Vaccination against bovine piroplasmosis

AIlSTRACTS AND REPORT.

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Conradi·Drigalski Agar.-Abundant growth of blue, glassy, dewdrop-like colonies causing no alteration in the agar. Milk.-No coagulation, clearing up after fourteen days, and the appearance of a yellow colour. Neutral Red Agar. - Gas"forma:tion, fluorescence, yellow colouration. Grape-sugar Brotlt.-Uniform turbidity, sedimentation, and great evolution of gas. For the exact identification of the organism numerous agglutination tests were carried out with typhoid, paratyphoid A, paratyphoid .H, and Ga::rtner immune sera, the dilutions being f : 25,000 in the case of the first, I : 3000 in the case~ of the second and fourth, and I : 5000 in the case of the I hird. Twenty-hour agar cultures wue used, and the results were inspected after two hours' incubation. The results were the same with all the cultures obtained from the calves. The results were negative in I: 100 with the typhoid and paratyphoid B sera, and with the paratyphoid A and Ga::rtner sera positive up to I : 3000. Pathogenicity.-Agar cultures proved fatal to mice in two to five days, and the post-mortem appearances were the same in all cases. Slight ha::morrhagic enteritis and swelling of the spleen. With a lens ha::morrhages in the serous membranes and minute grey areas in the liver were found in some cases. Short rods with rounded ends were found in smears of blood. Cultures on agar, endofuchsin agar, and Conradi-Drigalski agar grew with the characters of the paratyphoid Ga::rtner group. Agglutination tests with the cultures obtained from the heart blood gave negative reactions in dilutions of I : 100 with the typhoid and paratyphoid B sera, and positive reactions up to I : 3000 with the paratyphoid A and Ga::rtner sera. From these facts the opinion might at first sight be formed that meat poisoning in man must be common, but the author draws attention to the following points : I. Veal is rarely or never eaten raw. 2. In many cases only a few organisms are present in the muscular tissue, and such infected meat could only develop its full toxicity if kept under unfavourable circumstances (great heat in summer). 3. If only a few people became ill the poisoning would not be recognised and would not become known. The author refers to a case in which he had sent to him some veal which had been partaken of by a number of people who had afterwards sho\\'n sudden and unaccountable symptoms, diarrhrea being very severe. The melt was derived from a calf that had been slaughtered on account of a sudden illness. Organisms were isolated which in culture showed the characters of the paratyphoid-Ga::rtner group. Bacteriological investigations indicated that the infection was not carried to the veal from beef by means of the butchers' knives, etc. Numerous investigations carried out by the author. have in no case demonstrated the presence of Ga::rtner's bacillus in carcases of full-grown animals. (Winzer, Zeitsclzr. f Fleisclz- und Milchhyg., Vol. XXII, No. 3, December 19TI, pp. 81- 8 4.) .

VACCINATION AGAINST BOVINE PIROPLASMOSIS. AFTER giving some historical detai's regarding the disease and pointing out the difference in susceptibility in young and adult animals, the author indicates the two chief plans that are adopted in the campaign against

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ABSTRACTS AND REPORT.

the disease, viz., destruction of ticks and vaccination. His paper deals only with the latter. He refers briefly to the methods that have up to the present been advised by various authors, and draws the conclusion that none is quite satisfactory, although the best results are obtained, especially among calves, with the injection of blood from a recovered animal. He then points out that one species of piroplasm does not vaccinate against another, and that for a method of vaccination to be acceptable it must be practically without danger even for adults, capable of being applied at any season of the year, and absolutely certain in its results. Such a vaccine the author claims to have discovered. Publication of the method has been withheld, because it was desired to test it thoroughly on a large number of animals and over a long period. The author's work in connection with the disease commenced in 1897, and two years later he submitted to a conference a report bearing out all the important facts discovered by Smith and Kilborne, but adding nothing fresh regarding the subject of vaccination against the disease. A year later a fresh method of vaccinating was submitted to a commission of authorities in France. The following is a resume of the steps leading up to the discovery of the vaccine. The author observed that the intravenous injection of blood taken from diseased animals and kept at a low temperature instead of setting up a serious attack caused only a transient indisposition, which was followed by immunity. It was also observed that blood very rich in parasites caused only abortive attacks if kept at a low temperature sufficiently long. The author claims to have been able to show that blood taken from diseased cattle at a time when it contained a maximum number of parasites and kept at 5° to 8° C. for thirty days, when injected intravenously, frequently caused a mild form of the disease, which was followed by immunity. The parasites present in this blood were found by the author to be smaller than normal, spherical, and apparently mUltiplying. An intravenous injection of 10 cc. of this blood caused about the seventh day a general reaction which lasted for one or two days. This first vaccine retained its vitality for at least eight days. Ten days after this first dose the author injected 1 cc. of blood equally rich in piroplafms but which had been kept for fifteen days only at a temperature of 5° to 8° C. This acted as a test inoculation. The proof that the first vaccine was living and virulent was that if, instead of being injected intravenously, it was injected under the skin or into the muscles it often caused a fatal attack. It was this double vaccine that was tried at Buenos Ayres and Paris in 1900, and it was found that it always left animals immunised against an inoculation with virulent blood. At this time the piroplasma bigeminum was the only one known, and the author believed that the immunity conferred by the vaccine would be effectual against the natural disease. With the object of testing the method a number of animals were vaccinated and sent into infected areas. These vaccinated cattle were resistant to the piroplasma bigeminum however it might be inoculated into them, but in infected areas they suffered from severe piroplasmosis caused by the bites of ticks. The cause of this phenomenon was not discovered by the author for a long time, but he finally found that the parasite in this case was a different one from the piroplasma bigeminum. This parasite he named the piroplasma argentinum, and he discovered that while the piroplasma argentinum was effectual (or vaccination against the piroplasma bigeminum the converse did not

ABSTRACTS .AND REPORT.

hold good. He claims to have shown that animals that are very resistant to the piroplasma bigeminum may be very susceptible to the piroplasma argentinum, and that this parasite may even prove fatal. The author attempted to apply the same method of vaccine preparation to this second parasite, and found that it did, not form a vaccine when treated in this way but retained its full virulence. This is considered by the author as further evidence of the non-identity of the parasites. The difficulty of preparing a vaccine was overcome, however, by taking advantage of a partial immunity left by the piroplasrpa bigeminum against the other parasite. At this time the author commenced to prepare a triple vaccine. This involved the following operations. The first dose was one composed of blood very rich in the piroplasma bigeminum which had been preserved at SO to 8° C. for thirty days. Ten days later a subcutaneous injection was given of blood rich in the same parasite which had been preserved for two weeks only. Finally the animal received I cc. of blood containing the piroplasma argentinum. There was a more or less severe reaction after each of these vaccines, depending upon the susceptibility of the animal and the quality of the vaccine. Any strain of piroplasms that the author encountered that was more virulent than the strain he already possessed was incorporated in the· vaCCIne. Vaccination oj Calves.-From experiments carried out upon calves the author was able to conclude that animals up to six months old stood the subcutaneous injection of the second and third vaccines well. These injections were found to be far superior to those made with blood from a recovered animal, because the piroplasms contained were fully virulent, and. immunity was conferred with certainty. Further, the injections are polyvalent; they contain not only the two principal types of piroplasm found in the Argentine, but each type is represented by several strains of known, pathogenic power. Later the author found that the vaccines containing the two types of piroplasm could be mixed together, thus enabling one to give calves immunity by a single subcutaneous inoculation with polyvalent material. Vaccine jar Adults and Metfwd oj Preparation.-The author found that if his first vaccine were used upon adults and well-bred cattle there was likely to be a severe or even dangerous reaction. He also found that the best results were obtained by using blood which was very rich in, piroplasms, but which during preservation had become deep purple in colour. In the majority of cases the inoculation of the first vaccine was followed by a severe reaction associated with hremoglobinuria, but the animals, recovered within a day or two without having sensibly lost condition. The second injection was borne well, while the third caused a somewhat severe reaction. There appeared to be considerable danger in the use of the first vaccine, and some losses were experienced. Further, if by mistake the injection was made into the subcutaneous tissue there was almost certain to be a very severe reaction. One of the difficulties of the method is to decide whether a slight reaction is due to the method adopted, to a high degree of resistance on the part of the animal, or to an accidental attenuation of the parasite. The piroplasma bigeminum recently taken from an affected animal varies considerably in its power~, depending upon the age, breed, and condition, of the animal vaccinated, and upon the time of year. The author continued his investigations and attempted to make his

ABSTRACTS AN D REPORT.

first vaccine more safe. He found that, contrary to what he at first thought, the first vaccine was more effective when the parasites were greatly reduced in virulence. He showed by two experiments that it was not necessary for the piroplasms to multiply in the blood, and, in fact, that when the virulent blood rapidly becomes purple in colour and is filtered through a sufficiently porous filter the filtrate is capable of conferring a certain amount of immunity when injected intravenously, but not if the injection be subcutaneous. This result is only obtained if the blood filtered is very :rich in the piroplasma bigeminum. The author is inclined to think that the material which passes through the filter is the hremolytic toxin of the piroplasm, to which the red corpuscles gradually accllstom themselves and thus become more or less refractory. 0 The author found that blood that had been dried in vacuo at 37 to 400 c., and which consequently contained no living piroplasms, was capable of ·conferring a certain amount of immunity. The immunity so obtained is not immediate but is appreciable only after several days, a fact which is held ·by the author to confirm the existence of a toxin. The intravenous injection of this toxin does not give the animal a real immunity, but it increases its resistance, enabling it to withstand inoculation with virulent material, which always causes serious disturbance in control animals. A third method of ohtaining a virulent material rich in the products of the piroplasms is the freezing of defibrinated blood, keeping it on ice and salt fur seven to eight hours, and then allowing it to thaw at laboratory temperature. The deeply pigmented liquid so obtained confers a certain amount of immunity when injected intravenously. This constitutes the author's first .vaccine. The second and third vaccines remain the same. T/te Choice and Quality of the Vaccines.- The activity of the first vaccine ·depends upon the number and virulence of the organisms in the blood. The best time to take the blood is when the urine is just tinted (colour of port wine). Unfortunately the first vaccine prepared under the action of cold must be used within two or three days, as after that its action is uncertain. The toxin is apparently destroyed by free hremoglobin, or rather methremoglobin. Even the parasites are affected by this, for if one mixes one part of virulent blood with three parts of first vaccine five to ten days old, or with blood rich in piroplasms that has been kept in a refrigerator for more than forty days, 5 cc. of the mixture may be inoculated without causing the ·disease or setting up immunity. The author believes that this is the reason why the blood contained in ticks is sterilised with comparative rapidity. He has been able to ascertain that while blood taken from a diseased animal retains its virulence for several days, especially at a temperature of f to 8 c., the blood contained in ticks loses its virulence after twenty-four hours. The author believes that the change from hremoglobin to methremoglobin is due to the piroplasms, and consequently the more rapid this change the greater number of parasites present and the more suitable the material as vaccine. If the vaccine be injected under the skin the toxin to which its vaccinating properties are due is destroyed before it reaches the blood-stream. The methods of preparing the vaccine from blood containing the piroplasma bigeminum by desiccation or freezing cannot be applied to the piroplasma argentinum, because this parasite retains both vitality and virulence after freezing. Since the introduction of the method 23,000 animals have been vaccinated, and the vaccine is in every-day use. 0

ABSTRACTS AND REPORT.

A great advance would be made if one could simplify the method by avoiding the intravenous injection of the first vaccine. In preparing the vaccines the author thinks that the particular piroplasms met with in the country to which the vaccinated animals are to be sent should be incorporated in the vaccines, and the piroplasms used must be at least as virulent as those against which the vaccination is directed. Vaccinated animals should not be exposed to infection until at least a month after the last injection, and revaccination is unnecessary in the case of animals that are infested with ticks every year. It is advisable, however, in the case of animals that remain outside infected areas for more than two years if it is desired to send them into such areas again. (Lignieres, Rev. Gen. de MM. VH, No. 213, 1st November 1911, pp. 489-506.)

TREATMENT OF CONTAGIOUS PNEUMONIA (BRUSTSEUCHE) OF THE HORSE WITH SALVARSAN. THE experiments were carried out at two stud-farms, Gudwallen and Trakehnen, under the auspices of the Minister for Agriculture, and the salvarsan used was supplied by Ehrlich. At Gudwallen experiments were also made with Lorenz serum and tallianine. The following is the substance of Puschke's report regarding the investigations at Gudwallen. The outbreak of pneumonia occurred on 9th July 1911 among the stallions. The first animal attacked was a young stallion bought during the spring. This was followed by the second case fourteen days later. The total number of stallions affected by 21st October was ninety-one, and of these one died on 11th August. The animal first attacked had severe bilateral pleuro-pneumonia, and the only treatment adopted was the application of Priessnitz poultices to the thorax. The disease followed a normal course and the animal's temperature was normal in eight days. A week later there was inflammation of the tendon sheaths in both forelegs associated with severe lameness. From the beginning of September the animal commenced to become convalescent. Twelve stallions were treated with Lorenz serum, quantities up to 450 ce. being injected. These injections had no effect on the temperature. In three of the animals the lungs remained healthy. Twelve animals were also treated with tailianine, the quantity injected amounting in some cases to 130 cc. This also had no effect on the fever. Recovery took place as before in about eight days. Only three of the animals treated had any pneumonia, and one of these died. Sixty-five stallions were treated with salvarsan, and of these fifty-five were free from fever within five days, at the longest, after a single injection of 3 grammes. The other ten animals received a second dose of 2 grammes on the fifth day of the disease, and the temperature of these was normal on the eighth day from the onset of symptoms. No untoward symptoms followed the injection of salvarsan, and it was very striking how quickly the animals treated with the drug recovered. Three stallions were given an oil emulsion of salvarsan-joha. The first had been treated with tallianine, and its temperature was practically normal by the eighth day. On the ninth day the temperature rose again, and 7'5 cc. of joha, containing 3 grammes of salvarsan, were injected into