Eimerian life cycles: the patency of eimeria vermiformis, but not Eimeria pragensis, is subject to host (Mus musculus) influence

The ability of Eimeria vermiformis and Eimeria pragensis to produce oocysts in primary infections is influenced by host factors. Oocyst production and the duration of patency were determined for each species in: NIH mice, normal or immunosuppressed (by X-irradiation or injection of cortisone acetate), and strains of mice: nu/+, nu/nu; BALB/c, C57BL/6 known (E. vermiformis), or suspected (E. pragensis), to be of contrasting susceptibility to infection. The effect of induced or genetic susceptibility on the reproduction of E. vermiformis resulted in increases in both oocyst production and the duration of patency. Eimeria pragensis was less affected, with smaller increases in the numbers of oocysts produced and no significant prolongation of patency. The implications of these findings with respect to eimerian life cycles are 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 acervulina, E. brunetti, and E. maxima: immunity in chickens with low multiple doses of mixed oocysts.

Young chickens inoculated with multiple low doses of mixed oocysts of Eimeria acervulina, E. brunetti, and E. maxima had a high level of resistance to reinfection with a mixed challenge dose on Day 28, Day 84, or Day 140. Immunity was enhanced when the number of immunizing doses was increased from three to four. Resistance was also high in birds maintained on a proprietary mixture of amprolium, ethopabate, and sulphaquinoxaline (Pancoxin-Merck, Sharp and Dohme Ltd.) during immunization, although immunity to E. acervulina was lower in these birds. Oocyst production was lower in birds given mixed infections as compared with that of birds given pure infections with similar doses of oocysts. Competition between species did not inhibit the development of immunity in birds given low doses of mixed oocysts.     

The effect of cyclosporin A on the development of Eimeria in non-specific hosts.

The role of T lymphocytes on the development of turkey coccidia in chickens was investigated to further document the involvement of immune mechanisms in the reactions of non-specific hosts to infections with species of Eimeria. All chickens treated with the T cell-specific immunosuppressive drug, cyclosporin A (CsA), 1, 2 or 3 days before or for 3 consecutive days immediately before inoculation with sporulated oocysts of Eimeria from the turkey, shed oocysts in their feces 7–12 days after inoculation. Those chickens treated with CsA after inoculation produced no oocysts although in one experiment a small number of oocysts were discharged by birds injected with CsA 1 day after inoculation. High serum levels of CsA were maintained in the CsA-treated chickens for at least 24 h post-inoculation with the oocysts. Immunosuppressive activity of the drug was determined by measuring lymphocyte blastogenesis in response to concanavalin A. These results demonstrate that the suppression of T lymphocyte activity in a non-specific host early during an infection with a heterologous species of Eimeria permits the complete intracellular development of the parasite. Our findings are suggestive of a role for T lymphocytes in preventing the development of Eimeria in non-specific hosts possibly via a lymphokine-mediated mechanism.     

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.     

Pathological changes and immunity associated with experimental Eimeria vermiformis infections in Mus musculus

Pathological changes and immunity induced by Eimeria vermiformis (Ernst, Chobotar & Hammond, 1971) were studied in outbred Swiss mice inoculated with 5000, 10,000, 20,000, or 40,000 oocysts. Cross immunity to E. ferrisi was also studied. In the case of E. vermiformis, mortality was dose dependent; most deaths were observed in the intermediate-dose groups. Most deaths also correlated with peak oocyst output. Histopathologic changes consisted of an early neutrophil and mononuclear cell infiltration in the small intestine. Later, villus atrophy and crypt hyperplasia caused a decrease in the villus-crypt ratio. During the acute phase (8-10 days after inoculation), villus tips were eroded and parasites with necrotic debris filled the cryptal and intestinal lumina. Vacuolar changes were observed in epithelial cells of the small intestine. Neither parasites nor significant pathological changes were observed in extra-intestinal organs. Mice were totally immune to reinfection with E. vermiformis 30 and 105 days after inoculation. Cross immunity was not observed between E. vermiformis and E. ferrisi.     

Eimeria acervulina,E. brunetti,E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens

Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acervulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. acervulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completely immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatrix. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection.     

Eimeria tenella: effects of immunity on sporozoites within the lumen of the small intestine

The effect of immunity on the numbers of sporozoites of Eimeria tenella recoverable from the lumen of the small intestine 1 hr after an oral challenge inoculum of oocysts was examined. The experiments were carried out in chickens which had been given an immunizing inoculum of oocysts 9 or 18 days earlier, and the results were compared with those obtained in a control, unprimed, but similarly challenged, group. Similar numbers of "challenge" sporozoites were found in the intestinal washes of control and 18 day primed chickens but there were fewer in the 9 day primed groups. The titers of antisporozoite IgA antibodies (measured by indirect fluorescence) were higher in the gall bladder bile of the 9 day primed groups but resistance to reinfection (measured by the output of oocysts in the feces after challenge with oocysts orally or with sporozoites intracecally) was greater in the 18 day primed group. Although fewer in number, the challenge sporozoites recovered from the intestinal washes of 9 day primed chickens appeared to be morphologically normal when examined by light microscopy. Also, they were as infective as sporozoites recovered from unprimed control, or 18 day primed, groups when injected intracecally into naive chickens. The findings indicate that, whereas reduction of the number of sporozoites of E. tenella in the lumen of the small intestine (presumably caused by the action of secreted antibodies) can be a means of reducing the effective challenge inoculum, this mechanism does not play a major role in the expression of immunity.     

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.     

Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella

The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella.     

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.     

Eimeria acervulina: evaluation of the cellular and antibody responses to the recombinant coccidial antigens in B-congenic chickens

The roles of major histocompatibility complex (MHC) and non-MHC-linked genes in the genetic control of disease susceptibility and the development of protective immunity to Eimeria acervulina infection were investigated in six 15I5-B congenic and four different strains of chickens characterized for the MHC. When oocyst production was assessed, wide variations were noted following initial and challenge infections among the strains of chickens tested. In general, 15.N-21, 15.P-13, B21, B19, SC, and FP chickens were protected following challenge infection whereas 15I5, 15.P-19, 15.7-2, and 15.6-2 chickens were not. Strains of chickens sharing a same B haplotype on different genetic backgrounds did not show comparable levels of protection. These results lead to the view that non-MHC-linked genes have a profound influence on the outcome of the host response to E. acervulina infection. Chickens infected twice at 1-month intervals by an oral inoculation with E. acervulina developed both coccidial-specific antibody and T-cell responses. E. acervulina infected chickens showed T-cell-mediated immune responses to the intact sporozoites as well as to recombinant proteins, p130 of sporozoites and p150 of merozoites. Both p130 and p150 antigens have been identified and characterized previously. Sera obtained from all infected chickens recognized the p150 merozoite protein, but not the p130 sporozoite protein in immunoblots. In general, the cellular response, but not the antibody response to the p150 recombinant surface merozoite antigen correlated with the degree of protection following the challenge infection. These results suggest that the strains of chickens having improved protection against challenge infection demonstrate higher T-cell responses to the recombinant surface merozoite protein, p150.     

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.     

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.     

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.     

Stable transfection of Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the life cycle

The obligate intracellular apicomplexan parasite Eimeria tenella, one of seven species of Eimeria that infect chickens, elicits protective cell-mediated immunity against challenge infection. For this reason, recombinant E. tenella parasites could be utilised as an effective vaccine vehicle for expressing foreign antigens and inducing immunity against heterologous intracellular microbes. A stable line of E. tenella expressing foreign genes is a prerequisite, and in this work an in vivo stable transfection system has been developed for this parasite using restriction enzyme-mediated integration (REMI). Two transgenic populations of E. tenella have been obtained that express YFP-YFP constitutively throughout the parasite life cycle. Southern blotting and plasmid rescue analyses show that the introduced exogenous DNA was integrated at random into the parasite genome. Although the life cycle of the transgenic populations was delayed by at least 12 h and the output of oocysts was reduced 4-fold relative to the parental BJ strain of E. tenella, the transgenic parasites were sufficiently immunogenic to protect chickens against challenge with either transgenic or parental parasites. These results are encouraging for the development of transgenic E. tenella as a vaccine vector and for more detailed investigation of the biology of the genus Eimeria.     

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.     

Immunity patterns during acute infection by Eimeria bovis

Cellular and humoral responses were investigated following gavage inoculation of 6-wk-old bull calves with 35,000-40,000 oocysts of Eimeria bovis. At 3-4-day intervals for 40 days after inoculation (DAI), blood was taken and assessed for serum IgG against merozoites and sporozoites of E. bovis. Proliferative responses of peripheral blood lymphocytes were measured following stimulation with either concanavalin A (Con A) or a soluble antigen derived from E. bovis oocysts (EbAg). Serum IgG against merozoites and sporozoites reached a peak of activity between 10 and 20 DAI, coinciding with oocyst shedding on days 17 to 24. Serum antibody titers had dropped to base levels by 40 DAI, although anti-merozoite titers remained elevated for the duration of the study (i.e., from days 12 and 20 to day 40). Con A stimulation of lymphocytes was not affected by infection; there was no evidence of suppressed or augmented responsiveness. Lymphocyte responses to EbAg had reached a maximum by day 20 and remained elevated throughout the study. These results indicate (a) that sporozoites and merozoites share antigens recognized by serum IgG, (b) that there is no episode of marked immunosuppression during acute infection, and (c) that cellular immunity is probably more important in resistance against reinfection than humoral immunity.     

Effect of F-2 and T-2 fusariotoxins on experimental Cryptosporidium baileyi infection in chickens

The course of Cryptosporidium baileyi infection in chickens fed with different doses of fusariotoxins was compared with that of control groups. F-2 toxin levels of 0.187–1.5 mg kg⁻¹ and T-2 toxin levels of 0.187–6.0 mg kg⁻¹ were investigated. The experimental amimals were orally infected with 6 × 10⁵ C. baileyi oocysts at 1 week of age. Total daily oocyst output was monitored by a quantitative method. Acquired immunity was tested at the age of 4 weeks, by ELISA and by a challenge infection with an equal number of oocysts, upon recovery from the primary infection. The results show that in chickens kept on the lower doses of F-2 and T-2 toxins, the parasite infection ran a similar course to that in the control groups, and the animals became resistant to re-infection. However, when higher doses (2.0–6.0 mg kg⁻¹) of T-2 toxin were used, a depression of weight gain was observed with some other physiological parameters (PCV, weight of bursa, weight of thymus, skin thickness in PHA-P skin test) also indicating toxic effect and, simultaneously, the oocyst output decreased significantly and the patent period was slightly prolonged. Although certain modifications of the immune response could be revealed, the chickens became resistant to re-infection. Only early (1 week of age) parasite infection and 6 mg kg⁻¹ T-2 toxin in the feed significantly depressed body weight gain and immunity.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.