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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]

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]

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]

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]

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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]

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]

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]

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]