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Anticoccidial vaccines comprising living oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima, and Eimeria acervulina are used to control coccidiosis. This study explored the potential of IL-1β to act as a molecular adjuvant for enhancing the immunogenicity of Eimeria necatrix and mucosal immunity. We engineered E. necatrix to express a functional chIL-1β (EnIL-1b) and immunized chickens with oocysts of the wild type (EnWT) and tranegenic (EnIL-1b) strains, respectively. The chickens were then challenged with EnWT oocysts to examine the immunogenicity-enhancing potential of chIL-1b. As expected, the oocyst output of EnIL-1β-immunized chickens was significantly reduced compared to those immunized using EnWT. No difference in body weight gain and lesion scores of EnIL-1β and EnWT groups was observed. The parasite load in the small intestine and caeca showed that the invasion and replication of EnIL-1β was not affected. However, the markers of immunogenicity and mucosal barrier, Claudin-1 and avian β-defensin-1, were elevated in EnIL-1binfected chickens. Ectopic expression of chIL-1β in E. necatrix thus appears to improve its immunogenicity and mucosal immunity, without increasing pathogenicity. Our findings support chIL-1β as a candidate for development of effective live-oocyst-based anticoccidial vaccines. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical Abstract

The purpose of the research is study of chicken parasitic associations in small farms of Kotayk region, and analysis of the determined factors. Materials and methods. Research was conducted in 2021 to 2022 in small poultry farms of Kotayk Marz. Poultry feces samples were examined at the Laboratory of Parasitology of the Research Center of Veterinary Medicine and Veterinary Sanitary Expertise, Armenian National Agrarian University. Totally 210 fecal samples were examined. Eimeria spp. oocysts and nematode eggs were detected by means of method after Darling. The results of the research were processed by statistical methods. Results and discussion. Results of the research have shown that poultry eimeriasis had a wide prevalence in Kotayk Region (52.86%). Three species of Eimeria spp. have been detected: E. acervulina, E. tenella, and E. necatrix. E. acervulina was the most prevalence species (34.29%). The highest intensity of the infection has been registered in Nairi and Kotayk Regions of the region. The above-mentioned species of Eimeria spp. have been occurred both in mono- and poly-infection, with various combinations of the species. E. acervulina + E. tenella combination was the most common. Poultry eimeriasis occurred as a mixed-infection with ascaridiasis, heterakiasis, and capillariasis. The highest extensiveness of the poultry infection has been registered in Kotayk and Hrazdan Regions of the region (65.71% in both cases). Geographical location, as well as natural and climatic conditions of the region promote the prevalence of the mixed infection of poultry.

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. [ABSTRACT FROM AUTHOR]

Abstract Background Eimeria parasite infection occurs via ingestion of oocysts. The robust, bilayer oocyst wall is formed from the contents of wall-forming bodies (WFBs), WFB1 and WFB2, located exclusively in macrogametocytes. Eimeria necatrix gametocyte proteins 22 and 59 (EnGAM22 and EnGAM59) have been found to localize to WFBs and the oocyst wall. However, the exact localization of these two proteins is not clear. Methods WFBs of E. necatrix were extracted from purified gametocytes using a cutoff filter and the extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Then, the localization of EnGAM22 and EnGAM59 proteins was determined using an indirect immunofluorescence assay. Finally, the development of macrogametocytes and the oocyst wall of E. necatrix was analyzed using laser confocal microscopy and scanning electron microscopy. Results Purified WFBs had the same shape and size as those observed in macrogametocytes. EnGAM22 protein localized to WFB1, whereas EnGAM59 protein localized to WFB2. Both EnGAM22 and EnGAM59 native proteins were detected in the extracts of WFBs and gametocytes. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface of the macrogametocyte to form an anti-EnGAM22 positive layer. WFB2 then appeared to give rise to the inner layer, which was anti-EnGAM59 positive. Conclusions EnGAM22 and EnGAM59 proteins localized to WFB1 and WFB2 and were involved in the formation of the outer and inner layers of the oocyst wall of E. necatrix, respectively. The processes of macrogametogenesis and oocyst wall formation of E. necatrix are similar to other Eimeria parasites. The anti-EnGAM22 antibody could be used as a tool to track the transport and secretion of proteins in WFB1 during oocyst development. Graphical Abstract

Background: Eimeria parasite infection occurs via ingestion of oocysts. The robust, bilayer oocyst wall is formed from the contents of wall-forming bodies (WFBs), WFB1 and WFB2, located exclusively in macrogametocytes. Eimeria necatrix gametocyte proteins 22 and 59 (EnGAM22 and EnGAM59) have been found to localize to WFBs and the oocyst wall. However, the exact localization of these two proteins is not clear. Methods: WFBs of E. necatrix were extracted from purified gametocytes using a cutoff filter and the extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Then, the localization of EnGAM22 and EnGAM59 proteins was determined using an indirect immunofluorescence assay. Finally, the development of macrogametocytes and the oocyst wall of E. necatrix was analyzed using laser confocal microscopy and scanning electron microscopy. Results: Purified WFBs had the same shape and size as those observed in macrogametocytes. EnGAM22 protein localized to WFB1, whereas EnGAM59 protein localized to WFB2. Both EnGAM22 and EnGAM59 native proteins were detected in the extracts of WFBs and gametocytes. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface of the macrogametocyte to form an anti-EnGAM22 positive layer. WFB2 then appeared to give rise to the inner layer, which was anti-EnGAM59 positive. Conclusions: EnGAM22 and EnGAM59 proteins localized to WFB1 and WFB2 and were involved in the formation of the outer and inner layers of the oocyst wall of E. necatrix, respectively. The processes of macrogametogenesis and oocyst wall formation of E. necatrix are similar to other Eimeria parasites. The anti-EnGAM22 antibody could be used as a tool to track the transport and secretion of proteins in WFB1 during oocyst development. [ABSTRACT FROM AUTHOR]

The study of the prevalence of chicken eimeriosis in private poultry farms was carried out in the Aragatsotn region of the Republic of Armenia. Three species of Eimeria were discovered, of which Eimeria acervulina is the most common. The highest rates of infection with Eimeria were recorded in the foothills zone in the spring months, and the lowest in the high mountain zone in the winter. A low extensiveness and intensity of invasion was recorded, which is due to the geographical location and climatic conditions of the region. [ABSTRACT FROM AUTHOR]

Abstract Background Poultry necrotic enteritis (NE) is an economically important disease caused by C. perfringens. The disease causing ability of this bacterium is linked with the production of a wide variety of toxins. Among them, necrotic enteritis B-like (NetB) toxin is reported to be involved in the pathogenesis of NE; in addition there is some circumstantial evidence that tpeL toxin may enhance virulence, but this is yet to be definitely shown. The situation becomes more complicated in the presence of a number of predisposing factors like co-infection with coccidia, type of diet and use of high protein diet. These co-factors alter the intestinal environment, thereby favoring the production of more toxins, leading to a more severe disease. The objective of this study was to develop a successful animal model that would induce clinical signs and lesions of NE using C. perfringens type G strains obtained from field outbreaks. A separate trial was simultaneously considered to establish the role of dietary factor with coccidial co-infection in NE. Results The results have shown that use of net-B positive C. perfringens without predisposing factors induce moderate to severe NE (Av. Lesion score 1.79 ± 1.50). In a separate trial, addition of fish meal to a feed of C. perfringens challenged birds produced higher number of NE cases (Av. Lesion score 2.17 ± 1.28). However, use of less virulent E. necatrix strain along with fish meal in conjunction with net-B positive strain did not alter the severity of NE lesions in specific pathogen free chicken (Av. Lesion score 2.21 ± 1.13). Conclusions This study suggests that virulent C. perfringens type G strains can induce NE lesions in the absence of other predisposing factors. Birds in the clostridia challenged group showed moderate to severe NE lesions. Use of less virulent coccidia strain contributed to a lesser extent in increasing the severity of disease. Maize based diet along with fishmeal (1:1) increased the severity of lesions but statistically it was non-significant. The NE lesions in all experimental groups were found to be present more frequently in the duodenum. In this way, this study provided an effective model for in vivo production of NE in poultry birds.

Disease investigation was made in a native chicken farm with 150 numbers of 12 weeks old Aseel chickens kept in Iruvelpattu village, Villupuram district of Tamil Nadu. Clinical signs like dullness, inappetance, brownish diarrhoea in few birds and mortality of 5 birds within a span of 3 days were reported (3.33%). Necropsy examination of dead birds revealed that emaciated carcass, soiling of vent with reddish droppings, swelling of small intestine with haemorrhages and also duodenum with cylindrical worms. Examination of small intestinal contents and mucosal scrapings confirmed numerous unsporulated oocysts of Eimeria sp. and eggs of Ascaridia galli. The adult roundworms were identified as Ascaridia galli. Examination of droppings from live birds also confirmed of Eimeria (23.33%) and Ascaridia galli eggs (26.66%). Based on the lesions produced in mid-small intestine, oocysts morphometry and sporulation time involved, it was identified as Eimeria necatrix. The present study reports for concurrent parasitic infections of Eimeria necatrix and Ascaridia galli and importance of specific treatment and deworming for endoparasites in native chickens.

Abstract Background Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical Abstract

Abstract Background Eimeria necatrix, the most highly pathogenic coccidian in chicken small intestines, can cause high morbidity and mortality in susceptible birds and devastating economic losses in poultry production, but the underlying molecular mechanisms in interaction between chicken and E. necatrix are not entirely revealed. Accumulating evidence shows that the long-non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are key regulators in various infectious diseases. However, the expression profiles and roles of these two non-coding RNAs (ncRNAs) during E. necatrix infection are still unclear. Methods The expression profiles of mRNAs, lncRNAs and circRNAs in mid-segments of chicken small intestines at 108 h post-infection (pi) with E. necatrix were analyzed by using the RNA-seq technique. Results After strict filtering of raw data, we putatively identified 49,183 mRNAs, 818 lncRNAs and 4153 circRNAs. The obtained lncRNAs were classified into four types, including 228 (27.87%) intergenic, 67 (8.19%) intronic, 166 (20.29%) anti-sense and 357 (43.64%) sense-overlapping lncRNAs; of these, 571 were found to be novel. Five types were also predicted for putative circRNAs, including 180 exonic, 54 intronic, 113 antisense, 109 intergenic and 3697 sense-overlapping circRNAs. Eimeria necatrix infection significantly altered the expression of 1543 mRNAs (707 upregulated and 836 downregulated), 95 lncRNAs (49 upregulated and 46 downregulated) and 13 circRNAs (9 upregulated and 4 downregulated). Target predictions revealed that 38 aberrantly expressed lncRNAs would cis-regulate 73 mRNAs, and 1453 mRNAs could be trans-regulated by 87 differentially regulated lncRNAs. Additionally, 109 potential sponging miRNAs were also identified for 9 circRNAs. GO and KEGG enrichment analysis of target mRNAs for lncRNAs, and sponging miRNA targets and source genes for circRNAs identified associations of both lncRNAs and circRNAs with host immune defense and pathogenesis during E. necatrix infection. Conclusions To the best of our knowledge, the present study provides the first genome-wide analysis of mRNAs, lncRNAs and circRNAs in chicken small intestines infected with E. necatrix. The obtained data will offer novel clues for exploring the interaction mechanisms between chickens and Eimeria spp.

Anticoccidial vaccines comprising living oocysts of Eimeria tenella, Eimeria necatrix, Eimeria maxima , and Eimeria acervulina are used to control coccidiosis. This study explored the potential of IL-1β to act as a molecular adjuvant for enhancing the immunogenicity of Eimeria necatrix and mucosal immunity. We engineered E. necatrix to express a functional chIL-1β (EnIL-1β) and immunized chickens with oocysts of the wild type (EnWT) and tranegenic (EnIL-1β) strains, respectively. The chickens were then challenged with EnWT oocysts to examine the immunogenicity-enhancing potential of chIL-1β. As expected, the oocyst output of EnIL-1β-immunized chickens was significantly reduced compared to those immunized using EnWT. No difference in body weight gain and lesion scores of EnIL-1β and EnWT groups was observed. The parasite load in the small intestine and caeca showed that the invasion and replication of EnIL-1β was not affected. However, the markers of immunogenicity and mucosal barrier, Claudin-1 and avian β-defensin-1, were elevated in EnIL-1β-infected chickens. Ectopic expression of chIL-1β in E. necatrix thus appears to improve its immunogenicity and mucosal immunity, without increasing pathogenicity. Our findings support chIL-1β as a candidate for development of effective live-oocyst-based anticoccidial vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Duan, Abudureheman, Wang, Suo, Yu, Shi, Liu, Salama, Srivastav, Gupta and Suo.)

Background: Poultry necrotic enteritis (NE) is an economically important disease caused by C. perfringens. The disease causing ability of this bacterium is linked with the production of a wide variety of toxins. Among them, necrotic enteritis B-like (NetB) toxin is reported to be involved in the pathogenesis of NE; in addition there is some circumstantial evidence that tpeL toxin may enhance virulence, but this is yet to be definitely shown. The situation becomes more complicated in the presence of a number of predisposing factors like co-infection with coccidia, type of diet and use of high protein diet. These co-factors alter the intestinal environment, thereby favoring the production of more toxins, leading to a more severe disease. The objective of this study was to develop a successful animal model that would induce clinical signs and lesions of NE using C. perfringens type G strains obtained from field outbreaks. A separate trial was simultaneously considered to establish the role of dietary factor with coccidial co-infection in NE. Results: The results have shown that use of net-B positive C. perfringens without predisposing factors induce moderate to severe NE (Av. Lesion score 1.79 ± 1.50). In a separate trial, addition of fish meal to a feed of C. perfringens challenged birds produced higher number of NE cases (Av. Lesion score 2.17 ± 1.28). However, use of less virulent E. necatrix strain along with fish meal in conjunction with net-B positive strain did not alter the severity of NE lesions in specific pathogen free chicken (Av. Lesion score 2.21 ± 1.13). Conclusions: This study suggests that virulent C. perfringens type G strains can induce NE lesions in the absence of other predisposing factors. Birds in the clostridia challenged group showed moderate to severe NE lesions. Use of less virulent coccidia strain contributed to a lesser extent in increasing the severity of disease. Maize based diet along with fishmeal (1:1) increased the severity of lesions but statistically it was non-significant. The NE lesions in all experimental groups were found to be present more frequently in the duodenum. In this way, this study provided an effective model for in vivo production of NE in poultry birds. [ABSTRACT FROM AUTHOR]

Coccidiosis in chickens is an intestinal parasitic disease caused by protozoan parasites named Eimeria spp. In some Eimeria infections, intestinal lymphocytes are known to highly express chicken NK-lysin (cNK-lysin), an antimicrobial peptide with anticoccidial activity. Therefore, this study aims to investigate the expression of cNK-lysin in E. necatrix-infected chickens and its role in E. necatrix infection. The expression of cNK-lysin transcript was significantly increased in E. necatrix sporozoites-treated lymphocytes. In E. necatrix infection, cNK-lysin transcript was induced in intestinal lymphocytes but not in the spleen. The recombinant cNK-lysin exhibited anticoccidial activity against E. necatrix sporozoites as well as immunomodulatory activity on macrophages by inducing proinflammatory cytokines. These results indicated that E. necatrix infection induces high local expression of cNK-lysin and the secreted cNK-lysin helps protect coccidiosis. [ABSTRACT FROM AUTHOR]

Background: Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods: Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results: SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions: We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. [ABSTRACT FROM AUTHOR]

• Matrine regulates intestinal stem cell functions to repair eimeria Necatrix-induced intestinal injury. • Matrine standardizes the differentiation fate of intestinal stem cells into secretory cells after eimeria necatrix infection. • Matrine hinders the hyperactivation of wnt signaling to rescue intestinal stem cell activity following eimeria necatrix infection. Coccidiosis is a rapidly spreading and acute parasitic disease that seriously threatening the intestinal health of poultry. Matrine from leguminous plants has anthelmintic and anti-inflammatory properties. This assay was conducted to explore the protective effects of Matrine and the AntiC (a Matrine compound) on Eimeria necatrix (EN)-infected chick small intestines and to provide a nutritional intervention strategy for EN injury. The in vivo (chick) experiment: A total of 392 one-day-old yellow-feathered broilers were randomly assigned to six groups in a 21-day study: control group, 350 mg/kg Matrine group, 500 mg/kg AntiC group, EN group, and EN + 350 mg/kg Matrine group, EN + 500 mg/kg AntiC group. The in vitro (chick intestinal organoids, IOs): The IOs were treated with PBS, Matrine, AntiC, 3 μM CHIR99021, EN (15,000 EN sporozoites), EN + Matrine, EN + AntiC, EN + Matrine + CHIR99021, EN + AntiC + CHIR99021. The structural integrity of chicks jejunal crypt-villus axis was evaluated by hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM). And the activity of intestinal stem cells (ISCs) located in crypts was assessed by in vitro expansion advantages of a primary in IOs model. Then, the changes of Wnt/β-catenin signaling in jejunal tissues and IOs were detected by Real-Time qPCR,Western blotting and immunohistochemistry. The results showed that dietary supplementation with Matrine or AntiC rescued the jejunal injury caused by EN, as indicated by increased villus height, reduced crypt hyperplasia, and enhanced expression of tight junction proteins. Moreover, there was less budding efficiency of the IOs expanded from jejunal crypts of chicks in the EN group than that in the Matrine and AntiC group, respectively. Further investigation showed that AntiC and Matrine inhibited EN-stimulated Wnt/β-catenin signaling. The fact that Wnt/β-catenin activation via CHIR99021 led to the failure of Matrine and AntiC to rescue damaged ISCs confirmed the dominance of this signaling. Our results suggest that Matrine and AntiC inhibit ISC proliferation and promote ISC differentiation into absorptive cells by preventing the hyperactivation of Wnt/β-catenin signaling, thereby standardizing the function of ISC proliferation and differentiation, which provides new insights into mitigating EN injury by Matrine and AntiC. Proposed mechanism of Matrine and AntiC alleviating EN-induced intestinal damage in chicks. Matrine and AntiC rescue intestinal stem cells' proliferation and differentiation function by blocking the hyperactivation of the Wnt/β-catenin signaling pathway, which further repairs the intestinal barrier function. [Display omitted] [ABSTRACT FROM AUTHOR]

Disease investigation was made in a native chicken farm with 150 numbers of 12 weeks old Aseel chickens kept in Iruvelpattu village, Villupuram district of Tamil Nadu. Clinical signs like dullness, inappetance, brownish diarrhoea in few birds and mortality of 5 birds within a span of 3 days were reported (3.33%). Necropsy examination of dead birds revealed that emaciated carcass, soiling of vent with reddish droppings, swelling of small intestine with haemorrhages and also duodenum with cylindrical worms. Examination of small intestinal contents and mucosal scrapings confirmed numerous unsporulated oocysts of Eimeria sp. and eggs of Ascaridia galli. The adult roundworms were identified as Ascaridia galli. Examination of droppings from live birds also confirmed of Eimeria (23.33%) and Ascaridia galli eggs (26.66%). Based on the lesions produced in mid-small intestine, oocysts morphometry and sporulation time involved, it was identified as Eimeria necatrix. The present study reports for concurrent parasitic infections of Eimeria necatrix and Ascaridia galli and importance of specific treatment and deworming for endoparasites in native chickens. [ABSTRACT FROM AUTHOR]

Background: Eimeria necatrix, the most highly pathogenic coccidian in chicken small intestines, can cause high morbidity and mortality in susceptible birds and devastating economic losses in poultry production, but the underlying molecular mechanisms in interaction between chicken and E. necatrix are not entirely revealed. Accumulating evidence shows that the long-non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are key regulators in various infectious diseases. However, the expression profiles and roles of these two non-coding RNAs (ncRNAs) during E. necatrix infection are still unclear. Methods: The expression profiles of mRNAs, lncRNAs and circRNAs in mid-segments of chicken small intestines at 108 h post-infection (pi) with E. necatrix were analyzed by using the RNA-seq technique. Results: After strict filtering of raw data, we putatively identified 49,183 mRNAs, 818 lncRNAs and 4153 circRNAs. The obtained lncRNAs were classified into four types, including 228 (27.87%) intergenic, 67 (8.19%) intronic, 166 (20.29%) anti-sense and 357 (43.64%) sense-overlapping lncRNAs; of these, 571 were found to be novel. Five types were also predicted for putative circRNAs, including 180 exonic, 54 intronic, 113 antisense, 109 intergenic and 3697 sense-overlapping circRNAs. Eimeria necatrix infection significantly altered the expression of 1543 mRNAs (707 upregulated and 836 downregulated), 95 lncRNAs (49 upregulated and 46 downregulated) and 13 circRNAs (9 upregulated and 4 downregulated). Target predictions revealed that 38 aberrantly expressed lncRNAs would cis-regulate 73 mRNAs, and 1453 mRNAs could be trans-regulated by 87 differentially regulated lncRNAs. Additionally, 109 potential sponging miRNAs were also identified for 9 circRNAs. GO and KEGG enrichment analysis of target mRNAs for lncRNAs, and sponging miRNA targets and source genes for circRNAs identified associations of both lncRNAs and circRNAs with host immune defense and pathogenesis during E. necatrix infection. Conclusions: To the best of our knowledge, the present study provides the first genome-wide analysis of mRNAs, lncRNAs and circRNAs in chicken small intestines infected with E. necatrix. The obtained data will offer novel clues for exploring the interaction mechanisms between chickens and Eimeria spp. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Stable transfection of E. necatrix was established. • Second generation of merozoites were used for the nucleofecting. • EYFP expresses throughout the life cycle of transgenic E. necatrix. Abstract Eimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1 , consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocysts was noticed with more than 90% EYFP + oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine-delivery vehicle. [ABSTRACT FROM AUTHOR]

Coccidiosis, also known as Eimeriosis, is a highly prevalent parasitic disease affecting sheep in nearly all sheep-rearing nations across the globe. Currently, there is a scarcity of literature documenting the specific lesions in sheep resulting from coccidia infection. This study aimed to investigate these characteristic lesions through necropsy, microscopic observation, and molecular biological techniques. As a result, Eimeria granulosa was identified as the causative agent, which induced distinct pathological alterations in the small intestine of lambs as observed during necropsy. Notably, E. granulosa manifested as small scattered petechiae and white spots, visible through the serous membrane of the small intestine, akin to the pathology observed in E. necatrix. Therefore, this study provides valuable insights for the accurate diagnosis of coccidiosis in sheep. [Display omitted] • Necropsy, microscopic and molecular techniques to diagnose coccidiosis in sheep. • E. granulosa manifested as small scattered petechiae on the small intestine. • Molecular identification and ITS-1 rRNA gene sequence analysis. [ABSTRACT FROM AUTHOR]

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (En sag -CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG -CAP). The full length En sag -CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG -CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG -CAP could be recognized by anti-rEnSAG -CAP monoclonal antibody (anti-rEnSAG -CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG -CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG -CAP polyclonal antibody (anti-rEnSAG -CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG -CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG -CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of En sag -CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG -CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG -CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG -CAP. Chickens immunized with rEnSAG -CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG -CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine. [Display omitted] • A novel E. necatrix sag gene was cloned, encoding a protein with a CAP domain. • EnSAG is anchored to the membrane of parasites via a GPI anchor. • EnSAG might participate in sporozoite invasion into MDBK cells. • EnSAG could be used as a candidate antigen to develop a recombinant coccidiosis vaccine. [ABSTRACT FROM AUTHOR]

Eimeria necatrix is one of the most pathogenic parasites causing high mortality in chicken older than 8 weeks. Eimeria spp. possess a coccidian lifecycle including both sexual and asexual stages. Sexual differentiation and development occupies a central place in the life cycle of the Eimeria parasite. However, our knowledge of the sexual differentiation and gametocyte development of Eimeria is very limited. Here using RNA sequencing, we conducted a comparative transcriptome analysis between third-generation merozoites (MZ-3) and gametocytes (GAM) of E. necatrix to identify genes with functions related to sexual differentiation and gametocyte development. Approximately 4267 genes were differentially expressed between MZ-3 and GAM. Compared with MZ-3, 2789 genes were upregulated and 1478 genes were downregulated in GAM. Approximately 329 genes in MZ-3 and 1289 genes in GAM were further analyzed in the evaluation of stage-specific genes. Gene Ontology (GO) classification and KEGG analysis revealed that 953 upregulated gametocyte genes were annotated with 170 GO assignments, and 405 upregulated genes were associated with 231 signaling pathways. We also predicted a further 83 upregulated gametocyte genes, of which 53 were involved in the biosynthesis of the oocyst wall, and 30 were involved in microgametocyte development. This information offers insights into the mechanisms governing the sexual development of E. necatrix and may potentially allow the identification of targets for blocking parasite transmission. [ABSTRACT FROM AUTHOR]

Eimeria species are intracellular obligate parasites, among the most common pathogens affecting the intensive poultry industry. Oxidoreductases are members of a class of proteins with redox activity and are widely found in apicomplexan protozoans. However, there have been few reports related to Eimeria species. In this study, total RNA was extracted from the gametocytes of E. necatrix Yangzhou strain to amplify the En OXIO 1 gene using reverse-transcription polymerase chain reaction. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-En OXIO 1 was constructed and transformed into Escherichia coli BL21(DE3), and the recombinant protein rEnOXIO1 was expressed by induction with isopropyl ß-D-1-thiogalactopyranoside. The full length En OXIO 1 gene was 2535 bp encoding 844 amino acids, and the EnOXIO1 protein had a molecular weight of about 100 kDa and was mainly expressed in inclusion bodies. Western blot analysis indicated that the rEnOXIO1 protein had good antigenicity and cross-reactivity and was specifically recognized by a 6 ×HIS labeled monoclonal antibody, mouse anti-recombinant protein polyclonal antibody, and recovery serum from chickens infected with E. necatrix , E. acervulina , and E. tenella sporulated oocysts. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein was mainly located on the wall-forming bodies in gametocytes and played an important role in the formation of the oocyst wall. Quantitative PCR analysis revealed that transcript levels of En OXIO 1 were highest in the gametocyte stage. Protein expression levels of En OXIO 1 were higher in the gametocyte stage than in other developmental stages according to western blot analysis. Vaccination of chickens against E. necatrix was achieved with recombinant protein rEnOXIO1, which triggered humoral immunity and antibody production, increased average body weight gain, reduced oocyst output and alleviated lesions after E. necatrix infection. The highest ACI value (172.36) was observed in chickens that received 200 μg rEnOXIO1 compared with other immunization groups. • A novel GMC oxidoreductase gene En OXIO 1 from E. necatrix is cloned and characterized. • En OXIO 1 is highly expressed during gametogenesis and involved in oocyst wall formation. • En OXIO 1 may be used to develop recombinant subunit vaccines against coccidiosis. [ABSTRACT FROM AUTHOR]

Eimeria necatrix is an obligate intracellular parasite that has a complex life cycle and causes significant economic losses to the poultry industry. To better understand the cellular invasion mechanism of E. necatrix and develop new measures against its infection, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis to investigate protein abundance across different life cycle stages, including unsporulated oocysts (UO), sporozoites (SZ) and second-generation merozoites (MZ-2). Our analysis identified a total of 3606 proteins, among which 1725, 1724, 2143 and 2386 were annotated by the Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and InterPro (IPR) databases, respectively. We also identified 388, 300 and 592 differentially abundant proteins in SZ vs UO, SZ vs MZ-2 and MZ-2 vs UO, respectively. Further analysis revealed that 118 differentially abundant proteins were involved in cellular invasion and could be categorized into eight groups. These findings provide valuable insights into protein abundance across the different life cycle stages of E. necatrix and offer candidate proteins for future studies on cellular invasion and other biological processes. Eimeria necatrix is an obligate intracellular parasite results in huge economic losses to the poultry industry. Understanding proteomic variations across the life cycle stages of E. necatrix may offer proteins related to cellular invasion of E. necatrix , and provide resources for the development of new treatment and prevention interventions against E. necatrix infection. The current data provide an overall summary of the protein abundance across the three life cycle stages of E. necatrix. We identified differentially abundant proteins potential related to cellular invasion. The candidate proteins we identified will form the basis of future studies for cellular invasion. This work also will help in the development of novel strategies for coccidiosis control. [Display omitted] • A total of 3606 proteins were identified in the three developmental stages. • 388, 300 and 592 differentially abundant proteins were identified in SZ vs UO, SZ vs MZ-2 and MZ-2 vs UO, respectively. • 118 differentially abundant proteins were defined as being involved in cellular invasion. [ABSTRACT FROM AUTHOR]

Multiplex polymerase chain reaction (PCR) was optimized for detection of 2 important species of Eimeria (Eimeria tenella and E. necatrix). This optimized protocol was used to screen 100 fecal samples collected from broiler birds in Lahore periphery. Out of 100 samples we found 6 tenella and only 1 E. necatrix positive by this method. The detection limit of oocysts by this method was as leastas 16 oocysts. This optimized multiplex PCR method can be used as routine diagnostic tool for detection of E. tenella and E. necatrix and can be extended up to the detection of 7 Eimeria species in future.

Micronemes are secretory organelles of the invasive stages of apicomplexan parasites and contain proteins that are important for parasite motility and host cell invasion. Even though coccidiosis is a complex disease that can be caused by any combination of mainly seven species, most of the molecular researches concerning characterization of host and parasite genes and proteins have been limited to Eimeria tenella. The present study describes isolation and purification of Eimeria necatrix oocysts that can be used for an inexpensive and simple total RNA extraction method to partial sequence determination of a cDNA encoding microneme 5 protein. Using the extracted total RNA as template and oligo(dT) as primer, cDNA was synthesized. In order to amplify cDNA encoding the micronem 5 protein (EnMIC- 5), RT-PCR was applied with the specific primers based on the known EST sequence.Following amplification, the unique and thick 758 bp fragment was seen. Domain analysis of EnMIC-5 revealed that the sequence contains the conserved domain of PAN/APPLE superfamily between amino acid resides 130 to 201. This domain has strong similarity to the adhesive plasma pre-kallikrein. Despite sequence similarity of EnMIC-5 with those sequences in database, differences may represent some allelic polymorphism.

The excretion frequencies of cecal and intestinal droppings of Chinese Lingnan yellow chickens were observed for 10 consecutive days. The chickens were then orally inoculated with a precocious line of Eimeria necatrix , and the oocysts present in the cecal and intestinal droppings were separately collected and monitored using the McMaster method. The results showed that the excretion frequency of cecal droppings was significantly lower than that of intestinal droppings, and the oocysts of E . necatrix were distributed primarily in the cecal droppings. This distribution affects the homogeneity of the second and third generation of oocysts ingested by the chickens and therefore affects the immune effect observed during E . necatrix immunization. To artificially strengthen the immunologic homogeneity against E . necatrix , a method of artificially strengthening the second immunization was applied, and the immune effect was evaluated based on oocyst excretion, body weight gain, fecal scores, intestinal lesion scores and survival percentages. The results showed that no significant intestinal damage was caused by immunization reactions in the chickens. In addition, the number of excreted oocysts in the immunized chicken groups could be significantly increased, and the immunologic homogeneity of the immunized chickens could be improved by artificially strengthening the second immunization, which could in turn improve the immune protective effect. [ABSTRACT FROM AUTHOR]

• Ninety eight out of 680 E. necatrix protein spots were recognized by immune sera. • Out of the 56 spots selected for MS analysis, 50 proteins were identified. • Of the 50 proteins, 8 were previously characterized. • The proteins have a wide range of structures, cellular locations and functions. Eimeria necatrix , an apicomplexan protozoa of the genus Eimeria, causes intestinal coccidiosis that can reduce growth performance of poultry and result in high mortality in older chickens. In this report, the whole sporozoite proteins of E.necatrix were studied by two-dimensional electrophoresis (2-DE) and Western blotting using hyper-immune chicken serum containing E.necatrix -specific antibodies. Approximately 680 protein spots for E.necatrix sporozoite were detected by 2-DE with silver staining, where 98 spots were cross-reacted with the E. necatrix- specific immune sera. Out of the 56 spots that were selected for MALDI-TOF-MS/MS analysis, 50 unique proteins were identified using the MASCOT software, 8 proteins were identified as known E.necatrix proteins and the rest were all putative proteins. These proteins have a wide range of known or predicted structures, cellular locations and functions, including proteins in category nuclear location & function, multifunctional- or multifunctional motifs-containing proteins, cellular transport and structure-related proteins, proteins of enzymatic activities, motor proteins-related, cell surface and organelle-related proteins. These new findings will enhance our understandings of parasite immunogenicity and immune evasion mechanisms of E. necatrix and facilitate the discovery phase of highly effective vaccine candidates. [ABSTRACT FROM AUTHOR]

Intracellular infections by parasites require a functional anti-apoptotic mechanism for parasite survival within the host cell. The intracellular cycle of Eimeria tenella and Eimeria necatrix in chicken intestinal cells involves the maturation of schizonts within the epithelial cells lining the crypt lumen of the ceca (E. tenella) and jejunum (E. necatrix). After invasion, these cells detach from the epithelial layer and migrate into the underlying connective tissue, where maturation of second-generation schizonts takes place. However, the detached epithelial cells that harbour the parasite and localize in the lamina propia do not undergo apoptosis despite the fact that they are parasitized cells and are located in an inappropriate microenvironment. In this study we consider the hypothesis that E. tenella and E. necatrix may inhibit the host cell apoptosis that accompanies parasite-mediated transformation during late schizogony. To that end, the expression of both NF-κB, a transcriptional factor that blocks parasite-induced apoptosis, and bcl-xL, an anti-apoptotic protein induced by NF-κB, were studied in the host cell during the maturation of second-generation schizonts. In addition, the expression of the phosphorylated inhibitor of NF-κB, p-IkBα, was also studied to further confirm NF-κB activation. Immunocytochemical techniques, flow cytometric and blott analysis were applied by using polyclonal antibodies that specifically react with bcl-xL, p-IkBα, and NF-κB to detect these anti-apoptotic proteins in the parasitized cell. Our results offer evidence that both these coccidial species first induce NF-κB activation to protect the transformed parasitized cells from apoptosis, allowing the second-generation schizonts to mature, and later, after complete schizonts maturation, cause NF-κB inhibition to trigger host cell apoptosis in order to facilitate the escape of merozoites. To determine whether inhibition of the NF-κB pathway would induce apoptosis of the host cell, a protease inhibitor (TPCK), which induces apoptosis by mediating inhibition of IkB phosphorylation, was administered to parasitized chickens. [Copyright &y& Elsevier]

Paracox anticoccidial vaccine was administered to a 7-day-old flock of commercial broiler breeder stock subsequently reared to point-of-lay in the same house. For comparison, three subgroups of another flock of broiler breeders were also vaccinated with Paracox at 7 days of age, reared to 42 days and then transferred to new litter on another farm until point-of-lay. The first subgroup received no further treatment, but the second and third each received a second vaccination with Paracox, either immediately after transfer to the new litter or 42 days after transfer. Using an Eimeria necatrix model, protective immunity was demonstrated by virulent challenge of samples of birds from all groups by the age of 37–40 days (30–33 days after the first vaccination), and was maintained to at least 122–125 days of age, whether the birds remained on the same litter or were transferred to another farm, and whether they received one or two anticoccidial vaccinations. Therefore, there is no disadvantage in transferring birds onto new litter 35 days after a single Paracox vaccination, nor is there any advantage in giving a second vaccination after such a transfer. Vaccinated birds seeded the new litter with oocysts, despite being clinically immune to coccidiosis. A supplementary laboratory experiment showed that birds vaccinated at 8 days of age passed almost no oocysts after a second vaccination at 43 days of age. This indicated that they were not only protected against clinical coccidiosis, but were almost solidly immune to a homologous infection 5 weeks after a single vaccination. Nevertheless, oocysts appeared in the litter of all four groups of commercial breeders throughout the trial, showing that wild-type heterologous infections occurred whether the birds were transferred to new litter or not, but these did not overwhelm the acquired protective immunity and cause clinical coccidiosis. [ABSTRACT FROM AUTHOR]

Coccidiosis in chickens is an intestinal parasitic disease caused by protozoan parasites named Eimeria spp. In some Eimeria infections, intestinal lymphocytes are known to highly express chicken NK-lysin (cNK-lysin), an antimicrobial peptide with anticoccidial activity. Therefore, this study aims to investigate the expression of cNK-lysin in E. necatrix-infected chickens and its role in E. necatrix infection. The expression of cNK-lysin transcript was significantly increased in E. necatrix sporozoites-treated lymphocytes. In E. necatrix infection, cNK-lysin transcript was induced in intestinal lymphocytes but not in the spleen. The recombinant cNK-lysin exhibited anticoccidial activity against E. necatrix sporozoites as well as immunomodulatory activity on macrophages by inducing proinflammatory cytokines. These results indicated that E. necatrix infection induces high local expression of cNK-lysin and the secreted cNK-lysin helps protect coccidiosis.