Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.     

Transport of Eimeria necatrix sporozoites in the chicken: effects of irritants injected intraperitoneally

Light and electron microscopic observations confirmed that Eimeria necatrix sporozoites first enter villous epithelial cells of the chicken small intestine and are transported to the crypts by mononuclear cells. Ultrastructurally, these cells resemble granulated intraepithelial lymphocytes (IEL) rather than macrophages, as suggested previously. The injection of chickens intraperitoneally (i.p.) with a variety of irritants, including proteose peptone, at the time of oocyst inoculation or up to 12 hr postinoculation (PI) resulted in a delay in the arrival of sporozoites at the crypt. Significantly fewer sporozoites had arrived at the crypt by 24 hr PI in i.p.-injected birds as compared to controls. This delay in the arrival of sporozoites at the crypts was reflected by a delay in the development of intestinal lesions and in peak oocyst production. However, there was no significant decrease in the total numbers of oocysts produced by these birds as compared to controls, indicating that no significant loss of sporozoites occurs during the possible rerouting of the parasites. The presence of infective stages in extraintestinal sites was detected by transferring various tissues to coccidia-free recipients. Infection was transferable by gut, liver, and spleen from irritant-injected and control birds at all time intervals studied (12, 24, 36, and 48 hr PI). Infection was also transferable with blood and kidney, but not consistently. A small number of oocysts was passed by the recipients of peritoneal wash from irritant-injected birds at 12 hr PI. In all transfers, the prepatent period was normal, suggesting that the migrant stages are sporozoites.     

The Migration of Eimeria acervulina Sporozoites to the Duodenal Glands of Lieberkühn

SYNOPSIS. The establishment of Eimeria acervulina sporozoites in the duodenal glands of Lieberkühn of the chicken is described. Sporozoites were found to enter the tips of the villi and pass into the lamina propria, or core, of the villus. Within the lamina propria, sporozoites were engulfed by macrophages and taken to the glandular epithelium.
Data are presented which indicate that macrophages serve as a defense against infection as well as a mode of transportation.     

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.     

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.     

Immune responses to infections with coccidia in chickens: gut hypersensitivity.

Sporulated oocysts or oocyst hulls of Eimeria acervulina given via the crop, and oocyst juice or sporozoites inoculated directly into the intestinal lumen, were tested for their effects on the hypersensitivity of the intestines of immunized and susceptible birds. Only viable sporozoites consistently caused an increase in the permeability of the immunized intestine to macromolecules, shown by the leakage of intravenously injected dye. Repulsion of epithelium and disruption of the villi were seen more often in immunized than in susceptible chickens but there was no correlation with treatment or with dye leakage. The hypersensitive response of immune intestines challenged with a heterologous organism was approximately half as intense as the response to homologous challenge. The significance of these findings in relation to immunity to coccidiosis is discussed.     

B-lymphocyte responses in the large intestine and mesenteric lymph nodes of mice infected with Eimeria falciformis (Apicomplexa)

B-cell responses of 3 immunoglobulin isotypes (IgA, IgG, and IgM) were investigated in the large intestine and mesenteric lymph nodes (MLN) of naive or immune mice after inoculation of oocysts of Eimeria falciformis. Primary and anamnestic IgA and IgG lymphocyte responses to E. falciformis occurred in the large intestine of nonimmune and immune mice, respectively. IgA-containing lymphocytes (IgAc) were the largest population of responding B cells in the large intestine. In infected mice, IgAc accumulated in the apical portion of the lamina propria, whereas IgG-containing lymphocytes (IgGc) were more numerous at the base of the lamina propria. No significant increase in the number of IgM-containing lymphocytes (IgMc) was observed in the lamina propria of the large intestine. Primary but no anamnestic B-cell responses occurred in the MLN, and immune mice actually had reduced numbers of IgAc and IgGc in the MLN when compared with naive mice. IgGc were the largest population of responding B cells in the MLN. Thus, IgAc appear to accumulate preferentially at the site of parasite development, whereas IgGc are primarily localized deeper in the lamina propria of the large intestine and in the draining lymph nodes of mice infected with E. falciformis.     

The life cycle and pathogenicity of coccicium Eimeria nocens (Kotlán, 1933) in domestic goslings

Twenty-four 10-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 1.0x10(5)-1.0x10(6) sporulated oocysts of Eimeria nocens, and killed at intervals from 30 to 336 hr postinoculation (PI). Parts of the visceral organs, including intestines, kidney, liver, gallbladder, and spleen from inoculated goslings, were fixed and sectioned. The life cycle of E. nocens and histologic changes during infection were examined microscopically. The results showed that at least 3 generations of meronts developed in the endogenous stage of the life cycle of E. nocens. Two types of meronts were found. The first completed maturation at 54 to 78 hr PI. These meronts were the first generation, with each forming about 12 merozoites. The second completed maturation at 102 to 240 hr PI. These meronts were the second or third generations, with each meront forming about 24 merozoites. Development of gamonts began at about 198 hr after infection. The prepatent period was 9 days and discharge of oocysts continued for 4 days. Sporulation of oocysts occurred in 60-72 hr at 25 C. Eimeria nocens invaded the posterior jejunum, ileum, caecum, rectum, and cloaca. Developmental stages were localized within the epithelial cells of villi and crypts, and in lamina propria. Marked histological changes, including desquamation and necrosis of intestinal epithelium, submucosal edema, hemorrhages, infiltration of inflammatory cells, and villous atrophy, were seen during the periods of late merogony, gamogony, and oocyst shedding. They were most pronounced in the ileum and the regions nearby. The infected goslings showed severe diarrhea, bloody feces, anorexia, emaciation, and even death, suggesting that E. nocens is highly pathogenic for goslings.     

Coccidiosis in common wombats (Vombatus ursinus).

Eimeria arundeli is a widespread coccidian parasite of the common wombat (Vombatus ursinus), and has been considered to be nonpathogenic. We describe disease in two captive juvenile wombats ascribed to infection with E. arundeli. One animal had diarrhea, the second had mucoid soft feces and lost weight over several weeks prior to death. Masses of coccidial gametocytes in hypertrophic cells in the lamina propria distended villi, causing grossly visible raised pale thickened regions over extensive areas of the mucosa of the small intestine in both animals. Neutrophils infiltrated affected mucosa, and there was an inflammatory exudate into the intestinal lumen in case one. In case two, neutrophils infiltrated the lamina propria of villi focally, crypts were distended by necrotic debris, and epithelium on villi was extremely attenuated. No bacterial pathogens were isolated from lung and intestine in case one; case two was not cultured. Oocysts consistent with E. arundeli were present in large numbers in floatations of diarrheic feces in both cases.     

Interactions in vitro between sporozoites of Eimeria tenella and host peritoneal exudate cells: electron microscopal observations

The interactions of sporozoites of Eimeria tenella with peritoneal cells from normal and from immunized chickens were examined in vitro. Although the uptake of sporozoites by cells from immunized birds was greater than by cells from susceptible birds, no differences were apparent in their appearance in electron micrographs. Entry into both macrophages and heterophils (comparable to mammalian neutrophils) was by phagocytosis. The findings are discussed.     

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.     

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.     

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.     

Cellular Aspects of the Resistance of Chickens to Eimeria tenella Infections*

SYNOPSIS. Sporozoites of Eimeria tenella were injected into the peritoneal cavity of normal chickens and chickens immunized against E. tenella. In some experiments normal scrum and serum from resistant chickens were injected prior to the injection of sporozoites. After 15 or 30 minute periods of intraperitoneal incubation, exudates were harvested and the occurrence of intracellular sporozoites was determined. Only macrophages and degranulated granulocytes were observed to contain sporozoites. There was no significant difference between the number of macrophages obtained from normal chickens (normal macrophages) which contained sporozoites and the number of macrophages obtained from immune chickens (immune macrophages) which contained sporozoites. Significantly fewer immune macrophages treated with immune serum contained sporozoites than untreated normal or immune cells, normal macrophages treated with either serum, or immune macrophages treated with normal scrum. Sporozoites in untreated normal macrophages did not appear to be harmed by the intracellular environment, based on structural observations. The majority of sporozoites in macrophages from all other groups were difficult to distinguish within the cytoplasm and were visibly distorted. It is hypothesized that the presence of fewer infected macrophages in exudates of immune chickens and serum-treated normal chickens was caused by an enhanced ability of these cells to destroy the parasite. Similar observations were noted in the case of sporozoites within degranulated granulocytes of experimental groups. The lack of understanding of the degranulation phenomenon makes it difficult to interpret these findings.     

Anticoccidial drugs: Effects on infectivity and survival intracellularly of Eimeria tenella sporozoites

The effects of various anticoccidial drugs on extracellular and intracellular sporozoites were studied in cell culture and in chickens. Treatment of freshly excysted, extracellular sporozoites of Eimeria tenella for 18 hr with monensin, decoquinate, or robenidine at 100 ppm had no effect on oocyst production 7–10 days after the sporozoites were rinsed free of drugs and fed to chickens. Treatment of cultures of E. tenella in chick kidney cell monolayers with monensin (0.001 μg/ml), decoquinate (0.01 μg/ml), zoalene (20.0 μg/ml), or robenidine (0.01 μg/ml) had no effect on intracellular sporozoites at 4 hr following introduction of sporozoites and drugs into the culture. A significant reduction of intracellular parasites occurred at 24 hr in the cultures treated with monensin or zoalene. Remaining intracellular sporozoites in monensin-treated cultures were morphologically abnormal or degenerate, while sporozoites in other cultures appeared normal. The number and condition of sporozoites in the nontreated cultures were unchanged at 24 hr postinoculation. These results indicate that sporozoites undergo changes subsequent to penetration of host cells that render them susceptible to drug action.     

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.     

Eimeria maxima: characteristics of attenuated lines obtained by selection for precocious development in the chicken

Both the Weybridge strain and a mixture of six laboratory and field strains of Eimeria maxima have been attenuated by selection for early maturation of oocysts during serial passage in chickens. The prepatent times of the resultant precocious lines produces after selection were reduced from approximately 120 hr to less than 107 hr, and their reproduction and pathogenicity were less than those of the parent strains. Chickens which had been inoculated with a small number of oocysts of the Weybridge precocious or mixed field isolate precocious lines were immune to subsequent challenge with the parent strains. In a comparison made between the endogenous development of the precocious lines and their parent strains, it was found that asexual multiplication of the precocious lines was reduced, due to the earlier onset of gametogony. The results of this study also indicate that the parent (i.e., normal) strains of E. maxima probably undergo a minimum of four generations of schizogony.     

Immunomodulatory effects of feed-borne Fusarium mycotoxins in chickens infected with coccidia

The potential for Fusarium mycotoxins to modulate immunity was studied in chickens raised to 10 weeks of age using an enteric coccidial infection model. Experimental diets included: control, diets containing grains naturally contaminated with Fusarium mycotoxins, and diets containing contaminated grains + 0.2% polymeric glucomannan mycotoxin adsorbent (GMA). Contaminated diets contained up to 3.8 microg/g deoxynivalenol (DON), 0.3 microg/g 15-acetyl DON and 0.2 microg/g zearalenone. An optimized mixture (inducing lesions without mortality) of Eimeria acervulina, E. maxima and E. tenella was used to challenge birds at 8 weeks of age. Immune parameters were studied prior to challenge, at the end of the challenge period (7 days post-inoculation, PI), and at the end of the recovery period (14 days PI). Total serum immunoglobulin (Ig) A and IgG concentrations in challenged birds fed the contaminated diet were higher than controls at the end of the challenge period. Serum concentration of IgA, but not IgG, was significantly decreased at the end of the recovery period in birds fed the contaminated diet. The percentage of CD4+ and CD8+ cell populations in blood mononuclear cells decreased significantly at the end of the challenge period in birds fed the control or the contaminated diet compared to their percentages prior to challenge. The pre-challenge percentage of CD8+ population was restored at the end of the recovery period only in birds fed the control diet. Interferon-gamma (IFN-gamma) gene expression in caecal tonsils was up-regulated in challenged birds fed the contaminated diet at the end of the challenge period. No significant effect of diet was observed on oocyst counts despite the changes in the studied immune parameters. It was concluded that Fusarium mycotoxins modulate the avian immune system. This modulation involves alteration of gene expression but apparently does not enhance susceptibility or resistance to a primary coccidial challenge.     

Cultivation of Eimeria tenella in Japanese quail embryos (Coturnix coturnix japonica).

Complete development of Eimeria tenella in Japanese quail embryos was observed. Sporozoites were inoculated into the allantoic cavity of 7-day-old Japanese quail embryos (Coturnix coturnix japonica), after which the infected embryos were incubated at 41 C. In the chorioallantoic membrane mature first generation schizonts, mature second generation schizonts, and gametes were detected at 48 hr postinoculation of sporozoites (PI), 84 hr PI, and 126 hr PI, respectively. Mature gametes and zygotes were found at 132 hr PI, and oocysts were detected at 138 hr PI. Mortality of embryos increased with increment of inoculum size of sporozoites. LD50 was 1.7 x 10(2) sporozoites. Oocyst production was also dependent on inoculum size. Oocysts harvested from embryos sporulated. The oocysts were inoculated into 13-day-old chickens, and oocysts, capable of sporulating normally, were recovered from ceca 7 days after inoculation.