Studies on the adaptations for survival and invasion by Cryptosporidium and Eimeria tenella

Cryptosporidium and Eimeria are two coccidian parasites that infect a wide range of animals including humans for Cryptosporidium, resulting in disease and, in some cases, death of the host. Infection involves three stages; ingestion, excystation and the subsequent invasion of the epithelial cells lining the gastrointestinal or respiratory (as can be the case for Cryptosporidium) tract. The mechanisms employed by the parasites to withstand the hostile environment of the host, and to penetrate to and invade a host cell are not clearly understood. The objective of this study was to provide more information on these three steps to infection, to elucidate the mechanisms involved for Cryptosporidium and Eimeria tenella, and to establish the extent to which these features are shared by the parasites. An in vitro procedure for inducing Cryptosporidium excystation was optimised. Anaerobic reducing conditions produced the highest excystation rates, with oocysts from different species excysting most efficiently at the pH values akin to their in vivo locations in the gastrointestinal tract. Oocyst age affected the excystation process, with the number of sporozoites released declining with increasing age. A novel method for determining the viability of sporozoites was developed. This employed the use of the vital stains acridine orange and bis-benzimide using fluorescence microscopy. Sporozoite survival during incubation with different pH buffers varied with the species of parasite, exemplifying that each species is adapted for sui'vival at the pH similar to their infection site in vivo. Inhibitors of mitochondrial function had little effect on either C. parvum or E. tenella. These data are consistent with C. parvum lacking a mitochondrion, and E. tenella sporozoites not requiring the function of this organelle. C. muris was, to an extent, affected by some of the metabolic inhibitors, suggesting that this species either relies in part on a mitochondrion, or that the inhibitors were affecting other processes in the parasite. Biotinylated probes and antibodies raised against Leishmania mexicana cysteine proteinases were used to analyse the lysates of C. parvum, E. tenella and Toxoplasma gondii for the presence of proteinases. Evidence was found for the presence of both cysteine and serine proteinases in all of the coccidia, with the apparent localisation of a cysteine proteinase on the surface of E. tenella sporozoites. Stage-specific differences were observed when sera against L. mexicana cysteine proteinases were used to analyse unsporulated oocysts and sporozoites of E. tenella. A survey of a range of enzyme activities showed the presence of a number of enzymes and some apparent variations between life cycle stages. However attempts to detect sialidase in Cryptosporidium and Eimeria using fiuorogenic substrates were not successful. A system was developed to study the penetration of mucus layers. The results showed that C. parvum sporozoites and E. tenella sporozoites and merozoites were able to penetrate the layers and, through the use of specific enzyme inhibitors, that motility, cysteine and serine proteinases, polyamines and sialidase were involved in mucus penetration by the parasites. Host cell invasion by C. parvum sporozoites and E. tenella sporozoites and merozoites was investigated using Madin Darby Bovine Kidney (MDBK) cells and specific enzyme inhibitors. The results suggest that cysteine, serine, metallo- and aspartic proteinases play a role in sporozoite invasion, as do polyamines, whereas sialidase was not important. Invasion by merozoites also involved cysteine proteinases and polyamines, but sialidase did appear to have a role. The MDBK cell cycle was analysed using bromodeoxyuridine (BrdU) labelling and 4,6,-diamino-2-phenylindole (DAPI) staining and its influence on parasite invasion was investigated. Sporozoites of both E. tenella and C. parvum invaded the host cells at the highest rate when the host cells were four hours into S phase. It was also discovered that more MDBK cells from cultures infected with E. tenella were in S phase than in control cultures. Thus the studies performed provide information on how Cryptosporidium and Eimeria survive within the hosts intestinal tract and penetrate to and invade a host cell. The two parasites appear to employ some similar mechanisms to facilitate the infection process, yet there are stage-specific features of each parasite.