Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

A modified method for purification of Eimeria tenella sporozoites.

Coccidiosis is an economically important gastrointestinal disease in domestic fowl. Eimeria species are the causative agents of avian coccidiosis. Current challenges in management and prevention of eimeriosis enhance the need for research in this field. Sporozoite purification is a necessary step for Eimeria spp. in vitro infection models. Current alternatives such as DE-52 anion exchange chromatography and Percoll gradient require time and resources. We present a modified protocol consisting on vacuum filtration of sporozoites using a disposable 5-µL filter. Yield percentages were similar to those reported for Percoll gradient purification. By reducing time and efforts during sporozoite purification, it could be possible to increase resources in other areas of Eimeria studies.

The role of the American cockroach (Periplaneta americana) as transport host of Eimeria tenella to chickens.

The role of the American cockroach, Periplaneta americana as transport host for Eimeria tenella was evaluated. Twenty-four cockroaches were orally fed with sporulated oocysts of E. tenella. Their feces and digestive tract were examined for oocysts by sugar centrifugal flotation technique and PCR. Infectivity of the oocysts recovered from the digestive tract of infected cockroaches as well as from their feces was evaluated by orally inoculating them into Boris Brown chickens. E. tenella oocysts were found in the digestive tract and feces of infected cockroaches up to day 4 after ingestion of oocysts. Furthermore, oocysts that were recovered from the digestive tract and feces of cockroaches remained infective for 4 and 3 days after ingestion of oocysts, respectively. Presence of oocysts in the feces of chicken that had been inoculated with either digestive tract or feces of P. americana demonstrated the infectivity of E. tenella oocysts from digestive tract or feces, suggesting that P. americana may play a role in the transmission of E. tenella among chicken and between chicken flocks.

Use of discriminant analysis for the evaluation of coccidiosis resistance parameters in chickens raised in hot humid tropical environment.

Coccidiosis endemicity remains a major challenge in poultry production in the tropics and all over the world. In order to develop predictive tool for identification of chickens that are at risk of coccidiosis among Nigerian indigenous chickens, body weight gain (BWG) and hematological variables were determined for chickens infected with Eimeria tenella (female = 60, male = 63) and uninfected (female = 51, male = 45). The hematological variables analyzed include the following: packed cell volume (PCV, %), white blood cells (WBC, × 106/µl), and red blood cells (RBC, × 106/µl), as well as differential leucocyte percentages of neutrophils, lymphocytes, monocytes, and eosinophils. Body weight gain was determined at days 3, 6, 9, 12, and 15. Of the 12 variables analyzed, BWG at day 3, monocyte, PCV, and WBC in males and BWG at days 6, 9, and 12, PCV, and WBC in female chickens showed significant (P ≤ 0.01) difference between the infected and uninfected. Stepwise discriminant analysis evolved a model that could distinguish uninfected from Eimeria-infected chickens. Packed cell volume, WBC, BWG at day 3, and lymphocytes emerged the most discriminant between uninfected and Eimeria-infected chickens in male chickens. In female chickens, PCV, RBC, and BWG at day 3 were identified as most discriminant variables in separating the uninfected from Eimeria-infected chickens. Therefore, this study suggests that routine blood test and estimates of body weight gain could serve as a useful tool for identifying chickens that may be at risk of coccidiosis, enabling improvement of preventive measures.

Mitochondrial pathways are involved in Eimeria tenella-induced apoptosis of chick embryo cecal epithelial cells.

Accumulating evidence suggests that Eimeria tenella severely damages the intestinal mucosa in infected poultry, resulting in deadly haemorrhagic typhlocolitis and major economic losses. Damage to host tissue is believed to arise mainly from apoptosis, which is, in general, intimately related to mitochondrial function. However, it is unclear whether mitochondria-dependent apoptotic pathways are specifically involved in parasite-induced apoptosis of chick embryo cecal epithelial cells. Because the mitochondrial permeability transition pore (MPTP) and caspase-9 are important elements in these pathways, we studied the effects of their respective inhibitors (i.e., cyclosporine A [CsA] and Z-LEHD-FMK, respectively) in primary cultures of chicken embryonic cecum epithelial cells using histopathological techniques, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays, flow cytometry (FCM) and ELISA. Results indicated that the inhibitors significantly decreased (p < 0.01) DNA injury, apoptosis and caspase-9 and caspase-3 activity of chick embryo cecal epithelial cells at 24, 48, 72, 96 and 120 h after E. tenella infection. Thus, our data supported that mitochondria-dependent apoptotic pathways were involved in apoptosis of parasitised chick embryo cecal epithelial cells.

Genetic differences in the body weight and haematological traits of Nigerian indigenous chickens infected with Eimeria tenella.

In an effort to shed more light on the tolerance of indigenous chickens to coccidiosis, we compared the body weight, faecal oocyst load and haematological parameters based on sex and genotypes of Eimeria tenella-infected chickens. Three hundred chicks from three genotypes (normal-feathered, frizzle-feathered and naked-neck) of Nigerian indigenous chickens which comprised 100 birds per genotype were raised for 6 weeks. At 3 weeks old, each chick was weighed and faecal, and blood samples were collected before inoculation. Subsequently, the birds were weighed and faecal samples collected at days 1, 3, 6, 9, 12 and 15 post-inoculation. Blood samples were collected from 50 chicks per genotype at 3 and 5 weeks post-inoculation. Blood parameters were determined and faecal samples subjected to McMaster egg counting technique. Results showed genotype, and sex had significant effects on body weight from day 1 to 15 post-inoculation. Normal-feathered chicks had the highest body weight while frizzle-feathered chicks showed lowest body weight at post-inoculation. E. tenella was identified in caecal and lower intestinal mucosa of the genotypes, but genotype had no significant effect (p > 0.05) on the lesion score. There were no significant differences in haematological values among genotypes (p > 0.05) except for lymphocytes where naked-neck chicks had the highest lymphocytes' count (1.83 ± 0.02 %), followed by normal-feathered (1.79 ± 0.02 %) and the frizzle-feathered (1.68 ± 0.02 %). The present values of body weight, faecal oocyst and haematological parameters obtained seemed not to be convincing enough to suggest that the genotypes were different in terms of tolerance to coccidiosis.

QTL detection for coccidiosis (Eimeria tenella) resistance in a Fayoumi × Leghorn F2 cross, using a medium-density SNP panel.

BACKGROUND: Coccidiosis is a major parasitic disease that causes huge economic losses to the poultry industry. Its pathogenicity leads to depression of body weight gain, lesions and, in the most serious cases, death in affected animals. Genetic variability for resistance to coccidiosis in the chicken has been demonstrated and if this natural resistance could be exploited, it would reduce the costs of the disease. Previously, a design to characterize the genetic regulation of Eimeria tenella resistance was set up in a Fayoumi × Leghorn F2 cross. The 860 F2 animals of this design were phenotyped for weight gain, plasma coloration, hematocrit level, intestinal lesion score and body temperature. In the work reported here, the 860 animals were genotyped for a panel of 1393 (157 microsatellites and 1236 single nucleotide polymorphism (SNP) markers that cover the sequenced genome (i.e. the 28 first autosomes and the Z chromosome). In addition, with the aim of finding an index capable of explaining a large amount of the variance associated with resistance to coccidiosis, a composite factor was derived by combining the variables of all these traits in a single variable. QTL detection was performed by linkage analysis using GridQTL and QTLMap. Single and multi-QTL models were applied. RESULTS: Thirty-one QTL were identified i.e. 27 with the single-QTL model and four with the multi-QTL model and the average confidence interval was 5.9 cM. Only a few QTL were common with the previous study that used the same design but focused on the 260 more extreme animals that were genotyped with the 157 microsatellites only. Major differences were also found between results obtained with QTLMap and GridQTL. CONCLUSIONS: The medium-density SNP panel made it possible to genotype new regions of the chicken genome (including micro-chromosomes) that were involved in the genetic control of the traits investigated. This study also highlights the strong variations in QTL detection between different models and marker densities.

Evaluation of the protective efficacy of the anticoccidial vaccine Coccivac-B in broilers, when challenged with Egyptian field isolates of E. tenella.

The present study was performed to explore the efficacy of the commercial anticoccidial vaccine (Coccivac B®) in broiler chickens using five field strains of Eimeria tenella that were isolated from five provinces in Egypt. This study also analyzed the ITS-1-rDNA sequence of these five strains and its corresponding sequence in the vaccine. In a floor pen experiment, 216 one-day-old commercial broiler chicks were classified into vaccinated and non-vaccinated groups. Each main group was classified into six subgroups. The chicks were challenged on the 28th day of age with 10(4) sporulated oocysts of one of the five field strains of E. tenella. Our results indicated that Coccivac B® produced variable degrees of protection in the birds infected with the five different strains of E. tenella. Aligning the ITS-1 sequences from the five strains with the ITS-1 sequence of E. tenella from the vaccine revealed 96 % sequence similarity with the Kafer El-Sheikh strain, 94 % with the Gharbia strain, 90 % with the Alexandria strain, and 78 % with the Matrouh and Behera strains. While interesting, these similarity values were not useful for predicting the protection conferred by the vaccine against the five field isolates. However, based on the data reported here, we can conclude that Coccivac B® produced variable degrees of protection in the birds infected with the five different strains of E. tenella.

Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella.

Serine protease inhibitors (serpins) mediate many biological processes, including immune responses to pathogenic infection. In this study, a member of the serpin superfamily was identified from the common poultry parasite Eimeria tenella by expressed sequence tag analysis and the rapid amplification of cDNA ends technique. The full-length cDNA was 1,918 bp and had an open reading frame of 1,248 bp encoding a polypeptide of 415 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. Real-time quantitative PCR analysis revealed that the serpin gene was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). The sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector, and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was prepared and used to determine invasion inhibition capacity and localization; the results suggested that the serpin may play an important role in invasion and survival of the sporoziotes in the host.

Anticoccidial activity of traditional Chinese herbal Dichroa febrifuga Lour. extract against Eimeria tenella infection in chickens.

The study was conducted on broiler birds to evaluate the anticoccidial efficacy of an extract of Chinese traditional herb Dichroa febrifuga Lour. One hundred broiler birds were assigned to five equal groups. All birds in groups 1-4 were orally infected with 1.5 × 10(4) Eimeira tenella sporulated oocysts and birds in groups 1, 2 and 3 were medicated with 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respectively. The bloody diarrhea, oocyst counts, intestinal lesion scores, and the body weight were recorded to evaluate the anticoccidial efficacy. The results showed that D. febrifuga extract was effective against Eimeria infection; especially 20 mg D. febrifuga extract/kg feed can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion in comparison to infected-unmedicated control group.

Eimeria tenella infection perturbs the chicken gut microbiota from the onset of oocyst shedding.

Coccidiosis is a serious threat to the poultry industry, resulting in substantial economic losses worldwide. The effective development of alternative treatments for coccidiosis that does not involve chemotherapy drugs and does not result in antibiotic resistance relies on gaining a clearer understanding of the interaction between host intestinal microbiota and enteric coccidia. Here, we established an Eimeria tenella infection model in chickens and subsequently monitored the changes in the overall intestinal microbiome using 16S rRNA gene sequencing. We found that the gut (i.e. fecal) microbiota of infected chicken differed from that of uninfected naïve animals. Levels of non-pathogenic bacteria, including Lactobacillus and Faecalibacterium declined, whereas those of pathogenic bacteria, including Clostridium, Lysinibacillus, and Escherichia, increased over time in response to E. tenella infection. Similar dynamic changes of the fecal microbiota were observed in both Arbor Acres broilers and White Leghorn chickens, indicating that the perturbation of the microbiota was directly induced by E. tenella infection. Our findings could be used to further elucidate the serious damage to host health caused by coccidia infection, leading to the development of new effective treatment options for coccidiosis.

Application of a new PCR-RFLP panel suggests a restricted population structure for Eimeria tenella in UK and Irish chickens.

Eimeria species cause coccidiosis, most notably in chickens where the global cost exceeds US$3 billion every year. Understanding variation in Eimeria population structure and genetic diversity contributes valuable information that can be used to minimise the impact of drug resistance and develop new, cost-effective anticoccidial vaccines. Little knowledge is currently available on the epidemiology of Eimeria species and strains in different regions, or under different chicken production systems. Recently, 244 Eimeria tenella isolates collected from countries in Africa and Asia were genotyped using a Sequenom single nucleotide polymorphism (SNP) tool, revealing significant variation in haplotype diversity and population structure, with a marked North/South regional divide. To expand studies on genetic polymorphism to larger numbers of E. tenella populations in other geographic regions a cheaper and more accessible technique, such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), is desirable. We have converted a subset of SNP markers for use as PCR-RFLPs and re-analysed the original 244 isolates with the PCR-RFLPs to assess their utility. In addition, application of the PCR-RFLP to E. tenella samples collected from UK and Irish broiler chickens revealed a tightly restricted haplotype diversity. Just two of the PCR-RFLPs accounted for all of the polymorphism detected in the UK and Irish parasite populations, but analysis of the full dataset revealed different informative markers in different regions, supporting validity of the PCR-RFLP panel. The tools described here provide an accessible and cost-effective method that can be used to enhance understanding of E. tenella genetic diversity and population structure.

The mode of action of anticoccidial quinolones (6-decyloxy-4-hydroxyquinoline-3-carboxylates) in chickens.

The anticoccidial mode of action of quinolones (6-decyloxy-4-hydroxyquinoline-3-carboxylates) against Eimeria tenella and E. acervulina in chickens has been investigated, using decoquinate and M&B 15,584 as examples. The well known static effect on sporozoites of relatively high continuous drug concentrations in the food masked other components of the mode of action, newly described here. Lower concentrations of quinolones allowed sporozoites to continue their development. First-stage schizonts were susceptible to a secondary cidal effect, although later schizonts seemed to be rather refractory. Furthermore, the sporulation of oocysts produced by E. tenella that completed its life cycle in the presence of suboptimal concentrations of quinolones was inhibited: this probably reflects a drug effect on gametocytes. Quinolones were absorbed rapidly from the chicken intestine, probably in less than 1 h. Drug withdrawal experiments showed that quinolones persisted in chicken tissues at active concentrations for up to 48 h. Despite their static effect on sporozoites, they may nevertheless be expected to exert a therapeutic effect against drug-sensitive coccidia in interrupted regimes that allow the later cidal effect to come into play. This allows immunity to coccidiosis to develop in the presence of drug. These new results, with the previously available data have been combined in an updated account of the anticoccidial mode of action of quinolones in the chicken.

Protective oral immunization of chickens against Eimeria tenella with sporozoite surface antigens.

Antigens were extracted from the surface of Eimeria tenella sporozoites with a solution containing Triton X 100 (1%), sodium dodecyl sulphate (0.5%) Na deoxycholate (1%) and EDTA (1 mM). After removal of the detergents, these surface antigen preparations conferred an immunity that protected chickens against a subsequent infection (10(4) sporulated oocysts). The best results were obtained after two 250 micrograms injections of Al(OH)3 adsorbed antigens (oocyst output per g caecal material on Day 7 post infection: 2.39 x 10(7) +/- 0.32 x 10(7) oocysts for controls and 7.37 +/- 10(6) +/- 3.19 x 10(6) oocysts for vaccinated birds) and after four gastric intubations of liposome entrapped antigens (oocysts output on Day 7 postinfection: 2.75 x 10(6) +/- 2.02 x 10(6) g-1 caecal material). These results represented respectively 70 and 88% protection indexes. Studies on the systemic and local antibody response after one or several infections of chickens with the parasite indicated at least 20 different molecules in the detergent antigens which are classified after immunoblotting according to their properties.

Isolation and selection of ionophore-tolerant Eimeria precocious lines: E. tenella, E. maxima and E. acervulina.

Eimeria parasites were isolated from Nanhai Guangdong province (southern China) and studied in chickens in wire cages to evaluate their drug resistance against commonly used ionophores: monensin (100 mg/kg of feed), lasolacid (90 mg/kg), salinomycin (60 mg/kg), maduramicin (5 mg/kg) and semduramicin (25 mg/kg). Chinese Yellow Broiler Chickens were infected with 40,000 crude sporulated Eimeria oocysts at 15 days of age and prophylactic medication commenced a day prior to infection. Drug resistance was assessed for each ionophore drug by calculating the anticoccidial index (ACI) and percentage optimum anticoccidial activity (POAA) based on relative weight gain, rate of oocyst production and lesion values. Results revealed that Nanhai Eimeria oocysts comprising of E. tenella, E. maxima and E. acervulina, were resistant to monensin, sensitive to both salinomycin and lasolacid and partially sensitive to maduramicin and semduramicin. By selection for early development of oocysts during passage through chickens, the prepatent time of E. tenella, E. maxima and E. acervulina were reduced by 49, 36 and 22 h, respectively. The precocious lines are less pathogenic than the parent strains from which they were selected and conferred a satisfactory protection for chickens against coccidiosis. These ionophore-tolerant precocious lines could have wider applications in the development of anticoccidial vaccines for sustainable control of coccidiosis.

Quantifying risk factors of coccidiosis in broilers using on-farm data based on a veterinary practice.

A study was done to find and quantify risk factors for coccidiosis. The study population consisted of 4774 broiler flocks kept on 177 farms. Flocks were considered a case when at least one bird in the flock showed microscopic presence of oocysts in intestinal scrapings in a grow-out cycle. Other flocks were defined as controls. This was done for three types of Eimeria: Eimeria acervulina, Eimeria tenella and Eimeria maxima. Logistic regression was used to assess variables that influence the occurrence of Eimeria species. There were 49 variables, based on animal, flock or farm level. There was an enhanced risk of coccidiosis due to environmental and management factors that increase the risk of introducing contamination or that are related to hygienic measures. These include lack of use of overalls by visitors, a farmyard which is difficult to clean, bad hygienic status, personnel who might also be working on other farms, presence of other animals on the farm, and feeding and drinking systems which are more difficult to clean. Also, the presence of other diseases on the farm and Eimeria species found in the previous flock increased the risk of coccidiosis.

Eimeria tenella and E. acervulina: differences in ability to elicit cross-species protection as compared with the turkey coccidium, E. adenoeides.

Repeated oral inoculation of turkey poults with large doses (1 x 10(6) oocysts) of the chicken coccidia, Eimeria tenella or E. acervulina, failed to prevent weight loss, poor feed conversion, and intestinal pathology in turkeys challenged with the turkey coccidium, E. adenoeides. Invasion by E. tenella in turkeys was significantly greater than invasion by E. adenoeides in chickens; by 24 hr postinoculation (PI), the numbers of E. tenella and E. adenoeides sporozoites in the ceca had decreased markedly as compared with the numbers that initially invaded, and they did not differ significantly from each other. At 24 hr PI, however, transfer of cecal scrapings from chickens or turkeys inoculated with E. adenoeides produced infection in 53% of the recipient turkeys, but transfer of scrapings from either chickens or turkeys inoculated with E. tenella failed to produce infection in 20 attempts with recipient chickens. Cultured chicken peripheral blood monocytes (PBMs) that were inoculated with E. adenoeides sporozoites contained numerous vesicles that were recognized by the refractile body-specific monoclonal antibody 1209; the number of vesicles was markedly decreased in PBM cultures inoculated with gamma-irradiated E. adenoeides sporozoites. Very few vesicles were detected in the cytoplasm of turkey PBMs that contained E. tenella sporozoites, and none were detected in turkey PBMs containing E. adenoeides sporozoites. The survival of infective sporozoites, along with the secretion of refractile body antigen, may be more critical to the development of cross-species immunity than the number of sporozoites that initially invade the foreign host.

Adapting Eimeria tenella to grow in primary chicken kidney cells following repeated passages between cell culture and chickens.

The present study was undertaken to adapt a field isolate (F0) of Eimeria tenella to grow in primary chicken kidney cells (PCKCs) by selecting for characteristics of the parasite rather than modifying the culture and/or environmental conditions. Fourteen generations (F1 to F14) of E. tenella were produced following repeated passages between PCKCs and chickens. Although F1 yielded only a 28% increase in oocysts, in PCKCs compared with F0, F2 to F5 produced from 259% to 277% more oocysts, respectively. There was no significant increase in the percentage of oocysts produced in PCKCs by F6 to F14 compared with F5. Generations F1 to F14 demonstrated a greater propensity for multiple infections within the same host cell than did F0. For example, it was not uncommon to observe two, three, and occasionally four oocysts within a single PCKC. Chickens inoculated with F0 oocysts generally experienced greater pathogenesis by day 7 postinoculation than chickens inoculated with F14 oocysts as measured by decreased body weights, increased cecal lesions, and a higher mortality rate.

Response of unimmunized and sporozoite-immunized chickens to challenge with avian Eimeria species: effects of intraperitoneal injection of Sephadex.

Unimmunized chickens, given intraperitoneal injections of Sephadex at the same time that they were inoculated with oocysts of either Eimeria tenella or E. acervulina, had significantly lower lesion scores at 6 days postinoculation (PI) than unimmunized chickens that were injected with saline instead of Sephadex. Despite the difference in lesion scores, there was little effect on weight gain, except in one experiment, in which Sephadex-injected chickens gained significantly more weight than saline-injected chickens. In contrast, in chickens that were immunized by prolonged exposure to sporozoites of E. adenoeides, injection of Sephadex at the time of challenge with E. tenella did not reduce lesion scores or parasite development as compared with the uninjected chickens, and the weight gain of the Sephadex-injected challenged chickens fell to a level significantly lower than that of their saline-injected challenged counterparts. The data indicate that Sephadex injected at the time of oocyst inoculation 1) produces markedly different effects on lesion scores and weight gain in unimmunized and immunized chickens and 2) abrogates sporozoite-elicited immunity against E. tenella challenge.