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.     

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.     

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.     

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.     

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.     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

Anti-coccidial activity of 2-picoline, 6-amino-4-nitro-, 1-oxide

An investigational drug (2-picoline, 6-amino-4-nitro-, 1-oxide) was evaluated to characterize the anti-coccidial spectrum of the compound. Two concentrations of the drug (125 and 250 ppm) were evaluated for bioactivity; weight gain, survival, dropping, and lesion scores were the response variables utilized to ascertain activity. The activities of the picoline derivative were compared with monensin, maduramicin, and a narasin/nicarbazin (1:1) combination. The investigational drug had significant activity against Eimeria tenella and Eimeria necatrix, and the 250-ppm level was significantly more active than 125 ppm. At 250 ppm, the E. tenella activity of the picoline derivative was comparable to both monensin (120 ppm) and the 50-ppm narasin/nicarbazin combination, significantly less effective than maduramicin (6 ppm), and significantly more efficacious than 30 ppm narasin/nicarbazin. At the same level (250 ppm), the picoline derivative had significantly less E. necatrix activity than monensin (120 ppm), maduramicin (6 ppm), and narasin/nicarbazin (50 ppm), and significantly greater activity than 30 ppm narasin/nicarbazin. At best, only extremely weak Eimeria acervulina, Eimeria brunetti, and Eimeria maxima activities were noted with the investigational drug; higher concentrations of the picoline derivative may achieve greater anti-coccidial activity against these species. The efficacy of narasin/nicarbazin compared favorably with monensin and maduramicin; the 50-ppm level of the combination appeared significantly more efficacious than 30-ppm.     

Concatenated mitochondrial DNA of the coccidian parasite Eimeria tenella

Apicomplexan parasites of the genus Plasmodium, pathogens causing malaria, and the genera Babesia and Theileria, aetiological agents of piroplasmosis, are closely related. However, their mitochondrial (mt) genome structures are highly divergent: Plasmodium has a concatemer of 6-kb unit and Babesia/Theileria a monomer of 6.6- to 8.2-kb with terminal inverted repeats. Fragmentation of ribosomal RNA (rRNA) genes and gene arrangements are remarkably distinctive. To elucidate the evolutionary origin of this structural divergence, we determined the mt genome of Eimeria tenella, pathogens of coccidiosis in domestic fowls. Analysis revealed that E. tenella mt genome was concatemeric with similar protein-coding genes and rRNA gene fragments to Plasmodium. Copy number was 50-fold of the nuclear genome. Evolution of structural divergence in the apicomplexan mt genomes is discussed.     

Effect of coccidiosis on blood coagulation in broilers

Severe infection with Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella increased the prothrombin times in broilers compared with the times in uninfected birds. Recalcification time was not affected. The increase in prothrombin time was related to the severity of infection (as measured by lesion score), and was significant (P less than or equal to 0.05) only in the most severely infected birds. The increase was of short duration, lasting only 1 or 2 days, and first appeared on day 5 or 6 postinoculation. Restricting the feed intake of uninoculated birds to the amount of feed consumed by infected birds showed that the reduction in feed intake with coccidiosis was not responsible for the increase in prothrombin time.     

Analysis of transcripts from intracellular stages of Eimeria acervulina using expressed sequence tags

Coccidiosis in chickens is caused by 7 species of Eimeria. Even though coccidiosis is a complex disease that can be caused by any combination of these species, most of the molecular research concerning chicken coccidiosis has been limited to Eimeria tenella. The present study describes the first large-scale analysis of expressed sequence tags (ESTs) generated primarily from second-stage merozoites (and schizonts) of E. acervulina. In total, 1,847 ESTs were sequenced; these represent 1,026 unique sequences. Approximately half of the ESTs encode proteins of unknown function, or hypothetical proteins. Twenty-nine percent of the E. acervulina ESTs share significant sequence identity with sequences in the E. tenella genome. Additionally, EST hits seem to be much different compared with those of E. tenella. One of the differences is the very low number of ESTs that encode putative microneme proteins. This study underlines the potential differences in the molecular aspects of 2 Eimeria species that in the past were thought to be highly similar in nature.     

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.     

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.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Efficacy and cross-resistance studies on N, N'-bis (3,4 ditrifluoromethylphenyl) methylmalonamide, a novel anticoccidial agent.

N,N'-bis (3,4 ditrifluoromethylphenyl) methylmalonamide (Sch 18545) completely controlled a mild Eimeria necatrix infection at 50, 40 or 30 p.p.m. in the diet, and controlled E. tenella infections at 50 and 40 p.p.m. Slight oocyst passage was observed at each E. tenella treatment level with a marked increase at the 30 p.p.m. treatment level. Fifty p.p.m. were necessary to control E. acervulina infections; levels of 40 p.p.m. reduced E. acervulina oocyst production while 30 p.p.m. were ineffective. Evaluations of Sch 18545 using a mixed infection (Coccivac D) further suggested that activity with this compound was weakest against E. acervulina. Weight gains decreased with increasing concentration of drug in the diet of treated, infected birds and thus the compound showed an insufficient safety margin to be of practical value. Such 18545 administered at 35 p.p.m. in the diet was effective against amprolium, zoalene, aklomide or nicarbazin-resistant strains of E. tenella.     

Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants

Avian coccidiosis is an intestinal disease caused by protozoa of the genus Eimeria. To investigate the potential of recombinant protein vaccines to control coccidiosis, we cloned 2 Eimeria sp. genes (EtMIC2 and 3-1E), expressed and purified their encoded proteins, and determined the efficacy of in ovo immunization to protect against Eimeria infections. Immunogen-specific serum antibody titers, parasite fecal shedding, and body weight gains were measured as parameters of disease. When administered alone, the recombinant EtMIC2 gene product induced significantly higher antibody responses, lower oocyst fecal shedding, and increased weight gains compared with nonvaccinated controls following infection with E. tenella. Combined embryo immunization with the EtMIC2 protein plus chicken cytokine or chemokine genes demonstrated that all 3 parameters of vaccination were improved compared with those of EtMIC2 alone. In particular, covaccination with EtMIC2 plus interleukin (IL)-8, IL-16, transforming growth factor-beta4, or lymphotactin significantly decreased oocyst shedding and improved weight gains beyond those achieved by EtMIC2 alone. Finally, individual vaccination with either EtMIC2 or 3-1E stimulated protection against infection by the heterologous parasite E. acervulina. Taken together, these results indicate that in ovo vaccination with the EtMIC2 protein plus cytokine/chemokine genes may be an effective method to control coccidiosis.     

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.