The effects of selected gases on excystation of coccidian oocysts.

The mechanism of CO2 action in changing coccidian oocyst wall permeability was indirectly studied by substituting NO, NO2, N2O, H2S, SO2, CH4, NH3, and 8M urea in place of CO2 in an established excystation procedure. Changes in oocyst wall permeability of Eimeria stiedai, E. bovis, and E. tenella were determined by incubation in test gases and cysteine HCl followed by attempted activation of sporozoites by trypsin and bile and staining of intraoocyst components with methylene blue. The gases CH4, NO2, and N2O were negative for all 3 tests, as were SO2, NH3, and 8M urea which, in addition, were toxic to the oocysts. Both H2S and NO were capable of mimicking the action of CO2 and are related chemically to the reducing agent, and thus tend to underscore its importance in excystation. It now appears that the role of CO2 is that of an allosteric effector enhancing the action of the reducing agent.     

Demonstration of acid phosphatase in Eimeria spp.: partial characterization of the enzyme in E. vermiformis

Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporulated oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chromatography.     

Quantification of the crowding effect during infections with the seven Eimeria species of the domesticated fowl: its importance for experimental designs and the production of oocyst stocks.

The 'crowding effect' in avian coccidia, following administration of graded numbers of sporulated oocysts to na?ve hosts, is recognisable by two characteristics. First, increasing doses of oocysts give rise to progressively higher oocyst yields, until a level of infection is reached (the 'maximally producing dose') above which further dose increases result in progressive decreases in oocyst yields. Second, the number of oocysts produced per oocyst administered (the 'reproductive potential') tends to decrease as the oocyst dose is increased. The dose that gives the maximal reproductive potential is the 'crowding threshold' and doses exceeding this are 'crowded doses'. Graded doses of Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox or Eimeria tenella were given to chickens of the same breed, sex and age, reared on the same diet, under identical management. The two characteristics of the crowding effect were demonstrated graphically and, by interpolation, the estimated crowding thresholds were 903, < or =16, 39, < or =14, < or =16, < or =16 or 72 sporulated oocysts, respectively, for the seven Eimeria species enumerated above. This is apparently the first report of definitive experiments to quantify a crowding effect in E. brunetti, E. maxima, E. mitis, E. necatrix and E. praecox. Maximum experimental reproductive potentials were considerably lower than the theoretical reproductive potentials for all seven species. The interaction between availability of host intestinal cells and immunity contributing to the crowding effect is discussed. Standard curves obtained under specified conditions should be used to estimate appropriate infective doses for experimental designs or in vivo production of oocyst stocks. For experiments on effects of chemotherapy or immunisation on oocyst production, an infective dose lower than the crowding threshold should be used. For efficient production of laboratory or factory oocyst stocks, the maximally producing dose (which is greater than the crowding threshold), should be used.     

Partial protection against Eimeria acervulina and Eimeria tenella induced by synthetic peptide vaccine

Coccidiosis is a major parasitic disease of poultry industry and an ideal vaccine should induce long-lasting cross-species protective immunity. Broiler chickens (Cobb 500) were inoculated with single, double or triple injections of a synthetic peptide (derived from sequences of Eimeria acervulina and Eimeria tenella antigens) homogenized in Freund's complete and incomplete adjuvants. The immune responses to the vaccine were assessed by evaluation of antibody and lymphocyte proliferation responses, and the degree of resistance of vaccinated chickens to challenge with sporulated oocysts of E. acervulina or E. tenella determined by comparison of their oocyst output with those of control chickens. The results indicated that the synthetic peptide vaccine induced a high level of antibody and cellular responses associated with partial cross-species protection against challenge with sporulated oocysts of E. acervulina or E. tenella.     

Amino acids and their metabolites in Eimeria tenella (Coccidiida) oocysts

The amino acid composition of protein in the oocysts of Eimeria tenella has been studied in detail by using the new method of purification of the coccidial oocysts. 35 amino acids and their metabolites have been established for the first time at the exogenous stages of development of E. tenella. The oocyst sporulation is noted to be followed by quantitative changes of the majority of free amino acids and their metabolites.     

Characterization of Eimeria tenella unsporulated oocyst-specific cDNA clones.

A cDNA library was constructed with poly(A)+ RNA from unsporulated oocysts of Eimeria tenella in pUC18. After screening, 4 cDNA clones that hybridized to RNA of unsporulated and sporulating oocysts but not to RNA of either sporulated oocysts or second generation merozoites were isolated and characterized. Each of the cDNA clones is unique. The loci for 2 of the clones are on E. tenella chromosome 7, the site of the third is located on chromosome 6 and the last clone hybridizes, for the most part, to chromosome 5 but also to other E. tenella chromosomes. The cognate RNAs for each of the cDNA clones show differential patterns of hybridization during oocyst sporulation with the levels of RNA being low at the start of sporulation (0 hr), increasing to peak levels between 6.5 and 23 hr after the onset of sporulation and, in each case, decreasing to low hybridization levels at 48 hr after initiation of sporulation. These results establish that specific mRNA levels are differentially regulated during sporulation.     

Eimeria clethrionomysis sp. n., Eimeria gallatii sp. n., Eimeria pileata sp. n. and Eimeria marconii sp. n. from the red-backed vole Clethrionomys gapperi Vigors, from Pennsylvania.

Four new eimerian species are described from red-backed voles, Clethrionomys gapperi in Pennsylvania. Sporulated oocysts of Eimeria clethrionomyis sp. n. are ellipsoidal, 18.8 (16.5-21.5) x 14.9 (14.0-16.5) with elongate, ovoid sporocysts, 10.6 (9.5-12.0) x6.1 (5.5-7.0). The oocyst wall is smooth, with 2 layers, and thins, with terminal cap at one or both ends. Polar granules, dark Stieda body and sporocyst residuum are present. The oocyst residuum is absent. Sporulated oocysts of Eimeria gallatii sp. n. are ellipsoidal, 27.7 (21-32) x 19.3 (17-24) with ovoid sporocysts, 13.5 (12-15) x 8.8 (8-10). The oocyst wall is smooth, 2-layered, with a micropyle and thin wall at the end opposite the micropyle. Polar granules, Stieda body and sporocyst residuum are present. The oocyst residuum is atypical, of cobwebby material. Sporulated oocysts of Eimeria pileata sp. n. are subspherical to spherical, 25.2 (20.5-29.5) x 22.5 (19.5-25.5) with ellipsoidal sporocysts, 13.4(10.5-15.0) x 8.4 (7.5-9.5). The oocyst wall is rough, pitted, striated, 2-layered, with no micropyle. Polar granules, oocyst and sporocyst residuum, Stieda body and stiedal cap are present. Sporulated oocysts of Eimeria marconii sp. n. are ellipsoidal, 13.0 (10.5-15-0) x 10.6 (9.5-12.0) with elongate, ovoid sporocysts, 7.7 (7.0-8.5) x 4.2 (3.0-4.5). The oocyst wall is smooth, single-layered, with no micropyle. Polar granules, dark Stiedal body and sporocyst residuum are present. There is no oocyst residuum.     

Ultrastructural localization of antigenic sites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and Eimeria tenella by monoclonal IgG and IgM antibodies

Immunoelectron microscopy was used to study the localization of monoclonal IgG (13.9 and 15.84) and IgM (10.84) antibodies generated against Eimeria tenella sporozoites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and E. tenella. A uniform layer of ferritin was present on sporozoites of E. tenella fixed chemically before the addition of 10.84, 13.90, or 15.84 (called prefixed), whereas postfixed (fixed chemically after exposure to monoclonal antibody) sporozoites lacked ferritin, indicating that the latter had capped immune complexes. Patches of ferritin were present on prefixed and postfixed sporozoites of E. acervulina exposed to 15.84, indicating that immune complexes containing 15.84 were not capped. Sporocysts of E. tenella exposed to 10.84 had a uniform layer of ferritin on their outer surface; ferritin was localized in patches on those exposed to 13.90 or 15.84. In E. acervulina sporocysts exposed to 15.84, ferritin was widely scattered on the outer surface but formed a uniform layer on the inner surface of the sporocyst wall. Patches of ferritin occurred on the inner layer of the oocyst walls of E. tenella and E. acervulina exposed to 10.84, 13.90, or 15.84. These findings indicate the shared antigen detected by 15.84 differed in relative amount, spatial distribution, and structural location in sporozoites and sporocysts of E. acervulina and E. tenella.     

Coccidia (Eimeria tachyglossi n. sp., E. echidnae n. sp., and Octosporella hystrix n. sp.) in the Echidna, Tachyglossus aculeatus (Monotremata: Tachyglossidae)

Oocysts of Octosporella hystrix n. sp., Eimeria tachyglossi n. sp., and E. echidnae n. sp. are described from the feces of the echidna Tachyglossus aculeatus (Monotremata: Tachyglossidae) from Australia. Eimeria tachyglossi has subspherical oocysts, 26.4 × 23.7 μm in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 13.2 × 9.7, slightly pointed at one end, each containing two sporozoites. Eimeria echidnae has subspherical oocysts, 19.4 × 17.8 in size, with a single oocyst wall; no micropyle; four ellipsoidal sporocysts 9.8 × 7.8, blunt at both ends, each containing two sporozoites. Octosporella hystrix has ovoid or subspherical oocysts 32.9 × 29.7 in size with a thick outer and thin inner oocyst wall; no micropyle; eight sporocysts spherical or slightly subspherical 11.3 × 11.2 each containing two sporozoites lying in embrace, with an extensive granular sporocyst residuum about the equator of the sporocyst. Endogenous stages considered to be of E. tachyglossi at least, were recognized in the lamina propria and epithelium on villi in the small intestine of three echidnas.     

Improved Method for High-Yield Excystation and Purification of Infective Sporozoites of Eimeria spp.1

This report describes a new, gentle procedure for rapid and efficient excystation of large numbers of infective sporozoites of Eimeria vermiformis and Eimeria stiedai. Excysted sporozoites are purified using modifications of a previously described ion-exchange chromatography method. The procedure avoids physical breakage of oocysts and results in greater than 70% recovery of the sporozoites present as sporulated oocysts (i.e. 5–6 sporozoites per sporulated oocyst). The recovered sporozoites are greater than 95% pure and are infective in vivo. We routinely isolate greater than 2 times 10⁸ sporozoites without the use of specialized or expensive equipment.     

Eimeria stiedai in rabbits: the presence of an oocyst residuum

Observations on 5 different isolates of Eimeria stiedai from pure infections in laboratory-reared rabbits have confirmed the presence of an oocyst residuum. This structure was seen in 89--100% of the oocytes in cultures prepared both from the liver and from the faeces at different times during the patent period. The residuum consisted of 1--7 or more granules situated centrally in the oocyst and partially concealed by 4 sporocysts. The differential diagnosis of E. stiedai and E. coecicola is discussed.     

Eimeria tenella: immunogenicity of arrested sporozoites in chickens

Groups of chickens were medicated with the anticoccidial drug, decoquinate, and starting 1 day after this medication they were given daily inoculations of either 1 X 10(4) (Experiment 1) or 1 X 10(5) (Experiment 2) oocysts of a decoquinate-sensitive strain of Eimeria tenella. This assured the presence of large numbers of drug-inhibited sporozoites in the cecal tissues. The immunity arising from the presence of these inhibited sporozoites was assessed by challenging the medicated chickens with a 2.5 X 10(5) oocysts of a decoquinate-resistant strain of E. tenella. The response to challenge was assessed by weight gain, the severity of cecal lesions, hematocrits, and cecal oocyst numbers. The inhibited sporozoites promoted little (if any) immunity judged by clinical signs of disease. However, judged by body weight changes after challenge, the presence of inhibited sporozoites provided substantial protection against the body-weight-depressing effects of the challenge dose. These findings emphasize the importance of stage-specific antigen expression in Eimeria spp. infections and support the notion that immunogenicity is associated with tropic stages of the parasite.     

Eimeria clethrionomyis sp. n., Eimeria gallatii sp. n., Eimeria pileata sp. n. and Eimeria marconii sp. n. from the Red-Backed Vole Clethrionomys gapperi Vigors,from Pennsylvania§

SYNOPSIS Four new eimerian species are described from red-backed voles. Clethrionomys gapperi in Pennsylvania. Sporulated oocysts of Eimeria clethrionomyis sp. n. are ellipsoidal, 18.8 (16.5–21.5) × 14.9 (14.0–16.5) with elongate, ovoid sporocysts, 10.6 (9.5–12.0) × 6.1 (5.5–7.0). The oocyst wall is smooth, with 2 layers, and thins, with terminal cap at one or both ends. Polar granules, dark Stieda body and sporocyst residuum are present. The occyst residuum is absent. Sporulated oocysts of Eimeria gallatii sp. n. are ellipsoidal, 27.7 (21–32) × 19.3 (17–24) with ovoid sporocysts, 13.5 (12–15) × 8.8 (8–10). The oocyst wall is smooth, 2-layered, with a micropyle and thin wall at the end opposite the micropyle. Polar granules. Stieda body and sporocyst residuum are present. The oocyst residuum is atypical, of cobwebby material. Sporulated oocysts of Eimeria pileata sp. n. are subspherical to spherical, 25.2 (20.5–29.5) × 22.5(19.5–25.5) with ellipsoidal sporocysts, 13.4(10.5–15.0) × 8.4 (7.5–9.5). The oocyst wall is rough, pitted, striated, 2-layered, with no micropyle. Polar granules, oocyst and sporocyst residuum. Stieda body and stiedal cap are present. Sporulated oocysts of Eimeria marconii sp. n. are ellipsoidal, 13.0 (10.5–15.0) × 10.6 (9.5–12.0) with elongate, ovoid sporocysts, 7.7 (7.0–8.5) × 4.2 (3.0–4.5). The oocyst wall is smooth, single-layered, with no micropyle. Polar granules, dark Stieda body and sporocyst residuum are present. There is no oocyst residuum.     

Attempted Cross-Transmission of Coccidia Between Sheep and Goats and Description of Eimeria ovinoidalis sp. n.

SYNOPSIS.
Attempted infection of 2 young lambs with oocysts of Eimeria christenseni from a goat was unsuccessful. Negative results were obtained also when young kids were fed oocysts of Eimeria ninakohlyakimovae from sheep. There was no difficulty in infecting lambs with the sheep coccidium resembling E. ninakohlyakimovae nor goats with the goat coccidium E. christenseni. Oocysts from the goat measured 38.4 × 26.7 m, but were easily distinguished from Eimeria ahsata from the sheep by sporocyst size and shape, and from Eimeria ovina by oocyst size. Eimeria ninakohlyakimovae -like oocysts from sheep averaged 23.0 ×18.2 m and were morphologically indistinguishable from previously reported goat coccidia.
Since no cross infections of sheep and goats could be accomplished with oocysts of Eimeria sp. characteristic of one or the other host, I concluded that sheep coccidia previously known as E. ninakohlyakimovae are distinct from morphologically similar goat coccidia and therefore constitute a separate species. Since the name E. ninakohlyakimovae was first used for coccidia from the goat, the sheep coccidium is renamed Eimeria ovinoidalis with oocyst structure and endogenous stages similar to those previously described from the sheep.     

Effect of the anticoccidial arprinocid on production, sporulation, and infectivity of Eimeria oocysts

Medication of broilers with arprinocid [MK-302, 9-(2-chloro-6-fluorbenzyl adenine)] had 3 distinct effects on oocysts; (1) the number of oocysts produced was decreased, (2) fewer of the oocysts sporulated, and (3) those oocysts which did sporulate were less infective than those from unmedicated birds. The drug level necessary to prevent passage of oocysts depended on the species and strain of coccidia. To essentially eliminate oocyst production (less than 5% of controls) required medication with the following levels of arprinocid: 70 ppm with Eimeria maxima; 60 ppm with E. mivati, E. E. necatrix, and E. brunetti; and 50 ppm with E. tenella. With E. acervulina, oocysts were completely eliminated by 60 ppm of arprinocid with one field strain but were still numerous at 70 ppm with a second field strain. Oocysts recovered from birds on medication often failed to sporulate. No sporulation was seen at drug levels of 30 ppm or above with E. maxima and E. mivati. The level of arpinocid required to prevent sporulation with other species depended on the strain being studied, but varied from 30 ppm to 70 ppm. The oocysts of E. acervulina, E. mivati, E. tenella, and E. brunetti recovered from medicated birds that subsequently sporulated, were less infective when inoculated into susceptible birds, than oocysts from unmedicated birds. Oocysts from low medication level with E. necatrix (30 ppm) and E. maxima (10 ppm), once sporulated, were as infective as oocysts from unmedicated control birds, even though the numbers produced were less. No differences were detected in the time oocysts were produced between medicated and unmedicated birds infected with E. acervulina, E. maxima, E. brunetti, and E. tenella.     

Attempted cross-transmission of coccidia between sheep and goats and description of Eimeria ovinoidalis sp. n.

Attempted infection of 2 young lambs with oocysts of Eimeria christenseni from a goat was unsuccessful. Negative results were obtained also when young kids were fed oocysts of Eimeria ninakohlyakimovae from sheep. There was no difficulty in infecting lambs with the sheep coccidium resembling E. ninakohlyakimovae nor goats with the goat coccidium E. christenseni. Oocysts from the goat measured 38.4 X 26.7 microns, but were easily distinguished from Eimeria ahsata from the sheep by sporocyst size and shape, and from Eimeria ovina by oocyst size. Eimeria ninakohlyakimovae-like oocysts from sheep averaged 23.0 X 18.2 microns and were morphologically indistinguishable from previously reported goat coccidia. Since no cross infections of sheep and goats could be accomplished with oocysts of Eimeria sp. characteristic of one or the other host, I concluded that sheep coccidia previously known as E. ninakohlykimovae are distinct from morphologically similar goat coccidia and therefore constitute a separate species. Since the name E. ninakohlyakimovae was first used for coccidia from the goat, the sheep coccidium is renamed Eimeria ovinoidalis with oocyst structure and endogenous stages similar to those previously described from the sheep.     

Coccidia of wombats: correction of host-parasite relationships. Eimeria wombati (Gilruth and Bull, 1912) Comb. Nov. and Eimeria ursini Supperer, 1957 from the hairy-nosed wombat and Eimeria arundeli sp. n. from the common wombat.

Coccidial oocysts morphologically consistent with Eimeria ursini Supperer 1957, and E. tasmaniae Supperer 1957 were recovered from the feces of wild and captive hairy-nosed wombats (Lasiorhinus latifrons) in Australia. Eimeria arundeli so. n. was recovered from the feces of wild and captive common wombats (Vombatus ursinus). Eimeria arundeli oocysts are ellipsoidal to slightly ovoid 60.2--67.2 (63.7) X 40.6--47.6 (43.4); micropyle 3 in diameter usually visible; with oocyst wall granular, dark brown and occasionally opaque, 4--7 thick; inner oocyst wall clear, about 1.5 thick; small oocyst residuum present, four sporocysts ovoid 22.4--29.4 (25.8) X 12.6--15.4 (14.1) with protuberant Stieda body; opposite end of sporocyst also often slighly pointed; large granular sporocyst residuum obscuring sporozoites. Gametocytes of E. arundeli sp. n. and of an organism which is consistent with E. tasmaniae, are described developing in the lamina propria of villi in the small intestine. The stages in the hairy-nosed wombat are those described as Ileocystis wombati Gilruth and Bull 1912. It is suggested that the identification of the host of Supperer's E. ursini and E. tasmaniae as V. ursinus was in error and that the allopatric L. latifrons is the natural host. Eimeria tasmaniae Supperer 1957 is suppressed and E. wombati (Gilruth and Bull, 1912) comb. nov. is proposed and redescribed. No schizonts were identified among the endogenous stages, consistent with observations in the literature on other coccidia with similar gametocyte and oocyst structure.     

Eimeria ovata n. sp. and Other Coccidia of the Eastern Chipmunk Tamias striatus in Massachusetts

SYNOPSIS. Eimeria vilasi, E. wisconsinensis , and E. ovata n. sp. were found in the intestinal contents of eastern chipmunks Tamias striatus in Massachusetts. The oocysts of E. ovata are ovoid with mean dimensions of 26.1 × 16.7 μ. The outer oocyst wall is rough, with small pits. Micropyle and oocyst residuum are absent. Sporulation time at room temperature is 7–8 days.