Insecticidal activity of fluralaner (Exzolt®) administered to Gallus gallus domesticus against triatomines (Hemiptera, Reduviidae, Triatominae).

BACKGROUND: Triatoma infestans, Triatoma brasiliensis, Triatoma pseudomaculata and Rhodnius prolixus are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Chickens serve as an important blood food source for triatomines. This study aimed to assess the insecticidal activity of fluralaner (Exzolt®) administered to chickens against triatomines (R. prolixus, T. infestans, T. brasiliensis and T. pseudomaculata). METHODS: Twelve non-breed chickens (Gallus gallus domesticus) were randomized based on weight into three groups: negative control (n = 4); a single dose of 0.5 mg/kg fluralaner (Exzolt®) (n = 4); two doses of 0.5 mg/kg fluralaner (Exzolt®) (n = 4). Nymphs of 3rd, 4th and 5th instars of R. prolixus, T. infestans, T. brasiliensis and T. pseudomaculata (all n = 10) were allowed to feed on chickens before treatment, and at intervals of 1, 7, 14, 21, 28, 35 and 56 days after treatment, with insect mortality determined. RESULTS: Treatment with two doses of fluralaner showed higher insecticidal efficacy against R. prolixus, T. infestans and T. brasiliensis compared to the single-dose treatment. Similar insecticidal efficacy was observed for T. pseudomaculata for one and two doses of fluralaner. Insecticidal activity of fluralaner (Exzolt®) against triatomine bugs was noted up to 21 and 28 days after treatment with one and two doses of fluralaner, respectively. CONCLUSIONS: The results demonstrate that treatment of chickens with fluralaner (Exzolt®) induces insecticidal activity against triatomines for up to 28 days post-treatment, suggesting its potential use as a control strategy for Chagas disease in endemic areas.

Effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum and ileum in broiler chickens.

Coccidiosis, an intestinal disease caused by Eimeria parasites, is responsible for major losses in the poultry industry by impacting chicken health. The gut microbiota is associated with health factors, such as nutrient exchange and immune system modulation, requiring understanding on the effects of Eimeria infection on the gut microbiota. This study aimed to determine the effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum (CeL and CeM) and ileum (IlL and IlM) at multiple time points (days 3, 5, 7, 10, and 14) post-infection. E. acervulina infection decreased evenness in CeL microbiota at day 10, increased richness in CeM microbiota at day 3 before decreasing richness at day 14, and decreased richness in IlL microbiota from day 3 to 10. CeL, CeM, and IlL microbiota differed between infected and control birds based on beta diversity at varying time points. Infection reduced relative abundance of bacterial taxa and some predicted metabolic pathways known for short-chain fatty acid production in CeL, CeM, and IlL microbiota, but further understanding of metabolic function is required. Despite E. acervulina primarily targeting the duodenum, our findings demonstrate the infection can impact bacterial diversity and abundance in the cecal and ileal microbiota.

Low levels of chicken body louse (Menacanthus stramineus) infestations affect chicken welfare in a cage-free housing system.

BACKGROUND: The chicken body louse is an obligate ectoparasite of domestic chickens. Chicken body lice feed on feathers, and infestation with this louse is linked to decreases in egg production, hen weight, and feed conversion efficiency. However, it is unknown how chicken body lice impact egg-laying chickens in cage-free environments. Welfare and behavior metrics were collected from flocks of egg-laying chickens either infested with chicken body lice or left uninfested. METHODS: In two trials, two flocks of cage-free commercial egg-laying chickens were infested with chicken body lice or maintained as uninfested controls. At three timepoints, behavior and welfare of all chickens was measured. On-animal sensors were used to quantify pecking, preening, and dustbathing behavior. Other animal-based welfare metrics included recording comb wounds and skin lesions. RESULTS: Birds infested with chicken body lice exhibited significantly more preening behaviors than uninfested birds, even at low louse levels. Moderate or severe skin lesions were detected on birds that were moderately infested with chicken body lice while skin lesions were never detected on uninfested birds. CONCLUSIONS: The welfare of chickens was impacted by the chicken body louse, a chewing louse that primarily feather feeds. Evidence of skin lesions on infested birds suggests that lice may cause more damage to birds than previously thought, and further evaluation of louse economic damage is necessary.

The Poultry Red Mite, Dermanyssus gallinae, travels far but not frequently, and takes up permanent residence on farms.

Management of Dermanyssus gallinae, a cosmopolitan hematophagous mite responsible for damage in layer poultry farming, is hampered by a lack of knowledge of its spatio-temporal population dynamics. Previous studies have shown that the circulation of this pest between farms is of strictly anthropogenic origin, that a mitochondrial haplogroup has been expanding on European farms since the beginning of the 21st century and that its local population growth may be particularly rapid. To refine our understanding of how D. gallinae spreads within and among farms, we characterized the genetic structure of mite populations at different spatial scales and sought to identify the main factors interrupting gene flow between poultry houses and between mitochondrial haplogroups. To this end, we selected and validated the first set of nuclear microsatellite markers for D. gallinae and sequenced a region of the CO1-encoding mitochondrial gene in a subsample of microsatellite-genotyped mites. We also tested certain conditions required for effective contamination of a poultry house through field experimentation, and conducted a survey of practices during poultry transfers. Our results confirm the role of poultry transport in the dissemination of mite populations, but the frequency of effective contamination after the introduction of contaminated material into poultry houses seems lower than expected. The high persistence of mites on farms, even during periods when poultry houses are empty and cleaned, and the very large number of nodes in the logistic network (large number of companies supplying pullets or transporting animals) undoubtedly explain the very high prevalence on farms. Substantial genetic diversity was measured in farm populations, probably as a result of the mite's known haplodiploid mode of sexual reproduction, coupled with the dense logistic network. The possibility of the occasional occurrence of asexual reproduction in this sexually reproducing mite was also revealed in our analyses, which could explain the extreme aggressiveness of its demographic dynamics under certain conditions.

Detection, Identification, and Characterization of Eimeria spp. from Commercial Chicken Farms in Different Parts of Iran by Morphometrical and Molecular Techniques.

PURPOSE: Coccidiosis of domestic chicken is an important disease caused by any of seven species of Eimeria which, by developing within the epithelial cells of the intestine, cause lesions therein. We carried out a study on poultry farms located in various regions of Iran to determine the incidence and spread of Eimeria species by employing a single PCR test. METHODS: A total of 64 fully confirmed clinically intestinal tracts were collected from different parts of Iran. From these 64 intestinal tracts, 82 samples were prepared from the different sites involved in the digestive tract. In morphological assessment, 23 samples could not be isolated and its information was not evaluated. RESULTS: Using morphological methods, the following seven species of Eimeria were identified: E. acervulina (15/59; 25.42%), E. tenella (30/59; 50.84%), E. maxima (12/59; 20.33%), E. praecox (1/59; 1.69%), E. necatrix (2/59; 3.38%), E. mitis (5/59; 8.47%), and E. mivati (2/59; 3.38%). Mixed infections were found in eight (13.55%) samples. In molecular assessment, 31 samples could not be isolated and its information was not evaluated. Totally, the following five species were identified using molecular methods: E. acervulina (35/51; 68.62%), E. tenella (33/51; 64.70%), E. maxima (6/51; 11.76%), E. brunetti (5/51; 9.80%), and E. necatrix (2/51; 3.92%). Mixed infections were found in 23 (45.09%) samples. CONCLUSIONS: The present study is an update on the situation of poultry coccidiosis in Iran and provides the first data on the molecular detection, identification, and characterization of Eimeria spp. in the poultry population of this country and confirmed the presence of different species of this parasite in this area. According to the results, E. acervulina and E. tenella, as the main disease-causing species, should be considered in control programs such as treatment and vaccination strategies.

Eimeria tenella pyrroline -5-carboxylate reductase is a secreted protein and involved in host cell invasion.

Chicken coccidiosis, which caused by Eimeria spp, is a parasitic protozoal disease. At present, control measures of this disease depend mainly on anticoccidial drugs and live vaccines. But these control strategies have drawbacks such as drug resistance and limitations in live vaccines production. Therefore, novel control approaches are urgently need to study to control this disease effectively. In this study, the function and characteristics of the pyrroline-5-carboxylate reductase of Eimeria tenella (EtPYCR) protein were preliminary analyzed. The transcription and translation level were analyzed by using qPCR and Western blot. The results showed that the mRNA transcription and translation levels of EtPYCR were higher in unsporulated oocysts (UO) and second generation merozoites (Mrz) than that in sporulated oocysts (SO) and sporozoites. Enzyme activity showed that the enzyme activity of EtPYCR was also higher in the UO and Mrz than that in the SO and sporozoites. Immunofluorescence localization showed EtPYCR was mainly located on the top of sporozoites and the whole cytoplasm and surface of Mrz. The secretion assay indicated that EtPYCR was secretion protein, but not from micronemes. Invasion inhibition assay showed that rabbit anti-rEtPYCR polyclonal antibodies can effectively inhibit sporozoite invasion of DF-1 cells. These results showed that EtPYCR possess several important roles that separate and distinct from its conversion 1-pyrroline-5-carboxylate (P5C) into proline and maybe involved in the host cell invasion and development of parasites in host cells.

Immunoprotective effects of DNA vaccine against Eimeria tenella based on EtAMA3 and EtRON2L2.

Eimeria tenella is the most pathogenic and harmful intestinal parasitic protozoan. Recombinant DNA vaccines open options for promising strategies for preventing avian coccidiosis, replacing chemical drugs and live oocyst vaccines. Two important antigenic proteins, EtAMA3 (also known as SporoAMA1) and EtRON2L2, act together to promote the invasion of E. tenella sporozoites. In this study, a recombinant DNA vaccine, designated pcDNA3.1(+)-AR, was constructed based on EtAMA3DII, EtRON2L2D3, and EtRON2L2D4. Chickens were intramuscularly immunized with different doses (25, 50, or 100 µg) of pcDNA3.1(+)-AR to evaluate its immunoprotective effects in vivo. The chickens in the 50 µg and 100 µg groups had higher cytokine concentrations (interleukin 2, interferon-gamma, and interleukin 10), and lesion scores (81.9% and 67.57%, respectively) and relative oocyst production (47% and 19%, respectively) reduced compared with the unchallenged group, indicating partial protection against E. tenella. These results suggest that pcDNA3.1(+)-AR is a promising vaccine candidate against avian coccidiosis.

Epidemiological Survey on Post Mortem Coccidiosis in Broiler Chicken in Bejaia Province, Northern Algeria.

AIM: The epidemiological survey was carried out to determine the prevalence of eimeriosis in broiler chickens slaughtered depending the season, to determine the different Eimeria species causing the coccidiosis in poultry farms; and to assess the impact of Eimeria parasite on histomorphological structure and oxidative stress parameters of the intestine. MATERIALS AND METHODS: The study was conducted from December 2018 to December 2019 in the province of Bejaia, Algeria. The intestines chickens (n = 366) were obtained immediately after slaughter, each cut into different segments (duodenum, jejunum, ileum, and caecum). Microscopic and parasitological examinations were performed according to standard procedures. Histomorphometric measurements of intestine were obtained using Image J software. Oxidative stress parameters were carried out from intestine tissue. RESULTS: Eimeria spp. were detected in 73.77% (95% CI 71.20-76.34) of broiler gut samples. The prevalence varied significantly according to the season, with the lowest rates in winter (42.81%, 95% CI 40.35-45.27) and the highest in autumn (97.92%, 95% CI 97.6-99.4). All seven Eimeria species were identified, most commonly E. necatrix (27.70%), E. brunetti (26.47%), and E. tenella (20.96%). The mean lesion score ranged from 1.51 ± 0.05 to 1.79 ± 0.04. Significant differences in VH/CD ratio of intestinal epithelium (P < 0.001) were observed in different intestinal portions of infested broiler chickens compared to non-infested. The mean MDA concentration of intestinal segments was significantly higher in Eimeria species infested broilers compared to non-infested (P < 0.05). The results show at least one difference in CAT, SOD, and ABTS-+ concentrations (P < 0.05) in both chicken's groups. CONCLUSION: Our results revealed that coccidiosis is extremely prevalent in slaughtered broilers, with an abundance of pathogenic Eimeria species. Also, it was concluded that infestation induces tissue structure alterations which coincides with the oxidative damage.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites.

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Genotyping of Toxoplasma gondii in domestic animals from Campeche, México, reveals virulent genotypes and a recombinant ROP5 allele.

Toxoplasma gondii has at least 318 genotypes distributed worldwide, and tropical regions usually have greater genetic diversity. Campeche is a state located in the southeastern region of México and has favourable climate conditions for the replication and dissemination of this protozoan, similar to those in South American countries where broad genetic diversity has been described. Thus, in this study, 4 T. gondii isolates were obtained from tissues of stray dogs and free-range chickens in Campeche, México, and were genotyped by Mn-PCR-RFLP with 10 typing markers (SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) and 5 virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5) to provide new information about the distribution and virulence prediction of T. gondii genotypes. Two isolates of T. gondii genotype #116 and 2 of genotype #38 were obtained from stray dogs and chickens, respectively. The parasite load found in these species was between <50 and more than 35 000 tachyzoites per mg of tissue. Virulence marker genotyping revealed a recombinant 1&3 ROP5 RFLP pattern in 2 ToxoDB #116 isolates with no prediction of virulence in a murine model, while in the 2 ToxoDB #38 isolates, the ROP18/ROP5 combination predicted high virulence. Considering all the typed markers, there is a predominance of type I and III alleles, as constantly reported for the isolates characterized in various regions of México. It is crucial to determine their phenotype to corroborate the genetic virulence profile of the T. gondii isolates obtained in this study.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

Comparative proteomic analysis across the developmental stages of the Eimeria tenella.

Eimeria tenella is the main pathogen responsible for coccidiosis in chickens. The life cycle of E. tenella is, arguably, the least complex of all Coccidia, with only one host. However, it presents different developmental stages, either in the environment or in the host and either intracellular or extracellular. Its signaling and metabolic pathways change with its different developmental stages. Until now, little is known about the developmental regulation and transformation mechanisms of its life cycle. In this study, protein profiles from the five developmental stages, including unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), sporozoites (S) and second-generation merozoites (M2), were harvested using the label-free quantitative proteomics approach. Then the differentially expressed proteins (DEPs) for these stages were identified. A total of 314, 432, 689, and 665 DEPs were identified from the comparison of SO7h vs USO, SO vs SO7h, S vs SO, and M2 vs S, respectively. By conducting weighted gene coexpression network analysis (WGCNA), six modules were dissected. Proteins in blue and brown modules were calculated to be significantly positively correlated with the E. tenella developmental stages of sporozoites (S) and second-generation merozoites (M2), respectively. In addition, hub proteins with high intra-module degree were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analyses revealed that hub proteins in blue modules were involved in electron transport chain and oxidative phosphorylation. Hub proteins in the brown module were involved in RNA splicing. These findings provide new clues and ideas to enhance our fundamental understanding of the molecular mechanisms underlying parasite development.

Prevalence of Eimeria Species, Detected by ITS1-PCR Immobilized on FTA Cards, in Future Laying Hens and Breeding Hens in Six Provinces in Northeastern Algeria.

PURPOSE: Avian coccidiosis is an important and widely distributed disease that affects global agricultural economies through losses. In Algeria, there is limited epidemiological and ecological knowledge about this disease and this hinders implementation of control strategies. A recent study, in Algeria, demonstrated a high prevalence and diversity of Eimeria species in broiler chickens. However, very little is known about the Eimeria species that exist on chicken farms raised on the floor and older than broiler chickens (for example, future laying hens and breeding hens) in Algeria. METHODS: Samples were collected from 32 poultry farms located in 6 northeastern Algerian provinces (Algiers, Batna, Bejaia, Bordj Bou Arréridj, Jijel, Mila). These included 22 pre-laying pullet farms, with hens aged between 11 and 17 weeks, and 10 breeding hen farms with older hens (over 20 weeks). FTA cards were used to capture DNA and internal transcribed Spacer 1 PCR (ITS1-PCR) was used to determine the prevalence and composition of Eimeria species in the chickens. RESULTS: This showed the presence of six species of Eimeria with a diverse prevalence range. Eimeria necatrix (63%) was the most common species, followed by E. maxima (53%), E. tenella (31%), E. brunetti (19%), E. acervulina and E. mitis (both 0.3%). Eimeria praecox was absent. Eimeria infection affected all farms studied where co-infections by different Eimeria species (63%) were more frequent than single infections (38%). The number of oocyts, per ml of enriched oocyst suspension was higher in breeding hen farms compared to pre-laying pullet farms. CONCLUSION: This study, taken alongside a previous study involving broiler farms, demonstrated that the infection with this parasite is a significant problem in Algeria.

Coinfection of the gut with protozoal and metazoal parasites in broiler and laying chickens.

The chicken business faces substantial economic losses due to the risk of parasitic coinfection. Because the current study aimed to investigate enteric parasitic coinfections problems among the suspected examined chicken farms, samples were collected during the field investigation from suspected freshly dead birds, clinically diseased, apparently healthy, and litter samples for further laboratory parasitological, histopathological, and immunological examinations. Variable mortalities with various clinical indicators, such as ruffled feathers, weight loss, diarrhea of various colors, and a decline in egg production, occurred on the farms under investigation. In addition, the treatment protocols of each of the farms that were evaluated were documented and the m-RNA levels of some cytokines and apoptotic genes among the infected poultry have been assessed. The prevalence rate of parasitic coinfection in the current study was found to be 8/120 (6.66%). Parasitological analysis of the samples revealed that they belonged to distinct species of Eimeria, cestodes, and Ascaridia galli. When deposited, A. galli eggs were nonembryonated and ellipsoidal, but cestodes eggs possessed a thin, translucent membrane that was subspherical. Eimeria spp. oocysts in layer chickens were identified as Eimeria acervulina and Eimeria maxima in broiler chickens. Our findings proved that coinfection significantly upregulated the IL-1ß, BAX, and Cas-3 genes. Conversely, the IL-10, BCL-2, and AKT mRNA levels were downregulated, indicating that nematode triggered apoptosis. The existence of parasite coinfection was verified by histological investigation of the various intestinal segments obtained from affected flocks. A. galli and cestodes obstructed the intestinal lumen, causing different histological alternations in the intestinal mucosa. Additionally, the lamina propria revealed different developmental stages of Eimeria spp. It was determined that parasite coinfection poses a significant risk to the poultry industry. It was recommended that stringent sanitary measures management methods, together with appropriate treatment and preventative procedures, be employed in order to resolve such issues.

Molecular characterization and epidemiology of Toxoplasma gondii isolates from free-range chickens in the southwest region of Goiás: new genotypes.

The purpose of this study was to isolate Toxoplasma gondii from tissues of free-range chickens in the southwestern region of Goiás, to detect and molecularly characterize the genetic material of the parasite, and to determine the seroprevalence of the protozoan parasite in these animals. A seroprevalence of T. gondii antibodies of 76% (19/25) was found among the chickens, while genetic material from their tissues was detected in 56% (14/25). A total of 14 isolates was obtained in the bioassay, ten of which were considered acute, eight were considered isolates of high virulence lethal to mice, and four of low virulence, considered non-lethal but with the ability to chronify the infection. Seven of the ten isolates showed significant morphometric differences from the RH strain, in terms of nucleus-complex-apical distance, length and width. Genotyping of the acute isolates was performed by RFLP-PCR, using 11 genetic markers: SAG1, SAG2 (3'SAG2 and 5'SAG2), alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and APICO. The results were compared and classified according to the genotypes listed on the ToxoDB Platform, where different profiles were observed indicating the presence of two known genotypes (#7 and #63) and five new genotypes (NEW 3, NEW4, NEW5, NEW6, NEW 7). The results showed high seroprevalence, isolation rate, molecular detection and genotypic variations of T. gondii in free-range chickens in the southwestern region of Goiás.

Identification of Eimeria spp. in domestic chickens raised in alternative poultry production systems in the State of São Paulo, Brazil.

The objective of this study was to identify Eimeria spp. in alternative poultry production systems (APPS) in the State of São Paulo, Brazil. Fecal samples (168) and DNA extracted from fecal samples obtained in APPS located in different Municipalities in the State of São Paulo (93) were examined by microscopy or genera-specific PCR (ITS-1 locus). Samples positive for Eimeria spp. were examined using Eimeria lata, Eimeria nagambie, and Eimeria zaria species-specific PCR protocols (ITS-2 locus) and another E. lata-specific PCR (candidate IMP1 genomic locus) followed by molecular cloning (E. lata and E. zaria ITS-2 amplicons) and genetic sequencing. All positive DNA samples were also submitted to genera-specific nested PCR (18S rRNA gene) followed by next-generation sequencing to identify Eimeria spp. Eimeria nagambie, E. zaria, and Eimeria sp. were identified by ITS2-targeted species-specific PCRs and genetic sequencing. Next-generation sequencing identified, in order of prevalence: E. nagambie; Eimeria acervulina; Eimeria mivati; Eimeria praecox; Eimeria brunetti; Eimeria mitis; Eimeria sp.; Eimeria maxima; E. zaria, and Eimeria necatrix/tenella. Our results confirmed, for the first time in Brazil, the identification of E. nagambie, E. zaria, and Eimeria spp. ITS-2 and 18S rRNA gene sequences not yet described in Brazil.

Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, "ERR296879") share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Comparative proteomic analysis of wall-forming bodies and oocyst wall reveals the molecular basis underlying oocyst wall formation in Eimeria necatrix.

BACKGROUND: The durable oocyst wall formed from the contents of wall-forming bodies (WFBs) protects Eimeria parasites from harsh conditions and enhances parasite transmission. Comprehending the contents of WFBs and proteins involved in oocyst wall formation is pivotal to understanding the mechanism of the oocyst wall formation and the search for novel targets to disrupt parasite transmission. METHODS: Total proteins extracted from WFBs and the oocyst wall of Eimeria necatrix were subjected to comparative proteomic analysis using tandem mass tag in conjunction with liquid chromatography tandem-mass spectrometry techniques. After functional clustering analysis of the identified proteins, three proteins, including E. necatrix disulfide isomerase (EnPDI), thioredoxin (EnTrx) and phosphoglycerate kinase (EnPGK), were selected for further study to confirm their potential roles in oocyst wall formation. RESULTS: A total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively. Among these proteins, 1102 were identified as differentially expressed proteins, of which 506 were upregulated and 596 downregulated in the oocyst wall compared to the WFBs. A total of 108 proteins, including compositional proteins of the oocyst wall, proteases, oxidoreductases, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport, were proposed to be involved in oocyst wall formation. The approximate molecular sizes of native EnPDI, EnTrx and EnPGK proteins were 55, 50 and 45 kDa, respectively. EnPDI was present in both type 1 and type 2 WFBs, EnTrx was present only in type 2 WFB2 and EnPGK was present only in type 1 WFBs, whereas all of them were localized to the outer layer of the oocyst wall, indicating that all of them participate in the formation of the oocyst wall. CONCLUSIONS: To the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix. The data obtained from this study form a basis for deciphering the molecular mechanisms underlying oocyst wall formation of Eimeria parasites. They also provide valuable resources for future studies on the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission.

The microneme adhesive repeat domain of MIC3 protein determined the site specificity of Eimeria acervulina, Eimeria maxima, and Eimeria mitis.

Understanding the determinants of host and tissue tropisms among parasites of veterinary and medical importance has long posed a substantial challenge. Among the seven species of Eimeria known to parasitize the chicken intestine, a wide variation in tissue tropisms has been observed. Prior research suggested that microneme protein (MIC) composed of microneme adhesive repeat (MAR) domain responsible for initial host cell recognition and attachment likely dictated the tissue tropism of Eimeria parasites. This study aimed to explore the roles of MICs and their associated MARs in conferring site-specific development of E. acervuline, E. maxima, and E. mitis within the host. Immunofluorescence assays revealed that MIC3 of E. acervuline (EaMIC3), MIC3 of E. maxima (EmMIC3), MIC3 of E. mitis (EmiMIC3), MAR3 of EaMIC3 (EaMIC3-MAR3), MAR2 of EmMIC3 (EmMIC3-MAR2), and MAR4 of EmiMIC3 (EmiMIC3-MAR4), exhibited binding capabilities to the specific intestinal tract where these parasites infect. In contrast, the invasion of sporozoites into host intestinal cells could be significantly inhibited by antibodies targeting EaMIC3, EmMIC3, EmiMIC3, EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4. Substitution experiments involving MAR domains highlighted the crucial roles of EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4 in governing interactions with host ligands. Furthermore, animal experiments substantiated the significant contribution of EmiMIC3, EmiMIC3-MAR4, and their polyclonal antibodies in conferring protective immunity to Eimeria-affiliated birds. In summary, EaMIC3, EmMIC3, and EmiMIC3 are the underlying factors behind the diverse tissue tropisms exhibited by E. acervuline, E. maxima, and E. mitis, and EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4 are the major determinants of MIC-mediated tissue tropism of each parasite. The results illuminated the molecular basis of the modes of action of Eimeria MICs, thereby facilitating an understanding and rationalization of the marked differences in tissue tropisms among E. acervuline, E. maxima, and E. mitis.

First detection and characterisation of Eimeria zaria in European chickens.

The global poultry industry has experienced dramatic growth in recent decades, increasing the significance of pathogens of chickens. Protozoan parasites of the genus Eimeria can cause the disease coccidiosis, compromising animal health and welfare, and incurring significant annual costs. Seven Eimeria species have long been recognised to infect chickens, supplemented by three new candidate species first reported from Australia in 2007/8. Named Eimeria lata, Eimeria nagambie and Eimeria zaria, one or more of these new species have been reported in Australia, several countries in sub-Saharan Africa, India, Venezuela, and most recently the United States of America, but none have been detected in Europe. Here, a panel of 56 unvaccinated broiler chicken farms were sampled in the final week of production from France, Greece, Italy, the Netherlands, the Republic of Ireland, and the United Kingdom to assess the occurrence of all ten Eimeria species using specific polymerase chain reaction (PCR). Overall, 39 of 56 (69.6%) farms were found to host at least one species. Eimeria acervulina, E. tenella, and E. maxima were most common, with E. mitis and E. praecox also widespread. Eimeria necatrix was detected on one farm in France, while E. brunetti was not detected. Eimeria zaria was detected for the first time in Europe, appearing in Greece and Italy (one occurrence each). New primers were designed to confirm detection of E. zaria and provide template for phylogenetic comparison with the reference isolate from Australia. Detection of E. zaria in Europe reinforces the importance of integrated control for coccidiosis given the lack of protection induced by current anticoccidial vaccines.