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.     

Anticoccidial activity of herbal complex in broiler chickens challenged with Eimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of different concentrations of the herbal complex of 4 plants (leaves of Azadirachta indica and Nicotiana tabacum, flowers of Calotropis procera and seeds of Trachyspermum ammi) in broiler chickens in comparison with amprolium anticoccidial. Three concentrations (2 g, 4 g and 6 g) of herbal complex were given to the experimental groups once a day and amprolium (at the dose rate of 125 ppm) was given orally in drinking water from the 14th to the 21st days of age. One group was kept as infected, non-medicated control and one as non-infected, non-medicated control. All groups were inoculated orally with 75,000 sporulated oocysts on the 14th day of age except the non-infected, non-medicated control. Among herbal complex medicated groups, the maximum anticoccidial effect was seen in the group medicated with 6 g herbal complex followed by 4 g and 2 g herbal complex medicated groups. Treatment with 6 g of the herbal complex significantly reduced the negative performance and pathogenic effects associated with Eimeria tenella challenge at a level that was comparable with amprolium when using a largely susceptible recent field isolate. In summary, concentration-dependent anticoccidial activity of the studied herbal complex suggests its use as an alternative anticoccidial agent to chemotherapeutic drugs for Eimeria tenella control.     

Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens

The efficacy of an oral DNA vaccine carrying the Eimeria tenella 5401 antigen gene delivered by attenuated Salmonella typhimurium was examined in an experimental challenge study. The DNA vaccine preparation was made by transforming the recombinant plasmid pcDNA3-5401 into the attenuated S. typhimurium strain (Dam(-) and PhoP(-)) (designated hereafter as ZJ111/pcDNA3-5401). The chickens were randomly divided into six groups, 50 per group. Group A were given PBS as control. Chickens in group B were fed with 10(8) colony forming units (CFU) of attenuated S. typhimurium carrying pcDNA3. Group C were immunised with 100 microg of the recombinant 5401 protein via intramuscular injection. Groups D to F orally received ZJ111/pcDNA3-5401 at doses of 10(7), 10(8) and 10(9)CFU per chicken, respectively. All immunisations were boosted 2 weeks later. The immunised chickens were challenged with 6x10(4) homologous sporulated oocysts 14 days after the second immunisation. No significant differences in body weight were detected between the groups before immunisation and at week 4 after the booster immunisation. The ZJ111/pcDNA3-5401 was eventually eliminated from the spleen and liver on week 6 post-immunisation. The plasmid pcDNA3-5401 was stably maintained in over 80% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunisation of chickens with ZJ111/pcDNA3-5401 elicited specific humoral responses and stimulated proliferation of peripheral blood lymphocytes. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control chickens. Antibody response was significantly lower in group C than in groups immunised with strain ZJ111/pcDNA3-5401. Vaccination with the strain ZJ111/pcDNA3-5401 at 10(8) (group E) and 10(9) (group F) CFU per chicken provided 55.0 and 57.5% protection against E. tenella challenge, respectively. These results have important implications for the development of DNA vaccines against avian coccidiosis by bacteria-vectored oral delivery system.     

Eimeria tenella: ginsenosides-enhanced immune response to the immunization with recombinant 5401 antigen in chickens

Three-day-old specific-pathogen-free chickens were subcutaneously immunized with Eimeria tenella recombinant 5401 antigen (100 microg per chicken) with (0.25, 0.5 or 1.0mg per dose) or without ginsenosides, and boosted with the same dosage 14 days later. The chickens were challenged with 6 x 10(4) homologous sporulated oocysts 14 day after the booster. The specific antibody response and lymphocyte proliferation in response to Con A were measured before and 7, 14, 21, 28, 35, 42 days after the immunization. Oocyst output, mortality, and lesion scores were measured to evaluate the protective effects of the immunization. The vaccine containing 0.5 or 1.0mg ginsenosides per dose induces higher antibody response and lymphocyte proliferation in response to Con A than the vaccine without ginsenosides or containing 0.25mg per dose. The oocyst output indicated that recombinant 5401 antigen with ginsenosides (0.5 and 1.0mg per dose) gave a protection rate of 59.38 and 62.5%, respectively. The lesion score in the group vaccinated with recombinant 5401 antigen with 0.5 or 1.0mg ginsenosides per dose were significantly lower than in group without ginsenosides or containing 0.25mg per dose. Therefore, we conclude that ginsenosides have strong adjuvant effects at a dose of 0.5 or 1.0mg when mixed with E. tenella recombinant 5401 antigen, and has a potential as an adjuvant in chicken vaccine.     

Genetic variability of resistance to Eimeria acervulina and E. tenella in chickens

Summary The genetic variability of 18 sire families of the Athens-Canadian randombred population infected with coccidiosis was assessed by examining the response variables of weight gain, packed red blood cell volume, mortality and coccidial lesions. A significant gain and PCV depression and high lesion scores for Eimeria tenella and E. acervulina were produced in the infected group compared to the noninfected group. Significant variation among the sire families was observed for all of the response variables except E. acervulina lesions and a significant sex x sire interaction was observed for weight gain. The heritability (h²) estimates for the response variables revealed that resistance to coccidiosis in chickens is moderately heritable. The h² estimates for gain and PCV increased with the coccidial infections indicating that maximum progress in selecting for resistance should be made when the population was exposed to coccidial infection. Gain was positively correlated to the other measures of resistance and thus selecting for coccidial resistance should not reduce growth rates. PCV was similarly correlated but had higher positive correlation with E. tenella lesion. Percent mortality which is the selection parameter in most coccidial selection programs was correlated with resistance to coccidiosis. The phenotypic and genotypic correlations demonstrated that chickens susceptible to E. tenella were also susceptible to E. acervulina. Total lesion scores were moderately to highly correlated with the other variables and would be a suitable variable to use in coccidiosis experimentation including a genetic selection program for resistance. This study shows that progress could be made in selecting for resistance to coccidiosis in chickens using one or a combination of these response variables.     

Synthesis of nucleic acids and proteins in the appendix of chickens infected with Eimeria tenella]

A comparative study was carried out of the synthesis of DNA, RNA and protein in blind appendages of the intestine of healthy and infected with E. tenella chickens. It was established that the inclusion of P32 into DNA and C14 - orotic acid into RNA increases in 2-10 days after the infection. An intensive inclusion of C14-lysine into protein of blind appendages was observed in 2-6 days after the infection of chickens. Under the effect of the parasite an activation of metabolism of DNA, RNA and protein takes place in blind appendages of the host's intestine.     

Development of CRISPR-CAS9 based RNA drugs against Eimeria tenella infection

Parasitic diseases are major trouble in many parts of the world. We consider that if a chemical can break a DNA barcode sequence, it might be used to develop a species-specific anti-parasitic agent. To examine this hypothesis, we constructed sgRNAs that target both the control (5.8S rDNA) and a DNA barcode (ITS) sequence in Eimeria tenella. In vitro experiment showed that Cas9 mRNA combined with sgRNAs could reduce the sporulation percentage of oocysts and the survival rate of sporulated oocysts and sporozoites. Quantitative real-time PCR showed that the DNAs of parasites exposed to Cas9 mRNA and sgRNAs were significantly affected, regardless of whether they were exposed to a combination of two sgRNAs or just a single sgRNA. The DNA sequencing also indicated that the experimental group exposed to two sgRNAs mixed with Cas9-induced deletion of large parts and a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments. In vivo trial, the effect of sgRNA and Cas9 RNA on the pathogenicity of E. tenella in chicken showed less lesion score and oocysts score (P < 0.05) in experimental groups than control groups. The results and concepts presented in this research can lead to discovering novel nucleic acid therapeutic drugs for Eimeriasis and other parasitic infections, which provide insights into the development of species-specific anti-parasitic agents.     

Interaction of Eimeria tenella with intestinal mucin in vitro

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.     

Eimeria tenella: Host Specificity in Gallinaceous Birds

SYNOPSIS. Eight species representing 8 genera of gallinaceous birds were used: Alectoris graeca; Colinus virginianus; Coturnix coturnix; Gallus gallus; Meleagris gallopavo; Numidia meleagris; Pavo cristatus; Phasianus colchicus. Three-week-old birds were dosed with sporulated oocysts of Eimeria tenella Beltsville strain. At 4, 24, 48, 72, 96, 120, 144, and 168 hr after inoculation, 1-3 infected birds and uninoculated controls of each species were killed by cardiac exsanguination. Pieces of intestines were fixed and examined for stages of E. tenella as stained paraffin sections or indirect fluorescent antibody preparations. Oocyst counts were made in droppings collected for the first 6 days of the patent period. Sporozoites were found in the lamina propria of some birds of 5 species at 4 hr postinoculation, but no stages were found thereafter except in the breeds of G. gallus and A. graeca. At 144 and 168 hr postinoculation, a few macrogametes were found in the ceca of 2 A. graeca , but no oocysts were found in the feces. No statistical difference was found between the number of oocysts produced/bird in the breeds of G. gallus examined. It is evident from these observations that E. tenella did not complete its life cycle in several close phylogenetic relatives of G. gallus , even though in other studies this parasite was found to complete its life cycle in cell cultures derived from the same birds.     

Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis

Hybridoma cell lines, which secreted antibodies directed against two different strains of Eimeria tenella and one strain of E. mitis, were produced by fusion of spleen cells from sporozoite-immunized Balb/cByJ mice with P3-X63-Ag8 myeloma cells. The antibodies demonstrated at least eight different binding patterns on or in air-dried sporozoites as determined by the indirect immunofluorescent antibody (IFA) test. These patterns varied from a general internal fluorescence similar to that seen with sporozoites exposed to hyperimmune chicken serum, to fluorescence observed on the tip, pellicle, and refractile body of the parasite. Five cloned, antibody-secreting cell lines were successfully established. Four of these clones produced antibody that reacted only with various strains of E. tenella and cross-reacted with no other species of coccidia. The fifth clone produced antibody directed against only E. mitis and did not react with any other coccidial species.     

Eimeria tenella: anticoccidial drug activity in cell cultures

About a decade and a half after discovery of the presence in America of the Japanese beetle, Popillia japonica Newm., Glaser and Fox came upon sick and dying beetle grubs infected with a nematode. Steiner described and named it: Neoaplectana glaseri n. sp. A method for its cultivation in vitro, the first such success for a worm parasite, was devised by Glaser (1931). This led to testing the field introduction of infective stages of the nematode for beetle grub control. Mass culture techniques in the laboratory using, for instance, nonsterile ground veal pulp, produced harvests of infective larval forms in the millions. Killing effects of the parasite were verified as well as its persistence season to season in the field.In 1940 Glaser found a way to grow N. glaseri germ-free. Its sterile culture and that of other free-living and parasitic protozoa and metazoa by Glaser and colleagues led later to axenic culture in cell-free media.