Observations on Rinderpest

228

GENERAL ARTICLES.

OBSERVATIONS ON

By R.

RINDERPEST.*

DAUBNEY,

Veterinary Research Laboratory; Kenya Colony. I.-Introduction. Il.-The Virus in the Blood. IlI.-Attempted Cultivation of the Virus. IV.-Immunisation against Rinderpest. Vaccination with Formalised Virus. V.-Field Inoculations. VI .-Conclusions. I.-INTRODUCTION.

THIS paper has been compiled from the records of experiments carried out by the writer during the period from September, 1925, to December, 1927. A small uninfected stable was set aside for the housing of animals under experiment, and numbers of animals were from time to time allotted to the experiments under conditions that enabled infected animals to be utilised as virus producers by the serum-making division. The number of animals under experiment at one time was directly limited by the amount of stabling accommodation available, by the necessity for keeping that accommodation quite free from infection, and by the capacity of the serum-producing section to absorb infected animals or animals for which an immunity test with virulent blood was desired. With regard to the choice of a species for experiment, cattle alone have been used. Sheep and goats, in view of their varied individual susecptibility to the disease, do not appear to be satisfactory subjects for experimental work on rinderpest, particularly when questions relating to artificial immunisation are being investigated; and, although several attempts have been made by the writer to infect guinea-pigs and rabbits by subcutaneous, intravenous, intraperitoneal, or intradermal inoculation of the virus, no evidence of infection has been obtained. A further factor in the limitation of numbers has been the expense of purchasing susceptible cattle, costing £4 or £5 each, in a country where rinderpest is enzootic, and of transporting them to the laboratory and supplying them with food. The animals used throughout the experiments were high-grade cattle of 2t to 3 years old, drawn from farms in European occupation. The virus employed is the stock Kabete strain, maintained by the serum-producing section. With regard to the virulence of this strain of virus for the particular class of animal used in the experiments, the records of virus-producing animals retained after the seventh day of the disease furnish direct evidence as to pathogenicity. Of the virus-producers inoculated each week, one is retained over the week-end to supply any demands that may be made for virulent blood for the purpose of carrying out inoculations. The animal * Received for publication 11th May, 1928.

GENERAL ARTICLES.

229

selected is one which does not show red-water parasites in bloodsmears, and which, while reacting strongly, does not appear likely to succumb to the disease during the week-end. If the animal survives until the twelfth day, when the next supply of virus becomes available, it is usually given a therapeutic dose of serum. The records show that of the last 56 animals left over in this manner, 36 have died of rinderpest, a mortality of 64·3 per cent. This point is of importance in evaluating the results of vaccination, particularly where a partial immunity only has been conferred. The experiments were curtailed when the writer proceeded on leave at the beginning of 1928, and the opportunity has been taken to collect and publish the data already acquired. The more important results relate to the production of artificial immunity by the use of formalised vaccines, and the results here seem sufficiently promising to justify complete investigation of the possibilities of this method of immunisation by those countries in which the disease is still enzootic. The writer has much pleasure in acknowledging his indebtedness to Mr. E. Harrison, Deputy-Director of Agriculture, Kenya Colony, who obtained experimental animals and facilities for the work; to Dr. Henderson, of the Medical Laboratory, Nairobi, for the carrying out of electro-metric hydrogen-ion determinations; and to his laboratory assistant, Mr. E. Hall, for much valuable assistance during the progress of the experiments. H.-THE VIRUS IN THE BLOOD.

It is Common knowledge that the virus of rinderpest is present in the blood of an infected animal during the thermal reaction, and that the disease may readily be transmitted by the subcutaneous or intravenous inoculation of whole citra ted or defibrinated blood collected during this period. Drenching an animal with virulent blood is a less successful method of transmission, and much larger quantities of blood are required to bring about certain infection. According to Todd and White, blood is infective also when placed in contact with the conjunctival mucous membrane, but not via the mucous membrane of the vagina. This is in contrast with the action of lymph or of extract of organs, which are said to be infective both by the conjunctival and the vaginal routes. Survival of the virus at normal or low temperatures has been the subject of numerous observations and there is some disagreement as to its power of resistance, particularly at ordinary room temperatures. It is, however, the usual experience in Africa that, in the small doses generally employed to infect with fresh blood, blood that has been stored at room temperature or in the cool room for periods in excess of twenty-four hours is liable to produce a percentage of delayed reactions or even failures to infect. For this reason the observations recorded here have been made with fresh blood only. The minimal certainly-infective dose of blood is not very small; Todd and White placed it at between 0·002 c.c. and 0·0005 c.c. Schein (1915) found that a dose of 0·001 c.c. was invariably infective by subcutaneous inoculation, and in a series of dilution experiments he obtained some positive results with

230

GENERAL ARTICLES.

doses as small as 0·00004 C.c. This dose gave four infections out of eight animals inoculated. Higher dilutions failed to produce any infections. Hornby and Hall (1925) placed the normal infective dose at between ·02 and ·0002 c.c. The present writer has found that 0·001 c.c. of whole blood is a reliable small infective dose for experimental purposes. With this dose the incubation period of the disease is quite regular and is not longer than when a much larger dose, for example, 2·5 C.c., is employed. This is an important consideration when experiments are being carried out in an institute where rinderpest is maintained on the premises, since the much shorter incubation period of the inoculated disease enables one to detect at once any infections that are due to contact. The earlier workers were aware that the virus in the blood is practically confined to the cellular elements and that there is little or no virus circulating free in the plasma. Kolle (1899) found that after centrifuging defibrinated blood the supernatant fluid was not infective; and that the virus in the blood did not pass through either a Berkefeld or a Chamberland filter, even after hremolysis of the red cells by the addition of a hypotonic saline solution. Similar negative results were obtained in filtration experiments carried out by Todd (1907) and Ruedeger (1908). Nicolle and Adil Bey (1902) concluded that the virus was probably intraleucocytic but did not adduce any fresh evidence in support of the suggestion, while Baldrey (1911) adopted the view that the virus was contained in or adherent to the red cells; since in his experiments plasma stained with hremoglobin produced rinderpest in a dose of 1 c.c., while serum obtained by coagulation, which was quite unstained, failed to produce reactions in a similar dose. Todd and White were able to shew that plasma removed after double centrifuging was not infective, and attributed Baldrey's positive cases to contamination of the fluid as a result of disturbing the·sediment. By repeated centrifuging Todd and White were able to obtain a "buffy" layer consisting almost completely of white cells and platelets, the virulence of which was compared with that of red cells. Of the red cells 0·5 c.c. was necessary to produce the disease by subcutaneous inoculation, whereas the white cells were infective in a dose of 0·005 c.c. They were unable to obtain the leucocytes entirely free from the red cells, or apparently the red cells entirely free from leucocytes, but the fact that the " buffy " layer proved to be 100 times more infective that the red cells shews that the white cells or blood platelets constitute the main source of virus. Schein (1917) also made observations as to the comparative infectivity of the different blood elements. After centrifuging whole blood he took a small amount of the upper or " buffy " layer and diluted it 1-50,000, and a similar amount from the base of the deposit which he diluted 1-5,000. He was unable to obtain leucocytes alone from the upper layer. These two preparations were tested on two occasions and in no instance was the deposit of red cells infective at a dose of 0·0002 c.c., while with the dilution of white cells one goat out of three became infected with a dose of 0·00002 c.c. Schein also tested the virulence of blood plasma and obtained rather variable results: 1 C.c. of plasma is said to have immunised two animals and to have infected a third, 0·05 and 0·03 c.c. are said to have immunised occasional animals, and failed either to transmit infection to, or confer immunity upon, others. Schein concluded that plasma was extremely poor in the infective agent, containing only about 20 infective elements per cubic centimetre.

The present writer is able to confirm the findings of Kolle, Todd and White, Nicolle, Schein, and others, both as to the non-filtrability of the virus in the blood and a~ to its location in the white cells.

GENERAL ARTICLES.

231

Filtration through Berkefeld and Seitz E.K. Filters. Samples of (a) citrated and (b) defibrinated virulent blood were diluted and hremolysed by the addition of an equal volume of distilled water. Quantities of approximately 100 c.c. were then passed through standard Berkefeld coarse filters. Filtration was slow. The blood was proved to be infected before filtration by the inoculation of control animals with doses of 5 C.c. of the citrated blood and doses of 2t C.c. of the defibrinated blood. All the controls, four in number, developed rinderpest as the result of the inoculation. After 50 C.c. of the hremolysed blood had passed through the filter an alkaline buffered solution was drawn through the candle, and the first 20 C.c. of this washing fluid was retained for addition to the filtered blood used for inoculation. Two animals were inoculated subcutaneously with each sample of filtered blood. The dose given was 20 c.c., equivalent in the case of citrated blood to 5 C.c. of whole blood, and in the case of the defibrinated blood to 10 C.c. of whole blood. To the portion of blood used for each inoculation 10 C.c. of the alkaline filter-washings were added. None of the four animals inoculated with filtrate plus washings developed rinderpest, and all were proved to be susceptible to the disease by inoculation with virulent blood fourteen days later. 50 C.c. of defibrinated virulent blood was diluted with a double volume of distilled water to produce hremolysis and was filtered through a small Seitz E.K. Schichten filter, capacity 160 c.c. Filtration was still slow, though more rapid than in the previous experiment. The blood was again proved to be infective in doses of 2·5 C.c. by the inoculation of control animals. Cattle X826 and X836 were both inoculated with 30 C.c. of the filtrate and failed to react. Both animals when submitted to contact infection developed the disease after an incubation period of eleven days. A larger quantity (4 litres) of virulent blood was defibrinated and laked by the addition of an equal volume of distilled water. When laking was complete the blood was forced rapidly through a large Seitz four-press filter. The first litre of filtrate came through in 1 i minutes. The usual controls were inoculated to demonstrate the virulence of the blood. Bovines X1306 and X1308 were each inoculated with 20 C.c. of filtrate taken from the first litre to pass the filter. Both animals failed to react as a result of the inoculation, and when submitted to a test inoculation with 2t C.c. of citrated virulent blood fourteen days later both contracted typical rinderpest and were utilised as virus producers for serum-making. In these experiments filtered virulent blood proved to be noninfective in doses corresponding roughly to 10 or 20,000 minimal infective doses of whole blood. Although the precaution of washing the Berkefeld filter was taken, it was not thought at all likely that the failure of the virus to pass the filter could be to any appreciable degree attributed to the adsorbing power of the filter, which, incidentally, was a new one. The explanation is to be found in the fact that the virus is firmly linked to cellular elements which are not broken down

232

GENERAL ARTICLES.

during ordinary hremolysis. The failure of virus in the blood to pass ordinary Berkefeld or Seitz filters is in remarkable contrast with its occasional filtrability in peritoneal fluid or in nasal discharge. It seems likely that these fluids usually contain considerably more free virus than is present in blood.

The Infectivity of Plasma and Cells. Cells from defibrinated virulent blood were deposited by centrifuging and were subsequently washed three times in normal saline. The cell deposit was then made up to the original volume of whole blood and was tested against dilutions of the plasma removed after the first centrifuging. Animal X1355 was inoculated with 0·002 C.c. of plasma and failed to react; fourteen days later this animal reacted to a test inoculation of virulent blood and contracted typical rinderpest. Animal X1384 was inoculated with 0·004 C.c. of plasma and failed to react; fourteen days later this animal reacted to a test inoculation of virulent blood and contracted typical rinderpest. Animal X1327 was inoculated with ·001 C.c. of suspension of washed cells corresponding in concentration to whole blood. This animal reacted in four days, developing clinical rinderpest, and was bled to death for serum-making. Post-mortem lesions of rinderpest were found.

The Infecti'vity of Leucocytes. Two attempts were made to demonstrate the presence of the virus in the leucocytes and its absence from red cells. Advantage was taken of the technique of Fleming (1926), which depends upon the property of the leucocytes to adhere to the surface of such materials as fibre. glass-wool, cotton wool, gelatine, or filter paper. Non-absorbent cotton wool is tightly packed in a glass tube of approximately 8 mm. internal bore with a constriction at the lower end to retain the wool in posltlOn. Blood is then forced through the plug of wool under positive pressure. If a U~tube with constrictions at each end of the wool plug is used, it is possible to make the blood traverse the wool several times in opposite directions. At the first attempt it was not found possible to remove all the leucocytes from citrated blood by rapid filtration through a plug of wool about four inches in length. The leucocyte count before filtration was approximately 3,700 per cubic mm. of blood diluted with equal parts of citrate solution, and after citration some 200 per cubic mm. Bovine X1577 was inoculated with 0·002 C.c. of the filtered blood, which amount would contain approximately 400 leucocytes. The necessary dilutions were carried out with the greatest care, using large quantities of diluent in order to reduce as far as possible the chances of error. This animal contracted typical rinderpest after the usual incubation period of four days. Post-mortem lesions of rinderpest were present. The control animal inoculated with 0·001 C.c ..

GENERAL ARTICLES.

233

of whole blood also reacted on the fourth day, and was bled to death on the seventh dav. In a second attempt: the size of the cotton wool filter was increased to ten inches in length. Leucocyte count here before filtration was 3,400 per cubic mm. After filtration no leucocytt"s could be detected in the blood, although several hremocytometer preparations in a dilution of equal parts were examined. Thick stained smears also failed to shew any leucocytes at the margin of the film. The red cell count increased after filtration from 3,500,000 to 7,500,000; the rather low figure before filtration is explained by the dilution of the blood with citrate and also by the fact that the animal had been subjected to two heavy bleedings before the hlood for the experiments was taken. The apparent increase in the number of red cells after filtration was no doubt due to the retention of some plasma by the filter. The red cells were normal in appearance after filtration, and the blood platelets were apparently undiminished in numbers. Bovine X1620 was inoculated with 0·004 c.c. of filtered blood. This animal did not react to inoculation and when submitted to the usual test inoculation of whole blood contracted typical rinderpest, which was confi.med by post-mortem examination. Bovine X1629 was inoculated with 0·002 c.c. of filtered blood. This animal also did not react to the inoculation and when submitted with X1620 tc a test inoculation of virulent blood contracted typical rinderpest, which was confirmed post-mortem. Bovine X1624 was inoculated with 0·001 c.c. of whole blood taken from the same sample before filtration, and it reacted typically after the usual incubation period of four days. Lesions of rinderpest were demonstrated on post-mortem examination. It may be remarked that the retention of plasma by the cotton wool plug had the effect of more than doubling the number of red cells in a given quantity of blood, so that the doses received by animals X1620 and X1629 are, in terms of the red cells present, respectively eight and four times greater than that received by X1624. Virus is removed from the blood by a filtration method that retains only the white cells. The high antigenic value of formalised spleen tissue and of the pulp of other organs rich in lymphoid tissue which is discussed later in this paper, and the lack of such property in formalised blood, are confirmatory evidence in favour of the view that the virus is attached to, or present in, the white cells. Attempts to Detach the Virus from the Leucocytes.

It was thought possible that the virus might be simply adsorbed to the white cells and that physical changes, particularly those affecting the reaction of the suspended fluid, might result in its liberation; and that in this respect the attachment of the virus might prove analogous to that of the virus of foot-and-mouth disease, which has been shewn to be attached normally to small particles of protein in the vesicle fluid. The apparatus necessary to submit virulent blood to cataphoresis not being available, resort was made to the simple

284

GENERAL ARTICLES.

expedient of replacing the blood plasma by lightly buffered saline solutions, more acid or more alkaline than ordinary plasma. Defibrinated virulent blood was centrifuged and the plasma removed. The corpuscles were subsequently washed three times in saline solution, and were finally suspended in buffered phosphatesaline solution adjusted to the required pH immediately before use. The cells were allowed to remain suspended in this fluid at roomtemperature for three hours and were then again sedimented by the centrifuge. In the first experiment the suspension fluid chosen was at pH 4·5 when added to the cells. On removal three hours later, the pH ascertained by the colorimetric method was 7·6. The following inoculations were carried out.

Material Inoculated and Dosage.

Number of Animal.

' Result

of

of test InocuI Result I nocula- Ilation with tion.

I

!,

; Virulent I Blood.

------~-------------------------I---------------

(1) Plasma removed by first centrifuging, 0·004 c.c. (2) Fluid from third cell-washing, 0·004 c.c. ... (3) Suspending fluid, pH 4·5, removed after 3 hours' contact, 0·004 c.c. ...... Suspending fluid, pH 4·5, removed after 3 hours' contact, 0·002 c.c. ...... Suspending fluid, pH 4·5, removed after 3 hours' contact, 0·001 c.c. ... ... . .. (4) Corpuscles, after removal of the acid suspending fluid, 0·001 C.c. ... ... ... (5) Corpuscles after removal of plasma: resuspended in saline, concentration equal to that of whole blood, 0·001 c.c. ... ...

X 1384 X 1386

I Negative

, Postive

Negative

I Positive

X 1395

Negative

Positive

X 1402

Negative

Positive

X 1334

Negative,

Positive

X 1311

Negative

Positive

X 1327

Positive

This experiment, one of several carried out, shows that the virus is not liberated by suspension of the cells in an acid medium at pH 4·5. Apparently gases are liberated which have the effect of readjusting the medium to the normal for blood plasma. The acid reaction of the solution may be responsible for the destruction of the virus in the cells, as evidenced by the failure of X1311 to contract the disease. Similar results were obtained when less acid solutions, viz., pH 5·5 and pH 6·5 were employed, but in the latter case the infective power of the treated cells was still sufficiently great to infect animals in a dose of 0·001 c.c. In every instance but one where an alkaline solution of pH 9,5, 10·5, or 11·5 was employed inoculation of the suspending fluid gave negative results. In the one exception, animal X1578 failed to react to the inoculation of fluid and proved immune when subsequently tested with a large dose of virulent blood. One other animal, purchased by the laboratory in the same batch, X1574, also failed to react to an inoculation of 0·001 C.c. of virulent blood, and failed subsequently to react to 2t C.c. of virulent blood. It is possible that both these

GENERAL ARTICLES.

235

animals had acquired immunity as the result of an attack of rinderpest during calfhood. The results of the experiments upon this point indicate that it is not possible to detach the virus from the white celh simply by changing the hydrogen-ion concentration of the fluid in which the cells are suspended; but further observations as to possibility of liberating the virus by cataphoresis might be of value, although it is probable that they would yield equally negative results. The destruction of the virus in the cells by contact with acid solutions in the neighbourhood of pH 4· 5 is of added interest when it is compared with the rapid loss of the antigenic power of formalin-treated virus suspended in solutions of similar pH range. This point is referred to later in the paper (p. 247). IlL-ATTEMPTED CULTIVATION OF THE VIRUS.

Boynton in 1914 published a preliminary account of what purported to be successful cultivation of the virus of rinderpest, but no further communication has yet appeared in confirmation of the claims. Boynton's best results are stated to have been obtained with Bass and Johns' medium for malarial parasites. The source of virus for the inoculation of cultures was defibrinated virulent blood, and subcultures were made every three or four days. In one series a culture was found to be infective at the sixth transfer, in what was computed to be a dilution of 1 : 2832921, and it was contended that this was the strongest evidence that multiplication of virus had taken place. Minctt (1923), carefully working over the ground covered by Boynton's experiments, failed to obtain any evidence of growth and suggests that Boynton's apparent success was due to other factors of which perhaps the chief was the probability of considerable error in the carrying out of dilutions. It is shewn below that an occasional infection is produced by what are justly regarded as sub-infective doses, and that in other cases immunity is produced by such doses without the occurrence of any actual symptoms of infection; cf. animals X 1260, X 201, X 42, and X 161. Numerous attempts to cultivate the virus of rinderpest have been made by the present writer, and included in these are attempts in which Bass and Johns' and other similar media have been used, but in no case has any evidence of growth been obtained. Experiments which were designed to test the possibility of cultivating the virus by the method of Gye are perhaps of more general interest, in spite of the fact that the efforts at cultivation here also were unsuccessful. The report of Gye and Barnard (1925) upon the cultivation of the virus of chicken sarcoma and other neoplasms aroused considerable interest, and numerous workers have endeavoured to repeat Gye's observations, with somewhat contradictory results. While it is 2.sserted, on the one hand, by Mackenzie and. Ellingworth (1926), and Baker (1926) that results similar to those of Gye have been obtained, other workers, notably Murphy (1926), Flu (1926), and Cori (1926), have failed to obtain any evidence of growth. Flu and Cori have demonstrated that activation of chloroform-treated virus by non-specific substances is possible, but only when the chloroform-treated virus is itself in large doses capable of producing the disease. In other words, the suggestion is that the chloroform-treated virus of Gye (used by him as specific activator) itself contained a sub-infective dose of living virus, and that the presence of

236

GENERAL ARTICLES.

substances irritant to the subcutaneous tissue (e.g. embryonic tissue) in the inoculum enabled these sub-infective doses of virus to set up an infection. The cultivation experiments that were carried out (chiefly in 1925, and the early part of 1926) in this laboratory were designed to examine the possibility of cultivating rinderpest virus by Gye and Barnard's method, and were in no senSe an attempt to investigate the validity of the results already reported with neoplasms. The results are, however, of general interest since they demonstrate the possibility of producing occasional infection or immunisation with extremely small doses of virus. Spleen tissue of infected animals was chosen as the source of virus, on account of its richness in white cells. The tissue for cultivation was removed with sterile precautions and a small piece weighing 2 grams was pulped in a mortar and transferred to a tube containing 24 C.c. of serum broth. The broth used was a trypsin-digest medium made from lean beef, horse-meat, or ox-spleen (Hartley's formula with potassium chloride), and the serum of a horse or a susceptible bovine was added in the proportion of 1: 5. Fragments of normal bovine spleen were added to the tubes used for subculture, and to one batch of tubes there was the further addition of small fragments of rabbit-embryo. Definite amounts of broth were transferred by pipette when subculturing so that the approximate dilutions in the final subculture could be calculated. Spleen was also utilised in the preparation of the specific substance. A 5 per cent. suspension of spleen tissue in Ringer's fluid was made by pulping spleen with sterile sand in a mortar and subsequently filtering through pulped filter-paper and sand. The filtrate was practically cell-free, and its virus-content was not therefore likely to be very high. In fact, the regularly infective dose of this extract proved to lie between 0·1 c.c. and 0·01 c.c. The extract was inactivated by incubation in test-tubes for 3t hours with 10 per cent. chloroform. It was then transferred to Petri dishes and the chloroform finally removed by Geryck pump. The power of the chloroform-treated extracts to activate subinfective doses of blood .or untreated spleen pulp extract was tested, and also its power to activate cultures prepared by the methods outlined. In addition to the inoculations tabulated above, -inoculations of culture preparations alone were carried out from time to time, although none are included in this series. Appearances suggestive of growth-faint opacity in liquid media, and accretions superficially resembling colonies on solid media--were not infrequently observed; but in every instance the preparation when inoculated into cattle failed either to infect or to produce a reaction, and not one of these animals proved to be immune when tested later with virulent blood. The experiments on Group IV. are sufficient to indicate that rinderpest virus which has been inactivated by chloroform is not capable of reactivating cultural preparations of rinderpest virus prepared by this method. The results in Group I demonstrate further that the

'"

GROUP I.

- -

8 c.c. *40 c .c. "" 10 c.c.

X 1266 X 2188 X2178

c.c. c.c . c.c. c.c. c.c. c. c.

8 4 8 4 8 4

101 11 155 131 189 190

X X X X X X

Dose.

GROUP II.

SUBINFECTIVE Dos s.

Material used.

..

...

, ... Spleen extract, filtered through paper pulp an I diluted 1 :500 .. , ... Spleen extract filtered through paper pulp an ' diluted 1 : 100 .. . ... Spleen extract filtered through paper pulp an diluted 1 : 10 Spleen extract filtered through paper pulp and diluted with equal parts of broth ... ... 5 % Splcen extract, filtered through paper pulp, u ndiluted

LARGER DOSES.

...

- - - - - -- - --_ ._ -- - -- - --- -- ------- _._ - --_ ._- - ---- - ---

,

,

...

X 1592 X 1594 X 87 X 100 XIH X217

X X X X X

94 42 136 138 1260

No.

--

/{

I

I

I

i

i{ .. I ... ... ...

..

I

I

I

!

c.c. c.c. c.c. C.c. c.c.

1 c.c. 1 c.c. 1 c.c. 1 c.c. 2 c.c . 2 c.c .

1 1 2 2 2

Dose.

ANIMALS INOCULATED WITH DILUTIONS OF UNTREATED VIRUS (SPLEEN EXTRACT) ALONE.

5% Spleen extract, filtered through paper pulp an I diluted 6 X 10-'

5% 5% 5% 5%

{

{

{

f

No.

""These doses therefore contained roughly 8 and 2 grammes of spleen tissue respectivel y .

5% Spleen extract, filtered through paper pulp and :liltlted with Hardey's broth 6 X 10-·.

-- ---

5% Spleen extract filtered through paper pulp and treated CHCLa for 3! hrs. at 37° C. 1 : 4 Spleen pulp dilution in T yrode's solution . Treated with CHCLa 10% for 72 hrs . at 37° C. .

5% Spleen pulp dilution in Ringer's Fluid, not filtered. Treated with CHCL· for 3t hrs. at 37° C.

5% Spleen extract, filtered through paper pulp. Treated with CHCL. for 3! hrs. at 37 ° C.

5% Spleen extract, filtered through paper pulp. Treated with CHCL. for 3t hrs. at 37° C.

Material Used.

I

N egative Negative Rinderpest Rinderpest Rinderpest Rinderpest

Negative Negative Negative Negative Rinderpest

Reaction to inoculatino .

Negative N ega tive Negative

Negative Negative Negative Negative Negative N egative

Reaction to inoculation.

Rinderpest Rinderpest Rinderpest

Rinderpest Rinderpest Rinderpest Rinderpest Rinderpest Rinderpest

Subsequent test of immunity with virulent blood. Dose 2·5 c.e.

-

-

-

Rinderpest Rinderpest

-

Rinderpest Immune Rinderpest Rinderpest

J.

l~

O:J -'I

:>-

~r

r;; Z

~ Subsequent test of im- I p t%l munity with en virulent blood. Dose 2·5 e.c.

ANIMALS INOCULATED SUBCUTANEOUSLY WITH COMPARATIVELY LARGE DOSES OF CHLOROFORM: TREATED VIRUS ALONE .

f L

5% Spleen and diluted 6 X 10-'. Received -also 2 C.c. of 5% spleen extract filtered through paper pulp and treated CHCLa for 3t hrs. at 37° C.

1328 1329

X216 X 201

I

1 c.c. 1 c .c .

1 c .c . 1 C.c.

1 c.c. 1 C.c. Negative (?) Positive. Temperature reaction only. Negative Negative

Negative Negative

Rinderpest Rinderpest

Immune

Rinderpest

Immune Rinderpest

...

...

...

..

Culture in Hartley's broth + serum + spleen tissue, 4th subculture at 3 day intervals . Dilution approximately 6 X 10- 5 • Culture sown originally with pulped spleen tissue, unfiltered . Received also 2 c.c . of 5 % Rinderpest Spleen extract filtered through paper pulp and treated with CHCLa for 3t hrs. at 37° C. ... ... ... . .. Culture in Hartley's broth + serum + spleen tissue, 4th subculture at 3 day intervals. 5 Dilution approximately 6 X 10. • Culture sown originally with whole blood. Received also 2 C.c. of 5 % R. spleen extract, filtered through paper pulp and treated with CHCLa for 3t hrs . at 37° C. Culture in Hartley's broth + serum + spleen tissue and rabbit embryo, 4th subculture '- at 3 day intervals. Original sowing from spleen pulped in mortar, unfiltered. Received also 2 C c. . of 5 % R. spleen extract, filtered through paper pulp and treated with CHCLa in 3t hrs. at 37 ° C. ... ... ... ... ... ... .. . Culture in broth prepared by tryptic digestion of spleen pulp, with the addition of ox serum. 3rd subculture at 4 day intervals. Dilution approximately 6 X 10". Cultures originally sown with spleen pulp. ... ... .

j

~

I

l ~ J

I}

1375

1298

X 195

X 209

X 137

X 192

X202

I

I

I I

2 c.c.

1 C.c.

2 c.c.

1 C.c.

1 c .c.

2 c.c.

1 C.c .

Rinderpest

I

!

I

i

Negative

Negative

I ! Negative

Negative

----

Rinderpest

Rinderpest

Rinderpest

Rinderpest I

Rinderpest

I Negative I Negative

Rinderpest

Negative

I

GROUP IV. ANIMALS INOCULATED WITH CULTURE PREPARATIONS, TOGETHER WITH COMPARATIVELY LARGE DOSES OF VIRUS TREATED WITH CHCL a. Subsequent test of imDose. No. Reaction to munity with Material Used. inoculation. virulent blood Dose 2·5 c.c.

L

{

5% Spleen extract, filtered through paper pulp and diluted 6 X 10. 5. Received also 2 c.c. of 5% spleen extract filtered through paper pulp and treated CHCLa for 3t hrs. at 37° C. 5% Spleen, and diluted 6 X lO-5. Received also 2 c.c. of 5% spleen extract filtered through paper pulp and treated CHCL3 for 3t hrs . at 37° C. X 161 X 145

GROUP III. ANIMALS INOCULATED WITH SUPPOSEDLY SUBINFECTIVE DILUTIONS OF VIRUS, TOGETHER WITH VIRUS TREATED WITH CHCL a. - - --- - - - - -- -Subseque1it-test of imNo. Dose. Material Used. Reaction to mUllity with inoculation. virulent blood. Dose 2·5 C.c. ~

t:l

~

~

!

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239

GENERAL ARTICLES.

virus is easily destroyed by exposure to chloroform, and that virus so killed does not appear to retain any antigenic value. When the groups II and III are considered, comprising (II) animals inoculated with supposedly sub-infective dilutions alone, and (III) animals inoculated with similar dilutions plus virus treated with CHeL 3 , it is found that four of the animals in these two groups behave differently from the remail1ing seven. One animal, XI260, contracted rinderpest as the result of inoculations of a dilution alone. lOT to6

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The incubation period of five days (see Chart) is too short for contact infection, which generally takes from 8 to 14 days to develop. Moreover,there was no case of rinderpest in the building at this time, nor had there been any for some weeks. Other animals in the same loose-box did not contract the disease, e.g., XI266 in Group I which was inoculated on the same day with material from the same spleen and which served as a control also for destruction of the virus by chloroform: XI328 and XI329, which were both inoculated with portions of the same dilution of spleen pulp, plus virus treated with chloroform. A second animal in Group II, X42, and one in Group III, XI6I, which failed to react to the original inoculation, proved immune when tested after an interval of seven days with virulent blood. A companion animal to X42, viz., X94, which also did pot react to inoculation of the same dilution, was susceptible to rinderpest when tested with X42. Similarly, XI45, a companion animal to XI6I, inoculated with a portion of the same material, did not react to the inoculation and proved susceptible to rinderpest when tested with XI6l. The other controls inoculated on this day, namely XIOI and XlI in Group I, and X87 and XIOO in Group II, all behaved normally and were susceptible to the disease. The record of X20I in Group III, is also of considerable interest. This animal shewed a definite thermal reaction after inoculation (see Chart), but did not exhibit any symptoms of rinderpest. When tested by the inoculation of virulent blood it proved to be immune. The companion animal X216 behaved normally, as did also X155, XI3I, XI36, XI38, and XI44, inoculated on the same day, and with material from the same spleen. It may be argued that XI6I and X42 may both have been immune to rinderpest, possibly as a result of an unobserved attack of the disease at an early age; but in that case there still remains to be explained the developmen~ of rinderpest by XI260 under conditions that exclude,

240

GENERAL ARTICLES.

as far as is possible, any question of contact infection, and the thermal reaction of X201 which was followed by immunity to rinderpest. With reference to the latter animal, it is of value to compare the chart of X201 with those of animals X2263 and X2271 reacting to an inocula-

tion of formalised vaccine. Reactions of this type are a fairly constant feature in vaccination with spleen-pulp treated with very weak concentrations of formalifl, and during the temperature reaction the blood of such animals is infective. It is also significant that neither immunity nor reaction was produced in any of the animals of Group I (chloroform-treated virus alone) or Group IV (cultures and chloroform-treated virus), although the first seven animals in Group I received either 2 or 4 times the dose of chloroform-treated virus given to animals in Group III, and the last two X2188 and X2178 received respectively 80 and 20 times that dose without filtration. One must conclude, therefore, (a) that rinderpest virus is readily and completely destroyed by chloroform, and that virus so killed fails to retain any of its immunising value; (b) that what are normally sub-infective doses of fresh rinderpest virus can occasionally produce active immunity, sometimes with the accompaniment of a temperature reaction, or (c) that, still more rarely, such doses may give rise to an ordinary rinderpest infection. The results of the application of Gye's technique to the cultivaiton of rinderpest virus differ from those obtained by Flu (1926), Cori (1296), and Murphy (1926) with the viruses of Rous sarcoma, vaccinia, and fowl-pox, in that all the rinderpest virus in the treated tissue is killed by the action of chloroform, whereas in the case of the other viruses mentioned there is evidence that small quantities of virus survive the treatment. IV.-!MMUNISATION AGAINST RINDERPEST.

More than 170 years have elapsed since the earliest attempts were made immunise cattle against rinderpest. Soon after the contagious nature of the disease was recognised it was placed on record by several observers that an attack of the disease conferred a high and lasting immunity upon any animal that recovered. An analogy was at once conceived to exist between cattle plague and small-pox, for which arm-to-arm vaccination was then largely practised, and advocates of some similar method of inoculation in the cattle disease were not wanting. [Layard (1757) and others.] The first actual notice concerning artificial inoculation appears to be that of Dobson (1754), whose letter to a to

GENERAL ARTICLES.

241

friend was quoted in the Country Gentleman's Magazine. Dobson, upon the recommendation of a Yorkshire physician (unnamed), inoculated animals by making an incision in the dewlap, into which was inserted a piece of tow "dipped in the morbid matter discharged from the nostrils and eyes of an infected beast, and this was allowed to remain till the symptoms of the distemper appeared." This inoculation was taken up by Camper, Noseman, de Monchy, and Fleming and others in England and in the Netherlands, with, according to contemporary evidence, extremely variable and on the whole unpromising results. The countries of Western Europe very soon abandoned attempts to inoculate and, following the lead of England, adopted the" slaughter and quarantine" policy first advocated by Bates, with results that are selfevident to-day. Attempts to inoculate were still continued in Eastern Europe, and some success was claimed for the method in Russia, where it was stated that attenuation of the virus followed a long series of passages through cattle. The more recent experiences in serum-institutes where virus has been propagated in cattle over a long period of years fail to show any confirmatory evidence that attenuation results from passaging in cattle. The extensive outbreak of the disease in South Africa (1896-8) was the ,occasion for the introduction of another method of active immunisation, " the bile-method," and was also successful in attracting renowned scientific workers to the investigation of the problem. Koch, Theiler, Kolle, Kohlstock, and others investigated the immunising properties of the bile of infected animals. The bile taken from an infected animal on the 6th, 7th, or 8th day of the disease was injected in quantities of 10 c.c. into susceptible animals. In a few cases an attack of rinderpest resulted from the inoculation, but the immunity, even without such an attack, was said to last from ten days to four months, apparently according to whether a passive or active immunity was conferred. The bile was supposed to contain immune bodies the transference of which gave some temporary protection, and virus which produced the active immunity The chief disadvantages of the bile method were the difficulty of obtaining an ample supply of the material and the variability of the immunity produced. In preliminary observations carried out by the present writer bile taken on the 7th day of the disease failed to immunise animals in doses of 10 c.c. and attempts to exploit the bile method were abandoned. It is possible that the variability of the results can be attributed largely to the different virus content of bile on different days during the course of the disease. Subsequent to the use of the simple bile method attempts were made to destroy the virus in the bile by treatment with glycerin (Edington), or by filtration (Rogers), and to confer a passive immunity by means of a larger dose; but it was necessary to follow the bile inoculation with one of virulent blood to produce the active immunity. The bile method, which had a moderate success, was finally abandoned in favour of the simultaneous serum and virus method. The first observations as to the protective power of the serum of a recentlyrecovered animal appear to have been those of Semmer (1893), but the practical investigations of Theiler and Turner, and Kolle and others, form the basis of our present practice with regard to serum immunisation. In the earlier work the serum of recovered animals had been exclusively employed, but the South African workers in particular sought to increase the potency of the serum by repeated injections of virulent blood into the serum-producing animal. The serum was used to produce a passive immunity alone, or to produce an .active immunity when combined with a simultaneous inoculation of virulent blood or with natural infection by mixing with diseased animals.

242

GENERAL ARTICLES.

At present the simultaneous method is in general use in countries where rinderpest is still enzootic, e.g., Kenya, Egypt, Sudan, India, Indo-China. A few infected countries prefer to rely on the serumalone method, but the main disadvantage here is the short duration of the passive immunity and the expense of repeated inoculations with large doses of serum. The serum-simultaneous method when employed under carefully controlled experimental conditions is almost without risk, and may justly be claimed to be one of the most efficient methods of artificial immunisation in use to-day. When employed upon a large scale under field conditions as a general administrative measure, however, certain disadvantages attach to the method. In the first place, the reproduction in many animals of an attack of the disease creates new centres of infection, and the inoculations have accordingly to be planned district by district in order to avoid exposure of neighbouring herds to the risk of infection from animals reacting to inoculation. The severity of the reactions is supposed to depend entirely upon the potency and quantity of the serum injected to control the reaction, but, in fact, when doses of serum short of the amount required to produce a complete" block-out" of the reaction are used, the severity of the reaction depends quite largely upon such factors as the age and breed of animals, and perhaps more than all upon their physical condition. Animals in poor condition will frequently succumb to double-inoculation reactions, in spite of having received extremely large doses of serum. Veterinary officers and farmers both realise the importance of good condition, and this means that they are reluctant to undertake inoculatiori except at those periods when pasture, and consequently condition, is good. It is doubtful if a method of immunisation which creates new centres of infection will ever furnish a means of entirely eradicating the disease from countries where it is enzootic, and where there is a large population of susceptible game animals subject to little or no control by fencing. The risk of spreading infection necessitates a close control of immunisation, which is entirely in the hands of the veterinary administration; in fact, the routine rinderpest inoculations occupy such a large proportion of the time of the field staff that they interfere seriously with the opportunities of the service to give due attention to problems relating to other diseases of stock. To the high initial cost of serum there is thus added the cost of maintaining an expensive professional staff employed almost solely upon couble inoculation. A further difficulty arises in connection with the supply of virus, for which the only satisfactory vehicle so far has been found to be blood. In some countries virulent blood is forwarded from a central laboratory in sealed containers either at ordinary temperatures or upon ice, but the survival of virus under these conditions is disputed by many experienced workers, and it is certain that when stored blood is used considerable variation in the reactions is to be expected. One result of the use of stored virus may be failure of virus to infect, producing an apparent "block-out" of reaction when no active immunity has in reality been conferred. As a result, most services prefer to

GENERAL ARTICLES.

243

employ fresh virulent blood obtained by infection of an animal placed in a centre convenient for the inoculations. Here again difficulties arise; the animal is usually infected by contact with one previously infected by inoculation with virulent blood, and delay is involved if the contact animal fails to react to schedule. One of the greatest disadvantages of this method is risk of transmitting piroplasmosis, anaplasmosis, trypanosomiasis, and other blood-borne infections, which may be a very serious matter in a country where dipping is practised with partial success. Examination of animals from herds that have been dipped at short intervals show that a small proportion of these dipped cattle are not susceptible to " red water," and that while showing no parasites in blood films taken for microscopic examination the blood of such animals will transmit Babesia bigemina to susceptible animals. Virus-producing animals for the purpose of double inoculation on farms where dipping is in force are usually selected from the dipped herd, but should the inoculation officer be unfortunate enough to choose one of the few transmitting animals the inoculation of virulent blood into the remainder of the herd will be accompanied by considerable loss both from " red water" and rinderpest. For some years now there has been a desire on the part of the veterinary administrative services to be equipped with a method of conferring active immunity which does not involve the creation of new centres of infection and which is free from risk of transmitting blood-borne diseases such as piroplasmosis, trypanosomiasis, heartwater, etc. The occasional successes in immunising with sub-infective doses of spleen-pulp, referred to earlier in this paper, indicated spleen-tissue as likely to be useful for the preparation of a non-infective vaccine. Suspensions of spleen-pulp, however, in which the virus was destroyed by heating to 55° C. for one hour failed to immunise animals when injected subcutaneously in doses containing 5 to 10 grammes of tissue, and it has already been shown that comparatively large doses of spleen-pulp treated by exposure to the action of chloroform had no antigenic value. The successful immunisation of cattle and guinea-pigs by Vallee, Carre, and Rinjard (1926), with the formalised virus of foot-and-mouth diesase and the subsequent reports of Bedson, Burbury, and Maitland, and of Minett, on the preparation and use of such a vaccine in that disease, encouraged the writer to test the effect of similar formalised preparations of spleen in rinderpest. Spleen was naturally chosen as the source of virus in view of the results already obtained with extracts of that organ. During the progress of the experiments attention was drawn by abstracts in the Tropical Veterinary Bulletin to reports of successful immunisation against rinderpest by Kakezaki, Nakanishi, and Ozumi (1926), and Curasson and Delpy (1926). The publication of the Japenese workers was not available until the writer's return to England, in Februray, 1928, and the abstracts, unfortunately, did not furnish any information as to whether any chemical agent was used for inactivation or the virus was simply

244

GENERAL ARTICLES.

allowed to perish in vitro.* Curasson and Delpy were induced by the success of Vallee and his colleagues in foot-and-mouth disease to try a formalised vaccine made from rinderpest virus. Their vaccine was prepared by submitting a suspension of pulped spleen to the action of formalin, 1: 250, at room temperature in darkness. The vaccine was used after a lapse of at least 48 hours. In all, seven animals were inoculated with vaccine in doses of from 1 to 100 grammes, and were submitted to a test inoculation with virulent blood after an interval of twelve days. Four of these animals, receiving doses of from 12 to 100 grammes, proved to be immune; one animal, receiving 5 grammes, ·gave a thermal reaction only when tested; and the remaining two animals, receiving 1 and 2 grammes respectively, developed clinical rinderpest.

Vaccination 'lcith Formalised Spleen Pulp. Spleens were removed with sterile precautions from infected animals that had bled to death under chloroform anrestheisa on the seventh day after inoculation with virulent blood. The spleens were cut into small pieces and passed through a sterile vaccine-pulper of the Latapie type. The pulp was collected in sterile conical flasks and weighed. Tyrode's solution, Ringer's fluid, or some other suitable diluting fluid, was added in the proportion of four parts of fluid to one of spleen tissue. The formalin was added to the diluent before mixing with the tissue and the quantities referred to in the text or in the tables indicate the concentration of neutral commercial formalin (formaldehyde 40 per cent.) in the fluid. The mixture was shaken repeatedly for periods up to an hour and finally transferred to the incubator or cool-room for a definite period. Complete pulping could not be effected, so that the preparation consisted of a suspension of relatively -gross particles of spleen tissue in a saline fluid. The particles of tissue were, however, sufficiently fine to pass through a coarse hypodermic needle. In future experiments it is hoped to make use of some machine that will reduce the spleen pulp to a more or less homogeneous mass, and it is possible that more uniform results will be obtained in this fashion. The concentration of formalin used in these experiments is generally considerably weaker than that employed for the sterilisation of vaccines in foot-and-mouth disease, rabies, black-quarter, etc., since it was found at the outset that the weaker concentrations furnished a better antigen. With these weak concentrations, 1 : 1,0002,000, it is necessary to take precautions to avoid gross contamination during the preparation, and it is also of advantage to prepare the 'it Perusal of the original paper (1926) still leaves one in doubt as to the actual method --of preparation employed, but spleen was the source of virus, and the tissue was probably glycerinised after dilution and allowed to remain at room temperature for long periods (circa 2 years). The dose of this vaccine was 0·5 c.c. per Kwan, repeated after an interval of 7 days. Similar results were obtained with a vaccine prepared by treatment with 10 per cent. toluene and retained for 20-24 months. Lymphatic gland tissue was the only other material that furnished a satisfactory vaccine. Ether, iodine, and eucalpytol were also used in the preparation of spleen vaccines, the two latter furnishing fairly satisfactory prepartions. Virus inactivated by heat is also said to retain its antigenic ,powers.

24.'1

GENERAL ARTICLES.

vaccine in conical flasks, in which the suspension forms a comparatively shallow layer with an extensive surface at the bottom of the flask. It was repeatedly found . that a concentration of 1 : 1000 formalin failed to sterilise the material when an ordinary bulb-shaped flask was filled to the neck, while other preparations from the same organs in partially filled conical flasks became sterile in the ordinary manner. In the columns that denote the response to vaccination or to test-inoculation of virulent blood I means immune, T signifies a thermal reaction unaccompanied by any other symptoms of infection, and R denotes rind erpest. The animals marked R include all animals showing a ny symptoms o f illness whatsoever that could possibly be re ferred to a rinderpest infection apart from the simple temperature reactions, even if the symptoms merely consisted of lachrymation. Since it is not thought necessary to reproduce charts, clinical case records, and post-mortem sheets of all animals, an attempt is made to indicate the severity of the infection, if any, under the column headed" remarks." The four animals included in Table I were inoculated on the same day with preparations from the same spleen. The temperature charts of animals X1430 and X1602 are appended. It will be observed that the reaction of X1602 is weaker and of shorter duration than that

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of X1430 ; this may be considered as mainly due to the effect of the larger dose of vaccine received by Xl602, but perhaps also partly to the transmission of red-water to X1430. The urine of X1430 was port-wine coloured on the evening of the fourth day, and Babesia bigemina was demonstrated in blood-smears on each day following until the eleventh day, although hremoglobinuria was not observed after the fourth day. This animal was no doubt partially immune to red-water since the reaction was much weaker than is usual in susceptible animals. Apart from the temperature reactions, and in the case of X1430 the transient hremoglobinuria, neither animal showed any symptoms of illness; the coat remained bright, the eyes clear and dry, with healthy conjunctivre and the appetite unimpaired. It may conveniently be stated here that the injection of virulen t blood into a vaccinated animal may cause no reaction whatever, as in the case of X1432 and X1467, or it may produce a reaction ranging in severity from a simple thermal reaction, such as that displayed by X1602, to a fatal attack of rinderpest. All gradations bet\veen these two extremes are seen and there is sufficient evidence in the

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8 gr. 8 gr. 2 gr. 8 gr. 8 gr.

2 gr. 8 gr. 2 gr.

X 1941 X2001 X 2006

X 1916 X 2000

X 1990

X 2002 X2005

Liver pulp, diluted 1: 4 and formalised 1: 1,000, incubated at 37° C. for 72 hrs.

Pulp of lymphatic glands diluted 1 : 4, formalised I: 1,000, incubated at 37° C. for 72 hrs.

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Nil Nil Nil

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Subsequent Test of Immunity.

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Remarks.

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No symptoms. Temperature reaction commencing 4th da YI and lasting 6 days. No symptoms.

No symptoms. 1 Temperature reaction commencing 5th daY'1 and lasting 4 days. No symptoms. Reaction after incubation period of 4 d~ ys. Bled to death for production of virus.

I I

'~~ I ') Clinical Rinderpest after incubation per od o~ 4 days. Bled to death for productior of Virus.

J

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Bled to death for product

No symptoms.

Temperature reaction commencing on the 5th day and receding completely on the seventh. No clinical symptoms of Rinderpest. Temperature reaction commencing on the 4th day and receding from 9th to 14th day. B. bigemina and clinical red-water present during reactions. No clinical symptoms of Rinderpest. No symptoms.

Remarks.

~ Clinical Rinderpest after incubation per

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Subsequent Test of Immunity. :

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Interval in Days.

TABLE II

Nil

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8 grams tissue. 2 grams tissue.

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Reaction to Vaccine.

8 grams tissue.

X 1410

I

Number I

IAnf~als.

I

II

Dose.

Defibrinated blood diluted with equal parts of Tyrode's solution and formali sed 1 : 1,000.

Material Inoculated.

X 1467

Spleen pulp 1:4 Tyrode's solution formalised 1: 1,000 and incubated at 37" C. for 72 hrs.

X 1432

X 1602 X 1430

Spleen pulp 1: 4 in Tyrode's solution formalised 1 : 500 and incubated at 70° C. for 72 hrs.

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GENERAL ARTICLES.

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records included in this paper for the assertion that, other things being equal, the severity of the reaction to a test inoculation depends directly upon the degree of immunity conferred by vaccination. Naturally, animals in weak and under-nourished condition, or impaired physically by intercurrent infections while reacting to a rinderpest infection, will react more severely than sound animals. Hornby and Hall have endeavoured to distinguish between ability or failure to withstand completely the initial shock of infection, which they term susceptibility; and the varied response to an infection once started, which they call resistance. According to them susceptibility in a given breed of animals of approximately the same age is constant, since approximately the same minimal dose will produce infection in all the animals; and the infection set up by the minimal dose will take a varied course in these animals, depending upon their individual powers of resistance. The theory of these writers in reality amounts to a statement that immunity in rinderpest is absolute; they do not recognise any lower degree of immunity which might permit the establishment of an infection and subsequently control the response to the virus. There does not appear to be sufficient evidence to warrant this distinction, and it is shown here that any degree of immunity varying from complete resistance on the one hand to a degree which permits the development of a relatively mild and non-fatal attack of the disease, may be produced by vaccination; and that the immunity provoked varies, subject of course to the influence of minor individual idiosyncrasies, directly with the dose and antigenic efficiency of the vaccine employed. A large number of animals have been inoculated with doses of antigen sufficiently small to permit the development of an attack of clinical rinderpest after test inoculation, but the fact that the great majority of these animals recover, in contrast with the high mortality of the disease in unvaccinated animals, indicates that the course of the disease has been controlled by the partial immunity resulting from the vaccination. Immunisation with Formalised Blood and the Pulp of Other Organs.

Table II illustrates the comparative values of formalised blood, kidney, liver, and lymphatic glands. With reference to the sources of the material used in this experiment, the blood used in the preparation of vaccine for animals X1410 and X1440 was proved to be virulent in the fresh state by the inoculation of controls for virusproduction. The liver-pulp for X1941 and the lymphatic glands for X1916 were each a mixture from the same three animals, and the result in the case of X1916 indicates that the mixture was infective. The liver-pulp for X2001 and X2006, the lymphatic glands for X2000 and Xl990, and the kidney pulp for X2002 and X2005 were also each a mixture from three animals killed on the same day. The spleens of these three animals were also used in the preparation of vaccines for animals X1983 and X2045, Table III, and X1997 and X2041, Table V. (See continuation of article in next number for Tables III. and V.)

248

GENERAL ARTICLES.

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The charts of animals X2000 and X2005 are appended. Morning temperatures only are given on the chart of X2000 because this animal was out of doors during the day-time and its evening temperatures might possibly have been affected by exposure to the sun. Some difficulty was experienced in obtaining sterile perparations of liver tissue. About 75 per cent . of the flasks prepared with formalin 1 : 1000 become grossly contaminated and unfit for use. This experience has been repeated on several occasions, and it has never been possible to obtain a sterile preparation from liver-pulp with concentrations of formalin weaker than 1 : 1000, although several attempts have been made. Neither kidney nor lymphatic gland yield entirely satisfactory preparations, the former on account of the difficulty presented by the removal of adherent fat, and the latter because pulping in a vaccine pulper fails to break the tissue into sufficiently small particles . It is possible that with better methods of pulping both these organs may prove serviceable in the manufacture of vaccine; they might be utilised as a preliminary vaccine before the inoculation of a stronger preparation. Conclusions.

I.- Neither formalised blood nor liver-pulp appears to possess any antigenic value, although both are ordinarily infective in the fresh state. 2.-Formalised lymphatic gland tissue and formalised kidney pulp are both c~pable of conferring an immunity, but are apparently not equal in antigenic value to similar preparations of spleen. (To be continued.)