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.     

Disaccharidase activity in the intestinal tissue of broilers infected with coccidia.

Maltase and sucrase activities were measured in the intestine of broilers inoculated with sporulated coccidial oocysts. Infection with Eimeria acervulina, E. maxima, E. necatrix, and E. brunetti decreased disaccharidase activity in the intestinal region in which maximum infection was found compared with the activity in uninoculated controls. The maximum reduction occurred on the first or second day of patency followed by a rapid recovery in activity. Disaccharidase activity was inversely proportional to the inoculum dose.     

Oocyst production of Eimeria lettyae in northern bobwhites following low-dose inoculations

Inoculation of northern bobwhite quail ( Colinus virginianus ) with low doses of Eimeria lettyae oocysts stimulates a protective immune response, suggesting immunization may be an option for controlling coccidiosis. However, the oocyst production of inoculated birds could be considerable, leading to subsequent outbreaks. To determine the oocyst production following inoculation with E. lettyae, we orally infected 12-wk-old bobwhites with 100, 1,000, or 10,000 sporulated oocysts. Fecal materials were collected on days 5-9 post-inoculation, and total oocyst production was counted in McMaster chambers. Oocyst production/bird was 49.75, 89.5, and 436 × 10(6) for 100, 1,000, or 10,000 oocysts administered, respectively. Estimated oocysts produced/oocyst administered was 49.75, 8.95, and 4.36 × 10(4) for 100, 1,000, or 10,000 oocysts administered, respectively. These findings not only illustrate the crowding effect of larger oocyst inocula but also illustrate the fecundity of E. lettyae at low doses. This suggests that successful immunization of bobwhites against coccidiosis with live vaccines might require attenuated strains with reduced reproductive potential.     

Eimeria spp. of domestic fowl: the migration of sporozoites intra- and extra-enterically

Chickens were dosed orally with sporulated oocysts of Eimeria acervulina, E. brunetti, E. maxima, or E. praecox and the subsequent presence, in various tissues, of parasites capable of inducing patent infections was detected by transferring the tissues to coccidia-free recipients. Similar results were obtained with each of the 4 species studied, irrespective of whether initial development occurs in the superficial (E. praecox, E. brunetti) or crypt (E. acervulina, E. maxima) epithelium. Infection was transferable by gut scrapings and liver homogenates at all time intervals (3, 6, 12, 18, 24, and 36 hr postinoculation) studied. Infection was also transferable with blood and with splenic homogenates but not consistently. Transfers made within a short time of the inoculation of donors were more successful in producing patent infections in the recipients. In all transfers the prepatent period was normal for the species. These findings suggest that sporozoites enter the mucosa very shortly after inoculation, and some of them pass to the liver and spleen and then leave these tissues at a somewhat slower rate, possibly to reenter the mucosa. Sporozoites in the lamina propria of the gut were found within host mononuclear cells in all 4 species studied. Most of the cells harbouring E. maxima and some of those with E. praecox were identified as intraepithelial lymphocytes while all others could only be identified as agranular mononuclear cells that were not characteristically macrophages.     

Characteristics of a line of Eimeria necatrix after 16 successive passages of oocysts collected after peak oocyst production

A line of Eimeria necatrix was selected by repeated passages of oocysts that were collected after peak oocyst production from feces or cecal contents of previously infected chickens. When compared with the parent strain, the new line of E. necatrix after 16 successive passages had the following characteristics: (1) the peak of oocyst production was delayed by 2 days; (2) the sizes of 9 endogenous development stages became larger; and (3) the reproductive capacity and the immunogenicity were both enhanced. This new line of E. necatrix may be used for the development of new coccidiosis vaccines.     

Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens

Chicken coccidiosis caused by members of the genus Eimeria causes significant economic losses worldwide. In the present study we sequenced the complete mitochondrial DNA (mtDNA) sequences of six Eimeria species and analyzed features of their gene contents and genome organizations. The complete mt genomes of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. tenella and E. praecox were 6179bp, 6148bp, 6169bp, 6214bp, 6213bp and 6174bp in size, respectively. All of the mt genomes consist of 3 genes for proteins (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes. The organization of the mt genomes is similar to that of Plasmodium, but distinct from Babesia and Theileria. The putative direction of translation for 3 genes (cox1, cox3, and cytb) was the same in all six Eimeria species. The contents of A+T of the mt genomes were 65.35% for E. acervulina, 65.43% for E. brunetti, 64.53% for E. maxima, 65.04% for E. necatrix, 64.98% for E. tenella and 65.59% for E. praecox. The AT bias has a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses using concatenated nucleotide sequences of the 2 protein-coding genes (cytb and cox1), with three different computational algorithms (Bayesian analysis, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support, indicating that the six Eimeria spp. represent six distinct but closely-related species. These data provide novel mtDNA markers for studying the molecular epidemiology and population genetics of the six Eimeria spp., and should have implications for the molecular diagnosis, prevention and control of coccidiosis in domestic chickens.     

Molecular characterization and phylogenetic analysis of Eimeria from turkeys and gamebirds: implications for evolutionary relationships in Galliform birds

In order to determine the evolutionary relationships among Eimeria species that parasitize birds of the Galliformes, the 18s rDNA gene and a portion of the cytochrome oxidase subunit 1 (cox-1) were amplified from Eimeria species isolated from turkeys, chukars, and pheasants. The phylogenetic analysis of these sequences suggests that species infecting chickens are polyphyletic and, therefore, do not all share a direct common ancestor. Both the 18s rDNA and the cox-1 sequences indicate that Eimeria tenella and Eimeria necatrix are more closely related to Eimeria of turkeys and pheasants than to other species that infect the chicken. It is, therefore, likely that the chicken Eimeria spp. represent 2 separate ancestral colonizations of the gut, one of which comprises E. tenella and E. necatrix that infect the ceca, while the other includes Eimeria acervulina, Eimeria brunetti, Eimeria maxima, and Eimeria mitis, which infect the upper regions of the intestine.     

Modelling host cell availability and the crowding effect in Eimeria infections.

Within-host mathematical models of Eimeria maxima and Eimeria praecox infections of the chicken are presented and used to investigate the role of host cell availability as a possible determinant of the so-called 'crowding effect'; whereby the fecundity of the parasites decreases as infectious dose increases. Assumptions about the number of available host cells, the average lifespan of these cells and the age structure within the host-cell population were made and mathematical models were constructed and combined with experimental data to test whether these conditions could reproduce the crowding effect in the two species. Experimental data demonstrated that crowding during in vivo infections was apparent following very low infectious doses, but none of the models could adequately reproduce crowding at the same doses while maintaining realistic estimates of the dynamics of the enterocyte pool. However, both the size and lifespan of the enterocyte pool were demonstrated to have substantial effects on the fecundity of the infections, particularly at higher doses. These data indicate that host cell availability cannot be solely responsible for the crowding effect. Alternative factors such as the influence of the primary immune response to the parasite may also be explored using within-host models and other applications of these models are discussed.     

Eimeria spp. of the domestic fowl: resolution of chromosomes by field inversion gel electrophoresis

The molecular karyotypes of five species of chicken coccidia, viz., Eimeria acervulina, E. brunetti, E. maxima, E. necatrix, and E. tenella, were determined using field inversion gel electrophoresis (FIGE). Each species has a distinctive set of resolvable chromosomes which range from about 1 to greater than 5.7 megabases. We were able to resolve at least 8 chromosomes for E. acervulina, 5 for E. brunetti, 10 for E. maxima, 6 for E. necatrix, and 9 for E. tenella. If the value of 67 megabases for the genomic DNA of E. tenella is accurate, then under the conditions used here only about 60% of its chromosomal complement has been resolved.     

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.     

Effect of gamma-irradiation on oocysts of Eimeria necatrix.

Effect of gamma radiation on oocysts of Eimeria necatrix was investigated. It was observed that oocysts exposed to 200 kR or above did not sporulate. Irratiation at 10-150 kR caused a progressive decrease in sporulation. Irradiation affected normal development of unsporulated oocysts as the zygote protoplasm divided into unequal masses or was shattered into granules. Increase in the intensity of irradiation of sporulated oocysts resulted in the progressive decrease in severity of the resultant infections in chicks and their effects - mortality, type of lesions developed, total oocyst production and immunity produced - were comparable with infections induced by decreasing the number of unirradiated oocysts. Infection produced by 1000 unirradiated oocysts was comparable with that resulting from 50 000 oocysts irradiated at 25 kR. Infection obtained with 20 000 unexposed oocysts approximated to that produced by 50 000 oocysts irradiated at 2-5 kR. It was concluded that irradiation abolished infectivity of the oocysts/sporozoites rather than bringing about attenuation of the parasite.     

Formation of sulfhydryl groups in the walls of Eimeria stiedai and E. tenella oocysts subjected to in vitro excystation.

Eimeria stiedai or Eimeria tenella oocysts were incubated in aqueous cysteine hydrochloride (cysHCl) under carbon dioxide (CO2), aqueous cysHCl under air, water under CO2 or water under air, and analyzed for sulfhydryl (-SH) groups. The cysHCl-CO2 treatment produced more -SH groups than the other treatments and was effective in allowing activation of intact and sodium hypochlorite (NaOCl)-treated E. stiedai oocysts as well as NaOCl-treated E. tenella oocysts. The CO2-cysHCl complex may act directly on the oocyst wall, especially in the micropylar region, to unmask lipid-shielded disulfide bridges, which are reduced to -SH groups. The reduction apparently disturbs the protein superstructure of the oocyst wall, promotes opening of the micropyle, and changes the impermeable state of the sporulated oocyst.     

Eimeria (Protozoa: Eimeriidae) of the Cottontail Rabbit Sylvilagus audubonii in Northeastern Colorado, with Descriptions of Three New Species

SYNOPSIS. All of 100 cottontail rabbits Sylvilagus audubonii were found to be infected with 1-6 species of Eimeria. Three new species, E. audubonii, E. neoirresidua and E. poudrei are described from this host. Sub-spherical oocysts of E. audubonii average 21.2 by 17.1 μ; polar body, micropyle, oocyst residuum and sporocyst residuum are all absent; ellipsoidal sporocysts average 12.9 by 5.8 μ. Ovoid to ellipsoidal oocysts of E. neoirresidua average 25.7 by 17.9 μ; polar body and oocyst residuum are absent; micropyle and sporocyst residuum are present; ellipsoidal sporocysts average 14.5 by 6.4 μ. Ovoid to ellipsoidal oocysts of E. poudrei average 26.0 by 18.1 μ; polar body is lacking; micropyle, oocyst residuum and sporocyst residuum are present; ellipsoidal sporocysts average 14.4 by 6.4 μ. Three species of Eimeria previously described in the literature, E. maior Honess, 1939, E. media form honessi Carvalho, 1943 and E. environ Honess, 1939 are redescribed. A detailed structural and statistical analysis of each species is presented with at least 200 sporulated oocysts measured in each instance. A host list and a key to the Eimeria of cottontails is given. The use of detailed studies of oocyst size and structure as a tool for specific diagnosis of the Eimeria of cottontails is discussed.     

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.     

Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.     

Phylogenetic relationships among rodent Eimeria species determined by plastid ORF470 and nuclear 18S rDNA sequences

Phylogenetic analyses for 10 rodent Eimeria species from different host genera based on plastid ORF470 and nuclear 18S rDNA sequences were done to infer the evolutionary relationships of these rodent Eimeria species and their correlation to morphology and host specificity. The phylogenies based on both data sets clearly grouped the 10 rodent Eimeria species into two major lineages, which reflect more their morphological differences than host specificity. Species in lineage A have spheroidal to subspheroidal sporulated oocysts, are similar in size (18-29 x 17-23; xbar = 22 x 20 microm), have an oocyst residuum and one-two polar granules; these include Eimeria albigulae (Neotoma), Eimeria arizonensis (Peromyscus, Reithrodontomys), Eimeria onychomysis (Onychomys) and Eimeria reedi (Perognathus). Species in lineage B, including Eimeria falciformis (Mus), Eimeria langebarteli (Reithrodontomys), Eimeria nieschulzi (Rattus), Eimeria papillata (Mus), Eimeria separata (Rattus) and Eimeria sevilletensis (Onychomys) have different shapes (ovoid, ellipsoid, elongated ellipsoid, etc.), differ greatly in size (10-27 x 9-24; xbar = 19 x 16 microm) and all lack an oocyst residuum. Thus, The oocyst residuum was the most determinant feature that differentiated the two lineages. The accession numbers of ORF470 of E. albigulae, E. arizonensis, E. falciformis, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis, E. langebarteli are AF311630-AF311639 and 18S rDNA of E. langebarteli, E. papillata, E. reedi, E. separata, E. sevilletensis are AF311640-AF311644.     

The immunity arising from continuous low-level infection with Eimeria maxima and Eimeria acervulina.

Chicks were given daily inoculations of 1 or 5 oocysts of Eimeria maxima or 5 or 20 oocysts of E. acervulina. The inoculations ceased after 20 days with E. maxima or 25 days with E. acervulina when oocyst production had stopped. The responses to subsequent heavy challenges showed that with both species the immunity arising from the serial inoculations was stronger and/or more enduring than that produced by single inoculations of comparable numbers of oocysts.     

Eimeria acervulina: The influence of inoculation dose on transmission between broiler chickens

The course and clinical appearance of an Eimeria species infection in chicken flocks depend on the response of an individual bird to infection and on population-dynamics of the infection in the flock. Differences in ingested numbers of oocysts may affect oocyst load in the flock and the subsequent infectious dose for not yet infected birds. To study the link between numbers of oocysts excreted by infected birds and transmission of Eimeria acervulina, experiments were carried out with 42 pairs of broiler chickens using inoculation doses with 5, 50, 500 or 50,000 sporulated oocysts. In each pair one bird was inoculated and the other bird was contact-exposed. All contact birds became infected, which occurred on average within 34 h after exposure to an inoculated bird. Although a higher inoculation dose resulted in higher oocyst excretion in inoculated and contact-infected birds, only small non-significant differences in transmission rates between groups were found.     

Comparison Between Effects of Standard Feed and Whole Wheat Supplemented Diet on Experimental Eimeria tenella and Eimeria maxima Infections in Broiler Chickens

The effects of experimental infections with Eimeria tenella (Experiment 1, n = 144) or E. maxima (Experiment 2, n = 216) in broiler chickens fed whole wheat, with or without access to grit, as compared to a standard pelleted feed were studied. Inclusion of whole wheat was gradually increased up to 30% at 3 weeks of age. Grit was given separately. The chickens were kept on litter in a parasite-free environment with free access to water and feed. At 3 weeks of age half the number of chickens were individually inoculated with 500 sporulated oocysts of E. tenella (Experiment 1) or 3 000 sporulated oocysts of Eimeria maxima (Experiment 2), and the remaining birds were kept separate as uninfected controls. Neither coccidiostats nor growth enhancers were used. Oocyst concentration was determined from each group separately. Intestinal lesions were scored on 6 birds per feed regime 7 d postinoculation, and on the remaining birds at slaughter. Diet had no significant effect on bird performance during infection. However, there was an indication that the E. maxima infection had more negative effect on weight gain in birds given standard feed than in those given whole wheat supplement, but the difference was not significant (p<0.09). The number of oocysts shed or mean intestinal lesion scores did not differ between diets in either experiment. In both experiments, the number of Clostridium perfringens was higher in the caeca of inoculated birds, but there were no differences between diets.