A reappraisal of the taxonomic status of Eimeria mivati Edgar and Seibold 1964, by enzyme electrophoresis and cross-immunity tests.

An examination of 2 strains of Eimeria acervulina var. mivati (since 1973 E. mivati has been regarded as a variant of E. acervulina) showed that previous confusion over the taxonomic status of E. mivati arose because the investigations were done using laboratory cultures of E. mivati which were contaiminated with E. acervulina. Electrophoretic analyses of enzymes, host specificity and cross-immunity tests have revealed that: (1) The 1971 Houghton strain of E. acervulina var. mivati was a mixture of 2 parasites. (a) Passage of this strain in embryonating eggs resulted in a selection against that parasite previously characterized as E. acervulina. (b) The parasite which did reproduce in eggs did not immunize chickens against subsequent challenge with E. acervulina. This parasite is most likely E. mivati. (c) E. mivati recovered from eggs did, however, immunize chickens against challenge with a new field strain which was morphologically identical to E. mivati and characterized by the same electrophoretic forms of 2 enzymes. (2) A strain of E. acervulina var. mivati from the USA was also a mixture of E. acervulina and E. mivati.     

Characterization in vitro and in vivo of resistance to ionophores in a strain of Eimeria tenella.

A field isolate of Eimeria tenella (FS139) was propagated several times in chickens medicated with 200 ppm of dietary monensin. In a laboratory test with 2-wk-old-chickens, the strain was resistant to monensin, salinomycin, and lasalocid given at double use level and was resistant to narasin and maduramicin at the normal use level. In comparison, a laboratory strain (WIS) was controlled by the normal use level of each product. When free WIS sporozoites were treated in vitro with 1.0 microgram/ml of monensin for 0.5 or 4.0 hr at 41 C and inoculated into primary cultures of chicken kidney cells the invasion was reduced by 35.6% or 96.3%, but invasion of FS139 sporozoites was increased by 18.5% by 0.5 hr treatment and was about the same as controls after 2 hr of treatment. Few sporozoites from the WIS strain developed into schizonts, but numerous sporozoites from the FS139 strain developed into normal first and second generation schizonts. The structure of free WIS sporozoites was distorted after 3 hr of treatment with 2.5 micrograms/ml of monensin at 41 C, as observed by light and scanning electron microscopy, whereas there was no change in structure of most treated FS139 sporozoites.     

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.     

Eimeria tenella: experimental studies on the development of resistance to amprolium, clopidol and methyl benzoquate.

The development of resistance by the Houghton strain of Eimeria tenella to the anticoccidial drugs amprolium, clopidol and methyl benzoquate has been studied. Resistance to amprolium and clopidol developed more readily in experiments where a large number of coccidia were exposed to the drug, either by increasing the number of oocysts in the inoculum or by increasing the number of birds in the group. When 45 birds were given 2.0 X 10(6) oocysts, resistance to amprolium and clopidol appeared after 6 and 7 passages respectively. In previous experiments, under similar conditions, resistance to robenidine developed after 6 passages, suggesting little difference between these three drugs. Resistance to amprolium and clopidol arose gradually as the concentration of drug was increased, but resistance to methyl benzoquate appeared in a single step from sensitivity to high-level resistance. Both amprolium and clopidol-resistant lines showed an 8-fold reduction in drug sensitivity. Attempts to measure the degree of resistance by calculation of the ED50 were unsuccessful.     

Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis

Hybridoma cell lines, which secreted antibodies directed against two different strains of Eimeria tenella and one strain of E. mitis, were produced by fusion of spleen cells from sporozoite-immunized Balb/cByJ mice with P3-X63-Ag8 myeloma cells. The antibodies demonstrated at least eight different binding patterns on or in air-dried sporozoites as determined by the indirect immunofluorescent antibody (IFA) test. These patterns varied from a general internal fluorescence similar to that seen with sporozoites exposed to hyperimmune chicken serum, to fluorescence observed on the tip, pellicle, and refractile body of the parasite. Five cloned, antibody-secreting cell lines were successfully established. Four of these clones produced antibody that reacted only with various strains of E. tenella and cross-reacted with no other species of coccidia. The fifth clone produced antibody directed against only E. mitis and did not react with any other coccidial species.     

Endogenous Cycle of the Swine Coccidium Eimeria debliecki Douwes, 1921*

SYNOPSIS. A pure strain of Eimeria debliecki (University of Illinois strain A) established from a single oocyst was used to determine the endogenous cycle. Young parasite-free pigs 2 weeks to 3 months old were used throughout the study. The endogenous cycle was found to take place in the small intestine where the parasites were located in the distal portion of the striated simple columnar epithelial cells of the villi. The first generation schizonts were found in only the jejunum (15% of small intestine). The second generation schizonts and gametes occurred in the jejunum and ileum (70% of small intestine), a slight posterior progression occurring with each stage. The entire cycle required 6.5 days. The schizogonous cycle comprised 2 generations. The first generation schizonts required 2.5 days to reach maturity, measured 8-12 μ, contained 16 merozoites measuring 12-15 μ and had a polar residual mass. The second generation schizonts required 2 days to reach maturity, measured 13-16 μ, contained 32 rotund merozoites measuring 6–8 μ, and had only a few granules of residual material. Gametogony took place in 1.5 days. The macrogametes measured 12-16 μ, and the microgametocytes measured 9-14 μ with microgametes measuring 5–6 μ.     

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.     

Enzyme variation in Eimeria species of the chicken.

A method for the biochemical identification of protozoa belonging to the genus Eimeria is described for the first time. Starch gel electrophoresis of the enzymes lactate dehydrogenase, glucose phosphate isomerase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase from parasite extracts revealed both intra- and inter-species differences when 11 strains representative of 6 species of Eimeria were examined. Oocysts were the most accessible parasite stage for investigation but sporozoites and merozoites of an embryo-adapted strain of E. tenella were also examined for enzyme activity.     

Eimeria dispersa and Eimeria gallopavonis: Infectivity, Survival, and Development in Primary Chicken and Turkey Kidney Cell Cultures

SYNOPSIS. Eimeria dispersa (turkey strain) and Eimeria gallopavonis sporozoites were inoculated into primary cultures of chicken kidney (CK) and turkey kidney (TK) cells. Eimeria dispersa sporozoites were more infective in either cell type than those of E. gallopavonis : at 4 hr, the percentage of infection was 67-98 for E. dispersa but only 23-56 for E. gallopavonis . E. dispersa also survived better in culture: at 2 days, losses of E. dispersa in both cell types were only 4-19%, whereas losses of E. gallopavonis were 35-47% in TK cells and 60–95% in CK cells. However, E. gallopavonis developed further than E. dispersa . Location and increase in numbers of intracellular stages at 4 days indicated that E. dispersa proceeded through 2 schizogonic generations before development stopped.     

Eimeria tenella: synergistic interaction of sulfaquinoxaline and t-butylaminoethanol in the chicken.

A significant (P < 0.001) synergistic interaction between sulfaquinoxaline and t-butylaminoethanol was demonstrated against a sulfaquinoxaline-resistant strain of Eimeria tenella in chickens. The t-butylaminoethanol is a dimethylaminoethanol- and choline-reversible anticoccidial whereas sulfaquinoxaline is a classic PABA-reversible anticoccidial.     

Eimeria malabaricas sp. n. and Eimeria bandipurensis from the South Indian Tree Squirrel,Funambulus tristriatus*

Synopsis. Oocysts of Eimeria malabaricas sp. n. and Eimeria bandipurensis from the South Indian tree squirrel Funambulus tristriatus collected in Kerala, India, are described. Sporulated oocysts of E. malabaricas were ellipsoid to subspherical measuring 39.8 (35–45) × 32.1 (29–37) m?m, with a thick (2.5–3.0 m?m), 2-layered wall. The outer layer was yellow-brown, striated, and rough. There was no micropyle. but a polar granule was present in 34% of oocysts. The sporocysts were ovoid, 16.0 (14.0–18.0) × 11.2 (11.0–12.0) m?m, with a Stieda body and a granular residuum. Excysted sporozoites were 21.8 (19.0–23.0) × 3.4 (3.0–4.0) m?m, with a large refractile body. The sporulated oocysts of E. bandipurensis are redescribed.     

The Biology and Pathogenicity of a Recent Field Isolate of Eimeria praecox Johnson, 1930

A recent isolate of Eimeria praecox, strain G, was obtained from Georgia and purified. Studies of the life history, pathogenicity, and cross-immunity of the isolate were conducted to verify its identity. In inoculated three-week-old chickens, the occurrence of merogony and gametogony was limited to the superficial epithelium of the upper intestine. Oocysts, 23 × 19.5 äm, with a shape index of 1.17 were first observed 83 h after inoculation. Mortality and morbidity were not observed in any of the experimental birds. However, there was a positive correlation between dose of oocysts, reduced weight gain, and the incidence of exudative diathesis. These studies showed that E. praecox depresses weight gains in chickens and may be of economic importance. Although complete immunity to avian coccidiosis is believed to be species specific, chickens immune to E. praecox (G) or E. acervulina had a degree of cross-immunity to a heterologous challenge. Electrophoretic analysis of glucose phosphate isomerase and lactate dehydrogenase prepared from the European strain of E. praecox and E. praecox (G) showed no differences, confirming the identity of the isolate as E. praecox.     

Speciation studies with Eimeria acervulina and Eimeria mivati.

Eimeria mivati was described as a new species of chicken coccidia in 1964 by Edgar and Seibold, but recently some British workers have relegated its status to that of a variety of Eimeria acervulina. Using strains supplied by Dr. Edgar, we have prepared lines of E. acervulina resistant to methyl benzoquate, sulfaquinoxaline and robenidine and a line of E. mivati resistant to methyl benzoquate. Genetic transfer of resistance between the various lines of E. acervulina to produce doubly-resistant coccidia has been demonstrated, but no such transfer could be obtained between E. mivati resistant to methyl benzoquate and the resistant lines of E. acervulina. Although some immunological relationship between E. acervulina and E. mivati has been demonstrated, we conclude that this failure of the 2 organisms to interbreed lends support to the status of E. mivati as a distinct species.     

Eimeria tenella: host specificity in gallinaceous birds.

Eight species representing 8 genera of gallinaceous birds were used: Alectoris graeca; Colinus virginianus; Coturnix coturnix; Gallus gallus; Meleagris gallopavo; Numidia meleagris; Pavo cristatus; Phasianus colchicus. Three week-old birds were dosed with sporulated oocysts of Eimeria tenella Beltsville strain. At 4, 24, 48, 72, 96, 120 and 144, and 168 hr after inoculation, 1-3 infected birds and uninoculated controls of each species were killed by cardiac exsanguination. Pieces of intestines were fixed and examined for stages of E. tenella as stained paraffin sections or indirect fluorescent antibody preparations. Oocyst counts were made in droppings collected for the first 6 days of the patent period. Sporozoites were found in the lamina propria of some birds of 5 species at 4 hr postinoculation, but no stages were found thereafter except in the breeds of G. gallus and A. gracea. At 144 and 168 hr postinoculation, a few macrogametes were found in the ceca of 2 A. gracea, but no oocysts were found in the feces. No statistical difference was found between the number of oocysts produced/bird in the breeds of G. gallus examined. It is evident from these observations the E. tenella did not complete its life cycle in several close phylogenetic relatives of G. gallus, even though in other studies this parasite was found to complete its life cycle in cell cultures derived from the same birds.     

Preparation and purification of merozoites of Eimeria tenella.

Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.     

The Sporulated Oocysts of Eimeria illinoisensis n. sp. and of Other Species of Eimeria of the Ox

SYNOPSIS. The sporulated oocysts of 12 species of Eimeria occurring in the ox Bos taurus in the United States are described and differentiated. They are E. alabamensis, E. auburnensis, E. bovis, E. brasiliensis, E. bukidnonensis, E. canadensis, E. cylindrica, E. ellipsoidalis, E. illinoisensis n. sp., E. subspherica, E. wyomingensis and E. zuernii. Two other species, not yet found in North America, which are recognized as valid are E. pellita and E. thianethi. The sporulated oocysts of E. illinoisensis n. sp. are ellipsoidal or slightly ovoid, 24–29 by 19–22 μ with a mean of 26.3 by 20.7 μ; their sporocysts are 13–16 by 6–7 μ with a mean of 15.3 by 6.5 μ. This species was found in 3 cattle from one farm in Illinois.     

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.     

A Redescription of the Life Cycle of Eimeria mitis Tyzzer, 1929

Ten-day-old broiler chickens were inoculated with oocysts of a characterized strain of Eimeria mitis , and tissues were fixed at 4, 8, or 24-h intervals after inoculation for histopathological examination. Tissue collections were initiated at the time of inoculation and extended up to 168 h postinoculation. The preferred site of development of E. mitis was found to be the ileum although more limited development of the parasite also took in the jejunum, cecal pouches, cloaca, and bursa of Fabricius. No distinctive and consistent intestinal lesions were macroscopically evident even in heavily parasitized chickens. The prepatent period was approximately 92 h postinoculation. The histopathological features of the E. mitis infections were characterized using conventional bright-field microscopy as well as both scanning and transmission electron microscopy. No extra-intestinal development of the parasite was observed.