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XI] lation. When the culture medium was kept at blood heat the parasites very rapidly degenerated and disappeared; but when placed in an incubator at 27° C. they lived for three or four days and multiplied. A lower temperature was found to be even more suitable. When temperature was lowered to 20°-22° C. the parasites multiplied more readily, acquired a considerable size, and finally assumed the elongated, motile, flagellated form. The N.N.N. culture medium (p. 158) gives excellent results, and in this, by transplanting at intervals of from twelve to fifteen days, the cultures can be kept alive up to at least four years. The presence of moulds does not affect the cultures, but bacterial contamination is fatal.

Animal experiments.—— The early attempts in India to communicate kala-azar to the lower animals failed, but Nicolle in Tunis, and, subsequently, many others elsewhere, have shown that dogs, cats, jackals, monkeys, rats, mice, and to a less extent guineapigs and rabbits, are inoculable, provided laige doses of the virus are injected into the liver or peritoneal cavity. For a dog, 2 to 4 c.c. of a thick emulsion of infected liver, spleen, or bone marrow in normal saline usually suffices. Intravenous injection is not so successful. Injection of cultures sometimes succeeds. Archibald succeeded, in Khartoum, in infecting a monkey by feeding it on kala-azar material.

Mode of transmission.—— This has not been definitely ascertained as regards India, but there is evidence that in the Mediterranean countries the dog* is a principal reservoir of the disease, and may be concerned with the endemicity. It has also been asserted, but not proved, that the dog-fleas, Pulex serraticeps and Ctenocepkalus canis, are the transmitters. So far as ascertained, wherever kala-azar occurs, there the dogs are infected with L. donovani in variable proportion from 1-8 per cent, to 81 per cent. In India Donovan has failed to find, except in a negligible proportion of instances, naturally