Isolation of second-generation schizonts of avian coccidia and their use in biochemical investigations

Abstract

SummaryA technique is described for the isolation of a pure suspension of second-generation schizonts ofEimeria necatrixandE. tenella. Most of the schizonts appeared viable as judged by their ability to exclude Trypan blue and the use of this criterion is examined. They were able to metabolize radio-isotope labelled glucose and accumulate thiamine against a concentration gradient. A formula allowing detailed consideration of the latter process is introduced. Crude extracts of these schizonts exhibited serine hydroxymethyl transferase activity which is significantly different from that of the host tissue in terms of optimal pH (8·3 for the parasite and 7·8 for the avian intestinal cells) and Michaelis constant (approximately 2·6 × 10−3mand 0·6 × 10−3mrespectively). Differences in response to potential inhibitors are also shown.

Anticoccidial drug resistance in fowl coccidia: the state of play revisited

“…Many field strains show reduced sensitivity to amprolium but resistance to this drug is difficult to develop in the laboratory. Amprolium competes for the uptake of thiamine by second generation schizonts of E. tenella (James, 1980a). Thiamine is converted to thiamine pyrophosphate in the cell and this coenzyme is involved in many important reactions of carbohydrate metabolism.…”

Thiamine uptake in isolated schizonts ofEimeria tenellaand the inhibitory effects of amprolium

“…When NaCl was omitted from this solution, osmotic pressure was maintained by mannitol. Reagents used in schizont preparation have been described in the previous paper (James, 1980). All birds were reared in a coccidia-free environment until inoculated or otherwise used.…”

“…Isolation of the schizonts is described in the previous paper. Any preparation having a final Trypan blue exclusion level of less than 50 % was not used, while above this a correction formula was applied (James, 1980). Chick intestinal cells were prepared by a modification of the method of Kimmich (1970).…”

“…The phenomenon of the movement of thiamine against a concentration gradient has been reported in a variety of preparations: for example, single cells (Kawaski, Miyata, Esaki & Nose, 1969), tissue slices (Sharma & Quastel, 1965) and ligated intestinal loops (Polin, Wynosky & Porter, 1963). That this occurs in secondgeneration schizonts of Eimeria tenella, isolated from the caecae of infected birds, has already been demonstrated (James, 1980). Warren (1968) showed that the coccidia require thiamine and it has been suggested that since certain thiamine analogues cannot be pyrophosphorylated and so act as co-enzymes (Wang, 1978) they exert their anticoccidial action upon an active uptake mechanism.…”