It is absolutely essential in culturing L. donovani or L. infantum that the blood agar or citrated blood be sterile, as any bacterial contamination prevents growth. With the parasite L. tropica, however, bacterial contamination does not inhibit development and statements have even been made that growth is favored by a staphylococcal symbiosis. L. tropica, it would seem, will develop into flagellated forms in cultures at 28°C. while it will be remembered that Rogers in his original experiments failed to obtain other than commencing signs of division at 27°C., 22°C. being the temperature necessary for the development of flagellate forms.
L. tropica from South American cutaneous leishmaniases seems to grow more luxuriantly on N. N. N. medium than does that of oriental sore of Asia and Africa.
Giugni tried N. N. N. media made with human, rabbit and dog blood, respectively. The parasites grew well on dog and rabbit blood media but not on that made with human blood. He found growth best when he added salt in quantity from 5 to 9 grams per liter. When red corpuscles are laked in a medium the growth is less favorable.
While differences in development on different culture media may obtain not only with different species but with different strains of the same species, it would appear that such variations cannot be utilized as a means of separating the three species.
Animal Inoculation.—With animal inoculations we formerly thought that the parasite of kala-azar could be differentiated from that of infantile leishmaniasis by the fact that dogs could not be infected with L. donovani, while they were susceptible to infections with L. infantum. Recently Donovan and Patton have successfully inoculated dogs with kala-azar splenic material. Patton found the parasites in the liver, spleen and lymphatic glands as well as bone marrow of the inoculated dogs. Consequently we cannot separate the two visceral leishmaniases from a standpoint of susceptibility of the dog. Monkeys are susceptible to both diseases. It is important to recognize the fact that animal inoculations, even with spleen-juice, rarely give rise to infection.
As regards separating oriental sore from the visceral leishmaniases Gonder has shown that white mice may be infected with both kala-azar and oriental sore, there being produced in each case a general infection with the presence of parasites in spleen and liver. A point of difference, however, is that the oriental-sore mice develop lesions on feet, tail and head which was not observed with the kala-azar mice. There are some reasons for thinking that in human cutaneous leishmaniasis a generalized infection may precede the local manifestations.
Dogs and monkeys can be infected with L. tropica as well as mice, but in them we have only cutaneous lesions produced. Inoculation should be made intraperitoneally.
A very interesting point is that the dogs in India never show a natural infection with L. donovani, while in the regions where L. infantum is responsible for human infections the natural infection of dogs is not uncommon, indeed many think the dog the reservoir of virus for both L. infantum and L. tropica. It has been suggested that the dogs of India, where kala-azar prevails, may be immune.