Helen Profous-Juchelka

Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences

Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimecia bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa: this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox. Eimeria maxima, and Eimeria brumetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.

MOLECULAR PHYLOGENY OF THE OTHER TISSUE COCCIDIA: LANKESTERELLA AND CARYOSPORA

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. bigenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector lambda gt11 from poly (A+) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.

NITROPHENIDE (MEGASUL") BLOCKS EIMERIA TENELLA DEVELOPMENT BY INHIBITING THE MANNITOL CYCLE ENZYME MANNITOL-1-PHOSPHATE DEHYDROGENASE

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul™), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of M1PDH was observed in vitro using purified native enzyme. Moreover, M1PDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.