Transcriptomic responses of rabbits to infections by precocious line and wild-type Eimeria media: revealing molecular signatures and pathway differences in liver and duodenum during the peak and terminal phases of oocyst production.

Eimeria media is a principal pathogen responsible for rabbit coccidiosis, targeting the rabbit's intestinal epithelial cells. This parasitism damages the intestinal mucosal barrier, initiating a systemic immune and inflammatory response that jeopardizes the sustainable growth of rabbit farming. To understand the implications of infection on the host's immune and metabolic responses, we employed RNA-Seq to analyze RNA from the liver and duodenum tissues of post-infected rabbits infected with both the precocious line and wild-type strain of E.media. Comprehensive transcriptomic analysis revealed that the two parasites exhibit divergent transcriptomic imprints on host tissues. While the precocious line predominantly modulates immune-centric pathways with significant differential gene enrichment, wild-type strain favors pathways that affect metabolism. In addition, our study pinpointed a set of genes that undergo significant modifications in response to these effects. These revelations grant a fresh avenue to probe deeper into the symbiotic intricacies of the E.media and its rabbit host.

Cytokine production by bovine adipose tissue stromal vascular fraction cells upon Neospora caninum stimulation.

In bovines few studies addressed the contribution of adipose tissue to the host immune response to infection. Here we evaluated the in vitro response of bovine adipose tissue stromal vascular fraction (SVF) cells to the protozoan parasite Neospora caninum, using live and freeze-killed tachyzoites. Live N. caninum induced the production of IL-6, IL-1ß and IL-10 by SVF cells isolated from subcutaneous adipose tissue (SAT), while in mesenteric adipose tissue (MAT) SVF cell cultures only IL-1ß and IL-10 production was increased, showing slight distinct responses between adipose tissue depots. Whereas a clear IL-8 increase was detected in peripheral blood leucocytes (PBL) culture supernatants in response to live N. caninum, no such increase was observed in SAT or MAT SVF cell cultures. Nevertheless, in response to LPS, increased IL-8 levels were detected in all cell cultures. IL-10 levels were always increased in response to stimulation (live, freeze-killed N. caninum and LPS). Overall, our results show that bovine adipose tissue SVF cells produce cytokines in response to N. caninum and can therefore be putative contributors to the host immune response against this parasite.

Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in Ontario broodmares.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in broodmares in Ontario were investigated. Sixty of the 219 (27.4%) study broodmares were seropositive for N. caninum and 65/219 (29.7%) for N. hughesi with cut-offs of ≥1:40 and ≥1:160, respectively. Thirty-one of 63 participating farms (49.2%) had at least 1 broodmare seropositive for N. caninum. Thirty-three of the 63 (52.4%) participating farms had at least 1 broodmare positive for N. hughesi. Risk factors for N. caninum included presence of farm dogs (OR = 6.70; 95% CI = 2.14-20.97; p = 0.001), and high stocking density (OR = 2.83; 95% CI = 1.27-6.30; p = 0.011). Presence of livestock, excluding cattle, was associated with reduced risk of exposure (OR = 0.17; 95% CI = 0.06-0.53; p = 0.002). The only risk factor for exposure to N. hughesi was feeding hay on the ground in the paddock (OR = 4.31; 95% CI = 1.65-11.22; p = 0.003). This study demonstrated widespread exposure to Neospora spp. in broodmares in Ontario.

Construction of luciferase-expressing Neospora caninum and drug screening.

BACKGROUND: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. METHODS: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. RESULTS: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 µM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. CONCLUSIONS: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

Integrated application of transcriptomics and metabolomics provides insight into the mechanism of Eimeria tenella resistance to maduramycin.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

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.

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.

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.

Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens.

The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.

Neospora caninum peroxiredoxin 1 is an essential virulence effector with antioxidant function.

Neospora caninum, an obligate intracellular parasitic protozoan discovered by Dubey in 1988, is the pathogen of neosporosis, which causes neurological symptoms in dogs and abortions in cows. Since there is no effective drug or vaccine against N. caninum, a deeper understanding of the molecules critical to parasite survival inside host cells is necessary. This study aimed to determine the role of N. caninum peroxiredoxin 1 (NcPrx1) in maintaining redox homeostasis and virulence of N. caninum. By determining the localization of NcPrx1 protein and establishing NcPrx1 gene knockout strain (ΔNcPrx1), the roles of NcPrx1 in N. caninum for invasion, replication, growth, oxidative stress, as well as pathogenicity were investigated. Our results showed that a predicted Alkyl Hydroperoxide1 (AHP1) domain was found in the amino acid sequence of NcPrx1, which displayed a high degree of similarity to homologs of several protozoa. Immunofluorescence assay (IFA) indicated that NcPrx1 was a cytoplasmic protein in N. caninum tachyzoites. Compared to wild type (WT) strain, ΔNcPrx1 strain showed reduced plaque area, invasion and egress rates. Reactive oxygen species (ROS) and malondialdehyde (MDA) were accumulated, and total antioxidant capacity (T-AOC) was attenuated in ΔNcPrx1 tachyzoites, which indicated that ΔNcPrx1 strain was more sensitive to oxidative stress. Furthermore, ΔNcPrx1 strain-infected C57BL/6 mice showed improved survival rate, reduced parasite burden, alleviated pathological changes in tissues, and decreased secretions of IL-6, IL-12, TNF-α, and IFN-γ in serum compared to the WT strain group. These findings suggested that NcPrx1 was a virulence factor of N. caninum which played an important role in maintaining the redox homeostasis of the parasite.

Seroprevalence of Neospora caninum in pet cats, dogs and rabbits from urban areas of Poland.

BACKGROUND: Neospora caninum (N. caninum) has a broad intermediate host range and might cause multisystemic lesions in various species of animals. Dogs are both intermediate and definitive hosts of the parasite and play a crucial role in the horizontal transmission of this protozoan to other animals. Cats and rabbits could be sensitive to infection with N. caninum, however, clinical symptoms and the exact route of infection in these species are unknown. The epidemiology of N. caninum in cats and rabbits has been barely researched, and there is no published record of the seroprevalence of N. caninum infection in these species in Poland. Thus, the present study aimed to determine the frequency of seroreagents for N. caninum within pet dogs, cats and rabbits from urban areas of Poland and to identify possible risk factors for these animals. RESULTS: In total, serum samples from 184 cats (Felis catus), 203 dogs (Canis familiaris) and 70 rabbits (Oryctolagus cuniculus) were used in the study. The seroprevalence of anti-N. caninum antibodies in dogs and cats reached 1.0% (2/203; 95% CI: 0.3-3.5) and 3.3% (6/184; 95% CI: 1.5-6.9), respectively. No significant differences in seroprevalence regarding age group, gender, symptoms or sampling location were found. All 70 samples from pet rabbits were negative for anti-N. caninum antibodies. CONCLUSIONS: The seroprevalence rates of N. caninum in dogs and cats in the present study were low, however, our results confirmed N. caninum circulates among dog and cat populations in Poland, and neosporosis should be included in the differential diagnosis of neuro-muscular disorders in these species. This is the first serological survey of N. caninum in European pet cats and rabbits. The role of pet rabbits in N. caninum epidemiology and circulation in Poland is marginal.

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.

A NEW EIMERIA SPECIES (APICOMPLEXA: EIMERIIDAE) PARASITIZING COMMERCIAL CHUKAR PARTRIDGES (ALECTORIS CHUKAR) IN SOUTHERN ONTARIO.

A commercial producer hatching and rearing chukar partridges (Alectoris chukar) in Ontario, Canada had flocks experiencing coccidiosis. Microscopic analysis of Eimeria species isolated from a field sample indicated the presence of 2 distinct oocyst morphotypes; the most abundant species was determined to be Eimeria chapmani, based on oocyst morphology and sequence-based genotyping, and the less abundant, second Eimeria sp. was an undescribed parasite. Oocysts of the unknown Eimeria sp. were large and oval-shaped; dimensions averaged 27.9 µm by 17.0 µm (shape index = 1.65 µm). Oocysts contained at least 1 polar granule and 4 almond-shaped sporocysts with average dimensions measuring 12.5 µm by 6.9 µm (shape index = 1.83). Each sporocyst featured a Stieda body, sub-Stieda body, and sporocyst residuum; a sporocyst contained 2 sporozoites that each possessed a small anterior refractile body and a larger posterior refractile body. Virtually all oocysts sporulated after 24 hr when suspended in potassium dichromate at room temperature (22 C) on a rotary platform. Experimental infections with various doses of oocysts demonstrated elevated parasite shedding from birds gavaged with higher challenge doses; fecundity generally decreased in heavier infections. The approximate prepatent period of the parasite was 4-5 days (unsporulated oocysts observed histologically at 90 hr postinfection and in feces by day 5) and patency lasted until day 12 postinfection. To characterize the endogenous development of the Eimeria sp., tissues were collected at 8 regions along the intestinal tract (including the ceca and rectum) every 6 hr throughout the estimated prepatent period. Parasites were observed to infect the descending and ascending duodenum, midjejunum, proximal and distal ileum, and the ceca. The endogenous stages identified included intracellular sporozoites, 3 generations of merogony, and gametogonic stages. Sequences of the mitochondrial genome (GenBank MW934555) and nuclear 18S ribosomal DNA (GenBank MW934259) were obtained using polymerase chain reaction amplification for Sanger sequencing, and these were unique from all published sequences on GenBank. Molecular data, in conjunction with the unique biology of the Eimeria sp. isolated from the chukar partridge flock, support that this coccidium is new to science.

Serosurvey and associated risk factors for Neospora caninum infection in Egyptian water buffaloes (Bubalus bubalis).

Neosporosis is a parasitic disease that causes reproductive disorders in animals, making it a barrier to maximum efficiency. The purpose of this study was to determine the seroprevalence of Neospora caninum (N. caninum) antibodies in water buffaloes from four governorates in northern Egypt. A commercial indirect-ELISA test was used to detect antibodies against N. caninum in the serum of 450 water buffaloes. The total seroprevalence of N. caninum in water buffaloes from Egypt was 31.3%, and the highest prevalence was observed in Gharbia governorate. The identified risk factors for N. caninum infections in water buffaloes were sex (OR = 1.96, 95%CI: 1.22-4.17), buffaloes more than 4 years of age ( OR = 5.80, 95%CI: 2.26-14.86), abortion in second trimester (OR = 16.48, 95%%CI: 2.99-34.03), history of abortion (OR = 3.45, 95%CI: 1.58-7.52) and contact with dogs (OR = 2.55, 95%CI: 1.51-4.32). Thus, more studies are needed to determine the role of buffaloes in the epidemiology of neosporosis in Egypt.

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.

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.

Transcriptome profiling of Madin-Darby bovine kidney cells uncover differences in the susceptibility of cattle to Toxoplasma gondii and Neospora caninum.

Toxoplasma gondii and Neospora caninum are two major apicomplexan protozoan parasites with heteroxenous life cycles and worldwide distributions. The transplacental transmission of N. caninum causes bovine abortion, which resulting in serious economic losses to the dairy industry. Although T. gondii was also reported to cause abortions in pregnant woman and small ruminants, scarce cases about the symptom to the host cattle and the causality remains unknown. In this study, transcriptome analysis of Madin Darby bovine kidney (MDBK) cells infected with T. gondii and N. caninum was performed to uncover the differences in susceptibility of cattle to the two parasites. The results showed that 256 and 2225 differentially expressed genes (DEGs) were detected in cells infected with N. caninum and T. gondii, respectively. Moreover, significant biological differences were revealed by the functional analysis including GO and KEGG enrichment. One serpin peptidase inhibitor (SEPRINA14), which is associated with immunosuppression during pregnancy, was found to significantly decrease in cells infected with N. caninum and increase in cells infected with T. gondii-infected cells. Pattern recognition receptors TLR3 and NOD2 were also significantly upregulated in N. caninum-infected MDBK cells, but not in T. gondii. They could induce an increased inflammatory response leading to severe tissue damage. In addition, the interleukin 12 receptor subunit beta 2 (IL12ß2), which plays an essential role in Th1 and Th2 cell differentiation and inflammatory bowel disease, was also markedly upregulated in the N. caninum infected cells, which led to an imbalance in the Th1 and Th2 cells by promoting the Th1 cellular response. Altogether, our findings recognized a new understanding on the differences between T. gondii and N. caninum infection of MDBK cells, where SEPRINA14, TLR3, NOD2, and IL12ß2 may be the key genes that affect the difference in susceptibility of cattle to T. gondii and N. caninum, especially in pregnant animals. This study provides more clues as to why N. caninum is more likely to cause abortions in cattle.

Seroprevalence and associated factors with Neospora caninum infection in sheep and goats slaughtered in the state of Paraíba, Brazil.

The objective was to describe the seroprevalence of anti-Neospora caninum antibodies in goats and sheep slaughtered in the state of Paraíba and to identify possible associated factors with the infections. Two hundred twenty-nine samples from goats and two hundred five from sheep were analyzed by Indirect Immunofluorescence Reaction (IFAT) using a cutoff point of 1:50. The presence of anti-N. caninum antibodies was identified in 28.4% (65/ 229; 95% Confidence Interval: 22.6-34.2) of the goat samples and in 12.7% (26/ 205; 95% CI: 8.2 - 17.2) of the sheep samples. Contact between goats and dogs (Odds ratio 4.81; CI 1.13 - 2.67; p = 0.041) and cattle (OR. 1.87; CI 1.13 - 2.67; p = 0.002) was identified as a risk factor for goats and contact between sheep and dogs (OR 2.32; CI 1.58 - 3.14; p = 0.026) and history of abortion (OR 1.94; CI 1.28 - 2.90; p = 0.001) was considered a risk factor for sheep. The study revealed a high seroprevalence of anti-N. caninum antibodies in slaughtered goats and sheep in Paraíba. Risk factors such as contact with dogs/cattle and abortion history underscore the need for preventive measures to control infection and enhance animal health management.