Growth of Piscirickettsia salmonis to High Titers in Insect Tissue Culture Cells

ABSTRACT Piscirickettsia salmonis was grown in established insect, frog, and fish tissue culture cells. The yield of P. salmonis in Sf21 cells was up to 100 times that obtained in CHSE-214 cells, and virulence for Atlantic salmon was retained. The ceiling temperature for growth of P. salmonis in Sf21 cells was 24°C.

Isoenzyme characterization of trypanosomes of the subgenus Herpetosoma

SUMMARYIsoenzyme analysis was used to characterize 6 species of trypanosomes of the subgenus Herpetosoma using 13 different enzyme systems. The species studied were Trypanosoma lewisi, T. musculi, T. grosi, T. microti, T. evotomys and T. nabiasi which cannot be distinguished on morphological grounds. Extracts for thin-layer starch-gel electrophoresis were prepared from cultures of insect forms in either Schneider's Drosophila or Grace's insect tissue culture media with foetal calf serum or a nutrient agar medium. Extracts of T. lewisi and T. musculi bloodstream forms were also run for comparison. All parasites gave distinct patterns which enabled them to be differentiated on one or more enzyme systems. Two types of computer analysis were used to group the parasites; using these techniques the murine parasites T. lewisi, T. musculi and T. grosi fell into one broad group, and T. microti and T. evotomys of microtine rodents formed another. These findings are in accord with earlier observations on the behavioural characteristics of these parasites in their mammalian host and their vector (fleas). The clear differences observed provide the basis for the application of other biochemical and immunological techniques for differentiation within this subgenus of trypanosomes.

Effect of mutagen on cultured Schistosoma mansoni

Although lightly homogenized three-week-old Schistosoma mansoni incubated in Mistuhashi and Maramorosch insect tissue culture medium or the medium of Weller and Wheeldon produced adherent cell layers, continued growth of these cells did not occur. Non-adherent cells obtained by trypsinization also failed to produce long term cell cultures even after the addition of a range of growth factors. The possibility of producing tumour-like schistosome cells by the use of the mutagen ethyl methane sulphonate was therefore examined. Four-hour exposure of three-week-old schistosomes caused in some worms (a) large fluid filled ‘ballooning’, which also occurred in adult males, (b) enlargement of the gut, (c) increase in numbers of large round cells within the worms and (d) tissus outgrowths. It is suggested that these effects of mutagen offer new approaches to obtaining permanent schistosome cell cultures.

Tetrahymena dimorpha sp.nov. (Hymenostomatida: Tetrahymenidae), a new ciliate parasite of Simuliidae (Diptera) with potential as a model for the study of ciliate morphogenesis

A new species of hymenostome ciliate, Tetrahymena dimorpha sp.nov., is described. This ciliate occurs as a parasite in the haemocoel of larval, pupal and adult Simuliidae (Diptera). In larval hosts the total number of parasites never exceeds about 240 and the infection is benign. Within larval hosts the ciliates are large and broadly oval and possess an unusually wide range of somatic kineties, from 30 to 66; moreover a variable proportion of these kineties are characteristically disorganized, being incomplete, meandering or branched. Metamorphosis of the host to the adult fly is accompanied by a dramatic increase in the number of ciliates, which reach pathogenic intensity. Adult hosts may contain up to 19000 ciliates and the flies soon die from this heavy burden. Associated with ciliate population growth during host metamorphosis is a startling morphological transformation of the ciliates themselves. In adult hosts the ciliates are smaller and pyriform in shape and the cortex is greatly modified; the total number of somatic kineties is considerably reduced and has a limited range of 19—22. Most significantly, the kineties are ordered with typical tetrahymenine precision. By application of appropriate culture conditions to ciliates isolated from any host stage, either of the distinctive morphological forms of T. dimorpha may be selectively induced in vitro . In bacterized infusions, ciliates are produced that have the general form and cortical characteristics of those found naturally in adult hosts. Sterile culture in serum-supplemented Mitsuhashi and Maramorosch insect tissue culture medium produces a population showing features characteristic of ciliates from larval hosts. Sterile culture in proteose-peptone-yeast-extract medium results in populations exhibiting concurrent dimorphism, even after cloning. The extreme nature and multiple facets of the dimorphism together with the ease of its manipulation in vitro afford opportunities for the experimental investigation of many problems, particularly those related to cell surface patterning in ciliates, and these possibilities are discussed in relation to current concepts of ciliate morphogenesis.