Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Eimeria falciformis extracellular vesicles differentially express host cell lncRNAs.

Long noncoding RNAs (lncRNAs) are regulatory transcripts during protozoan infections in the host intestinal epithelial cells (IECs). Apicomplexan Eimeria falciformis sporozoite extracellular vesicles (EVs) contain virulence factors that modulate host IECs pro-inflammatory genes and immune responses. In this study, E. falciformis sporozoites were made to interact with inactivated host cells, and the parasite EVs were separated from total secretome by ultracentrifugation and purified on density gradient medium. Dose-dependent bio-activity of E. falciformis EVs was investigated by RNA sequencing, functional annotation and quantitative PCR. It was found that E. falciformis EVs induced mRNA, circRNA, and lncRNA expressions in mouse IECs. Of 38, 217 lncRNAs assembled, 157 and 152 were upwardly and downwardly expressed respectively. Differentially expressed lncRNAs were associated with cytokines, pyroptosis, and immune signaling pathways including FoxO, NF-κB, MAPK, and TGF-ß. In essence, E. falciformis EVs altered host cell RNA expressions during the interaction with host IECs. Also, differentially expressed lncRNAs are potential diagnostic transcripts during Eimeria infections.

Identification of sheep eimerian parasites, Eimeria crandallis and Eimeria faurei, employing microscopic and molecular tools.

Coccidiosis, caused by apicomplexan Eimeria species, is a protozoan disease that affects various species of wild and domestic animals. However, data available on Eimeria diversity in ruminants in Saudi Arabia is meagre. Therefore, this study was designed to investigate some eimerian parasites infecting sheep (Sawakni and Harrie breeds) using microscopy and molecular methods for the first time in Saudi Arabia. Twenty-four fecal samples were collected from sheep farms. Based on the floatation technique, eimerian oocysts were observed in 8 of the 24 (33.33%) fecal samples. The coccidian-positive samples were subjected to fecal culture in a shallow layer of 2.5% potassium dichromate (K2 Cr2 O7 ). Detected eimerian oocysts were described micromorphometrically as the basis for traditional oocyst identification. Morphologically, the sporulated oocysts were similar to those of sheep eimerian parasies; Eimeria faurei and Eimeria crandallis. PCR products from the two eimerian species detected from Sawakni and Harrie breeds were sequenced and were found to be distinct from each other with mutations at five positions. One of them clustered with E. crandallis with 99.8%-100% identity with sequences available in GenBank. E. crandallis was obtained from two Sawakni sheep and two Harrie sheep. The other sequences grouped with E. faurei with 99.8% identity with the only sequences available in GenBank. E. crandallis was detected from both Sawakni and Harrie breeds whereas E. faurei was detected only from Sawakni sheep. The findings of this study have implications for the importance of morphometric identification with advanced molecular tools to confirm the identities of sheep Eimeria species and to address the taxonomic study of this eimeriid parasite at the species level.

Identification of Eimeria acervulina (Apicomplexa: Eimeriidae) infecting the broiler chicken Gallus gallus domesticus through morphology and molecular analyses.

Coccidiosis is an intestinal protozoan disease that affects the poultry industry worldwide. The severity of this disease varies depending on the identity of the infectious agents. Therefore, this study was carried out to identify the Eimeria species that affect broiler chickens, Gallus gallus domesticus, through morphological and molecular phylogenetic analyses. Twenty-five faecal samples were collected from the broiler chickens in a commercial poultry farm in Riyadh (Saudi Arabia). Using the floatation technique, faeces were examined microscopically for the Eimeria occurrence. Identification of Eimeria species was performed based on morphological criteria and molecular tools (DNA amplification for the partial small subunit ribosomal RNA (18S rRNA), internal transcribed spacer (ITS)-1, and mitochondrial cytochrome c oxidase I (COI) genes. In this study, 32% (8 out of 25) of collected samples were found to be positive for coccidiosis. After sporulation in potassium dichromate (K2Cr2O7), the sporulated oocysts were observed as ovoid and measured 18.37-23.19 µm (19.87) long and 15.07-18.67 µm (16.46) wide, with the anterior location of a polar granule and absence of micropyle. These Eimeria oocysts were assumed to size and shape characteristics of Eimeria acervulina. Molecular analysis was conducted on the sequences of the polymerase chain reaction products from the three genes studied (18S rRNA, ITS-1, and COI). At the three genes, results showed that the resultant sequences clustered with E. acervulina from different regions confirming morphological description. This study highlighted the importance of molecular techniques to detect avian Eimeria species more than the traditional morphology-based tool to optimise the appropriate anticoccidial strategies for long-term control in the studied area.

Comparative molecular analyses of Eimeria Schneider (Apicomplexa: Eimeriidae) species from rock ptarmigan in Iceland, Svalbard-Norway, and Japan.

The rock ptarmigan (Lagopus muta) has a Holarctic breeding distribution and is found in arctic and sub-arctic regions. Isolated populations and glacial relicts occur in alpine areas south of the main range, like the Pyrenees in Europe, the Pamir mountains in Central Asia, and the Japanese Alps. In recent decades considerable effort has been made to clarify parasite infections in the rock ptarmigan. Seven Eimeria spp. have been reported parasitizing rock ptarmigan. Two of those species, E. uekii and E. raichoi parasitizing rock ptarmigan (L. m. japonica) in Japan, have been identified genetically. Here we compare partial sequences of nuclear (18S rRNA) and mitochondrial (COI) genes and we detail the morphology of sporulated oocysts of E. uekii and E. raichoi from Japan, E. muta and E. rjupa, from the rock ptarmigan (L. m. islandorum) in Iceland, and two undescribed eimerian morphotypes, Eimeria sp. A, and Eimeria sp. B, from rock ptarmigan (L. m. hyperborea) in Norway (Svalbard in the Norwegian Archipelago). Two morphotypes, ellipsoidal and spheroidal, are recognized for each of the three host subspecies. Our phylogenetic analysis suggests that the ellipsoidal oocyst types, E. uekii, E. muta, and Eimeria sp. A (Svalbard-Norway) are identical and infects rock ptarmigan in Japan, Iceland, and Svalbard-Norway, respectively. Eimeria uekii was first described in Japan in 1981 so that E. muta, described in Iceland in 2007, and Eimeria sp. A in Svalbard-Norway are junior synonyms of E. uekii. Also, phylogenetic analysis shows that the spheroidal oocyst types, E. rjupa and Eimeria sp. B (Svalbard-Norway), are identical, indicating that rock ptarmigan in Iceland and Svalbard-Norway are infected by the same Eimeria species and differ from E. raichoi in Japan.

New coccidian Eimeria iyoensis n. sp. (Apicomplexa: Eucoccidiorida: Eimeriidae) in farmed ring-necked pheasants (Phasianus colchicus L.) in Ehime, Japan.

Eight Eimeria spp. (Apicomplexa: Eimeriidae) have been isolated from the ring-necked pheasant (Phasianus colchicus Linnaeus), native to the temperate zone of Asia and eastern regions of Europe. Enteric coccidiosis has become a major issue associated with the breeding of farmed pheasants for game bird release or meat production. In this study, 35 fecal samples were collected from two-to-three-month-old ring-necked pheasants from four pheasant-rearing farms in Ehime Prefecture, Japan. Microscopic examination using a saturated sugar solution technique detected numerous subspherical oocysts from the samples of one farm and ellipsoidal Eimeria phasiani Tyzzer, 1929 oocysts from the three other farms. The subspherical oocysts were artificially sporulated and measured 18.6 µm by 15.7 µm with a 1.18 shape index (n = 150). Each oocyst contained four 10.7 µm × 5.8 µm sporocysts (n = 30) and one coarse refractile polar granule; no micropyle or residua were detected. Each sporocysts contained two sporozoites with one large and one small refractile body and sparsely distributed residua. The complete, 1,443-bp cytochrome c oxidase subunit I gene (cox1) of this isolate exhibited low sequence identity with published Eimeria spp. sequences including E. phasiani that was previously recorded in the same area. Meanwhile, the oocyst morphology most closely resembled that of Eimeria tetartooimia Wacha, 1973, but with distinct refractile polar granules and sporocyst residua. The available GenBank cox1 sequence of E. tetartooimia exhibited a sequence identity of < 94.5% with the study isolate. Here, the coccidian isolate identified in this study represents a new Eimeria iyoensis n. sp. capable of infecting ring-necked pheasant.

Morphological and molecular characterization of Eimeria haematopusi n. sp. (Apicomplexa: Eimeriidae) in an Australian Pied Oystercatcher (Haematopus longirostris) (Aves: Charadriiformes).

A novel Eimeria Schneider, 1875 species is described from an Australian pied oystercatcher Haematopus longirostris Vieillot, in Western Australia. The pied oystercatcher was admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC), Perth, Western Australia in a poor body condition, abrasion to its right hock and signs of partial delamination to its lower beak. Investigation into potential medical causes resulted in a faecal sample being collected and screened for gastrointestinal parasites. Unsporulated coccidian oocysts were initially observed in the faeces and identified as Eimeria upon sporulation. The sporulated oocysts (n = 20) are ellipsoidal, 20-21 × 12-13 µm in shape and have thick bi-layered walls which are c.2/3 of the total thickness. Micropyle is present, robust and protruding, and occasionally has a rounded polar body attached to the micropyle. Within the oocyst, a residuum, in addition, two to five polar granules are present. There are four ellipsoidal sporocysts 9-11 × 5-6 µm with flattened to half-moon shaped Stieda bodies. Sub-Stieda body and para-Stieda body are absent. The sporocysts contain sporocyst residuums composed of a few spherules scattered among the sporozoites. Within the sporozoites, anterior and posterior refractile bodies are present, but the nucleus is indiscernible. To further characterise the novel Eimeria species from H. longirostris, molecular analysis was conducted at the 18S ribosomal RNA locus, using PCR amplification and cloning. Two cloned sequences from the novel Eimeria were compared with those from other Eimeria spp. with the highest genetic similarity of 97.6% and 97.2% from Clone 1 and 2, respectively with Eimeria reichenowi (AB544308) from a hooded crane (Grus monacha Temminck) in Japan. Both sequences grouped in a clade with the Eimeria spp. isolated from wetland birds, which include Eimeria paludosa (KJ767187) from a dusky moorhen (Gallinula tenebrosa Gould) in Western Australia, Eimeria reichenowi (AB544308) and Eimeria gruis (AB544336) both from hooded cranes. Based on the morphological and molecular data, this Eimeria sp. is a new species of coccidian parasite and is named Eimeria haematopusi n. sp. after its host H. longirostris.

Protective efficacy of multiepitope vaccines constructed from common antigens of Eimeria species in chickens.

Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4+ and CD8+ T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.

Next-generation sequencing amplicon analysis of the genetic diversity of Eimeria populations in livestock and wildlife samples from Australia.

Eimeria is an important coccidian enteric parasite that infects a wide range of hosts and can cause substantial economic losses in the poultry and livestock industries. It is common for multiple Eimeria species to infect individual hosts, and this can make species identification difficult due to morphological similarities between species and mixed chromatograms when using Sanger sequencing. Relatively few studies have applied next-generation amplicon sequencing (NGS) to determining the genetic diversity of Eimeria species in different hosts. The present study screened 408 faecal samples from a range of hosts including livestock and wildlife using a previously developed quantitative polymerase chain reaction (qPCR) at the 18S locus and conducted amplicon NGS on the positives using a ~ 455-bp fragment of the 18S locus. A total of 41 positives (10.1%) were identified by qPCR from various hosts and NGS was successful for 38 of these positives. Fifteen Eimeria species and three genotypes were detected by NGS: E. ferrisi, E. kanyana, E. potoroi, E. quokka, E. setonicis, E. trichosuri, E. reichenowi, E. angustus, E. ahsata, E. auburnensis, E. bovis, E. brasiliensis, E. christenseni, E. crandallis, E. ovinoidalis, Eimeria sp. (JF419345), Eimeria sp. (JF419349) and Eimeria sp. (JF419351). Mixed infections were detected in 55.3% (21/38) of positive samples. The most striking finding was the identification of the same species in different hosts. This could be due to contamination and/or mechanical transmission or may provide support for previous studies suggesting that Eimeria species can infect not just closely related hosts but different genera and further research is required. This is also the first study to audit Eimeria populations in livestock (sheep and cattle) by NGS and could be applied in the future to determine the extent of pathogenic species and outcomes of Eimeria control strategies.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Molecular characterisation and additional morphological descriptions of Eimeria spp. (Apicomplexa: Eimeriidae) from brown kiwi (Apteryx mantelli Bartlett).

Brown kiwi (Apteryx mantelli Bartlett), a ratite endemic to New Zealand, is currently listed as "Vulnerable" under the IUCN classification system due to predation by introduced mammals. Operation Nest Egg (ONE) raises chicks and juveniles in predator-proof enclosures until they are large enough to defend themselves. These facilities experience an environmental accumulation of coccidial oocysts, which leads to severe morbidity and mortality of these kiwi. Four species of coccidia have been morphologically described from sporulated oocysts with additional opportunistic descriptions of endogenous stages. This research continues the morphological descriptions of these species of Eimeria with an additional novel morphotype also morphologically described. It also provides the first genetic characterisation targeting the mitochondrial cytochrome c oxidase I (COI) gene. Based on these findings, it was determined there are at least five morphotypes of Eimeria that infect brown kiwi and co-infections are common at the ONE facilities surveyed. The COI amplicon targeted for this study was sufficient to provide differentiation from other members of this genus. Sanger sequencing yielded ambiguous bases, indicating the need for more in-depth sequencing.

Genetic selection of Eimeria parasites in the chicken for improvement of poultry health: implications for drug resistance and live vaccine development.

Apicomplexan parasites of the genus Eimeria are widespread in poultry flocks and can cause the intestinal disease coccidiosis. Early studies, concerned with intraspecific variation in oocyst morphology, indicated that phenotypic changes may be induced by selection experiments conducted in vivo. Genetic selection driven by targeted selection for specific phenotypes has contributed to our understanding of the phenomenon of drug resistance and the development of live attenuated vaccines. Our present knowledge regarding genetics of Eimeria is largely based upon the utilization of such selected strains as genetic markers. Practical advantages of working with Eimeria spp. in the chicken are discussed. The selection of drug-resistant strains by serial propagation has provided useful information regarding the mechanisms of drug resistance and likely longevity of anticoccidial drugs when introduced in the field. Selection experiments to develop precocious strains of Eimeria and growth in chicken embryos have contributed to the development of safe and effective live attenuated vaccines for the control of coccidiosis. Establishment of protocols for genetic complementation by transient or stable transfection of Eimeria is now supporting direct manipulation of parasite genotypes, creating opportunities to expand the range and value of live parasite vaccines. Procedures for developing drug-resistant and precocious lines of Eimeria and/or genetic markers described here are likely to prove useful for researchers investigating the propensity for resistance development to novel compounds and the development of new attenuated vaccines. Such investigations can be helpful in providing a better understanding of biochemical and molecular aspects of the biology of these parasites.

Observations on an Eimeria sp. (Apicomplexa) from the green kingfisher Chloroceryle americana (Gmelin, 1788) (Coraciiformes) in Southeastern Brazil: an example of how the ecological aspects of the host can be essential for the identification of its coccidians.

Parasitism in kingfishers is very little reported and predominantly related to hemoparasites, helminths, and ectoparasites. The present study provided a morphological and genotypic study of an Eimeria sp. recovered from a green kingfisher Chloroceryle americana (Gmelin, 1788) captured in the Marambaia Island, on the coast of the state of Rio de Janeiro, Southeastern Brazil. The coccidial density, some morphological aspects of its oocysts, the molecular results, and, mainly, the ecological niche of C. americana in the mangrove of the Marambaia Island suggest that this coccidian species is a pseudoparasite.

Eimeria spp. (Eimeriidae) in the migratory whooper swan (Cygnus cygnus) Linnaeus, 1758 (Anatidae) from Sanmenxia Swan Lake National Urban Wetland Park in the middle reaches of the Yellow River in China.

This study i dentifies four Eimeria spp. recorded from fecal samples of migratory whooper swans (Cygnus cygnus) in Sanmenxia Swan Lake National Urban Wetland Park in Sanmenxia city in the middle reaches of the Yellow River, China. Eimeria hermani, Eimeria nocens, Eimeria stigmosa, and Eimeria magnalabia were compatible in all characteristic features with their respective original descriptions. In addition to the preliminary morphological identification, this study provides a preliminary genotypic identification of these four Eimeria spp. via sequencing of the 18S rRNA, 28S rRNA, and COI gene loci that are suitable for the genotypic differentiation of these coccidia. This is the first report of molecular data for the four Eimeria spp. in migratory whooper swans. Finally, this study discusses the environmental risks of these coccidia for migratory whooper swans in Sanmenxia Swan Lake National Urban Wetland Park.

Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors.

Coccidiosis is one of the most notable diseases in chickens having a high economic impact on the poultry industry worldwide. The present study is the first attempt to epidemiologically investigate Eimeria spp. distribution and associated risk factors under different housing and production systems in three major regions in Greece. Faecal samples were obtained from 42 operations (broilers, floor housed, free range and organic layers, backyard farms). A questionnaire was obtained from included operations to acquire additional information regarding farm management, location, production rate and diseases history. Positivity level was 85.7%. All seven Eimeria species were identified, and the most prevalent ones were E. acervulina (79.3%) and E. tenella (65.5%). Single-species and mixed infections were detected in 20.7% and 79.3% of the flocks, respectively. Flock size, type of outdoor area, production system and presence of respiratory disease proved significant risk factors. Flock size up to 10,000 birds correlated strongly (p = 0.02) with higher E. tenella quantities. A very strong correlation (p < 0.001) was found between the presence of respiratory disease and the average OPG level in broiler farms. Organic flocks showed higher prevalence of E. tenella (p = 0.023), while presence of vegetation at the outdoor area correlated strongly with E. brunetti (p < 0.001). Molecular analysis and correlation results in this survey give strong indications although more studies are needed to further understand the involvement of different Eimeria species in various husbandry, production and management systems, to gain more knowledge about the sustainable control of coccidia in poultry.

Molecular identification of Eimeria species in liver and feces of naturally infected rabbits in Japan.

Among the 11 species of Eimeria in rabbits, some of which are known to be pathogenic and cause enteritis, E. stiedae induces severe liver lesions resulting in elevated mortality. Unlike in other countries, the incidence and prevalence of the parasites in rabbits have not been reported in Japan. In the present study, we histopathologically analyzed hepatic coccidiosis in a rabbit and attempted several primers to genetically identify the parasites and investigated the prevalence of Eimeria species at the same farm. In the liver of the affected rabbit, we observed fibrosis and edema around multiple bile ducts and epithelial cell hyperplasia of the bile ducts. Large numbers of developing parasites of Eimeria spp., mainly oocysts, were present in the bile ducts. PCR and sequencing analyses with the published primers for Cyclospora and Eimeria spp. were used to successfully identify the parasites in the liver as E. stiedae. The oocysts of Eimeria spp. were detected in 13 out of 20 fecal samples collected from other rabbits at the farm, and five Eimeria spp. (E. perforans, E. flavescens, E. exigua, E. magna, and E. vejdovskyi) were genetically confirmed. Our results provide the first indication that Eimeria spp., including highly pathogenic species, are present in Japan and the primer set used herein can be a useful tool for the identification of rabbit Eimeria spp.

Prokaryotic Expression of Eimeria magna SAG10 and SAG11 Genes and the Preliminary Evaluation of the Effect of the Recombinant Protein on Immune Protection in Rabbits.

Eimeria magna is a common coccidia in the intestines of rabbits, causing anorexia, weight loss, diarrhea, and bloody stools. This study cloned and determined the expression levels of four Eimeria surface antigens (EmSAGs) at different developmental stages and showed that EmSAG10 and EmSAG11 are highly expressed at the merozoite stage. Rabbits were immunized with rEmSAG10 and rEmSAG11, and then challenged with E. magna after 2 weeks. Serum-specific antibodies and cytokine levels were detected using ELISA. Immune protection was evaluated based on the rate of the oocysts decrease, the output of oocysts (p < 0.05), the average weight gain, and the feed: meat ratio. Our results showed that rabbits immunized with rEmSAG10 and rEmSAG11 had a higher average weight gain (62.7%, 61.1%), feed; meat ratio (3.8:1, 4.5:1), and the oocysts decrease rate (70.8%, 81.2%) than those in the control group, and also significantly reduced intestinal lesions. The specific IgG level increased one week after the first rEmSAG10 and rEmSAG11 immunization and was maintained until two weeks after the challenge (p < 0.05). The TGF-ß, IL-4, and IL-10 levels in the serum increased significantly after the secondary immunization with rEmSAG10 and rEmSAG11, while the IL-2 levels increased significantly after the secondary immunization with rEmSAG11 (both p < 0.05), suggesting that rEmSAG10 can induce a humoral and cellular immunity, while rEmSAG11 can only induce a humoral immunity. Therefore, rEmSAG10 is a candidate antigen for E. magna recombinant subunit vaccines.

Prevalence and molecular characterization of Eimeria species affecting backyard poultry of Jammu region, North India.

The present study was conducted from January 2018 to December 2019 to know the prevalence of coccidiosis in backyard poultry in Jammu, Samba, and Udhampur districts of Union Territory of Jammu and Kashmir, North India. A total of 600 pooled fecal samples collected from backyard poultry were examined for presence of Eimeria oocysts. Morphometry and Polymerase Chain Reaction (PCR)-based amplification of ITS-1 gene was carried to characterize the Eimeria species infecting the backyard poultry of the study area. An overall prevalence of 28.5% Eimeria spp. infection among backyard poultry birds was recorded. Among the seasons, highest prevalence was recorded during rainy season (32%) with significantly (p < 0.05) high oocyst excretion (1.77 ± 0.01) and lowest during summer (19.3%) with low oocyst excretion (0.17 ± 0.006). Young birds up to 3 months of age were found to be more susceptible to infection than older birds, with a significantly (p < 0.05) high prevalence percentage of 38.02. Morphometry with COCCIMORPH software revealed presence of Eimeria tenella, Eimeria necatrix, Eimeria acervulina, and Eimeria maxima species with prevalence rates of 27.6%, 21.3%, 16.5%, and 3.6%, respectively. The amplified fragments of ITS-1 gene presented different sizes of Eimeria spp. viz. E. acervulina (321 bp), E. tenella (278 bp), E. maxima (145 bp), and E. necatrix (383 bp). The study concluded that although backyard poultry did not show clinical form of coccidiosis, it may act as source of potential reservoir.

Prevalence of Eimeria species, detected by ITS1-PCR, in broiler poultry farms located in seven provinces of northeastern Algeria.

Coccidiosis is an important global chickens' disease which can cause serious economic losses in the poultry industry worldwide. Little is known about the extent of infection or diversity, of the causative agent Eimeria spp., in Algeria. A priority, therefore, is to determine the prevalence and species composition to inform strategies on treatments and control measures. Samples were collected from 187 broiler farms, located in 7 Northeastern Algerian provinces (Jijel, Constantine, Skikda, Mila, Setif, Batna, Bordj bou-Arreridj), and Internal Transcribed Spacer 1 PCR (ITS1-PCR) was used to determine the prevalence and composition of Eimeria species in chickens. The survey revealed the presence of all seven species of Eimeria at different prevalences (E. maxima (69%), E. acervulina (68.4%), E. necatrix (11.2%), E. tenella (8%), E. praecox (4.3%), E. mitis (2.1%), E. brunetti (2.1%). Multiple infections, with up to 4 different Eimeria species present on a single farm, were the most frequent situation in our samples (51.9% mixed infections versus 47.6% single infections). All farms revealed infected samples, and we conclude that this parasite is a significant problem in these provinces.

Rhomboid protein 2 of Eimeria maxima provided partial protection against infection by homologous species.

Rhomboid-like proteases (ROMs) are considered as new candidate antigens for developing new-generation vaccines due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2). This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima). Firstly, Western blot assay was conducted to analyze the immunogenicity of rEmROM2. The result showed that rEmROM2 was recognized by chicken anti-E. maxima serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay revealed apparent transcription and expression of EmROM2 at the injection site. qRT-PCR (quantitative real-time PCR), flow cytometry and indirect ELISA indicated that vaccination with rEmROM2 or EmROM2 DNA significantly upregulated the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-ß and TNF SF15), the proportion of CD8+ and CD4+ T lymphocytes and serum IgG antibody response. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against E. maxima. The result revealed that vaccination with rEmROM2 or pVAX1-EmROM2 significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima. In summary, vaccination with rEmROM2 or pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.