COMPARATIVE STUDIES OF HERPETOMONADS AND LEISHMANIAS : I. CULTIVATION OF HERPETOMONADS FROM INSECTS AND PLANTS

Nine strains of herpetomonads have been isolated in pure culture from eight varieties of insects, and three strains from two species of plants. Four of the cultures were derived from latex-feeding insects (Oncopeltus fasciatus, Oncopeltus sp. ?, Lygaeus kalmii) and three from latex plants (Asclepias syriaca, Asclepias nivea), two from mosquitoes (Culex pipiens and Anopheles quadrimaculatus), one from the house fly (Musca domestica), and two from bluebottle flies. In addition impure cultures have been obtained from Oncopeltus cingulifer and from its plant host, Asclepias curassavica. The flagellates cultivated, all of which belong to the genus Herpetomonas, have been identified chiefly by their biological relationships, which will be described in detail in Part II of this report. The seven strains from latex-feeding insects and latex plants represent two distinct species, which have been designated H. oncopelti and H. lygaegorum. The two strains from mosquitoes proved to be the same organism and have been called Herpetomonas culicidarum. The culture obtained from Musca domestica contained larger individuals than those of any other strain, and the organism is morphologically distinct from either of the Calliphora strains. None of the fly flagellates cultivated could be identified with the. species H. muscae domesticae or H. calliphorae, and hence they have been given new names, Herpetomonas muscidarum, H. media, and H. parva. Blood agar plates were used for initial cultivation of the strains from insects and the semisolid leptospira medium for isolation of the plant flagellates. A number of the strains were purified by plating on acid blood agar, a procedure which reduces considerably the growth of bacterial contaminants. The Barber technique was utilized for isolation of the flagellates from flies, because of the very large number of bacteria found with them in these insects, and, in one or two instances, for the purification of impure cultures. Once they have been obtained in culture, all the strains grow well on leptospira medium, as well as on blood slants. Growth takes place both at 26 degrees C. and at 37 degrees . The morphology of the organisms is considerably modified by cultivation. This is especially true of the plant flagellates. In the latex they have ribbon-like bodies, often twisted, and comparatively short flagella; the protoplasm is clear, almost hyaline. The flagellates seen in the gut and feces of insects are usually large, slender organisms, with flagella as long as or even longer than the body, which contains numerous volutin granules in the cytoplasm. In cultures under parallel conditions the flagellates from both these sources become shorter and thicker, the plant forms no longer appear flat and ribbon-like, and in general the organisms approach one another in morphological features. Even in the case of the least modified insect flagellates, i.e. those from flies, there is never exact correspondence between the natural and the cultivated forms. The morphological features of the cultivated flagellates vary according to the medium on which the organisms are grown and the age of the culture. The flagellates grown on the surface of blood slants are pyriform, with truncated anterior portion, and short flagellum; in the condensation water, however, the individuals are elongated and have long active flagella. On the leptospira medium the slender active forms with long flagella predominate. In the presence of fermentable carbohydrate, or in medium containing considerable acid, peculiar bifurcated or multifurcated individuals are seen. Similar forms have been seen under natural conditions. Cultures of Leishmania behave in the same way under the conditions described. There is a striking difference in rapidity of growth between the organisms isolated by us and the leishmanias, H. ctenocephali, and T. rotatorium. While the stock cultures of the group first mentioned multiply rapidly at 37 degrees C., growth becoming visible within 24 hours, the latter group grow scarcely at all at 37 degrees and only slowly at 25 degrees , 1 to 2 weeks being required for growth to become macroscopically demonstrable. While the flagellum of the leishmanias, as also of H. ctenocephali, is long, serpentine in its movements, and heavy, having the appearance of being enveloped by a sheath throughout its entire length, that of the recently isolated strains is thin, less flexible, and without the sheathlike appearance. The only exceptions to this rule are the flagellates from Musca domestica and Calliphora No. 1, which have a long flagellum not unlike that of the leishmanias. As the foregoing observations indicate, morphological differentiation of the flagellates studied, while not impossible, is subject to error by reason of the variations due to age and cultural conditions. The flagellates of the latex-feeding insects, the plants, the flies, and the mosquitoes can readily be distinguished from Leishmania by their rapid growth at 37 degrees C., but their differentiation from one another is possible only by serological and fermentation reactions.