution—
haematoxylin orange—20 cm. irom scolex) :—

(a) Total length of cell from

insertion in cuticula to

attachment to fibrilla.. 57” 426 364 42 604 5le
(6) Greatest width of same cell 34 454 64 44 44 4+
(c) Length of nucleus. . .. 64 64 5e 5e 5e 64
(7) Breadth of nucleus . BF 454 454 44 44 34

Only such cells were measured whose insertion in the cuticula and
attachment to the fibrilla could be made out. The attachment to the
fibrilla is only able to be found in cells lying entirely in the plane of the
section.

Between the just described subcuticula cells and the cuticula, two
layers of muscle fibrillae are to be found running parallel to the cuticula.

The outermost of these runs horizontally and forms an outer ring
muscle layer; the inner one runs at right angles to it in the direction of
the long axis of the worm. The outermost longitudinal fibres pass between
the circular fibres. These systems are best studied on sections parallel to
the surface of the cuticula, but can be made out also on transverse sections.

Calcareous Corpuscles.
Stilesia centripunctata does not seem to possess any calcareous bodies.

118

Nervous System.

The two lateral nerves, which in the strobila lie midway between the
ventral canal and the lateral margin, on entering the scolex converge
somewhat and pass in well within the rhomb-shaped area enclosed by
the suckers. On transverse sections shortly after the cavity of the
suckers is reached a rhomboidal commissure is visible, whose angles pro-
ject towards the interacetabular spaces and form ganglia there.

Directly anterior to the rhomboidal commissure, transverse commis-
sures connect. the four ganglia of the rhomboidal commissure ; these two
transverse commissures are fused in the centre and form a cross.

From each of the four ganglia in the angles of the rhomboidal com-
missure four nerves originate, one laterally going to each of the two
adjacent suckers, the other two ascending, at first towards the summit
of the scolex, then bending over and forming loops with each other, the
right ventral fusing with the right lower (ventral) lateral, the upper (dorsal)
right lateral with the right dorsal, the left dorsal with the upper left
lateral, and the lower lateral with the left ventral.

A large median plate, poor in ganglion cells, appears to le further
anteriorly, just below the anterior loops of the lateral canals, but I have
not been able to trace the connection of the plate with any of the eight
nerves (four loops).

The lateral nerves on transverse sections fixed with silver nitrate
appear to consist of a dark network, containing many small black granu-
lations (precipitate of silver ?) on the meshes. The meshes become rather
denser towards the margins. There is no membrane between the nerve
and the parenchyma. Small cells, glia cells, are frequent, especially on
the circumference of the nerve; some are, however, situated in the depth.
These glia cells are very poor in protoplasm, only their vesicular nuclei
with a large nucleolus being visible. The nucleus measures about 4:5
by 3“. Where the nerve is not cut transversely, but obliquely or longi-
tudinally, the network is seen to consist of more or less parallel fibres.
Large ganglion cells are very frequent in the rhomboidal and transverse
commissures and in the nerves originating there, and also in the body
of the suckers. (Fig. 3.)

The ganghionic cells in the nerves are elongate, fusiform, and in some
cases appear to be bipolar.

Their long axis always lies parallel to the axis of the nerve. These
cells vary in size, their apparent total length in a section, of course,
depending on the angle in which the plane of the section has cut them.

The processes originating from these cells can often be traced for a
long distance in the nerve. The protoplasm of these nerve-cells is almost
invariably filled with short rod-shaped bodies, arranged serially, each
series more or less parallel to the next, staining dark blue with haema-
toxylin (tigroid bodies). |These structures also occur for some distance
along the nerve processes. The nucleus is oval, vesicular, staining very

119

lightly with haematoxylin, 9 # long by 4-5 « wide; it contains a large,
round, dark nucleolus, not quite 3 “ in diameter, and up to four smaller
bodies, staining equally dark.

The total length of one of the fusiform nerve-cells is 30 +, its fibril
can be followed on one side for further 36 +, breadth about 9 ». These
cells are evidently conducting elements. Larger, more or less rounded,
ganglion cells are found in the ganglia at the origin of the nerves. I can-
not state definitely whether they are multipolar or unipolar. In these
cells the tigroid bodies appear to be absent, the protoplasm staining blue
with haematoxylin, and presenting a spongy or reticulated appearance,
dark meshes enclosing a lighter ground.

The nucleus is round, rather pale, but contains a very large dark
nucleolus and several (six or more) smaller dark staining bodies. One
such cell measured 27 in diameter, its nucleus 12 4, the nucleolus 3 +.

Large ganglion cells are also found in the body of the suckers, lying
between the bundles of radial fibres, usually closer to the body side than
to the cavity of the sucker, and often form an almost definite layer.
(Fig. 4.) These ganglion cells do not contain tigroid bodies; their protoplasm
has the same reticulated or spongy appearance as the ganglion cells of the
scolex ganglia. The nuclei are vesicular, their membrane very distinct,
their body not taking stain, their shape is round to oval. The size of one
of these cells measures 18 » across, the nucleus 9 ¢ by 6 #. nucleolus 3 +.
Besides the nucleolus there are a few (two or three) smaller dark staining
chromatine bodies, often very closely approached to each other.

These cells and the bipolar nerve-cells in the commissures described
above are evidently the ‘ Myoblasts”’ of Pinter and Zschokke. They
are evidently not muscle cells; their appearance is, if possible, even more
typical for nerve elements in Anoplocephala magna, Abilg., than in Stilesea,
owing to their larger size. (Fig. 5.) One specimen measured in Anoploce-
phala magna: nucleus 12 » long by 9 + broad, vesicular, nucleolus distinct.
round, 4 “ in diameter. Protoplasma staining dark with haematoxylin,
reticulated or spongy in appearance, distally the meshes arrange them-
selves more and more to parallel threads and finally lose themselves in
typical neurofibrills, which can be traced some distance. No cell mem-
brane. Total length (exclusive fibrillae) 75 +, breadth 15 +; the neuro-
fibrillae could be traced as a definite thread for 36 / on one side, 60 4 on
the other. The fibrillae of the longer side could be seen to divide at their
end and to connect with other similar ganglionic cells. In Anoplocephala
as in Stilesia, these cells are restricted more or less to a definite zone, near
the middle of the radial fibres, and form a definite nervous system in the
suckers.

In the strobila on horizontal sections one sees occasionally nerves
proceeding from the lateral nerve towards the lateral margin, accompany-

‘ing the genital ducts. They are, however, hard to find, and cannot be
followed for any distance.

120

THE MUSCULATURE.

The transverse and longitudinal muscles between the cuticula and
subcuticula have already been mentioned, and it has already been stated
that I consider the subcuticula itself to belong to the contractile elements.

The longitudinal, transverse, and dorsoventral systems of muscles
(Parenchym muskeln, Braun) are found in characteristic arrangement
in the strobila. The longitudinal muscles are the most developed of the
three systems. They lie in a single layer between the subcuticula and
the transverse muscles, being divided into rather indistinct bundles by
the dorsoventral muscles. The longitudinal muscle layer is broken laterally
on both sides.

The fibres run continuously, through more than one segment, parallel
with each other. They are roundish on transverse sections, often enclos-
ing a hollow, appearing then annuliform. Myoblasts are fairly frequent.

The transverse muscles form a much weaker layer than the longi-
tudinal, consisting both of fewer and of thinner fibres. The dorsal and
ventral plates of transverse fibres meet laterally, but the fibres of either
plate do not pass round into the other plate. Myoblasts are frequent.

The dorsoventral is the weakest system, being composed of single
fibres; the fibres are thickest in the lateral fields. The fibres of this
system appear to attach to the fibrillae already mentioned as continuing
the subcuticular cells. Myoblasts are very frequent.

The musculature of the scolex is more complicated, but consists of
fibres derived from these three systems. At the base of the scolex the
longitudinal fibres of dorsal and ventral series approach so close as almost
to meet laterally.

The transverse muscles form a much thicker and denser layer, and
the dorsoventral muscles become more prominent and more frequent.
Entering the scolex their arrangement and direction becomes changed,
owing to the other organs contained in the scolex. Without going into
detail, the following systems of muscles can be mentioned as occurring in
the scolex :—

(1) A diagonal cross system anterior to the terminal loops of the
longitudinal canals ; it covers the entire anterior surface of the
scolex, passing from the front of the left ventral to the right
dorsal, and from the right ventral to the left dorsal sucker.

(2) A second diagonal cross system composed of four bundles, each
consisting of a few fibres; it is situated just behind the ter-
minal loops of the longitudinal canals and runs from sucker to
sucker in such a way that (1) fibres from the lateral face of
the right ventral sucker run to the lateral face of the left dorsal
sucker; (2) fibres from the lateral face of the right ventral
sucker run to the median face of the left dorsal sucker; (3)
fibres from the median face of the left ventral sucker run to the

121

lateral face of the right dorsal sucker; and (4) fibres from the
lateral face of the left ventral sucker run to the median face
of the right dorsal sucker.

(3) A third cross system orthagonal, running dorsoventral and
latero-lateral, is situated just behind the second diagonal
system. Behind this third muscle-cross the great nerve com-
missures occupy the central space between the suckers.

(4) A fourth cross system is found (behind the great nerve com-
missures) at the base of the suckers; the fibres of this system
insert on the suckers in such a way that : (1) the median faces
of the two right suckers, (2) the median faces of the two left
suckers, (3) the lateral faces of the two dorsal suckers, and
(4) the lateral faces of the two ventral suckers are connected.

(5) Behind this there appears to be a muscular connection between
(1) the right sides of the ventral suckers; (2) the left sides of
the ventral suckers; (3) the right side of the dorsal suckers ;
(4) the left sides of the dorsal suckers. J did not notice this
system to connect the dorsal and ventral suckers.

Tue SUCKERs.

The suckers are large and are pointed diagonally outwards and for-
wards. A ring formed by a fold of the subcuticula projects beyond the
margin of the suckers, giving them greater depth. Externally they are
covered with cuticula, similar to that covering the rest of the worm;
internally they are separated from the scolex by a very thin membrane.

The muscle fibres composing the suckers are arranged in the usual
manner. The radial fibres are well developed, arranged in small bundles
in the middle zone; these bundles spread out slightly both exteriorly and
interiorly. The ganglion cells already referred to are mostly situated at
from the middle to the inner third of the radial fibres.

A thick layer of muscles is found on the membrane separating the
suckers from the scolex. These muscles run parallel to the membrane
and are seen lengthwise on transverse sections; the radial fibres pass
between these fibres to attach themselves to the membrane. Above this
layer proceeding away from the delimiting membrane, one finds cross-
sections of muscles running also parallel to the membrane, but at right
angles to the first layer. These muscles all show a hollow in their middle
(or appear annuliform on section). About midway between the two
curved surfaces of the sucker (still examining a transverse section) one
finds very fine fibrillae, probably muscular, which run from margin to
margin of the sucker. They do not keep the exact middle between the
cuticula and membrane, but in the centre of the sucker approach the
membrane.

A ring muscle, or sphincter, is seen at the margins of the sucker. On
transverse sections one only finds cross sections of its muscle-fibres. This

1
muscle seems to be a further development of the second layer from the
membrane mentioned above, as on transverse sections it is continued into
it. Its fibres are also found under the cuticula, fewest in the middle of
the sucker, in greatest abundance at the margins.

All the muscles of the suckers are embedded in parenchym. A layer
of cells, with small round nuclei, measuring on an average about 4°5 », 1s
found between the radial fibres quite close to the cuticula. These cells
are rather poor in plasma, their nucleus is vesicular, contains a fairly large
nucleolus and one or two chromatin bodies. These cells may represent a
much modified subcuticula, for which their arrangement in a definite layer
under the cuticula and the cuticular muscles (ring muscle) would seem to
speak; on the other hand, their plasma bodies do not resemble the normal
subcuticular cells of this worm.

Tue LONGITUDINAL CANALS.

The longitudinal canals have already heen shown to be very con-
spicuous in the posterior part of the strobila. It is, however, only the
ventral canals which one sees; the dorsal canals become rudimentary at
50 cm. from the scolex, though they can be traced as a solid line of tissue
throughout the strobila. In the proglottids, the dorsal canal gets dis-
placed and runs laterally and internally to the ventral canal. In the hase
of the scolex we find them lying dorsal and ventral to each other; even
here the difference in calibre is very apparent.

The left ventral canal on reaching the level of the base of the suckers
turns at right angles and proceeds horizontally to the base of the left
dorsal sucker, rising vertically and laterally to the sucker to just above
the first sections showing the lumen of the sucker; it then turns over
and proceeds downwards parallel to its ascent ; arrived near the base of
the sucker it gives off a short branch which terminates abruptly in about
the same height as the loop just mentioned (i.e. after the first sections
showing the Jumen of the sucker).

The main canal, however. twists around, internal to its initial hori-
zontal portion and almost parallel to it, crossmg the scolex externally to
the dorsal canal; arrived at the base of the left ventral sucker, it ascends
the median side of the sucker until the external opening of the sucker is
reached, it then runs downwards parallel to its ascent, until the base of
the sucker is reached ; passing round the external face of the base of the
sucker and forming a few spiral loops at its base, it ascends the lateral
inner side of the sucker as far as the cross commissure, then it sends off
a branch which proceeds a short distance to the outer surface of the dorsal
sucker, where it terminates abruptly. The main canal, however, continues
ascending just between the two suckers, after making a single spiral loop,
to the summit of the scolex, where it bends over, descends in the inner
angle formed by the two suckers until the cross commissure is reached ;
here it bends inwards, then upwards, and runs parallel and ventral to the

125

dorsal canal until the summit of the scolex is reached; here it again bends
over horizontally and dorsally to join the left dorsal canal in a simple

loop.

Explanation of Figure.—v.v.c. right ventral canal.
l.v.c. left ventral canal.
r.d.c. right dorsal canal.
l.d.c. left dorsal canal.
The scolex is seen obliquely, the left central sucker facing the observer.

The right ventral canal, having reached the base of the suckers, also
bends over dorsally and horizontally, running to the right dorsal sucker,
then it ascends the median internal face of the right dorsal sucker and
runs almost the whole length of the sucker, veering over more and more
to the right until it at last appears on the dorsal summit of the sucker ;
then it turns over, descends parallel to the direction it came, until the base
of the sucker is again reached ; it now turns and runs horizontally through
the scolex, remaining exterior to the dorsal canal but interior to its first
horizontal winding; arrived at the median internal base of the right
ventral sucker, it ascends and descends its internal ventral side, reaching
the height of the opening of the sucker. Passing around the base of the
right ventral sucker on its posterior surface, it ascends the outer angle of
the two right suckers; at about the same level as the left canal branches,
the right canal does the same, but unlike the left canal, it sends off two
ramifications, one to the outer surface of the dorsal, the other to the outer
surface of the ventral sucker. After the branches have been given off,
it ascends almost to the summit, bends over laterally and inwards, descends
in the inner angle of the right suckers until the cross commissure is
reached ; here it turns inwards, passes under the nerve commissure, then
proceeds in a straight line anteriorly, parallel, and ventral to the dorsal
canal; arrived at the summit it bends over dorsally and joins the dorsal
canal. The two dorsal canals ascend the scolex in a straight line until
they are joined to the ventral canals; the right and left dorsal canals are
connected at the summit by a short loop.

The course of these canals can be summed up as follows: The ventral
canals on entering the scolex pass over to the dorsal suckers, run up and

124

down their exterior surfaces, recross the scolex, run up and down the
median surfaces of the ventral suckers, pass round the base of the same
suckers, run up the outer lateral angle formed by the dorsal and ventral
suckers, cross over the anterior angle formed by the same suckers, run
down the inner angle, pass under the commissure, and then vertically to
the summit of the scolex parallel and ventral to the dorsal canal, joining
the dorsal canal at the summit. The dorsal canal runs straight through
the scolex, uniting at the summit with the ventral canals, and joined near
the summit by a short loop. Blind branches are given off by both night
and left canals (perhaps rudiments of a ring canal ?).

I have not found any trace of commissures between the lateral canals
in the proglottids ; in S. hepatica anastomosing canals connect the ventral
canals, these commissures lying at the end posterior of the segment.

The longitudinal canals, when cut transversely to their long axis are
more or less circular or oval in outline. Their diameter varies consider-
ably in various parts of the body, as can be seen from the following :

Dorsal. Ventral.
Scolex, at apex 2 nh a 72 x 72 pu
, at base oo ny ot 32 pe 128
Strobila, near scolex .. be . 164 824 94x 484
is 10 cm. from scolex « Wem 2428 324% x 804
i 20 cm. ms .. 2% xX 20» 96% x 96 &
4 30 cm. im . 4¢@ % 4e I2* x Pe
M 40 cm. ie .. obliterated 1200 * x 804
ie 50 cm. - Be 3 80 * x 110 &
i 60 cm. ss se es 84% x 48 &
re 70 cm. ; 160 * x 240 z

Segments with nearly ripe ova obliterated 192 , x 80. The walls
of the lateral canals are formed of a membrane, staining with orange g.
In the apex of the scolex, in sections tangential to the course of the canals
a very fine cross-striping becomes apparent; the stripes are 2 » across,
separated from each other by an almost invisible fine dark line. J have
been able to find this structure in material fixed in Zenker’s solution or
formaline, stained with haematoxylin and orange g. The walls of the
canals are covered with a layer of cells, probably belonging to the
parenchym. In the strobila the cross-stripes are much harder to make
out.

On sections in the proglottids the dorsal and ventral canals differ
considerably.

The ventral canals have thin walls, and the nuclei of the surrounding
parenchym cells lie quite close and tangentially to it. The dorsal canals,
on the other hand, have much thicker walls, often thicker than the lumen :
these walls have three different layers, the innermost very thin, dark; the
middle about 1 » thick staining orange, the outer about 2 » thick, staining
with haematoxylin; they are surrounded by closely radially arranged

125

club-shaped structures, thickest at the outer end, staining lightly with
haematoxylin, total length about 44 », thickness perhaps 4 « or less.
(Zenker, haematoxylin, orange g, 40 cm. from scolex.)

THE GENITAL APPARATUS.

The first trace of the genital apparatus can be seen in total prepara-
tions at a distance of about 1 cm. from the scolex; here one notices a
narrow unbroken band of darker stained tissue running down the chief
axis of the strobila; this band becomes more distinct by the time the
10th cm. from the scolex is reached. Between the 12th and 20th cm. the
testes begin to appear, filling the dorsal portion of the field between the
ventral canal and the lateral nerve, and between the ventral canal to just
bevond the dorsal canal; there is no space for testes dorsally to the
ventral canal. The central dark stripe also begins to lose its continuity
and to break up into a series of transverse lines. Between the 20th and
30th cm. a great deal of development takes place. The median dark
stripe disappears, the testicles become much more conspicuous, occupying
the outer quarter of the median field, but not passing the nerve laterally.
In each segment one now sees a dark strip of tissue, irregularly alternating
to right or left, commencing just before the median line, slightly swollen
at its internal end, clearly traceable as far as the lateral canal; very care-
ful examination shows these transverse lines of tissue to be continued to
the opposite side by a very fine canal, running to the testes (vas differens).
At 40 cm., the testes remaining as they were, these strips of dark staining
tissue have still further developed. Just to the right or left of the median
line, according to whether the segment opens to the right or left, very
distinct round clumps of tissue are seen (ovaria); to each side of these
clumps the dark tissue becomes fusiform and stains less darkly; it is con-
nected by a mere thread across the ventral canal to the anlage of the
vagina and cirrhus apparatus, which liesasa long, slightly pointed strip between
the ventral canal and margin, without, however, reaching the margin. At
50 cm. the chief difference seen is that the cirrus has somewhat approached
the margin, which is reached at 60 cm. At 70 cm. we notice the anlage
of the uterus crossing the median field. As the uterus becomes larger
and broader and fills with eggs, the ovary atrophies and finally disappears
from view on total preparations about 40 cm. further on. (The exact
distance cannot be given, as the specimens studied consist of various
fragments.) At about 95 cm. from the scolex a strip of dark staining
fibrillar connective tissue begins to develop between the uteri of every
two consecutive segments, firmly holding them in position. At 130 cm.
from the scolex, the uteri have increased still more in size, and in con-
tracted material three or more segments can be seen to pass over a single
uterus; that is to say, the uterus of each segment bulges into the two
adjacent and sometimes even into still more distant segments. At this
stage Wolfthiigel’s “ Faserknaeiiel ” (Stilesia hepatica, p. 10), is to be seen

126

on transverse sections, in typical arrangement as described by Wolffhiigel,
only that (in S. hepateca and in S. centripunctata) it is connected with the
uterus and not with the ovary. Out of this organ the egg-pouches develop;
the eggs leaving the uterus, which atrophies, finally collect in the egg-
pouches.

TOPOGRAPHY OF A SEGMENT AT Mae SEexuAL RIPENEsS.

The genital organs, as has already been explained, open irregularly
alternately to the right or left.

At the stage when the spermatozoa are maturing and the first have
reached the receptaculum seminis, the genital organs, which all lie in the
transverse plain, are composed as follows :—

There are three, four, or more testes on each side of both nght and
left ventral canals, lying slightly dorsally; all contain spermatozoa and
also spermatoblasts. Vasa efferentia can be found upon very careful
search; they appear to run from the testicle direct to the vas deferens.
The vas deferens crosses the dorsal side of the median zone, quite close to
the transverse muscles, from the aporose to the pore side. Just before
reaching the cirrhus pouch, laterally to the ventral canal, it becomes much
dilated and crammed with spermatozoa and is no longer straight, but
wound in a few spirals. Having entered the cirrhus pouch it is wound
up in a number of convolutions, still filled with spermatozoa; the terminal
portion is straight, easily distinguishable by the dark blue staining of the
ciliated layer next to its lumen. The length of this ciliated portion is
63 ~; breadth about 3». The genital pore is extremely small, probably
no longer functional. The end of the cirrhus bends over and joins the
vagina; it is not introduced into the vagina, but appears to be fused to
the end of it. (Fig. 6.) There is a short, very narrow connection between
vagina and pore.

SCHEMATIC DIAGRAM OF THE PRINCIPAL ORGANS AT MALE SEXUAL MATURITY.

c Cp rs vd ve ve

re
at
v at ve ac tod o u
Leplanation of Figure.
v. vagina. t. testicle. r.s. receptaculum seminis.
c.p. cirrhus pouch. o. ovarium. n. nerve.
g.o. genital aperture. o.d. oviduct. v.c. ventral canal.

yd. vas deferens. ud. uterine duct. d.c. dorsal canal,
v.e. vas efferens. u, uterus.

127

The vagina runs straight from the pore to dorsal of the lateral canal
and testes. On transverse sections I find that on one side of the body
the cirrhus is dorsal to the vagina, on the other ventral to it. (The main
axis of these sections being uncertain, I cannot state which is right and
left.) Arrived in the median field it proceeds dorsally to the testes, but
ventrally to the vas deferens, forming a receptaculum seminis dorsal to the
most median testes. The receptaculum seminis is continued inwards by a
single canal, the fertilisation canal, which, however, soon branches, one
branch being the oviduct, the other the uterine duct.

The ovary is single, somewhat bean-shaped. There is [as in
St. (globipunctata and?) hepatica] neither vitelline gland nor shell gland.
The uterus at this stage forms a straight, cylindrical chamber, lying in
the transverse axis of the proglottis. At this stage the first eggs are
finding their way over into the uterus, so that it is evident that the testes
and the ovary ripen simultaneously ; in S. hepatica the ovary ripens before
the testes are quite mature.

SPERMATOGENESIS.

The spermatogenesis is rather difficult to follow for several reasons.
In the first place it has to be studied on section series, then it is rather
confusing that several stages can occur at the same time in one and the
same testis, and finally one has to distinguish between the development
of two different kinds of cells, but which both appear to have a common
origin.

As already remarked by Child (Amitosis in Moniezia), it is impossible
to distinguish the cells from which the testes arise from the parenchym
cells. The first stage at which one can feel certain is one at which two
or more cells are found lying together in definite arrangement and
apparently surrounded by a membrane.

The youngest cells seen (urogenital cells, wall and basal cells) form a
syncytiium of four nuclei in a testicle—diameter of testicle, 9 “; nucleus
6“ xX 44 & cytoplasm stains blue; nucleus blue, same depth of colour as
cytoplasm ; nucleolus minute, at the most 1 +, very dark; chromatin in
small round masses lying on the surface of the nucleus. The arrangement
of the nuclei not radial.

On the same sections one finds testicles which have developed further,
and on which the division into the two mentioned types cells have taken
place. These can be classed by their appearance into “ vellow ” or acid
cells and blue or basal cells, according to their staining properties. The
“yellow” cells generally stain yellow with orange g, they are devoid of
cytoplasma or nearly so, a very large nucleolus, measuring up to 3 # (some-
times smaller in younger stages). The chromatin in young stages in
globules, later band-shaped, finally absent. The nucleolus in sections first
stained with borax carmine, then haematoxylin and orange g, has a

128

decidedly ruby red tinge, not noticeable in my sections in any other type
of cell in the whole worm.

These vellow cells are found in the testicles during the younger stages
of development; they disappear before maturity and apparently are con-
cerned with the nutrition of the blue cells which finally produce the
spermatozoa.

The “ blue” cells differ very much in size during the various stages
they pass through ; their cytoplasm is, when present, alwavs blue (except
detritus remaining after formation of spermatozoa, which eventually turns
yellow). Their nucleolus never reaches the size of that of the “acid ”
cells.

For some time, and until the testes have reached a fair state of
development, the multiplication of both types of cells appears to take
place by amitotic division of the cells into two, the resultant cells not
arranging themselves radially (spermatogonia).

After these preliminary divisions, one finds both blue and yellow
cells undergoing division, by which the resultant new cells (spermagenes)
le arranged radially (Fig. 7), connected in the centre by thin filaments
of protoplasm, yellow in the case of “ acid” cells, blue in the case of the
normal spermagene.

This stage resembles the “ first phase of proliferation ’’ Bugnion and
Popofft saw in the spermatogenesis of Lombricius agricola (Bugnion and
Popoff, La Spermatogenése du Lombric, Zool. Congress, Berne, 1904). It
is followed as in the earth-worm by a phase of dissociation, especially
marked in the case of the acid cells which separate entirely. The blue
cells (spermatocytes 1), on the other hand, remain connected to the centre
of radiation by their protoplasmic processes for some time, increasing in
size; their nuclei becoming denser and darker as the cells increase. in
size, but without changing in magnitude (nuclei soon after proliferation
4% , after increase of cell 45 4). After proliferation chromatin in small
peripheral globules, after growth of cell in larger masses filling the nucleus.

The basal cells do not seem to divide again, but degenerate and finally
disappear.

Just before the second proliferation of the spermatocytes the chromatin
again changes, first forming small globules, then stretching itself out into
threads. The next change appears to be that the cells forming one of the
radial figures left after the first proliferation and dissociation fuse, the
nuclei resolve themselves into chromatin threads and wander into the
interior of the syncytium, all traces of the single component cells becoming
lost. The nucleoli remain visible until the chromatin has drawn itself
out into threads; it then also appears to form threads. The chromatin
then seems to break up into short lengths, each of which at the surface
forms the nucleus of a new cell (spermatid). These cells after further
division give rise to spermatozoa. A large quantity of cytoplasma and

12)

a few loops of the chromatin remain behind in the middle and form a
cytophore. The cytophore can persist for some time, eventually all trace
of its nucleus disappears, the protoplasm changes its reaction, staining
orange, and finally it disappears when the spermatozoa mature.

I have not been able to compare with any data given by other
observers than those quoted, owing to inaccessibility of the literature.

FurTHER DEVELOPMENT OF THE FEMALE ORGANS.

The Oogenesis in Stilesia would be doubly interesting to follow, for,
owing to the absence of vitelline glands, it differs from that of most other
cestodes.

In the earliest stages the ovarian nuclei form a syncytium and divide
amitotically as described by Child for Moniezia; the first mitoses were
observed at about 50 cm. from the scolex, at a stage at which the oviduct
and uterus were yet solid masses of tissue without apparent lumen.

At this stage, the cell boundaries are becoming distinct. As develop-
ment progresses, mitoses become more frequent, and finally all cell division
appears to take place by mitosis. In the ripe ovary (80 cm. from scolex)
mitoses are frequently observed, four chromosomes appearing to be the
normal number. In the case of the ovarian ova, the resultant cells seem
to separate as soon as the mitosis are over. Mature ovarian ova measure
approximately 20 » x 16 .; their nuclei are round and about 8 » in diameter.
Fertilisation probably takes place in (the oviduct or) the uterine duct.
The passage of eggs through the ovi-uterine duct must be a very quick one,
as in no case have I found an ovum traversing either of the two ducts;
this although many segments have been examined on sections in stages
in which the ovarium is in full function, and the uterus is filling with ova.

Arrived in the uterus mitoses commence again, but the resultant
nuclei remain embedded in a syncytium; such cells with two or four
nuclei are very common; the number of chromosomes seems to have
increased—I believe that there are eight—but they are very difficult to
count ; in any case four is no longer the normal, but a higher number is
always present. Young uterine ova measure approximately the same as
the mature ovarian ova, their nuclei however decrease in size ag division
progresses ; two in one young ovum measured 6 » in diameter. It may
here be stated, that I have sometimes found ova similar to those in the
ovary or to those just arrived in the uterus, free in the parenchym of the
body, occasionally in large numbers. Just at this part, my material
is unfortunately not sufficiently well preserved to enable the further
changes to the ova to be followed in detail.

The maturest ova which I have seen have a thin membrane, and
lie in egg-pouches, no longer in the uterus.

After the stage at which fertilisation takes place, several changes
occur in the female genitalia; in the first place a layer of dense fibrous
supporting (or connective) tissue is formed anteriorly to each uterus.

150

On sagittal sections, it sometimes appears to be both anterior and
posterior, but in such cases I believe that this is only apparently the case
and in reality the pads of fibrous tissue belong to separate segments. In
sagittal sections one often finds two pads between two adjacent uteri;
on a horizontal section it would be seen that the uterus of a third segment
has been crowded out to the side between the two uteri visible on the
section. Owing to bad preservation of my material just at this part of
the worm I cannot venture any statement as to the origin of the pads of
fibrous tissue.

The uterus after having received the fertilised ova undergoes several
changes ; at the time when first filling with ova, it is simply a wide trans-
verse tube. The ova at a slightly later stage appear to be surrounded by
septa formed by cells originating from the uterine walls. (Rather
macerated material.) With the disappearance of testicles and ovary
the segments lose breadth and contract latero-laterally, forcing the uterus
to become more and more globular.

At this stage an organ resembling “ Wolffhiigel’s Faserknaiiel” becomes
apparent; its early stages are unfortunately lost to me on account of the
maceration the material has undergone just at this part. In
S. centripunctata it has much the same appearance in certain stages as
in S. hepatica; in both species it is an outgrowth of the uterus, not of the
ovarum. On transverse sections (about 120 cm. from scolex) one notices
the following changes :—The ova all lie on that side of the uterus nearest
to the surface of the worm, in one segment perhaps dorsally, on the next
it may be ventrally, according to the position of the uterus, which again
depends on the pressure from the uteri proceeding and following it in the
strobila.

On the other side of the uterus is a mass of fibrous tissue with radial
or parallel fibres, which in its further development gives rise to the
Faserknatiel, which in turn develops into egg-pouches. (Fig. 8.) This
mass of fibrous tissue is in early stages arranged within the uterus con-
verging towards the side opposite to the ova; it passes beyond the uterus
into the parenchym of the body, still converging; the opening it passes
through is at first large, later this contracts, thus forcing the fibres to
form a double radiation, one in the uterus, the other outside it. At first
nuclei are very numerous, later on they become rarer. (Fig. 9.) The
portion outside the uterus grows larger and more globular, the fibres arrange
themselves so as to form a number of pockets, the uterus itself during
this process diminishes in width. (Fig. 10.) Then we see ova appearing
in the pockets of the fibrous organ. (Fig. 11.) As they disappear at the
same time from the uterus, there is no doubt from where they came.
Finally all the ova are in the pockets of the organ and the original uterus
atrophies. (Fig. 12.) The fibres of the egg-pouch form an outer cir-
cular layer around the inner pouches, which themselves consist of fibres.
In each case the fibres seem to be arranged so as to form lamellae.

131

Before leaving the uterus, the ova are already provided with their shell.
They are globular, measuring 24 , to 28 » in diameter (from specimen
macerated with 10 per cent. potash), and are devoid of projections or
spines of any kind. The pockets in which the eggs are finally enclosed,
resemble to a very great degree those figured by Lungwitz for Taenia
Ovilla (Thysanosoma giards).

INFLUENCE oF Host.

I have not observed any lesions attributable to the presence of the
worm at post-mortems of sheep infested with Stilesta centripunctata.
The suckers are, however, often filled with epithelial elements.

SpPeciric D1IaGnosis.

Stilesia centripunctata (Riv. 1874), Railliet, 1895. Head large,
varying from 1°5 mm. to 3-1 mm. long, by 1-0 mm. to 2-4 mm. broad.
Suckers, large, directed diagonally outwards. Strobila from 2 to 3 metres
long. Segments 10 cm. from scolex measure from 2 to 3 mm. wide at
20 cm. up to 3-2 mm.; growing narrower posteriorly, the strobila loses
its flatness and finally becomes almost round in section, measuring about
1 mm. in diameter terminally. The greatest width is often, but not
invariably, close to the scolex. Segments are always much thicker than
long, and anteriorly much broader then thick. Genital openings
irregularly alternate ; uterus transverse in median portion of field, mature
ova in egg-pouches arising out of fibrous organ. Testicles extend from
a short distance from each end of uterus to almost to the lateral nerve.
The genital canals pass dorsally to the nerve and to the dorsal and ventral
canals. The vas deferens passes dorsally across the segment, dorsal to
lateral canals, nerves, vagina spermiduct, receptaculum seminis, and uterus.
The cirrhus is becoming rudimentary, opening directly into the vagina.
No vitelline gland nor shell gland. Ova round, without spines or other
projections, measuring 24 to 28 ». Development unknown ; host sheep,
Italy and Africa; habitat in the small intestines.

Cirrhus pouch
Cirrhus

1. Scolex, half expanded (Formaline specimen).
2, Subcuticula and cuticula.
3. Ganglion in rhomboidal commissure. ‘

4, Section through acetabulum, showing ganglion cells.

5, Section of sucker of Anoplocephala magne Abilgaard
to show ganglion cells.

6. Genital openings, to show connection between cirrhus and vagina..

All figures (except 1) were from specimens fixed in Zenker's fluid and stained with Haematoxylin

(Ehrlich), counterstained with orange g ; average thickness of sections + #-

Testes
Fibrous
HSSuUe

\

£qg pouch

Ovarium.
7

Pad of oo £9q pouch.
16r0US
Hssue.

fibrous
tissue.

7. Testicles with various stages of spermato-gencsis and anlage of ovariun.
8, 9, 10, 11, 12. Consecutive stages in the development of the egg-pouches.

Notes on the Pathological Anatomy of
Pleuro-Pneumonia,

By Dr. K. F. MEYER.

The Pathological Laboratory.

The Bacteriological Laboratory.

Notes on the Pathological Anatomy of Pleuro-
Pneumonia Contagiosa Bovum.

By Dr. K. F. Meyer.

No attention has been paid to the lesions caused by the virus of
pleuro-pneumonia in the lungs and in the tissues generally, since the
classical investigations of Sussdorf,* Pourcelot,t M‘Fadyean,t and
Csokor.§ This is all the more astonishing, for since the discovery of
Nocard of the micro-organism of pleuro-pneumonia (which was made
since the publication of the papers cited above) we are now able to verify
the histological lesions with the aid of pure cultures.

Itis difficult to assign any reason why such investigations have not been
carried out. It may be that, thanks to energetic prophylactic measures,
this once so fatal disease has received such a check that suitable material
for investigation has become unobtainable; or else, that in consequence
of the mutual interdependence of modern research, which all seems to
tend in the same direction, workers have left this problem to take up
more sensational ones, which might be described as the fashion of the
day. I am, however, of the opinion, that it will be amply rewarded if
we take up the study of old, well established phenomena from the modern
points of view, especially considering the results and technique elaborated
by V. Prowazel| and Lipschutz]] for visible filterable micro-crganisms.

From the observation of various changes which are absolutely specific
for this disease, one is forced to draw conclusions as to the effect of the
noxes of the infection. These changes will accordingly be employable in
diagnosing the disease.

The macroscopic lesions in the lungs in pleuro-pneumonia (exudative
fibrinous pneumonia and pleuritis) cannot be used to diagnose the disease,
as they can appear primarily or secondarily as lesions due to other
diseases: the various histological changes on the other hand are quite
specific. This can be seen to a limited extent from the already existing
literature on this subject. It is very useful to be able to make a diagnosis
of the disease entirely from the histological lesions, without having to
wait for the result of cultures, especially here, where pleuro-puenmonia
is of fairly frequent occurrence.

*Deutsche Ztschrift f. Tiermedizin, 1879, VY. Bd., page 358,

flyvon Médical, 1881, page 145.

{The Journal of Comparative Pathology, 4/LO/TR9T, page 333.

§Wiener Klinische Wochenschrift, 1894, page 964, anil Lehrbuch der Gerichtlichen Tierheilkunde 2
HAlfte, Wien 1891, page 415.

|| Arbeitenaus dem Kaixerlich, Gesundheitsamt 1905-06, Munch. Mediz. Wochenschrift, 1908,

{ Centralblatt £. Bacteriologie, Bd. XLVITT, 1908, Heft 1, page 79.

156

It is our intention to show that histological diagnosis is both possible
and practical.

Both the pathological anatomical collections of the Government
Veterinary Bacteriological Laboratory, and material collected during the
investigation of an extremely virulent pleuro-pneumonia virus, offered
special facilities for the study of the histology of the lesions of the disease,
and the more recent cases have given us a specially valuable statistical
material. Unless otherwise stated, the material, on which this paper is
based, has been treated with Kayserling, or with Orth’s liquid (Formol-
Mueller), passed through alcohol to xylol to paraffin. And it may be
stated here that Orth’s method gives splendid results; provided that
the tissues have been sufficiently washed out they take all ‘the most recent
stains and give perfect illustrative preparations.

The study of the lesions is best carried out proceeding from the
already known to the new, and drawing one’s conclusions from recognised
data of the lungs to compare the other phenomena connected with pleuro-
pneumonia. Unless otherwise stated, corrections will only be necessary
with regard to microscopical details.

Ina paper “On Microscopically Visible Filterable Vira,” Lipschutz
deplores without reason the absence of histological records in pleuro-
pheumonia, and expresses the wish that investigations should be made with
the object to demonstrate the presence of “ Einschlusse”’ (inclusions in cells),
or of specific products of degeneration in epithelial cells. I have paid
special attention to this question as far as possible on hardened material.
I intend to return to this problem elsewhere.

My object in this paper is not to study the alterations in the cells
themselves, but the changes in the histological complexes. First taking
the microscopical lesions I will then go over to the microscopical] details.

Macroscopican Lesions IN iHE Lunes.

All the lobes of the lungs examined were preserved in Kayserling 3,
and consequently gave very clear insight into the structure of the morbid
changes. It would carry us too far should I analyse in detail each of
the pieces at my disposal, especially as we are dealing with well-known
data. The general lesions that can be described from about ten lungs
and from a similar number of pieces, most of which are taken from
chronic cases, or such with high fever, are as follows :—

The pleura has in most cases undergone the different known changes.
The fibrinous, plaque-hke, thick deposits on the pleura pulmonalis of the
anterior and middle lobe are most conspicuous ; their fibrous consistence
points to fibrine. They are easily ruptured, frequently white in the
middle, with filamentous surroundings, outlined with dark yellow, some-
times, however, only straw-coloured irregular contours, which are fre-
quently haemorrhagic infiltrated.

a!

13

The thickness usually varies from 0-5 mm. to 4 mm.; 6 mm. to
8 mm. is, however, no rarity. The subjacent pulmonary tissue is always
oedematous and atelectatic.

The pleura costalis was only in one case attached to the pleura
pulmonalis by fibres. The pleura diaphragmatica is only very rarely
attached in a similar manner to the diaphragm. The spongy tissue of
the fibrinous deposit is completely filled with a sero-yellowish fluid. The
pleural cavity itself is filled with a reddish- yellow serum, containing
scattered flakes of fibrine. This fluid, the virus par excellence, coagulates
fairly readily when in contact with the atmosphere, but expresses a large
quantity of serous liquid. On removing the fibrinous deposit, the pleura
appears as a much thickened, opaque, whitish membrane, which gradually
merges into the diffuse grey pleura of the whole lung; these discoloura-
tions are often pigmented, and streaked with small rarely ramificated
blood vessels. Wherever the septum mediastinale is preserved, one finds
yellow gelatinous masses, which separate the cava pleuralia in the form
of tumours. On incision a considerable quantity of fluid escapes. In
two cases the oesophagus was deflected by infiltration of solid masses of
fibrine, the exudative process having here also affected that organ. As
a consequence, a pericarditis fibrinosa per contiguitatem had arisen.

The changes are very varied in the cases under review. [Frequently
only small reticulations of fibrine and capillary ektasia are present, giving
the pleura the appearance of a simple pleuritis sicca. This can be con-
sidered to be, from the point of view of comparative anatomy, the secondary
consequence of the subjacent croupous pneumonia. Through Nocard’s
researches it was demonstrated that the affection of the interstitial tissue
is primary, and that the fibrinous pleuritis arises secondarily per con-
tinwitatem. It is therefore clear that when the fibrinous exudation has
reached the great extent it does, and the serous exudate has become
prominent, the process of the disease has already reached an advanced
stage. I paid special attention to the question if the portions adjacent
to the pleura showed an older stage of pneumonia than the pleura itself ;
this could be demonstrated to be the case in 80 per cent. of the cases.
IT cannot go into the question here, why the interstitial tissue of the
lungs or its lymphatic vessels must be the points of attack of the pleuro-
pneumonia virus, as has been demonstrated experimentally and com-
paratively. As shall be shown further on, the pleura behaves in just the
same manner towards the virus of pleuro-pneumonia, on account of its
wealth of lymphatics, as do the sercus membranes generally.

The morbid portion of the lungs is conspicuous on account of its great
roundness and tougher consistence, it is consequently very easv to
separate the diseased tumescent portions from the normally collapsed
tissues of the healthy parts. The diseased lobes attain dimensions such
as are otherwise never observed. The condition of the lungs remind me

138

very forcibly of those of horses suffering from the pulmonary form of
horse-sickness,

These mostly circumscript affections of the lungs are usually covered
smoothly bv the pleura. The parenchym is compact, extremely elastic,
and heavier than water. In some cases it is also very friable. The
changes in elasticity are so variable that in some cases the affected parts
are soft and pliable, in other cases so tough and hard that a distinct grating
sound is emitted on incision. A variable quantity of sanguineus fluid,
or of yellowish-reddish, slowly coagulating, serum-like hquid runs from
the section.

On the surface of the cut appears the well-known appearance of
interstitial pneumonia, specific in such dimension for pleuro-pneumonia.
Broad, whitish-grey bands of connective tissue surround interlobularly,
interalveolarly, and peribronchially the consequitive stages of croupous
pneumonia. Such an appearance 1s described in pathologic anatomy as
“marbled.” The name * pleuro-pneumonia marbling ” is generally applied
on account of the thickened lymphatic vessels traversing the hepatised
lung tissues like the veins in marble. Large and small dark and light
lobuli group themselves to an artistic mosaic, not without regularity,
although the point of departure of the infection is often hard enough to
locate. It must be considered of importance that in 80 per cent. of
the specimens, the stage of dark red friable, or haemorrhagic, hepatisation
has been found. Blackish-red lobuli are seen in the tough, atelectatic
tissue, only rarely penetrated by thick, thrombotised blood vessels. The
surface of the sections is usually coarsely granulated. Close by one
invariably finds lobuli in the stage of grey hepatisation. I cannot say
that it is difficult to find such places, as Sussdorf states. Such groups
of lobuli are often in dry necrosis. They are dark brownish-grey, and as
compared to the other lobuli conspicuously dry. I do not find that they
are always surrounded by dark lobules in my material. One frequently
finds patches (from 1 cm. to 20 cm. in diameter) showing oedematous
infiltration, and immediately alongside of them patches in advanced
necrosis. It is reserved for experiments to show the reason of such
differences. The primary condition (stage of ‘“‘engouement’’) of the lobuli
is always found; it is conspicuous that it is always arranged so as to
surround the dark red patches. Healthy lobules, or such with a slight
crackling oedema, are frequently interspersed among the diseased ones.
We possess specimens of lungs in which single dark red hepatised lobuli
are scattered on otherwise absolutely healthy tissue, but the expression
“absolutely healthy ’ must not be taken literally, because the inter-
stities are always somewhat enlarged and infiltrated. The older the
stages, the more frequent is necrosis and sequestration. It has been
demonstrated specific for pleuro-pneumonia that several lobuli, or even
a whole lobe, necrotises and becomes separated in consequence of a

139

demarcating inflammation by energetic capsulation from apparently
healthy or freshly hepatised tissue.

It is a peculiarity of the virus of pleuro-pneumonia and its toxines
to facilitate the closure of all nutrative channels by the thrombosing of
the blood vessels and the progressive-productive processes of the inter-
stitia, thus forcing necrosis to take place. Even if in certain portions,
through resolutio, a restitutio ad integrum takes place, bands of connective
tissue remain behind along the septa and bronchi. A sequester appears
as a brownish-grey mass surrounded by a granular slippery capsule, from
which portions of blood vessels and thick fragments of bronchi project.
Mummification of sequesters often takes place, and if bacteria from out-
side penetrate, fermentation processes can take place, and deliquation
often causes the whole to anneal.

Sussdorf observed such cicatrisation of small foci in protracted cases
of lung-sickness. Two such cases are in our collection. The sequester
is always intimately connected with the thickened septa. This stage of
pleuro-pneumonia corresponds to a Pnewmonia dissecans.

The bronchi are always more or less affected, the peribronchial tissues
are during the initial stages oedematous; later on changes take place in
their lymph vessels, with exudation of fibrine, followed the formation by
neoplastic connective tissue. For this reason the bronchi appear in
chronic cases as thick, rigid tubes, sometimes containing plugs of fibrine,
close beside them one usually finds blood vessels containing a thrombus,
whose walls present the same thickened appearance. I have not yet seen
any direct bronchioectasia as described by Sussdorf.

The mucous membranes of the largest bronchi are usually somewhat
swollen, with small superficial haemorrhages. The nearer they are to
the diseased lobuli, the more readily are they drawn into the morbid
process; they then contain slimy, crumbly, friable plugs.

Besides all these manifestations of exudative pneumonia, closer
attention must be given to the interstitium of the interlobular, inter-
alveolar, and of the peribronchial tissue.

Pleuro-pneumonia virus reaches the lungs by inhalation, as has been
demonstrated by experiment; arrived there, its pathogenic influence is
first felt by the lymph vessels. Without going into the question
whether the processes take their origin in the lymph vessels of the inter-
stities or of the pleura, it is certain that its dissemination is closely cor-
nected with their course. In the beginning of the disease, one finds the
interstities broadened, waxy, yellowish-white, greatly infiltrated, covered
with a reticulation of haemorrhages.

The margin of such parts are white, consolidated, but the interior is
filled with small cavities and crevices, containing a clear, amber-coloured
fluid or fibrinous coagulum. In later stages, which can often be found
on the same section, the walls of the cavities or varices, which are nothing

140

else than lymph vessels or ducts, have generally become thicker and
‘more callous. The rounded cavities are turgid with exudate, often even
filled with small, firm, friable, dark yellow thrombus. One can cut them
open and squeeze out the contents.

The septa look like rosaries in consequence of the transparent hollows.
As the processes becomes older the appearance also gets more uniform ;
the neoplastic connective tissue takes up most of the space, and the septa
becomes broader, opaquer, and more callous. Septa 3 cm. thick are not
rare. The pulmonary tissue is divided up into more or less square areas
by the interstities, so as to give a fanciful resemblance to a chess board.

The septa close up to the greyish-white, much-thickened subserosa
if the pleura is affected, and if its morbid changes take place pari passu
with those in the lungs. On removing the yellowish-white attached
exudate, one recognises bread opaque bands of connective tissues corre-
sponding to the outlines of the lobuli.

A remark on the modus of infection may here be interesting. In a
specimen of a lung of an ox (belonging to the herd of this laboratory) a cir-
cumscript serous fibrinous infiltration of the peribronchial lymphatic
vessels was found.

The processes seemed to have the tendency to spread from the major
bronchus to the interalveolar tissue. I feel inclined to beheve that most
pleuro-pneumonia infections originate in the bronchial and peribronchial
lymph vessels and spread per continuitatem to the rest of the lung. Be
that as it may, it is certain that in the initial stages we have before us
pathologic-anatomically an interstitial pneumonia due to serous fibrinous
lymphangitis. The inflammation, although originally a purely interstitial
disease, an uncomplicated pulmonal lymphangitis, soon spreads to the
neighbouring alveoli, giving the appearance in chronic cases of a croupous
pneumonia. Unmixed pleurogene pneumonia seem to be rare, that is
to say pneumonias in which the process has spread secondarily from the
pleura, although it has been postulated that only in such cases does one
find the well-known characteristic macroscopic lesions. It is certain that
the interstitial pneumonia of lung-sickness is not a recuperative reparatory
process, such as pneumonias caused by foreign bodies or traumatic
pneumonias or abscesses are, for the changes i in the septa take place at a
period when there can be no question of reconvalescence.

Deposits of fibrine in the vessels of the interstities are caused by the
influence of the exudation, which spreads from the lymph vessels to the
petivascular tissue; the blood vessels being very often thrombosed.
The same fate awaits the pulmonary blood vessels, which, compressed by
infiltrated lung tissues, can pass through all stages ot ‘thrombosis and
resolution. Dark red thrombus, attached to the intima are most frequently
surrounded by the adventitia and perivascular connective tissue as a
tough yellowish-grey ring. Fuiliform or lobular appendages of the intima

141

of the pulmonary veins as described by Sussdorf have not been observed
In my material.

Although the changes due to lung-sickness are not restricted to the
pulmonary tissue, yet the lesions in the lungs are of sufficient value to be
of great help in diagnosing pleuro-pneumonia macroscopically.

1. The interstitia, ie. the interalveolar, the interlobwlar, and_peri-
bronchial connective tissues are the seat of a primary inflammation of
serofibrinous character. The chief changes due to inflammation take
place in the connective tissue, therefore pleuro-pneumonia can rightly
be said to be an cnterstitial pneumonia.

2. The disease follows the course of the lymphatic vessels very
closely and affect the lymphatic vessels in the first place; having recourse
to the microscopic examination, we must diagnose a lymphangitis
serofibrinosa.

3. The thickening of the septa (which can reach 8 cm.), and the
so-called marbling of sections have certainly some diagnostic value, pro-
vided that the histological details are in agreement.

One will never find the described stratified arrangement of cavities
and fissures in traumatic pneumonia, so common in cattle in consequence
of perforation by foreign bodies; quite independently of the total differ-
ence in histological details.

4. After sequestration has taken place diagnosis is no longer possible
except by microscopical investigation.

MicroscopicaL LESIONS IN THE LUNGS.

The specimens which were preserved as already mentioned in
formaline or Orth’s liquid, were examined in paraffine or celloidin sections,
after staining with haemalum-orange, Van Gieson, May-Grunwald Fischer,
Heidenhains-haematoxyline, fibrine staining after Weigert and Kockel*
or after Fraenkel} with Best’s carmine for glycogen elastic fibrillae after
Fraenkel and Mallory. Hach section was carefully analysed in order to
facilitate comprehension and to be able to make comparisons.

In looking through many specimens, one finds great diversity in
details according to whether the sections were taken from pieces of recently
diseased lobuli, or from such of more or less chronic cases. However
they all have in common that thev are stages of a croupous pneumonia,
and show conditions that are known already and which have been
described elsewhere. It will be readily understood that the specificity
of the histological lesions of pleuro-pneumonia will not be found here,
although an observer, who has much to do with such specimens might
find out details which could help towards a diagnosis. It was shown in
the microscopical anatomy of pleuro-pneumonia that all three stages of

* Centralbl f. All. Patholog., Bel. 10.
+ Muench. Mediz. Wochenschrift No, 50, 1908, p. 2634.

142

croupous pneumonia are found, the height of the condensation or grey
hepatisation being the most numerous.

More or less homogenous loose fibrinous masses mixed with flakes
of albumen fill the alveoli, the fibrine threads being shown wonderfully
distinctly by fibrine staining. Near the septs, the alveolar plugs appear
denser, owing to the accumulation of polynuclear neutrophile leucocytes
and red blood corpuscles. —

Alveolar anastomoses are often found in such places near the septa
because the distribution of fibrine is more regular. In certain stages
of the disease the darkest and most nucleated alveoli are situated in the
immediate vicinity of the bronchioli, bronchi, and blood vessels, and in
degenerative processes which will be described further on. However,
one can state generally, that in classical cases, fibrine exudation always
takes the first place. Frequent emigration and desquamation in_ the
alveoli, which lie just aposed to the interstitial connective tissue, is so
great that one immediately recognises that the inflammation is most acute
there.

It is clear that these places are those which in advanced stages show
the first signs of degeneration or restitution. Detached and_ fatty-
degenerated endothelial cells are enclosed in the fibrine, and form the
more or less granular-flaky contents of the alveoli. In some of the
examined pieces of pulmonary tissue a diathesis with erythrocytes was
prominent, so that one could believe that what one saw was a direct
haemorrhage, on account of the engoument and effacement of the septs.
Such haemorrhages are poor in fibrine. These haematomes are separated
from the croupous infiltrated lung tissue by a dense wall of polynuclear
leucocytes and lymphocytes embedded in fibrine. In advanced stages
one find large tracts of the lung poor in chromatine and impossible to
stain.

Analyses show such places to be necrotic, and to contain only
scattered fibrine elements.

A demarcating inflammation is localised at the edges of such a focus :
Karyolytic nuclear elements distension of the capillaries, large collections
of leucocytes with phenomena of emigration.

That the process is of old standing is shown by voung granulation
tissue originating from the nearest septa. Direct organisation processes
of such places are rare, consisting of large protoplasmatic cells, which
errode the trabecular masses of the sequester. The alveolar exudates
become richer in connective tissue as organisation advances. The alveolar
epithelia merit special consideration. The latest researches of Lipschutz*
on filterable and visible -micro-organisms show certain histological
pecuharities which are Dees by the action of einen SIeasns on the

* Loco vit.

145

epithelial tissues. No inclusions similar to those found in variola and
pigeon pox were seen in the cells, despite the fact that all the proper
methods for staining them were used.

All attempts to render the germs visible in sections were in vain,
although they are easily stained and demonstrated in smears treated with
gentian violet. Some of the specimens, it is true, showed fine dust-like
inclusions in the cells, but before I have elaborated a specific method of
staining, I do not care to regard this as being the stained lung-sickness
microbe.

It is remarkable that the walls of the alveoles do not show any
inflammatory changes. In young stages, the alveolar capillaries are
filled with star-shaped fibrine deposits; in other parts, they are enlarged
and crammed with erythrocytes. The larger arteries and veins are mostly
choked with fibrine. Mixed or pure leucocyte-thrombus are frequent.
In advanced stages dam-like collections of leucocytes are observed, they
are peculiar to pleuro-pneumonia and will be described under the heading
interstitial connective tissue.

The bronchioh and bronchi which have become affected show strong
epithelial desquamation and infiltration of the mucosa and_ subserous
connective tissue by leucocytes. Thick, mixed fibrinous fillings are
rare. Distinct dendritic injections formed only in two cases the cause of
the stepwise advance of the inflammation. The alveoles have
characteristics very different and changeable. The structure of the
interstities of the alveoles however, is absolutely constant in pleuro-
pneumonia. I came to recognise this peculiarity not from examination
of the lungs in the first place, but through comparison with the lesions
in the other organs.

A rich comparative material of true croupous pneumonia and other
secondary pneumonias showed that the lesions in the structure of the
interstities of the alveoles were not the same as in pleuro-pneumonia.
The histological changes which principally take place around the blood
vessels are also found in the muscles, svnovial membranes, and serous
membranes.

In the beginning of the disease the endothelial tubiform lymphatic
vessels in the widened interstities of the lobuli and the peribronchial septa
are greatly dilated. Frequently thev are only filled with a finely granu-
lated albumen coagulum, frequently also strongly infiltrated with
fibrine. The walls of the vessels are absolutely free from pathological]
changes. The typical specific changes soon supervene. The walls of the
distended lymphatic vessels become filled with leucocytes and conse-
quently indistinct. An exudate rich in fibrine spreads along the lymph
ducts through the whole interstitium, and condensing, encircles
capillaries, greater arteries and veins, The distance from the walls of

144

the vessel to the outer aspect of the condensation in every direction
measures about 150 «.*

Fibrine tinction gives results which are so characteristic that it is
impossible to make a mistake in the diagnosis. All kinds of changes can
take place in the lymph vessels, independent of the typical lesions ; these
changes can present themselves as lymphothrombosis, or lymphangoitis
haemorrhagica; in the last case the vessels contain thrombus rich in
fibrine and erythrocytes. If such strictures of lvmph vessels take place
in peribronchial tissue. the capillaries and bronchioles become obstructed
by them, and all the lesions of a bronchiolitis sero-fibrinosa appear in
the latter.

The incrustations of fibrine around the blood vessels can be replaced
in later stages by thick fibrillae of connective tissue; this. however, does
not seem to occur frequently. More usually dense crowds of leucocytes
take the place of the fibrine, which in turn undergo regressive metamor-
phoses: karvorhexis, karyolysis, and protoplasmolysis change the whole
finely granular mass into untingable flakes of granules, which are in part
resorbed and carried away by emigrant leucocytes, and a regenerative
process sets in, by which the defect is replaced by fibrillae of connective
tissue.

Should a portion remain behind as detritus, it fills fissure between
the sequester formed as just described and the surrounding layer of
connective tissue. The connective tissue of the capsule usually merges
into that of the interstities; in the immediate vicinity of the sequesters
the characteristic alterations of the lymph vessels with the typical incrus-
tations of fibrine are however demonstrable.

Such conditions appear regularly in chronic cases of pleuro-
pneumonia, and in the light of what has been said already can well serve
as basis for diagnosis.

The walls of the lymph vessels merit special attention, for we have
to decide whether we have to do with a lymphangitis or not. This
question is the more important, as Csokor is of the opinion that it is a
productive meso and periarteriitis of the capillaries of the interstitial tissue,
of which the enormous stasis in the lymphatic vessels is a consequence.
Perfectly fresh foci from the lungs would be most suitable for the study
of this problem; hut as such stages are rare we are obliged to have
recourse to foci derived from other tissues, and to make our deductions
by comparative anatomical methods.

I had the good fortune to find a perfectly recent focus in one speci-
men, it being a dilation of a lymphatic capillary. Close beside one sees
in the lumina of the vessels slight exudation and coagulation together
with strong mn bipien ee enlargement of endothelial cells. The walls

*y Mikron,

145

of the capillaries being very thin, enables the process to spread easily all
round. The perivascular connective tissue is hyperaemic and_ is
depositing fibrine. The wall of the lymph vessel is infiltrated, and dense
crowds of lencocytes surround the lumina of the larger vessels. These
phenomena, which are found to have changed still further in older stages,
are true perilymphangitic processes. At the same time no changes have
taken place in the blood vessels or their capillaries. Only after the
exudation has reached huge dimensions and the supporting tissue has
been pushed apart, and after subsequent invasion of fibroplasts, we find
stasis in the blood vessels. Pari passu with the advance of the disease,
the capillaries of the interstitities become more and more dilated and
distended with erythrocytes.

The denser the neoplastic connective tissue of the perilymphangitis,
the more readily are the walls of the blood capillaries damaged by the
toxine of pleuro-pneumonia virus. Exudation of fibrine and formation of
thrombus are the consequence. Disturbances in the circulation and con-
sequitive lesions of the walls of the vessels are responsible for the develop-
ment of these phenomena. In the smaller vessels one usually finds
small mixed thrombus with layers of fibrine on the walls; in the larger,
trabecular masses fill the lumen.

The streaks of blood-platelets are flanked by walls or rings of leucocytes,
from which fibrine mixed with erythrocytes form loops connecting the
various streaks. In older stages one finds thick neoplastic connective
tissue around the pulmonary veins, perforated and penetrated by loose
fibrine and leucocytes and lymphocytes. The leucocytes soon fall a prey
to karyolysis and karyorhexis ; plasmorhexis is sometimes also observed.
When such changes are found one also sees organisatory processes in the
thrombus ; except in the thrombosised vessels where the intima is involved
in the organisatory process, one only finds the adventitia of all three layers
of the walls of the vessels affected.

Tt is more densely nucleated around the vasa vasorum, even lymphatic
elements being accumulated in such places. An increase in elastic fibres
is not rare. During the appearance of these disturbances, the media is
not in any way affected. In strongly thrombosised vessels one finds
small nucleated foci in this portion of the wall. It was interesting to
demonstrate Diirck’s* fibrilles by means of Weigert’s iron haematoxylin
staint ; they could be recognised running through the media, not unlike
telegraph wires. That these bristle-like fibrillae, which are so readily
damaged, are found intact shows that the walls of the vessels are affected
secondarily.

The basis of the foci of leucocytes is always a lymph space, although
the walls of the spaces are often hardly possible to demonstrate. Necrosis

*H, Diirck, Virchows Archiv, bd. 189, 1907, Heft 1, page 62.
tArt. Weigert, “ Nerveufasern,” Enzyklopadie der Mikroscopischen Technik, page 942,

146

in the walls of the vessels is very rare. The elastic tissues of the intima
and media are interrupted by the necrotic elements. Direct dissolution
of the media was observed in vessels near to large necrotic portions of
the lungs, here nucleated flaky masses, in which remains of elastic elements
stain faintly, are seen. In later stages these rudiments are replaced by
elements of connective tissue.

Reviewing the above, we find that an arteriitis as described by Csokor
is really present, but the arteriitis is due in the first place to the lymphan-
gitis, as 1s seen from the lesions in the adventitia and media. The disease
is restricted to these two layers and spreads along the course of the lymph
spaces of the vessels, demonstrating the inflammation per continuitateni.
It may be that the pleure- pneumonia virus acts primarily, or it may be
that small embolic infarcts in the lymph spaces of the walls of the capillaries
and blood vessels incite the described regressive processes.

The Pleura.

I intend to point out in short the changes which take place directly
in or underneath the intermediate zone of fibrine and pleural tissue. The
fibrine itself usually lies in small pitlike depressions, from which its fila-
ments grow outwards in great quantities, covering eventually the whole
of the pleura. In the zone between fibrine and pleura, one sometimes
find a epithelial layer with large nuclei, of this occasionally only the
detritus remains. The epithelial layer becomes more and more destroyed
as the pneumonic process spreads further over the pleura.

But we usually have to do with old pleuritis, and in such cases the
epithelium is replaced by a layer of tissue of loose tough texture contain-
ing nuclei, and attached to the serous membrane. Wide vessels crammed
with erythrocytes bore their way vertically to the surface through the
masses of fibrine. These vessels are often ramified and extremely thin
walled. The endothelia of these vessels have frequently suffered shght
imbibition, their nuclei are vesicular and the chromatine attached to the
nuclear membrane. The walls are interspersed by frequent polymorphous
leucocytes, rare fusiform connective elements, and lymphocytes; the
whole is held together by fibrillar interstitial tissue.

Towards the lungs the fibrinous layer becomes arranged in parallel
layers, which have probably been exudated and coagulated in situ.
Around these layers leucocytes collect and organisation commences. The
damaged epithelia are, as is to be expected, rich in cells. The blood vessels
are dilated, often much ramified and even tangled. Here too an incrus-
tation of fibrine mixed with leucocytes, undergoing karyolysis and
karyorhexis is found around the vessels.

As the process grows older more and more of the fibrine is dissolved
and replaced by organisatory tissues. The well-known retae of fibrine
merge slowly into the supporting tissue of the serosa. As consequence of

147

the opposition of connective tissue to the lymph vessels, a great thickening
of the pleura appears, which gradually goes over into the thickened inter-
stities of the lobuli.

The adjacent alveoli are in the. stage of carnification, or have only
reached stage one of pneumonia.

Contrary to what was seen in the interstities, there is a very great
number of lymphocytes in the spaces of the tissue, which must be regarded
as emigrants from the small subpleural lymphatic glands. :

PERITONEUM.

Almost the same lesions were found in the peritoneal walls of a sero-
fibrinose peritonitis which was experimentally produced by intraperitoneal
injection of a pleuro-pneumonia culture. Another specimen was taken
from case 9, which will be described below with its macroscopic lesions.
The stages observed by me had strong infiltration and regeneration of
the intermediate layers. In some places in the peritoneum granulation
tissues are found, the membrana propria and the endothelial layer have
disappeared ; instead of them there is a layer of fibrine undergoing
organisation, just as in the pleura. The subserosa and its lobules of fat
are extremely rich in capillaries and foci of infiltration; margins of
leucocyte emigrants are very frequent. The quantity of plasma cells is
more than normal.

I believe that I am right in stating that the action of pleuro-pneumonia
virus on serous membranes is the same as on the interstities of the lungs,
and that the lymph spaces and lymph vessels are the starting point of
the inflammatory process, in which all stages of lymphangitis can be
observed.

I will now quote several post-mortem reports, material collected at
which serve as basis for further histological investigations. It will be
shown that inflammatory processes of the nature of pleuro-pneumonia
can establish themselves in the muscles, in the subcutaneous connective
tissues, in the capsules of joints and in the synovialis, these processes
being really specific of the disease.

CASE 1.—Ox 671 was injected for experimental purposes in the right
side of dewlap with two different vira, originating from two different
spontaneous cases, separated in time and locality, but both from the
Transvaal. The animal was slaughtered on the 16th day, a large
oedematous swelling having appeared at the site of injection.

Post-mortem Report: On the right side of the dewlap, from the
spina scapulae to the fourth cervical vertebra and on the left side to the
fossa jugularis is a huge swelling. The skin over the swelling is drawn
tight ; there are excoriations and bald patches at the bottom of the dew-
lap. The tumour is hard, very turgescent. Impression of the finger leaves
a distinct pit in some places. On section a large quantity of limpid

148

yellowish fluid rich in albumen escapes which flows from the tumour as
water would from a sponge. The subcutaneous tissue is thickened and
separated from the fascias of the muscles ; in it one observes the swollen
lymph vessels. The muscles (mm. cleido-occipitalis, subclavius hom. ;
pectoralis profundus, biceps brachu, and a small part of the m. sternoce-
phalicus) are distended by a similar jelly-like trembling tissue. The
muscles themselves are very moist, tough, and rich in connective tissue,
or spongy and with a characteristic appearance on the section. The fibre
bundles contain localised yellowish-white cavities with gelatinous masses,
which send radial prolongations into the apparently healthy muscle tissue.
In other places one finds dense masses of connective tissue around large
thrombosised vessels. The following changes are frequent on transverse
sections. The perimysium internum is replaced by a spongy tissue, which
forces the primary fascules of muscles apart. In these layers of oedematous
tissue one observes long reddish-brown, frequently very sanguineus, stripes
and patches, or wavelike lines transversing the muscle. In such foci one
only rarely finds small thrombosised veins. The described haemorrhagic
stripes are regularly } mm. to 1 mm. broad, but vary in length. Around
the streaks the tissues are apparently condensed, and whitish-yellow
discoloured. The healthy muscles next to the diseased ones do not show
any capillary haemorrhagies, whereas the affected ones appear dotted
with small red dots. In the musculus sternocephalicus the changes are
most pregnant, as the thickness of the modified subcutaneous connective
tissue is greatest here. Large thrombus or actually necrotic foci are
absent. The left praescapular gland is enlarged 18 x 6} cm. and of rigid
consistence. A large quantity of yellowish serous fluid, mixed with blood,
flows from the section. The capsule is thickened and infiltrated, its
spongy connective tissue is 2} cm. thick. The vessels entering the hylus
are thrombosised. The adjacent sinus of the lymph gland are very
haemorrhagic. The medulla is granulated and extruding, single yellowish
white points being prominent. The vena circumflexa scapulae is throm-
bosised, and appears as a thick black cord with greyish-whitish surround-
ings. The lungs are normal, the liver is soft and shows a slight stasis in
the vena porta; the abdominal organs with the exception of the kidneys
are normal. The perirenal fat consists of a yellowish gelatinous mass,
the capsule is easily removed on section, the cortex is slightly oedematous,
glomeruli injected and slight hyaline degeneration.

Pathological-Anatonucal Diagnosis: Myositis serosa and haemorr-
hagica, thrombolymphangitis and plebitis, lymphadenitis haemorrhagica,
nephritis parenchymatosa as consequence of intoxication with pleuro-
pheumonia virus.

CASE 2.—Calf “ Dalton,” derived from a farm on which about forty
sucking calves and oxen died from pleuro-pneumonia in consequence of
prophylactic injection with collected pleuritic exudate. The strain was
very virulent, as was proved experimentally.

149

Post-mortem Report: Body well nourished, carpal and tarsal joints
extremely swollen, skin in tension over the joints, strong’ fluctuations
noticeable. The left carpal joint contains a large quantity of light yellow,
slightly slimy, fluid with small threads of fibrine. The articulatio ante-
bracheo-carpalis has slight defects of the cartilage, capillary ramifications
and easily removable deposits of fibrine. The intercarpeal articulation
contains larger flakes of fibrine which are specially located in places
where there is least friction. The articulatory capsules of these joints
are extremely moist and thickened; their synovial coating forms folds
in many places; the same lesions were found in other joints. In the
left talo-crural articulation the deposit of fibrine is membranaceous. The
cartilage under it has two round circumscript defects; 34 c.c. synovial
fluid could be collected from this joint alone by means of the syringe.
The ligaments of all the joints show serous imbibition to a great degree.
The tendovagina of the left musculus digitalis profundus is thickened and
rich in fluid. The internal coating has small punctiform haemorrhagies.
The atlantoepistrophical articulation is naturally strongly involved on
account of its frequent motion when the calf was sucking. The capsule
of the joint is thickened by a fibrinous exudate.

Pathological-Anatomical Diagnosis: Polysynovitis and Tendovaginits
serofibrinosa, as a sequel of pleuro-pneumonia.

CASE 3.—Ox 672 was injected on the right side of the dewlap with
5 c.c. fluid (one part fluid derived from the joints of the last case diluted
with ten parts of bouillon Martin). On the seventh day after injection
a large oedematous swelling appeared, synchronising with a rise of tem-
perature. On the tenth day two pieces of the base of the dewlap, from
surface and from depth, were excised to enable histological diagnosis to
be made. The animal was slaughtered on the twelfth day for experi-
mental purposes.

Post-mortem Report: The right side of the dewlap shows a tumour
50 cm. by 25cm. The subcutaneous tissue is spongy, filled with a yellowish
serous fluid which coagulates quickly. The muscles and intra-muscular
tissue shows the usual lesions. The humor of the deeper layers contains
flakes of fibrine. Abomasus hyperaemic, mucosa swollen and covered
with slightly bile-stained mucus; omasus normal; contents of reticulum
very dry; rumen normal, containing a small quantity of food. Coecum
and colon normal; ileum slightly congested ; jejunum, mucosa swollen,
diffuse ramifications of the blood vessels and hyperaemia of the folds.
Mesenterial lymphatic gland swollen, somewhat soft; mght lobe of lungs
in posterior part not collapsed ; pleura rather whitish, milky, tough. The
tissues of the other portions of the lungs normal. Foam in trachea. Heart
contracted, left ventricle empty, endocard normal; right ventricle empty,
endocard a few white patches. Liver normal; spleen slightly swollen,
pulpa soft, trabecles distinct. Kidneys slight hyperaemia of cortex, in
two lobuli a few small white points of the character of infarcts. Synovia

150

of trochanter, joints increased and mixed with fibrous filaments. In the
right carpal jomt-increase of synovia, two small defects in the cartilage.
Fetlock joint normal. Left elbow joint capsule infiltrated thickened,
synovia slightly increased. The left joint of acetabulum, synovia increased
with fibrous flakes. The ligamenta surrounded by haemorrhagic and
slightly swollen tissue. Left knee joint, synovia increased, capsule dis-
tended, strongly infiltrated, the intermediate cartilage thickened and
hyperaemic tendovagina musculi quadricipis distended with a yellowish
liquid. Right tarsal joint, synovia increased, cartilage normal. Left
knee joint, synovia slightly increased, capsule infiltrated, the neighbour-
ing intramuscular tissue shows the usual lesions. Left praescapular
lymphatic gland enlarged to five times normal size, with haemorrhagic
infiltration of the sinus. Capsule thickened 1:3 cm., lympoglandulae
cervicales caudales swollen and haemorrhagic.

Diagnosis: Myositis et synovitis serofibrinosa specifica.

CASE 4.—Ox 688 was injected at the tip of the tail with 0-5 c.c. of a
pure culture of pleuro-pneumonia. After fourteen days a rather small
local swelling at point of injection appears, when a small piece was removed

~by operation.

Macroscopically the piece of the tail showed a very loose structure
of the different tissues, in which a lymph-like fluid had collected. Between
muscles and subcutis were small lymph spaces, surrounded by dark,
bloodstained walls. The structure of the muscles was loose, they were
discoloured, often more or less grey or white. The whitish parts were
harder and more densely fibrous.

CASE 5.—Ox 675 injected with 2 c.c. pleuro-pneumonia culture 2 cm.
above the tip of the tail. Temperature rises. The lower end of the tail
was amputated on the sixteenth day and histologically examined.

Macroscopically the intramuscular tissue of the musculus coccygeus
was broadened, a fibrous tissue replaces muscular tissue in part, in places
small thrombus, bone normal.

CASE 6.—Ox 670, injected on the right side of the dewlap with 2 c.c.
Martin’s boullion culture which was made by rubbing in a mortar fibrine
flakes obtained from the talo-crural joint of calf “ Dalton.” On the
eighth day fever and swelling at the point of injection were observed,
on the twelfth day polyarthritis. The animal was slaughtered in collapse
temperature.

Post-mortem Report: On the dewlap a tumour 20 cm. x 10 cm.,
the skin above it is taut, the consistence hard, only in places fluctuating
and oedematous. On section an enormous quantity of clear yellow fluid
flows from the spongy but tough tissue. In -many places are small
dilations of the lymph vessels and large and small thrombus of the blood
vessels. The muscles (mm. pectoralis profundus, brachiocephalieus,
sternocleidomastoideus, and biceps brachii) are changed, on section tough,

151

and filled with many small cavities containing fluid. The muscle fascules
are separated and atrophic. The intramuscular tissue and the tissues
surrounding the trachea and oesophagus have the same lesions of pleuro-
pneumonia. In the musculus sternocephalicus, at 64 cm. from the
sternum, is a necrotic focus of the size of a hen’s egg. A reddish-yellow
compact mass of tissue is situated in the middle of a very humid tube
of muscle, which has all the various changes usual in myositis. A mixed
thrombus, 25 cm. long, attached to the wall, is situated in the vena
jugularis dextra. The musculus sternomandibularis is extremely haemorr-
hagic, the areas surrounding the blood vessels being especially con-
spicuous. The oesophagus is very oedematous, and its mucosa is loosened.
The mucosa of pharynx and larynx is swollen and covered with capillary
extasia. The retropharyngeal lymph glands are swollen, and dripping ;
so are the lymphoglandulae cervicales mediae et caudales. The right
praescapular lymph gland measures 12 x 4 cm. The capsule is
thickened, and the hylus vessels leading into is thrombosised. The sinus
are slightly dilated, the follicles and follicular trabeculae protude. The
yellowish jelly-like infiltration of the subcutis extends from the mandibula
along the neck of the tenth rib. In the omentum maius there are
irregular haemorrhages, the mucosa of the abomasus is swollen and
diffusely hyperaemic. The other parts of the stomach are normal.
Coecum empty, some haemorrhagic spots. Mesenterial lymph glands
swollen; colon pigmented with a few calcareous parasitic nodules or
such containing pus. [Ileum, mucosa slightly swollen; jejunum bloody,
jelly-like contents, and longitudinal stripes. Lungs in inspirium pleura
white. Severe oedematous infiltration of the mediastinum. On the
apex of the heart a few subepicardial ecchymoses. Kidneys, the capsular
fat slightly infiltrated, glomeruli injected. All joints, but especially
the left articulatio metacarpophalangea are swollen. The capsules are
distended by a light yellow slimy fluid containing flakes of fibrine. The
synovialis only slightly reddened, but thickened and folded. The surface
of the cartilages are everywhere intact. Some of the.vaginae of the
tendons are dilated and filled with fibrinous exudate.

Pathological-Anatomical Diagnosis: Myositis, synovitis, and tendo-
vaginitis serofibrinosa specifica. Intoxication by pleuro-pneumonia virus.

CASE 7.—Heifer 674, injected with 2 c.c. boullion culture at the tip
of the tail; considerable swelling of the whole tail; skin lifted off up
to.the third caudal vertebra; in places necrotic, and in others covered
with small abscesses. The left anal lymph gland is the size of a hen’s egg.
The tail was amputated at the second vertebra and the gland extirpated.

Macroscopically the amputated part showed a considerable thicken-
ing, the skin had peeled from the subcutis over a surface of 30 cm., the
hairs had fallen off in parts, and at the lower end of the tail they were
replaced by small holes filled with creamy pus. The tip was dry, leathery,

152

and at 15 cm. from its end was encircled by a distinct haemorrhagic
demarcation. On section the musculi coecygei showed pronounced
atrophy of the fasicules of muscles, sero-fibrinous imbibition of the intra-
muscular tissue, and in a few places slight superficial suppuration. The
vertebrae and the intervertebral cartilages are intact, a slight separation
of the periost 1s only remarked in two places directly under intramuscular
abscesses. Towards the base of the tail the lesions become gradually less
pronounced and the course of the affection along the lymph vessels of
the subcutaneous and intermuscular tissues is evident. The lymph gland
weighs 22-2 grams, its capsule is slightly haemorrhagic, infiltrated, and
thickened. A large quantity of fluid flows on section, the gland is other-
wise absolutely normal in appearance with exception of the dark red
infiltrated marginal sinus.

MicroscopicaL Lesions oF THE SUBCUTANEOUS TISSUES.

Specimens taken from cases 1 and 3 serve as a basis for my investi-
gations on the lesions in the dewlap; cases 5 and 7 for those in the
caudal apex. It is a notable fact that although the skin of the dewlap
is hardly thickened, measuring 8-5 mm., it measures often over | cm. on
the tail, which would normally be less than that of the dewlap. Bacteria
can be demonstrated, the cracks and ruptures due to the oedema gave
them access and a suitable substratum to multiply on, facilitating the
formation of abscesses.

As far as it is present the stratum corneum presents advanced
desquamation and flask-shaped masses in a fibrous tissue rich in leuco-
cytes. These masses consist of necrotic detritus, and lie in small holes
in the epidermis close to the stratum lucidum. Dense small celled foci
of infiltration and young deeply staining tissues separate them from the
intact cells of the stratum lucidum. The infiltration increases in pro-
portion to the suppuration. In places the whole of the coriuwm, together
with the epidermis, is necrotic; only a few stainable nuclei are seen in
the innermost layers of the stratum reticulare, surrounded by karyolytic
and plasmolytic elements. Externally this layer j is covered by a stratum
of faintly staining fibrine. Where the pleuro-pneumonia virus has acted
solely on the deeper layers, and the pressure caused by the oedema has
left the surface of the skin undamaged, the stratum basale contains a few
foci of leucocytes, as sign of a coming inflammation.

The corpora papillares are full of emigrants, the blood vessels dis-
tended, a few lymph spaces are remarkable on account of their great size
and coagulated contents, employing fibrine staining. The lesions become
more peculiar, but also more familiar in the deeper layers of the sub-
cutaneous tissue where the follicles of hairs become more frequent. Darkly
staining margins of leucocytes surround the capillaries, whose walls are
almost always slightly thickened ; between these emigrants, and closely
attached to them, is a fine reticulation of fibrine. The single fibres of

153

the subcutaneous tissue are separated by minutely granulated masses,
except in such places or around hair follicles. The cells are rich in proto-
plasm and enlarged. On sections through pieces of the tail the changes
in the muscles and in the fat are most prominent. As I intend to deal
with the muscles at greater length further on, I will now only make a
few remarks about the lesions in the adipose tissue. The margins of the
globular tissue consist of dense tissue filled with elastic fibres and very
many nuclei. In some sections the masses of leucocyts have the appear-
ance of a demarcating line, in other places they seem to originate from a
recent invasion. In the first case one finds fragments of nuclei and
fibrino, in the second case irregular tangled capillaries surrounded at
a distance of 20 » to 60 » by a ring of fibrine. Where the fat approaches
the fascia a layer containing a great number of nuclei separates the two
tissues, and the fascia itself is weakened by friable masses. It must,
however, be pointed out, that ischaemic necrosis is only found at the base
of the tail, a distinct lymphangitis is usual nearer the apex.

In accordance with the anatomical structure is the reticulation of the
papillary stratum; all processes of infection are passed through in this
layer of the skin. Later on one finds crowds of leucocytes around the
hair follicles. The necrotic parts are separated by the usual demarcatory
processes; in their immediate surroundings the capillary vessels are
thrombotised,. and frequently even in resorption. Such places are not
frequently found unmixed, usually there are regeneratory foci forming
neoplastic connective tissue. Quite close to them, young spindle-shaped
cells are forcing themselves between the leucocytes and deposits of exudate
which in later stages replace the described lesions by proliferation. In
such a condition it is difficult to say histologically what irritation has been
caused by the layers of connective tissue between and around the muscles.
The condensation of the nucleated tissue around the blood vessels in the
subcutaneous strata may perhaps show that an inflammatory process has
existed around the vessels.

It is interesting to demonstrate that whichever tissue may have been
the seat of injection of pleuro-pneumonia virus it always shows exactly
the same changes as have been described as being found in the interstities
of the lungs under natural infection. Practical experience shows that
after a period of incubation of twelve to fifteen days, the swelling creeps
along the course of the lymph vessels centripetally, starting from the point
of injection, and cause the usual complications, such as necrosis of the
apex of the tail, peritonitis, etc.

It has been stated that contamination of the vaccine is the cause of
complications. I quite disagree with such a notion, for, working with
absolute pure cultures and taking all aseptic precautions, we yet cannot
avoid having losses. Individual disposition, or weakening of the con-
stitution, form the cause of the misfortune.

154

In South Africa there are strains of cattle which, when inoculated
with pleuro-pneumonia vaccine, invariably succumb to a rapid progressive
lymphangitis and peritonitis. The practitioner must help nature by
cauterising the skin around the tail above the swelling caused by injection,
thus causing an acute inflammation of the blood vessels and stopping the
disease from progressing towards the body of the animal. Another proof
that the virus does not follow the blood vessels, for if it did, we would
expect that it would be favoured by the inflammation. Contamination
of the virus with bacteria can be shown to be of little importance ; from
a vaccine which produced absolutely local lesions, no less than twelve
species of bacteria were isolated.

Tue MicroscopicaL Lesions In THE MUSCULATURE.

Pieces of muscles of cases 1, 3, 4, and 6 were examined. In order
to obtain comparable specimens, sections were made in each case of the
musculus sternocleidomastoideus, preparations of other muscles were also
made, especially when the macroscopic lesions were in any way remark-
able. The first changes observed in any muscle are the great thickening
of the subfascial connective tissue, the next alteration observed is that
the perimysum externum becomes involved ; here one remarks bands of
a finely fibrillar tissue, which all stain with the acid components of the
tinction employed, and which can to some extent be demonstrated by
fibrine staining, crossing and recrossing the field of vision. In this spongy
coarsely reticulated tissue one remarks isolated deep staining complexes,
these last usually contain rarely ramified blood vessels distended with
blood corpuscles. The vessels are surrounded by polynuculear leucocytes
which are arranged radially around their axis. The size of the radius
is remarkably constant, it averages 50 ¥. On the edges of such complexes
the intermediate fibrine condenses to form a dense layer. In the meshes
of the fibrine the polynuclear leucocytes readily break up; in older stages
karyorhexis is very pronounced. The blood vessels are still perfectly
normal at this stage, except perhaps that the intravascular leucocytes
collect towards the walls and a slight diapedesis of the erythrocytes is
taking place. The perimysium is not alone the starting point of the
inflammatory processes in the muscles; the secondary muscle fasciles are
strongly invaded by leucocytes on their surface where they are in contact
with morbid tissue of the perimysium internum. The invasion proceeds
gradually towards the centre; however, one sometimes finds places
where leucocytes crowding in ‘between the secondary fasicles towards
the centre, surround a capillary and there produce the usual lesions. It
is remarkable that each muscle fibrilla is separated from its neighbours
by a typical finely fibrous tissue. Here and there are places where the
muscles have entirely atrophied, their former presence being indicated
by the hollows they have left behind. Those fasciles of the muscle lying

155

nearest to the perimysium are poorest in fibres, but are filled with young
connective elements derived from the perimysium. The component muscle
fibres are swollen and vacuolised, frequently hardly recognisable. The
muscle and connective nuclei are, on the contrary, well preserved. It
is interesting to find places where the thickness of the incrustation of
leucocytes around the capillaries can be measured. As the capillaries are
situated in the perimysium of the fibres, the emigration of leucocytes caused
by the inflammation must necessarily follow the fibre perimysium. An
invasion of leucocytes takes place between the fibres of the muscle radially
around the capillary, forming a ring whose inner diameter is 100 ., and
whose thickness is of greater or less dimensions according to the size of
the blood vessel. The dark, dense net of fibres readily undergoes regressive
metamorphosis and proliferation.

We have shown that under the influence of the oedema the muscle
fibres become vacuolised by the formation of fissures and cavities, which
are only products of a widening of the intercolumnar spaces: the lesions
all point to pronounced lymphangitis.

The plasmatic substance of the fibrillae, and the fibrillae themselves
to a certain extent, dissolves under the influence of pleuro-pneumonia
toxine corresponding to the yellow-brown foci of the macroscopic
lesions. In fresh specimens one notices here and there processes
of hyaline degenerations, and in such cases it is sometimes possible to
demonstrate fat by Sudan III staining. The yellow-brown foci, just
mentioned, prove on examination to be old stages of the lesions noted
above as occurring around blood vessels, but here the blood vessels have
proliferated on account of the action of the virus and now form com-
plexes of much twisted, recently formed, epithelial tubes. Between the
vessel and the incrustation there are a large number of young cells of
connective tissue, but the incrustation itself forms a kind of demarcation
in which a detritus of nuclei erythrocytes, pigments, blood-platelets, etc.,
transversed by a few connective fibrillae form a dividing line from the
spongy tissue described above. Muscle fibres are nowhere recognisable.
A few elastic fibres may perhaps have once belonged to the perimysium.

This stage is conspicuous on account of the great proliferation of
blood vessels and connective tissue. Whether the process could advance
further cannot be stated, because animals succumb to the infection at
this stage ; it continues in the tail until a cicatrice remains. These lesions
are however verv rare, usually necrosis takes place before they have time
to develop on account of the thrombus in the blood vessels. Around the
necrotic detritus, which is very easily stained with eosine or with picric
acid, and then appears absolutely structureless, circular layers of blood
pigment, and externally to them the usual circular or long oval incrusta-
tions of leucocytes are formed. The muscle lying nearest to the necrotic
portions are more filled with round cells than those further off.

156

It is now evident that the diseased process (phlegmon) which arises
under the influence of pleuro-pneumonia virus is absolutely specific.
Regeneration, which takes place so readily in other diseases of the muscles,
does not happen here, for all the phenomena following the myositis tend
towards cicatrisation. Interruption of circulation is here the cause of
all changes, and the tangled plexus of blood vessels represent nothing
else than collateralia seeking in vain (as far as can be seen on my sections)
for a connection.

Microscopic Lesions oF THE LyMPH-GLANDS.

The most prominent lesions in the lymph glands mentioned in cases
1,3, and 6 were in the capsule and the sinus, they compliment the observa-
tions made in the other organs in the most remarkable manner.

The capsules measure 4-6 to 6-1 mm.; their structure is fibrous and
stratified, and yet contains but few connective elements. The vasa
afferentia which normally increase in size in the capsule are enormously
enlarged, and their ramifications nearly fill the whole tissue; the cavities
thus formed contain fibrine and thick deposits of leucocytes on their walls.
The arteries which branch in the capsule are distended with erythrocytes
and surrounded with the small cellular deposit usual in pleuro-pneumonia.
The lesions would have been absolutely similar to those in the lungs and
the muscles had it not been that they were complicated by the sero- and
leucocytotactic inter-action of stratified thrombus on the hylus causing
a large deposit of pigment in the form of haemosyderin crystals, and had
it not been for the very small quantity of connective elements formed.
In the tissue of the glands the sinus of the cortex are remarkable ; these
are filled with granulated albuminous matter, provided irritation is not
caused by the proximity of an inflammatory process; if this is however
the case they form bands or domes of round cells around the lymph follicles.
The medulla becomes looser towards the centre of the gland and abnor-
malities become rarer at the same time. If a few of the blood vessels in
the trabeculae were not thrombosised, the lesions would be those of acute
lymphadenitis.

In consequence of these conditions we find changes around the blood
vessels as usual in pleuro-pneumonia. The changes taking place in the
lymph vessels are dependent on the conditions surrounding them; the
vessels in the capsule and those immediately below and above it are the
first to suffer. The typical incrustations around the blood vessels are not
wanting.

Tue MicroscopicaL LESIONS OF THE CAPSULES OF THE ARTICULATIONS
AND OF THE SYNOVIALIS.

Macroscopic lesions were present in the joints described, although
no suggestion was made as to their origin. Infection per continuitatem
is out of the question, except in one case in which it occurred in the
articulatio scapulo-humeralis; it must be supposed to take place by

157

lymphogene or haematogene channels in the majority of cases. Accord-
ing to Theiler* it is possible to produce acute pleuro- “pneumonia of the
peritoneum with all the characteristic lesions by intraperitoneal injection
of the blood of a calf suffering from the natural disease. Hutcheon} goes
a long way to prove the lymphogene or haematogene transportation of the
virus ; he states that if a calf suffering from pleuro-pneumonia be castrated
or ear marked, typical pleuro-pneumonia tumours will be found at the seat
of the wound.

Except for a slight desquamation of the epithelia of the synovialis
no remarkable lesions were present. A small deposit of fibrine on the
defects was demonstrable by staining. The plicae synoviales are free
from villi, but it is not possible to state whether the changes are constant,
as only a small portion of the synovialis was at my disposal. From the
physiological point of view this would be possible, for the negative pressure
in the articulation is reduced. Those capsules of joints which have been
invaded by the way of the blood vessels present quite different lesions.
Tn normal histology one learns that the stratum synovialis is easily destroyed,
and that it sooner succumbs to inflammatory processes than any other tissue.
The great quantity of capillaries and large number of cells it contains present,
of course, great possibilities for attack; the pleuro-pneumonia process
naturally acts on these membranes. A deposit of fibrine usually con-
taining crowds of leucocytes is exudated on the denuded membrane of the
synovialis. The plicae are reflected, often adhering to each other, or two or
more are enveloped in masses of exudate. The capillaries are distended, and
form dense reticulations and loops. These lesions are not found on all
parts of the synovial membrane. The underlying connective tissue is
loosened, filled with leucocytes and its otherwise regular arrangement is
disturbed, the fibrillae running in all directions. An invasion of emigrants
following the course of the capillaries takes place only in the lower stratae
of the synovialis, the upper layers remaining free. In the fibrosa the
lesions are nearly the same, only there is less infiltration. The parasy-
novia] tissue is not affected, so that the haematogenic nature of the
infection is evident. Here it becomes clear that, when pleuro-pneumonia
virus is exuded by any means, the accompanying lesions are totally
different to those we have hitherto studied. It is consequently easy to
understand that these changes readily heal, in contrast to the other
lesions, where the blocking of the blood vessels causes necrosis and requires
peripheric restitution.

Macroscopic LESIONS IN THE INTESTINES.

Shortly before completion of this paper I had the good fortune to
observe the following case.

CASE 10.—Ox 662, injected with 0-5 c.c. culture from virus “Dalton,”
2% cm. from apex of tail.

* The Transvaal Agricultural Journal, Vol. II, No.7 , 1904, page 360,
+_Vide Theiler, loco cit., page 361.

158

Clinical Symptoms: Fourteen days after injection a large swelling
was observed 10 cm. from the tip of the tail: on the 16th day the tumour
had progressed past the base of the tail and invaded the perianal tissue.
(See figure.) The anal mucosa was oedematous and painful, causing
incessant straining. Both anal lymph glands were swollen and hard.
It may here be mentioned that twenty-six control animals only showed
local lesions. Killed shortly before death. _

Post-mortem Report: Body in fair condition; tail greatly swollen,
skin of basal portion with many cracks, resembles bark. A large tumour
extends from the base of the tail to the regio pubica. Anus open, mucosa
jelly-like, with small superficial haemorrhagies. The subcutaneous
connective tissues form a tough layer containing large cavities, from which
a yellowish liquid flows which coagulates slowly. The tissue extends to
the musculus glutaeus, semimembranosus and semitendinosus, both muscles
having their perimysium internum extensively thickened. The lymph
glands on the tubera ischiadica are enclosed in a dense envelope of connec-
tive tissue, closely attached to the capsule. The glands, which equal
a hen’s egg in size, are sappy on section. From here, the oedematous
infiltration extends to the sphincter ani, passes upwards to the musculus
bulbo-cavernosus and ischiocavernosus, and disappears gradually in an
oedema of the subcutis or near the middle of the crura. The pelvic cavity
has following lesions. The urinary bladder projects into the peritoneal
cavity, surrounded by a thick ring of oedematous fat tissue, its diameter
is more than 30 cm. The serosa, the lower portions of the excavatio-
rectovesicalis and the parietal folds of the pelvis are covered with a dense
coating of fibrine, or with fibrous fibrine. The cervix vesicae is connected
to the rectum by a mass of fibrine. The ligamenta lateralia vesicae are
2 cm. thick and deeply covered with fibrine. The peritoneum is either
diffusely hyperaemic or shows arborisation of the capillaries. The bladder
contains 3 litres of a clear, dark yellowish urine. The mucosa of the
trigonium heutaudi is slightly oedematous and contains haemorrhagic
spots. The urethra is slightly dilated before the obstruction, its mucosa
is slightly swollen and streaked longitudinally. At the collicus seminalis
the pressure of the oedematous fibrinous perianal fatty tissue closes the
urethra. The mucosa is pale, with a few ecchymoses. The whole urethra
and its corpora cavernosa are covered with a spongy tissue of fibrous
character. The left kidney is raised out of the capsule fat, weighs 920 gr.,
some of the lobuli are irregularly swollen, the corresponding urethra
is slightly dilated. The capsule strips easily ; in one of the middle lobuli
is an irregularly outlined yellowish-white patch 7 cm. broad, carrying
in its centre a few haemorrhagic patches. This part proves on section to
be an intarct, with a remarkably strong injection of the glomeruli.
The cortex of the other parts of the kidney 1s oligaemic, the medulla
alone is slightly. congested. In the slightly flattened papillae: renales
whitish-yellow stripes converge towards the apex of the papillae.

159

Histo-chemicallv the white stripes prove to be infarcts of uric acid. The
pelves and calices renales are dilated, and contain friable concrementa.
The whole kidney is moist and friable. The right kidney weighs 760 grams,
has a few infarcts and the same signs of stasis as the left. The connective
tissue around the aorta is gelatinously infiltrated as far as the arteria
mesenterica anterior, and covered with fibrine deposits which gradually
merge into the perirectal fatty tissue. From the anus cranially over a
distance of 60 cm. the walls of the rectum are 8 cm. thick, 14 cm. of which
belong to the thickened serosa, 35 cm. to the muscularis and submucosa,
the remainder to the mucosa. This coarse tube, with its thick walls, hardly
resembles a rectum ; its walls are full of small caverns containing a yellow,
quickly coagulating exudate; the cavities are mostly situated between
the fasicles of the circular muscle. The mucosa is folded into six thick
swollen folds each 14 cm. high, perpendicularly above these are narrow
small folds, all strongly hyperaemic. This diseased portion passes
gradually into the healthy rectum. The lymph glands are greatly enlarged,
soft and succulent. Some blood vessels of the mesentery and of the serosa
are thrombotised. The anal musculature is strongly infiltrated, its
muscle fibres are smothered by fibrous tissue. The abomasus is
oedematous in the folds, and hyperaemic. The duodenum contains bile
stained jelly-like contents, its mucosa is swollen and has a few hyperaemic
patches. The jejunum is slightly swollen and congested, in the ileum
the mucosa is slightly reddish or yellowish with adhaerint mucus. Coecum
and colon are swollen and contain many haemorrhagic patches. Spleen
50 em. x 15 cm. X 5 em., irregularly swollen, margins round, on section
dark red, slightly protuding. The liver has in an old cicatrix on the
diaphragm side in the middle lobe of the capsula Glissoni a few fibrinous
flakes. Colour dark brown with groups of clay coloured lobuli, grates on
cutting, hard. Pleura not folded, rather whitish, slight oedema. All
other organs normal.

Pathological Anatomical Diagnosis: Pleuro-pneumonic inflamma-
tion of the rectum, of the tail, muscles of pelvis, perirectal and perianal
connective tissue, peritonitis fibrinosa, hydronephrosis, lymphadenitis
serosa, cirrhosis and fatty degeneration of the liver.

MicroscoPicaL LESIONS OF THE RECTUM.

Of all the layers of the intestine the mucosa is least altered, its
epithelium is nearly intact and consists for the greater part of mucous
goblet cells, whose granular contents are connected with the coating of
mucine by mucus almost free from epithelial elements.

Here and there a slight dissociation of the periglandular connective
tissue with a few swollen endothelial cells may be observed. The pro-
portional thickness of the mucosa and of the muscularis mucosae are,
however, absolutely normal. Under the epithelial layer are a few enlarged

160

capillaries, crammed with erythrocyctes. The submucosa is in progressive
stages of inflammatory swelling; one instantly recognises its being a
granulation tissue by its wealth in vessels and its dense network of con-
nective tissue. Cavities and fissures filled with plasmatic cells poor in
chromatin are situated directly below the muscularis mucosae; these
elements are evidently the overgrown, desquamated, endothelia of the
lymph vessels. Small crowds of leucocytes surround blood vessels close
to necrotic foci, whilst regeneratory processes are scattered here and there
with multiplication of the connective cells and lymphocytes, which last
originate from the follicular apparatus of the intestines as shall be demon-
strated later. The centre of the inflammation, namely, the plexus
containing lymph vessels between the circular and longitudinal layers of
muscles follows this relatively narrow zone; it is remarkable on. account
of the large number of cavities containing fibrine and leucocytes, and
measures 1-2cm.to 1-3cm.in diameter. Directly under the submucosa are
irregular groups of muscles and scattered muscle fibres, which can easily
be recognised as the remains of the circular muscles; cavities containing
more or less dense endothelial elements as already described form layers
around these. In places the epithelial accumulations are so compact that
at first sight they have the appearance of carcinomatous infiltration. It
is chiefly connective elements which surround in great quantities the large
cells in the denser parts; arteries and veins thickened by connective
tissue and encrusted with leucocytes are not rare. Dense tangles of
vessels or ramifications of such bring variety into the image.

Large lymph spaces and emigration phenomena, as found in the
subcutis, are seen embedded in fibrine and opposed to the longitudinal
musculature. Disregarding their contents, the large round cavities are
readily recognised on account of their walls to be lymph vessels. Dense
bands of leucocytes follow in waved lines the course of the blood vessels
around these lymph spaces. The longitudinal musculature is disrupted,
and its fasicles lie in a net-like tissue, which merges into the thickened
serosa. Similar cavities are found in the serosa, only here the tissue
immediately surrounding them is more packed with leucocytes and, con-
sequently, shows older stages of lymphangitis. The epithelium of the
serosa has totally disappeared, fibrinous deposits, in all stages as described
for the pleura, have taken its place.

These observations serve as further proof that pleuro-pneumonia
infection follows the course of the lymph vessels. The lymph plexus
between the two layers of muscles which is involved in all affections of
the intestine, is also here the centrum of the pathological changes. The
large protoplasmatic cells can, following Saltykov (Zeitschr. fur Heilkunde,
1900), be regarded as descendants of elements belonging to the lymph
vessels. As they throw light on problems discussed above, I reserve the
special description of them for a future paper.

161

CasuaL InrEcTIon or Various Oreans. (See Plate 1.)

In South Africa drenching is the favourite way for immunising. This
absolutely unscientific method consists of pouring pleuro-pneumonia virus,
either pure or diluted, from a bottle into the slightly opened month of
the animal. Since Nocard’s investigations have shown that pleuro-
pneumonia virus remains absolutely inactive when passed through the
stomach, it is strange that such a procedure should be employed and
confer a certain immunity. It has been possible to demonstrate that this
method is frequently accompanied by complications; sometimes pleuro-
pRenOnt was the result, sometimes pathological anatomical lesions
appeared, which were caused by the virus entering a defect in the tissue
and there producing the usual progressive process. Logically one can
expect the most dangerous pathological changes; I wish, however, to
draw attention to one case only, referred to already by Theiler* > a speci-
men from this case is preserved in our museum.

The specimen is a slice through the trachea, oesophagus, etc. The
virus must have entered near the base of the tongue, for according to the
post-mortem record, a huge oedematous swelling was found from the base
of the tongue, along the throat as far as the bifurcation of the trachea.
Clinically, the animal showed blocking of the oesophagus, which was seen
by the animal bringing up through its nostrils the water it had drunk.
From the specimen, following details have been made out:—One of the
rings of cartilage, probably the thirtieth, judging from the position of the
oesophagus, is surrounded in a loose spongy connective tissue, or better,
in a fibrous tissue. Single groups of fat lobules are intruded between the
gelatinous lymph spaces. The upper margin of the tracheal ring is inter-
rupted by a space of 1} mm., through which the peritracheal tissue
communicates by an isthmus with a tumour protruding 2-4 cm. into the
trachea and spreading to the middle of both of its sides. The tumour
consists of a coarse fibrous supporting tissue, containing broad lymph
spaces with thickened walls; the whole is covered by a strongly folded
epithelium-like layer. The mucosa of the trachea is slightly thickened,
its structure is similar, though not developed to the same degree. The
oesophagus is difficult to recognise as such, for everywhere a similar tumour
protrudes itself within the otherwise normally folded mucosa. The lesions
observed macroscopically in the mucosa are sponge-like plicae, covered
by a fine white membrane, below this a jelly-like filling. The muscularis
is thickened, its fibres are dissociated, in structure it appears honey-
‘combed and it is almost separated from the fibrosa by a few lymph spaces.
Two blood vessels close by are thrombosised; their walls are greatly
thickened.

The intermuscular tissue of the M. sternohyoideus and sternomandi-
bularis is dissociated in the usual manner. The tissues of the fossa

* Loco cit., pages 361 and 366.

162

jugularis are enlarged and metamorphosed into a gelatinous mass. Several
larger blood vessels are thrombosised here. It is evident that the specimen
is a portion of a peritracheal and perioesophagal oedema.

Microscopical Lesions.

The tumour in the trachea, the changes in the mucosa, the perichon-
drium and the cartilage offer most interest ; the cartilage is absolutely
normal, a few foci of leucocytes are remarked in the perichondrium ; the
peritracheal tissue is no longer recognisable, dense layers of a tissue
containing very many cells surround the cartilage, between the layers are
insulae of blood vessels and typical condensations of leucocytes and
fibrine. All stages of inflammation such as described as above can be
noted, likewise muscle atrophies and resorptions. Examined with a low
power, large round deeply stained rings or reticulations become visible.
The tumour, which half fills the trachea, consists of a stroma of connective
tissue in which pleuro-pneumonia lesions with all their peculiarity are
observed. The inner surface consists of a band composed of leucocytes,
fibrine, blood vessels, and connective cells; within this there is a layer of
transverse sections of vessels, connective tissue with reticulations of elastic
fibres fills the interior. The mucosa has entirely disappeared; the con-
nective elements appear to be derived from the perichondrium, as the
connective trabecles originate on the perichondrium to proceed into the
tumour, where they form reticulations. In the different layers of the
oesophagus one finds similar lesions. As the tumour only represents a
combination of all pleuro-pneumonia lesions on the connective elements
of various organs, the illustrations will demonstrate all the changes in
more eloquent manner than would a recapitulation of what has already
been said.

ConcLusions.—Pleuro-pneumonia virus has an absolutely specific
effect on the tissues, it chiefly attacks the connective elements, the lesions
which can be demonstrated histologically are characterised by the follow-
ing phenomena :—

(1) The virus multiplies in the lymph vessels after having invaded
them (lungs, muscles, subcutaneous connective tissue, lymph
glands, intestines and mucosae and serosae generally). It
causes an inflammatory process, which presents itself micro-
scopically as lymphangitis serofibrinosa, lymphothrombosis, emigra-
tion of leucocytes, ete.

(2) The process proceeding only along the lymph vessels (per con-
tinuitatem) involves also the lymph spaces of the walls of
blood vessels, a peri- and mesoarterlitis, with formation of
thrombus results. Thick rings of leucocytes with deposits of
fibrine surrounding the blood vessels at a short distance in
circular symmetrical arrangement give quite a characteristic
appearance to the microscopical lesions.

163

(3) The blocking of the blood vessels gives rise to ischaemic necrosis
(Cohnheim), in which all stages of necrobiosis, demarcation, and
of incipient regeneration can be demonstrated.

(4) The dead portion can undergo sequestration, a capsule of con-
nective nature being formed by reactive inflammation (lungs,
muscles), or the primary pleuro-pneumonia process comes to
a standstill, and the tissue become transformed into a cicatrice
by opposition of connective tissue (subcutaneous tissue).

(5) The lesions of pleuro-pneumonia having originated by metastasis
present microscopically the image of a corpuscular infiltration
with pronounced serofibrinous exudation, without there being
any localisation of the process within definite portions of the
tissue. It is in the nature of the virus to form metastases,
especially in the joints, the age of the affected animal being
apparently immaterial.

(6) The histological lesions around the vessels differ from those of
chronic productive pneumonias, and can be used with certainty
for diagnosis.

164

: DESCRIPTIONS OF PLATES.”
Plate I.

Intertracheal tumour of a calf, as a sequel of drenching. (Natural size. )

‘Plate ITI.

The incrustations of fibrine and leucocytes specific for pleuro-pneumonia.
Kockel’s fibrine stain, bordeaux red (1 : 62).

Plate IIT.
Fig. 1.—Interstitium of the lungs; lymph spaces with incrustations of leucocytes.
Parenchym i in different stages of croupous pneumonia (1 : 10).

Fig. 2 (Case 1).—Subcutaneous tissue with typical lymphangitis and periar teritis.
Fibrine stain (1 10).

Fig. 3 (Case 4).—Progressive stage of lymphangitis and periarteritis, incipient
cicatrisation. Van Gieson’s stain (1 : 70).

Plate IV. |
Fig. 4 (Case 7).—Traverse section through’a piece of the tail. Wide lymph

spaces, “dissociation of muscle fasicles, and in places changes in the blood vessels.
Haemalum and eosiné (1 : 4). ;

Fig. 5 (Case 7).— Portion of same section enlarged, shows tangles of capillaries
and incrustation of leucocytes (1 : 80). ;

Fig. 6 (Case 1).—Transverse section through a vinaele Shows incrustation of
leucocytes in the perimysium internum and around the blood vessels. Van Gieson
(l_ 60).

Fig. 7 (Case 1),-- Portion of same section with muscle fibres enlarged (1 : 10).

Fig. 8 (Case 6).—Changes of the tissue around necrotic muscles. The incrusta-
tion of leucocytes around blood vessels is in concentric rings. May-Gruenwald-Fischer

ds 4).

Plate V.

Fig. (Case 6).—External lesions of articulations. The large swelling on the
right fetlock joint is due to pleuro-pneumonia.

Fig. 10 (Case 1).—Lymphatic glands, shows thickening of capsule, thrombosis
of blood vessels of hylus, and serose infiltration of cortex sinus. Van Gieson (1 . 10).

Plate VI.
Fig. 11 (Case 10).—Traverse section of rectum. (Reduced to 0°6 natural size.)
Fig. 12 (Case 10).—Rectum laid open longitudinally. (Reduced to 0°6 natural size.)

Plate VII.

Fig. 13 (Case 10).—Lesions in the rectum, mucosa, muscularis mucosae, sub-
mucosa and the beginning of the intermuscular plexus. Van Gieson (1 4).

Fig. 14 (Case 10).—Lesions in rectum (same section as Fig. 13). Intermuscular
plexus, longitudinal muscles and serosa. Van Gieson (1 : 4).

Fig. 15 (same case as Plate 1)—Pleuro-pneumonia tumour in section of trachea.
Haemalum-eosine (1 : 4).

Plate |.

Plate I.

Plate 3. Fig. 3.

Plate +.

Plate 5.

Fig. 12,

Plate 6.

Fig. 13, Fig. 14.

Plate 7,

NORMAL SPLEEN.

(A) EOSINOPHILE.(B) NEUTRPOPHILE CRANULATIONS.
GIEMSA_ STAIN.

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reRe gantget?
‘

pr}
<
ar

ABNORMAL SPLEEN.

(A) HOCHS GRANULES.
GIEMSA STAIN.

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General View from the East

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The Quarters of the Staff,

The Hostel.

The Vaccine Lymph Laboratory.

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