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.     

Monosaccharide transport by Eimeria tenella sporozoites

Eimeria tenella sporozoites were incubated in the presence of 3 different [14C]-labeled sugars; D-glucose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose. The initial velocity, Vi, of uptake of D-glucose and 2-deoxy-D-glucose was similar, 41 micrograms/10(10) sporozoites/min and 46 micrograms/10(10) sporozoites/min, respectively; whereas that for 3-O-methyl-D-glucose was significantly lower, 17 micrograms/10(10) sporozoites/min. Initial velocity studies also revealed that glucose uptake was a saturable event, with an apparent KT of 20 mM and an apparent Vmax of 312 micrograms/10(10) sporozoites/min. Uptake was unaffected by exogenous sodium levels or the presence of ouabain. However, 0.1 mM phloretin significantly inhibited glucose uptake. Thus, it would appear that E. tenella sporozoites possess a Na-independent, phloretin-sensitive, carrier-mediated monosaccharide-transport system.     

Pathogenic mechanisms not operating in Eimeria necatrix infections.

Having investigated certain aspects of Eimeria necatrix coccidiosis in chickens, workers of the Hannover Veterinary School postulated "that death following a single inoculation of a large number of oocysts is due to an alarm reaction and not a specific pathogenic action of the parasites". Because this hypothesis is somewhat revolutionary in its consept, several pieces of evidence on which it is based and several logical deductions which can be made from it have been examined. It has been confirmed that injection of chichens with cysteamine or 5-hydroxytryptamine 30 min before inoculation of the birds with a lethal dose of E. necatrix oocysts reduces subsequent mortality; the reason for this, however, appears not to be the neutralisation of the proposed shock reaction, but rather an inhibition of the excystation process, brought about indirectly through the host. Inoculation of chickens with a non-lethal dose of E. necatrix oocysts 30 min before inoculation with a lethal dose of oocysts was followed by increased mortality rather than the decreased mortality which the hypothesis would predict. Treatment of chickens with sulphadimidine starting 48 h after inoculation resulted in survival of the birds rather than death which would ensue if in fact mortality was due to a shock reaction irreversibly initiated at the time of inoculation. A direct effect of sulphadimidine on the parasite has been shown both in vivo and in vitro.     

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.     

Extraintestinal sporozoites of chicken Eimeria in chickens and turkeys

Oocysts were found in the feces of chickens (recipients) dosed orally with whole blood, liver, lung, or heart homogenates from chickens and turkeys (donors) inoculated 3 and 4 days previously with a mixture of 3.5 X 10(6) oocysts of chicken Eimeria. No oocysts were found in the feces of recipients given spleen homogenates from these same chickens and turkeys and none were found in the feces of recipients given similar material from uninoculated donors. Intracellular sporazoites were found in the peripheral blood of a turkey inoculated with chicken Eimeria. The results indicate that a small number of sporozoites are capable of invading and surviving for at least 4 days in the peripheral blood of chickens and turkeys.     

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.     

The viability and survival of sporozoites of Eimeria in vitro

Some factors affecting excystation and viability of sporozoites of several species of Eimeria from chickens were examined in vitro. Chicken embryos or cultured kidney cells were inoculated with sporozoites in order to assess viability.Sporozoites of E. tenella survived in phosphate buffer (P.B.S.) containing 0·9 per cent NaCl for 14 days. Some sporozoites survived in solutions containing up to 16 per cent NaCl for 3 days at +4°C. Sporozoites of E. maxima and E. acervulina survived for only 27 h in phosphate buffer containing 1 or 2 per cent NaCl.Sporozoites of E. brunetti, E. maxima, and E. acervulina var: mivati were released rapidly from sporocysts in vitro, but survived for relatively short periods in PBS at 4°C. However, the addition of serum or gelatine to these solutions increased survival to at least 96 h.The viability of sporozoites after freezing and storing in liquid nitrogen was best when 12 per cent dimethyl sulphoxide (DMSO) was added to the sporozoite suspensions. P.B.S. with DMSO was less suitable than the other solutions used and serum or gelatine with the DMSO, was needed to increase survival. Increasing the density of sporozoites in the frozen stabilates did not increase survival.     

Flotillin-1 localization on sporozoites oF Eimeria tenella

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.     

Capping of immune complexes by sporozoites of Eimeria tenella

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.     

Some observations on the biology of five strains of Eimeria necatrix

Five laboratory strains of Eimeria necatrix were characterised with regard to the size of their oocysts, pathogenicity, reproduction, cross-immunity, ability to grow in embryonated eggs, and electrophoretic variation of enzymes. Three strains were highly pathogenic whilst two caused only few deaths and milder changes to the mean body weight gains of infected chickens. Cross-immunity was incomplete judged by scores of lesions after heterologous challenge, and electrophoretic variation of the enzymes lactate dehydrogenase and isocitrate dehydrogenase from oocysts of the five strains was also found. All the strains completed their life cycle in embryonated eggs but only a few oocysts were recovered.     

Eimeria necatrix virus: intracellular localisation of viral particles and proteins

The presence of the Eimeria necatrix virus was investigated in the following life cycle stages: sporocysts, sporozoites, merozoites, and macrogametes. Electron microscopy revealed virus-like particles (VLPs) in sporozoites, which were purified from sporozoite extracts and used to raise polyclonal antibodies. Viral proteins were identified as RNA polymerase (95 kDa) and the major capsid protein (80 kDa). Polyclonal antibody was used to detect the intracellular localisation of VLPs and proteins. Immunoelectron microscopy and immunohistochemistry identified a viral protein of 95 kDa in all the E. necatrix stages studied, whereas the 80 kDa protein was found only in sporocysts and sporozoites. In addition, no VLPs were found in sporocysts. These results indicate that the synthesis of viral capsid proteins takes place during the early events of sporulation, and is then packaged into novel viruses during the late events. No VLPs were seen and no capsid proteins were found in the merozoites and macrogametes, whereas the 95 kDa RNA polymerase was present in both these stages. In addition, no VLPs or proteins were detected in chicken tissues.