RESULT OF INOCULATION.

Control animal

CONCLUSIONS.

(1) A specific complement fixing body exists in the sera of cattle naturally infected with pleuro-pneumonia and in the sera of cattle which react to inoculation with pleuropneumonia virus.

(2) The conglutination test carried out according to the above described technique is of value in the diagnosis of bovine pleuro-pneumonia.

(3) A positive reaction indicates a pre-existing but not necessarily present infection.

(4) The specific complement fixing body can be demonstrated in the case of some naturally infected cattle for a period of at least approximately twelve months after infection, and in cattle which react to inoculation for at least seven months. (Further tests are necessary to determine the longest period during which the complement fixing body exists in both naturally infected and inoculated cattle.)

The writer acknowledges the assistance given by Mr. J. Ford, Laboratory Assistant, who showed much technical skill in the carrying out of the tests.

REFERENCES.

1 DUJARDIN BEAUMETZ (1906).-Annales de l'Institut Pasteur, Vol XX, 499. 2 HESLOP (1921).—Proc. Royal Society, Victoria, Vol. XXXIII (New series).

3 HESLOP (1922).—Proc. Royal Society, Victoria, Vol. XXXIV (New series), Pt. II.

6 SCHOCHOWSKY (1912).-Verbandt der Zusammenkunft der Veterinarbakterologen, S. 215.

7 MEYER (1914).-" Filterable Viruses," Report of the 10th International Veterinary Congress, London, Vol. III, 267.

8 TITZE & GIESE (1919).-Berliner Tierarz Wochenschrift, No. XXXII, Vol. XXXV, 281.

BORDET & GAY (1909).-On the relation of Sensitizers to Alexin, in Studies in Immunity," Bordet & Gay, 363.

10 BORDET & STRENG (1909).-The Phenomena of Adsorption of the Conglutinin of Bovine Serum, in Studies in Immunity," Bordet & Gay, 440.

A NOTE ON THE CULTIVATION OF FLAGELLATES OF THE TRYPANOSOMA THEILERI TYPE FROM THE BLOOD OF SOUTH AFRICAN CATTLE.

BY

E. M. ROBINSON, Dr. Med. Vet., M.R.C.V.S.,

Division of Veterinary Education and Research, Onderstepoort,

Pretoria.

With Plates XX, XXI.

Read July 13, 1923.

The object of the present article is to record the cultivation of the crithidial forms of Trypanosoma theileri in artificial media in South Africa. Flagellate bodies of the Crithidia type have been cultivated from the blood of cattle in most parts of the world, and it is generally recognised that they are forms of the large nonpathogenic trypanosome T. theileri occurring in the blood of cattle. This trypanosome has many synonyms and has been called T. americanum in the United States of America, T. wrublewski in Turkestan, T. franki in Germany, T. giganteum, himalayanum and indicum in India. There is little doubt that all these species are the same organism or at least are very closely related. Up to the year 1916 cultures of T. theileri had not been obtained from the blood of South African cattle, although Theiler as long ago as 1903 had described the trypanosome which bears his name as occurring in this country. In 1916, D. Kehoe, at that time officer in charge of the Research Laboratory of this Division in Pietermaritzburg, Natal, was able to obtain cultures of T. theileri in broth to which defibrinated blood of normal cattle on the station had been added. His cultures were incubated at room temperature, and flagellates were observed in them similar to those seen by other workers along the same lines. Kehce did not record his observations at the time in the form of a paper, and only a few notes in his laboratory reports now remain. Kehoe was able to cultivate crithidial forms of T. theileri from the blood of seven out of ten cattle at the laboratory in Pietermaritzburg. Judging from several slides made by him at the time and which are still preserved, the type of flagellate seen in the cultures was the same as was later seen by me.

During the time when I was stationed at the laboratory in Pietermaritzburg, from October, 1921, to February, 1923, bearing in mind Kehoe's observations, I carried out a few myself on the cultural flagellate forms of T. theileri in the blood of cattle there. My object originally was not to study these forms, but to attempt to obtain a sufficiently rich growth of these flagellates to allow an emulsion to be made from them for use as an antigen in serological tests in trypanosomiasis. At that time, I was engaged in carrying out complement fixation tests with the sera of animals infected with various types of trypanosomes, but more especially for horses infected with T. equiperdum (dourine) and cattle infected with nagana (T. congolense and T. brucei). thought that one might perhaps be able to substitute T. theileri culture antigen for that made from the dourine trypanosomes in the blood of infected white rats, but, as will be mentioned later, such antigen did not prove of any value.

It was

During my attempts to cultivate T. theileri in artificial media, I was able to obtain the usual cultural forms from the blood of most of the cattle at the laboratory. They were all exposed to the attacks of biting flies, most of them being out at grass day and night, but a few were stabled at night. My cultures were, however, usually made from two or three cattle only, which 1 knew from previous cultural tests were infected. This was done because I wished to be fairly certain that growths would be obtained, so that I could use the flagellates for making antigen.

The cultures were made, as a rule, by adding defibrinated. blood from an animal to ordinary nutrient broth in the proportion of about 2 c.c. of blood to 10 c.c. of broth. Sterility in manipulation is very important, as the cultures have to be kept under observation for periods up to a month, and, although a bacterial infection when only slight may not interfere with the growth of the flagellates, it will, when heavy, rapidly kill out the cultural forms of T. theileri. The proportion of blood to broth does not appear to be very important, but one should use at least 1 c.c. of blood to 10 of broth, as the trypanosomes may be rare in the blood of the animal. Cultures were made throughout the year 1922, and it was found, as observed by Crawley in the United States of America, that during the winter months it was difficult or impossible to obtain cultures from eattle which previously gave them. In two cases which I followed through all seasons of the year no cultures were obtained in the months of June, July and August. Whether the trypanosomes disappear from the body and a reinfection occurs with the onset of spring is an undecided question. If very large quantities of blood were used for cultures, one might find that the parasites were present in very small numbers. It is quite probable, on the other hand, that the trypanosomes temporarily disappear from the peripheral circulation during the cold weather. One of the cattle which I observed throughout the year was an animal from which Kehoe obtained cultures six years before, and it probably had remained infected

since then. I am inclined to think that the parasites do not really disappear from the body, as they are again cultivable before there are many biting flies about, but I cannot express a definite opinion on the point.

In common with Crawley' and other workers, I found that cultures would not develop at 37° C., but would remain alive at that temperature for a few days when a good growth was already established by incubation at room temperature.

In the course of attempts at obtaining good growths in artificial media, various different ones were tried. These included ordinary nutrient broth, 2 per cent. glucose broth, isotonic glucose solution (5.4 per cent.) in distilled water, Löffler's liquid bloodserum medium, potassium citrate solution 3.8 per cent., 1 per cent potassium oxalate solution, Locke's solution and normal salt solution. In addition, the defibrinated blood was allowed to settle by itself and examined to see if flagellates developed. Only in broth, glucose broth and isotonic dextrose solution were growths obtained. The dextrose solution gave a very good growth, quite as good as any in broth. Subcultures into broth to which defibrinated ox blood free from flagellates had been added fail: d to develop any growth, though they were made on several

occasions.

All attempts to infect rabbits, guinea-pigs, white rats and white mice with material from cultures failed, as they had done in the hands of many previous workers.

Attempts were made to obtain growths of flagellates in the medium of Nöller, which is a 1 per cent. dextrose agar made with weakly alkaline horse flesh broth. When this agar is to be used it is melted in a water bath and an equal quantity of defibrinated horse blood is added to it. Plates are then poured from it and allowed to set in an ice-chest. The plates are inoculated, inverted over a 2 to 3 per cent. sublimate solution to prevent evaporation and then incubated. Nöller obtained very good results with this medium, but unfortunately, owing to the laboratory at Pietermaritzburg being in the course of undergoing extensive alterations at the time I was making these plate cultures, they invariably became contaminated with moulds. Since my return from Pietermaritzburg in February, 1923, I did not again attempt to make cultures of T. theileri until May, and have so far not been able to obtain a growth, probably on account of the onset of the cold weather. It is probable that, until September or October, no growths will be obtainable from the blood of cattle here at Onderstepoort.

As regards the flagellate forms (Plates XX, XXI) seen in cultures at Pietermaritzburg, they correspond very closely with those seen by workers in other parts of the world. Usually flagellates were seen at the earliest on the third day after inoculation, and usually they had died out by the 25th day. Large numbers of the parasites were generally present between the 8th