IntroductionMycoplasma synoviae (M. synoviae) is an economically significant pathogen that causes respiratory infections, synovitis, and arthritis in chickens, inflicting substantial economic losses on the global poultry industry. Its frequent co-infection with other respiratory pathogens, often exacerbates the resultant pathogenic damage. As an important respiratory pathogen, it remains unclear whether Cryptosporidium baileyi (C. baileyi) can cause co-infection with M. synoviae in commercial large-scale poultry farms and what synergistic pathogenic pattern exists between them.MethodsA total of 1,118 choanal cleft swab samples were collected from commercial chicken farms across eight regions in Guangdong province for the detection of M. synoviae and C. baileyi. The extracted DNA was analyzed by qPCR for M. synoviae and nested PCR for C. baileyi, respectively. Furthermore, a total of 90 one-day-old chicks confirmed free of C. baileyi and M. synoviae were randomly divided into six groups to establish a co-infection model and investigate the synergistic pathogenic effect pattern of the two pathogens.ResultsThe overall positive rates of M. synoviae and C. baileyi were 41.32% and 17.80%, respectively, with significant regional, city-level, and age-related variations. Samples from Western Guangdong and chickens over 45 days old showed the highest infection risks for both pathogens. The co-infection rate was 10.55%, and a significant positive association was observed between the two pathogens (OR = 2.44, p < 0.001). Co-infection risk increased markedly with age, especially in chickens older than 45 days. Chicken co-infection model was established to explore synergistic pathogenesis between C. baileyi and M. synoviae. Co-infection did not alter the prepatent period of C. baileyi, but significantly increased oocyst shedding peak and prolonged excretion time. Meanwhile, C. baileyi markedly elevated M. synoviae loads in the choanal cleft at multiple time points. Gross and histopathological examinations showed that co-infection aggravated laryngotracheal lesions caused by C. baileyi, and exacerbated footpad, joint and air sac lesions induced by M. synoviae. Overall, C. baileyi and M. synoviae exert mutual promoting effects on proliferation and pathogenicity in chickens.DiscussionThe findings in the present study confirm the high prevalence of M. synoviae-C. baileyi co-infection in commercial poultry flocks and demonstrate that co-infection synergistically enhances the pathogenicity of both pathogens. These results fill an important knowledge gap in co-infection research, provide novel insights into the interaction mechanisms of multiple pathogens in poultry, and offer key scientific support for addressing complex disease challenges in modern poultry production.

This study characterized Bacillus subtilis (B. subtilis) BS-Q as a novel probiotic candidate against Clostridium perfringens (C. perfringens). Polyphasic analysis, including 16S rRNA gene sequencing (>99% similarity to reference B. subtilis strains), identified the isolate as BS-Q. It exhibited robust growth, and sporulation was observed. Its cell-free supernatant (CFS) demonstrated potent, broad-spectrum inhibition against C. perfringens types A, C, and G with a minimum inhibitory concentration (MIC) of 33.06 ± 0.53 μg/mL. The antibacterial activity was localized to the CFS and showed remarkable stability after exposure to harsh conditions: high temperature (100°C for 30 min), a broad pH range (3.0-11.0), and high salinity (up to 10% NaCl). Protease treatment markedly reduced the activity, confirming the proteinaceous nature of the antimicrobial compounds, which was supported by the presence of unique protein bands (25-35 kDa) in the CFS as revealed by SDS-PAGE. In conclusion, B. subtilis BS-Q produces highly stable, proteinaceous antimicrobials effective against C. perfringens. Its strong anti-clostridial activity and resilience position it as a promising probiotic and a potential antibiotic alternative for controlling necrotic enteritis in poultry.

Avian coccidiosis, especially infection caused by the highly pathogenic Eimeria necatrix, causes major economic loss in the poultry production. With the increasing limitations of anticoccidial drugs and live vaccines, there is an urgent need for novel, broad-spectrum control strategies. In this study, a reverse vaccinology workflow was used to screen the proteomes of five Eimeria species. Six highly conserved membrane protein families were selected: carbonic anhydrase (CA), mechanosensitive ion channel (MSC), cell division control protein 50 (CDC50), major facilitator superfamily transporter (MFS), ATP-binding cassette transporter (ABC) and lipid transfer protein (LTP). Antigenic fragments from these families were linked with EAAAK linkers to generate a chimeric multi-antigen fusion construct, EimeriaBig. Five prime-boost regimens were tested in chickens challenged with E. necatrix. The regimen of one protein prime followed by two DNA boosts showed the highest protection among the tested regimens, with moderate protective efficacy. Our results demonstrated that a heterologous strategy, comprising a recombinant protein prime (rEimeriaBig) followed by two DNA vaccine boosts (pcDNA3.1-EimeriaBig), conferred the best protection among the regimens tested, with moderate efficacy. In this group, cecal lesion score was reduced by 47.64% and oocyst output was reduced by 49.75%, and the anticoccidial index was 168.72. The same regimen produced higher antigen-specific IgY and increased transcription of IL-2, IFN-γ, and IL-10. These results support further optimization of this vaccine strategy for control of E. necatrix.

Increasing evidence suggests that regulatory noncoding RNAs (ncRNAs) modulate a series of epithelial responses following Cryptosporidium invasion. During infection, ncRNAs are involved in the activation of intracellular signaling pathways, the production of antimicrobial molecules, the expression of cytokines/chemokines, the release of epithelial cell-derived exosomes, and the feedback regulation of immune homeostasis. In addition, Cryptosporidium may have developed strategies to modulate host ncRNA-mediated cellular function for immune evasion. These findings indicate that ncRNAs may be important determinants of the infection resistance conferred by the host and the long-term latency established by the parasite. Here, we summarize recent progress on the role of ncRNAs in the regulation of host-Cryptosporidium interactions, relevant to the development of ncRNA-based drug therapeutics for cryptosporidiosis in the future.

Avian coccidiosis, caused by protozoan parasites of the genus Eimeria, poses a major threat to the poultry industry worldwide, leading to severe economic losses through reduced growth rates, poor feed efficiency, and increased mortality. Although the conventional management of this disease has relied on anticoccidial drugs, the overwhelming use of these agents has led to the rapid emergence and spread of drug-resistant Eimeria isolates, highlighting the urgent need for novel therapeutic approaches. This study employed computational approaches to identify novel inhibitors targeting Eimeria tenella prolyl-tRNA synthetase (EtPRS). Based on the virtual screening of a library of 3045 natural compounds, 42 high-confidence inhibitors were identified. Three compounds, including Chelidonine, Bicuculline, and Guggulsterone, demonstrated strong and selective binding to EtPRS through stable interactions within the active site. ADMET predictions revealed favorable safety profiles, while molecular dynamic simulations confirmed binding stability. Overall, this research established a solid framework for the development of effective anticoccidial agents targeting PRS, contributing to the advancement of therapeutic strategies for combating parasitic infections in the poultry industry.

Pentatrichomonas hominis is a protozoan parasite that infects the gastrointestinal tract of humans and mammals, causing abdominal pain and diarrhea. However, its presence in pigs and its potential as a pathogen causing diarrhea in piglets have not been well studied. This study aimed to investigate the prevalence of P. hominis in pigs and its potential for zoonotic transmission. A total of 406 pig fecal samples were collected from four pig farms located in Guangdong and Anhui Provinces. Fecal DNA extraction was carried out using a commercially available kit. A nested PCR methodology was employed to detect the presence of P. hominis infection. Samples that tested positive were subsequently subjected to sequencing, and the genetic characteristics of the 18S ribosomal RNA (rRNA) gene were analyzed. The overall infection rate of P. hominis was 34.98% (142/406), in Guangdong Province 28.47% (80/281), and in Anhui Province 49.60% (62/125). Among different age groups, suckling piglets had the highest infection rate at 40.24% (68/169). Genetic analysis of the P. hominis isolates showed that the PH-1 genotype was predominant and had a high degree of similarity to P. hominis sequences obtained from humans, cats, and pigs, indicating the potential for zoonotic transmission. The high infection rate and genetic diversity highlight the need for effective control measures in pig farming to reduce parasite transmission and zoonotic risk.

Intestinal health is vital for poultry production, and protein plays a key role in intestinal nutrition. The present study used 16S rRNA gene sequencing and serum metabolomics to investigate the effect of CAP on the cecal microflora structure and serum metabolites in 42-day-old broiler chickens. A total of 480 one-day-old Arbor Acres broiler chickens were randomly divided into four treatments with twelve replicates comprising 10 chickens each, evenly divided by sex. The four groups were basal diet group (CAP0), treatment group 1 (CAP2), treatment group 2 (CAP3), and treatment group 3 (CAP4). The broilers in the CAP0 group were fed a basal diet (without CAP), while those in the CAP2, CAP3, and CAP4 groups received diets containing 2%, 3%, and 4% CAP, respectively. Growth performance results showed that dietary CAP supplementation significantly ameliorated the feed conversion rate (FCR) of broilers at 42 days in the CAP3 and CAP4 groups (p < 0.05). Microbial results revealed that CAP did not alter the dominant microorganisms in the cecum at the phylum, family, and genus levels. LEfSe analysis showed significantly higher relative abundances of p_Desulfobacterota, f_Desulfovibrionaceae, and g_Ruminococcus in the CAP3 group compared to the CAP0 and CAP4 groups. Metabolomic analyses indicated that the effect of incorporating CAP into the diet on serum metabolites primarily focused on organic acids and their derivatives, small peptides, amino acid derivatives, and oxidized lipids. The addition of 3% or 4% CAP to the diet can enhance metabolic pathways such as the citrate cycle (TCA cycle) and arginine and proline metabolism. In summary, incorporating CAP into the diet can increase the relative abundance of beneficial bacteria in the cecum and improve the feed conversion efficiency of broilers by enhancing amino acid and energy metabolism.

Avian coccidiosis, caused by protozoan parasites of the genus Eimeria, is a globally prevalent and highly pathogenic disease that poses a serious threat to the poultry industry, resulting in significant economic losses. However, the mechanism by which Eimeria species invade host cells remains unclear. Previous studies have identified rhoptry neck protein 2 (RON2) from Eimeria tenella as a critical factor in host cell invasion, but a comprehensive understanding of the role of EtRON2 in host cell invasion and its relationship with E. tenella invasion is lacking. To address this gap, this study focused on the secreted protein EtRON2 from E. tenella and its interaction with host cell receptors. The receptor interacting with the EtRON2 protein was identified through a GST pull-down assay, followed by mass spectrometry, and the interaction was further validated through subcellular co-localization analysis. Furthermore, sporozoites and host cells were treated with a specific antibody targeting the EtRON2/annexin A2 interaction, and the invasion rate of E. tenella was assessed using RT-qPCR to analyze the effect of inhibiting this interaction on host cell invasion by E. tenella. Annexin A2 protein, located on the surface of the host cell membrane, was screened, and the results of the sporozoite invasion assay revealed that inhibiting the interaction between these two proteins reduced F-actin aggregation. Understanding the interaction between the EtRON2 protein and its receptor during parasite invasion could help elucidate the function of the EtRON2 receptor and provide a theoretical foundation for further studies on the invasion mechanisms of E. tenella and the prevention and control of avian coccidiosis.

Pig breeding is a crucial sector of the global economy, playing a significant role in meat production. However, the prevalence of Trichomonas spp., a group of parasites known to induce diarrhea in various hosts, presents significant challenges in breeding facilities. These parasites pose a substantial threat to the pig breeding industry. Furthermore, despite its prevalence, diagnosing Trichomonas spp. is often challenging, primarily owing to the presence of mixed infections involving different species within clinical samples. To address this concern, we developed a novel isolation method that combines a single-cell isolation culture technique with an antimicrobial drug susceptibility test. Trichomonas was isolated and cultured by using the established single-worm separation technology combined with antibacterial drug screening method, and it was identified as Pentatrichomonas hominis by molecular biological identification and morphological identification. The in vitro culture conditions of the isolate were optimized to establish a stable in vitro culture system. The method developed in this study was effective in successfully isolating a pure species of trichomonad from fecal samples obtained from weaned piglets in Guangdong Province. By optimizing important variables such as the culture medium, serum type, and inoculum quantity, we established a stable in vitro culture system utilizing a modified Diamond medium supplemented with 10

Sexual development is an important process in eukaryotic organisms that involves the fusion of gametes to produce genetically diverse offspring. In apicomplexan parasites, sexual development is crucial for their life cycle and transmission. This paper reviews historical advancements in the understanding of sexual development in these parasites and discusses future research directions. Key discoveries related to gamete production, fertilization, and the formation of infectious stages are explored. Additionally, potential avenues for further investigation are proposed, including the molecular mechanisms governing sexual development, the role of host factors, and the influence of environmental cues. This review aims to help future research and improve our understanding of the complex sexual development processes in apicomplexan parasites by highlighting past achievements and outlining future research directions.

Coccidiosis is a costly intestinal disease of chickens caused by Eimeria species. This infection is associated with high mortality, reduced feed efficiency, and slowed body weight gain. The diagnosis and control of coccidiosis becomes challenging due to the fact that chickens can be infected by seven different Eimeria species and often occur mixed-species co-infections. Grasping the epidemiology of Eimeria species is crucial to estimate the efficiency of poultry management. This study aimed to explore the distribution of Eimeria species in broiler chickens in China after administering live anticoccidial vaccines. A total of 634 samples were obtained, and the survey results showed that the prevalence of Eimeria was 86.12% (546/634), and the most common species were E. acervulina (65.62%), E. necatrix (50.95%), E. mitis (50.79%), E. tenella (48.42%), and E. praecox (41.80%). Most samples indicated mixed-species infections (an average of 3.29 species per positive sample). Notably, 63.98% of samples contain 3 to 5 Eimeria species within a single fecal sample. The most prevalent combinations were E. acervulina–E. tenella (38.96%) and E. acervulina–E. necatrix (37.22%). Statistical analysis showed that flocks vaccinated with trivalent vaccines were significantly positive for E. necatrix in grower chickens (OR = 3.30, p < 0.05) compared with starter chickens, and tetravalent vaccinated flocks showed that starter chickens demonstrated a higher susceptibility to E. tenella–E. brunetti (OR = 2.03, p < 0.05) and E. acervulina–E. maxima (OR = 2.05, p < 0.05) compared with adult chickens. Geographically, in the case of tetravalent vaccine-immunized flocks, a substantial positive association was observed between E. necatrix infection rates and flocks from eastern (OR = 3.88, p < 0.001), central (OR = 2.65, p = 0.001), and southern China (OR = 3.17, p < 0.001) compared with southwestern China. This study also found a positive association between E. necatrix (OR = 1.64, p < 0.05), E. acervulina (OR = 1.59, p < 0.05), and E. praecox (OR = 1.81, p < 0.05) infection and coccidiosis occurrence compared with non-infected flocks in tetravalent vaccinated flocks. This molecular epidemiological investigation showed a high prevalence of Eimeria species in the field. The emergent species, E. brunetti and E. praecox, might be incorporated into the widely-used live vaccines in the future. These insights could be useful in refining coccidiosis control strategies in the poultry industry.

Trichomonas gallinae is a protozoa that parasitizes the upper gastrointestinal and respiratory tracts of various animals and birds, including Columbidae, Passeriformes, and Falconiformes. Polymerase chain reaction-based T. gallinae ITS1/5.8S/ITS2 gene typing yields inconsistent results owing to methodological differences. To standardize the statistical analysis of T. gallinae genotype distributions, this study employed MEGA-X software with the Tamamura 3-parameter (T92) + G model in the neighbor-joining method, with 2,000 bootstrap replicates, to calculate a systematic evolutionary tree. The resulting tree comprised 12 branches, ITS-OBT-Tg-1 to ITS-OBT-Tgl, with similar phylogenetic relationships. Relevant literature review yielded T. gallinae prevalence data in Columbidae. Statistical analysis was conducted from two perspectives: non-biological and biological factors, using chi-square tests and ordered logistic regression analysis. T. gallinae positivity rates differed significantly across diverse regions (χ2 = 4,609.9, P = 0.000, df = 4) and at various times (χ2 = 2,810.8, P = 0.000, df = 3). However, temperature and precipitation did not significantly affect T. gallinae positivity rates. Additionally, T. gallinae positivity rates differed significantly among diverse hosts (χ2 = 2,958.6, P = 0.000, df = 14) and by host age (χ2 = 478.5, P = 0.000, df = 2) and sex (χ2 = 96.00, P = 0.000, df = 1). This comprehensive analysis aimed to control T. gallinae transmission, reduce economic and species resource losses, and provide a foundation for future related research.

Pentatrichomonas hominis is a common intestinal parasitic protozoan that causes abdominal pain and diarrhea, and poses a zoonotic risk. Probiotics, known for enhancing immunity and pathogen resistance, hold promise in combating parasitic infections. This study aimed to evaluate two porcine-derived probiotics, Lactobacillus reuteri LR1 and Lactobacillus plantarum LP1, against P. hominis infections in pigs. Taxonomic identity was confirmed through 16 S rRNA gene sequencing, with L. reuteri LR1 belonging to L. reuteri species and L. plantarum LP1 belonging to L. plantarum species. Both probiotics exhibited robust in vitro growth performance. Co-culturing intestinal porcine epithelial cell line (IPEC-J2) with these probiotics significantly improved cell viability compared with the control group. Pre-incubation probiotics significantly enhanced the mRNA expression of anti-oxidative response genes in IPEC-J2 cells compared with the PHGD group, with L. reuteri LR1 and L. plantarum LP1 significantly up-regulating CuZn-SOD、CAT and Mn-SOD genes expression (p < 0.05). The anti-oxidative stress effect of L. reuteri LR1 was significantly better than that of L. plantarum LP1 (p < 0.05). Furthermore, pre-incubation with the probiotics alleviated the P. hominis-induced inflammatory response. L. reuteri LR1 and L. plantarum LP1 significantly down-regulated IL-6、IL-8 and TNF-α gene expression(p < 0.05) compared with the PHGD group. The probiotics also mitigated P. hominis-induced apoptosis. L. reuteri LR1 and L. plantarum LP1 significantly down-regulated Caspase3 and Bax gene expression (p < 0.05), significantly up-regulated Bcl-2 gene expression (p < 0.05) compared with the PHGD group. Among them, L. plantarum LP1 showed better anti-apoptotic effect. These findings highlight the probiotics for mitigating P. hominis infections in pigs. Their ability to enhance anti-oxidative responses, alleviate inflammation, and inhibit apoptosis holds promise for therapeutic applications. Simultaneously, probiotics can actively contribute to inhibiting trichomonal infections, offering a novel approach for preventing and treating diseases such as P. hominis. Further in vivo studies are required to validate these results and explore their potential in animal and human health.

Trichomonas gallinae, a globally distributed protozoan parasite, significantly affects the pigeon-breeding industry. T. gallinae infection mainly causes yellow ulcerative nodules on the upper respiratory tract and crop mucosa of pigeons, impeding normal breathing and feeding and ultimately causing death. Real-time quantitative PCR (qPCR) is a crucial technique for gene-expression analysis in molecular biology. Reference-gene selection for normalization is critical for ensuring this technique's accuracy. However, no systematic screening or validation of T. gallinae reference genes has been reported. This study quantified the transcript levels of ten candidate reference genes in T. gallinae isolates with different genotypes and culture conditions using qPCR. Using the geNorm, NormFinder, and BestKeeper algorithms, we assessed these reference genes' stabilities and ranked them using RankAggreg analysis. The most stable reference gene was tubulin beta chain (TUBB), while the widely used reference genes TUBG and GAPDH demonstrated poor stability. Additionally, we evaluated these candidate reference genes' stabilities using the T. gallinae TgaAtg8 gene. On using TUBB as a reference gene, TgaAtg8's expression profiles in T. gallinae isolates with different genotypes remained relatively consistent under various culture conditions. Conversely, using ACTB as a reference gene distorted the data. These findings provide valuable reference-gene-selection guidance for functional gene research and gene-expression analysis in T. gallinae.

Background Coccidiosis is one of the most frequently reported disease in chickens, exerting a substantial economic impact on the poultry industry. This study aims to conduct an epidemiological investigation into the occurrence of Eimeria species and associated risk factors under intensive management conditions across four regions in Guangdong province, China. Results A total of 394 fecal samples were obtained from 89 broiler chicken farms, culminating in an overall positivity rate of 87.06%. The results showed that the identification of all seven Eimeria species, with E. acervulina (36.29%), E. mitis (35.03%), E. tenella (34.52%) and E. necatrix (30.96%) emerging as the most prevalent species. Remarkably, single-species infections were observed in 42.86% of instances, while two to three species mixed infections were detected in 39.94% of the samples. Moreover, brid age, farming practices, control strategies, farm locations, and the presence of necrotic enteritis (NE) proved significant risk factors. Notably, a strong correlation was observed between brid age, particularly in adult birds, and the occurrence of E. necatrix ( p < 0.001). A significant correlation was identified between the infection of E. necatrix or E. acervulina and the presence of NE in flocks ( p < 0.001). Flocks from northern Guangdong and Peal River delta displayed higher prevalence of E. necatrix ( p < 0.05). Flocks under the control programs incorporating live vaccines correlated strongly with E. tenella – E. brunetti infections ( p < 0.05). Conclusions Molecular analysis undertaken in this study, coupled with the correlation results, furnishes compelling evidence. Nevertheless, it is imperative to underscore the necessity for further surveys to delve deeper into the occurrence of different Eimeria species under intensive management conditions, which will contribute significantly to our knowledge of coccidia control in poultry.

Clostridium perfringens, a Gram-positive bacterium, causes intestinal diseases in humans and livestock through its toxins, related to alpha toxin (CPA), beta toxin (CPB), C. perfringens enterotoxin (CPE), epsilon toxin (ETX), Iota toxin (ITX), and necrotic enteritis B-like toxin (NetB). These toxins disrupt intestinal barrier, leading to various cell death mechanisms such as necrosis, apoptosis, and necroptosis. Additionally, non-toxin factors like adhesins and degradative enzymes contribute to virulence by enhancing colonization and survival of C. perfringens. A vicious cycle of intestinal barrier breach, misregulated cell death, and subsequent inflammation is at the heart of chronic inflammatory and infectious gastrointestinal diseases. Understanding these mechanisms is essential for developing targeted therapies against C. perfringens-associated intestinal diseases.

Avian trichomoniasis, caused by the protozoan parasite Trichomonas gallinae, is a prevalent and economically significant disease in pigeons. This study investigated the drug resistance of T. gallinae isolates in Guangdong Province, China. The results revealed that 25.3% (20/79) of the isolates were resistant to one or more of the four nitroimidazole drugs tested, namely, metronidazole, dimetridazole, secnidazole, and tinidazole. Secnidazole elicited the highest resistance rate (19.0%; 15/79), followed by tinidazole (17.7%; 14/79), metronidazole (17.7%; 14/79), and dimetridazole (13.9%; 11/79). An enormous majority of the resistant isolates (70.0%; 14/20) exhibited resistance to multiple drugs. Additionally, the resistance rate was significantly higher in isolates from birds aged < 30 days (53.3%; 8/15) than in those from older birds (23.1%; 12/52). Moreover, no drug resistance was detected in female pigeons. The genotype of the isolated strain was also associated with drug resistance. Specifically, 50.0% (15/30) of ITS-B genotypes exhibited resistance to drugs, while only 10.2% (5/49) of ITS-A genotypes demonstrated resistance. This study also found the growth characteristics of different Trichomonas isolates to be influenced by their genotypes and initial inoculum concentrations. These findings underscore the urgent need for effective measures to control and prevent drug-resistant T. gallinae infections in pigeons, thus ensuring the stable development of the pigeon industry.

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve’s slope of −3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method’s application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method’s robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Background The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. Methods In this study, we use the SV40 large T antigen ( SV40T ) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. Results This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. Conclusions An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite–host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract. Graphical Abstract

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