Wenyon, however, tried to infect two young dogs with great number of fleas which had previously fed on dogs infected with canine leishmaniasis and at autopsy, five or six weeks later, was unable to find parasites in smears from spleen, liver or bone marrow and did not succeed in obtaining cultures from this material inoculated into tubes of N. N. N. medium.
Basile states that a temperature of 22°C. is necessary for the development of the parasite in the flea and that negative experiments have been due to their not having been conducted in the winter. Patton has had fleas feed on a heavily infected dog, whose peripheral blood showed hundreds of parasites per film. These experiments were made in the winter and although examining 200 of these fleas he failed to find any evidence of the flagellates after eight hours.
Views have been entertained that the canine infection is one with a flea herpetomonad distinct from Leishmania, but as dogs can be infected with L. infantum and then show manifestations similar to canine leishmaniasis the parasites are probably the same.
Patton fed great numbers of fleas on a dog experimentally infected with L. donovani and found that the flagellates had entirely disappeared from the alimentary tract of fleas dissected after eight hours, although fleas dissected within four to six hours showed degenerating Leishmania.
As regards oriental sore Wenyon has found that bedbugs and Stegomyia will feed from the sores and take up parasites which develop into flagellate forms in the gut of the insects.
Proof of transmission by these agents however is lacking and others are inclined to suspect the house fly or some species of moth midge.
In Brazil there exists some evidence that the cutaneous leishmaniasis found there may be transmitted by species of the tabanid family.
It must be understood that there is always a suspicion that the flagellate forms noted in arthropod experiments may be those of nonpathogenic herpetomonad or crithidial species as such forms are common in arthropods and are difficult to distinguish from the flagellate stage of leishman bodies.
Cultural Forms.—Very definite is our knowledge of the cultural forms of Leishmania. Rogers first cultured material from splenic juice of kala-azar patients in 10% sodium citrate solution at a temperature of 17° to 24°C. The medium was slightly acidulated with citric acid. There was no satisfactory development at blood temperature. In forty-eight hours the oval parasites have developed into herpetomonad flagellates, from 20 to 22 microns long by 3½ microns broad, with a 20-micron flagellum which takes origin from the blunt anterior end of the body near the blepharoplast. The peripheral blepharoplast and centrally placed macronucleus are at a distance from one another as opposed to the approximation of the crithidial blepharoplast to the centrally placed nucleus in a body with pointed anterior end.
Formerly it was thought that there were differences in the three species of Leishmania from the standpoint of growth on various culture media, L. donovani not growing on N. N. N. medium while L. infantum grew well on N. N. N. medium but not in citrated blood. It is now known that both species will grow on these two media.