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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.

Background Coccidiosis, a prominent intestinal protozoan disease, carries significant economic implications for the poultry industry. The aim of this study was to evaluate the effects of Fengqiang Shengtai (BLES), a probiotics product, and coccidiosis vaccine in modulating the intestinal microbiome and providing insight into mitigating the occurrence and management of avian coccidiosis. Methods Broilers included in the study were divided into four pre-treatment groups: the Pre-Con group (commercial diet), Pre-BLES group (BLES supplement), Pre-Vac group (coccidiosis vaccination) and Pre-Vac-BLES group (combined vaccination and BLES). Body weight gain, feed consumption and feed conversion ratio were monitored from age 25 to 55 days. Cecum contents were collected at 8 and 15 days of age for comparative analysis of intestinal microbiomes. In the Pre-BLES and Pre-Vac-BLES groups, probiotics were administered at a dose of 0.01 g per chicken between ages 3 to 6 days and 10–13 days. At 3 days of age, chickens in the Pre-Vac and Pre-Vac-BLES groups were vaccinated with 1700 sporulated oocysts of the live coccidiosis vaccine per chicken. At the age of 25 days, Eimeria spp. challenge experiments were performed based on the aforementioned immunization strategy, and the oocysts per gram (OPG) in the feces, intestinal lesion score and intestinal pathological characteristics were evaluated. Specifically, 30 chickens were randomly selected from each group and orally administered 34,000 sporulated oocysts of Eimeria spp. per chicken, re-defined as Eimeria group, BLES-Eimeria group, Vac-Eimeria group and Vac-BLES-Eimeria group, respectively. Additionally, 30 chickens were randomly selected from the Pre-Con group and included as negative control without Eimeria spp. challenge. Intestinal microbiota was sequenced and analyzed when the broilers were 32 days old. Results A significant improvement was observed in body weight gain of the broilers in the Pre-BLES and Pre-Vac-BLES group at 45 days of age. Analysis of the intestinal microbiota revealed a positive correlation between the experimental groups receiving BLES and coccidiosis vaccines at 8 and 15 days of age with the Enterococcus genus and Lachnospiraceae NK4A136 group, respectively. In addition to the reduced lesion score and OPG values, the combination of coccidiosis vaccine and BLES also reduced the intestinal epithelial abscission induced by coccidiosis vaccines. The results of intestinal microbial function prediction demonstrated that N-glycan biosynthesis and ferroptosis were the prominent signal pathways in the Vac-BLES-Eimeria group. Conclusions Taken together, the results of the present study suggest that supplementation of BLES with coccidiosis vaccine represents a promising strategy for improving growth performance, alleviating clinical manifestations and inducing favorable alterations to the intestinal microbiota in broiler chickens affected by coccidiosis.

Background Cryptosporidium is one of the most prevalent parasites infecting both birds and mammals. To examine the prevalence of Cryptosporidium species and evaluate the public health significance of domestic chickens in Guangdong Province, southern China, we analyzed 1001 fecal samples from 43 intensive broiler chicken farms across six distinct geographical regions. Methods Individual DNA samples were subjected to nested PCR-based amplification and sequencing of the small subunit of the nuclear ribosomal RNA gene ( SSU rRNA). Analysis of the 60 kDa glycoprotein gene ( gp 60) was performed to characterize the subtypes of C. meleagridis . Results The overall prevalence of Cryptosporidium was 13.2% (95% CI 11.1–15.3) (24 of 43 farms), with C. meleagridis (7.8%), C. baileyi (4.8%) and mixed infections (0.6%). Using the gp 60 gene, three subtype families, IIIb, IIIe and IIIg, were identified, including six subtypes: one novel (IIIgA25G3R1a) and five previously reported (IIIbA23G1R1c, IIIbA24G1R1, IIIbA21G1R1a, IIIeA17G2R1 and IIIeA26G2R1). Within these subtypes, five known subtypes were genetically identical to those identified in humans. Conclusions This is the first report of C. meleagridis in chickens from Guangdong. The frequent occurrence of C. meleagridis in domestic chickens and the common C. meleagridis subtypes identified in both humans and chickens is of public health significance. Our study indicates that broiler chickens represent a potential zoonotic risk for the transmission of Cryptosporidium in this region. Graphical Abstract

Background Enterocytozoon bieneusi , a common opportunistic fungal pathogen, has a wide range of hosts. Limited epidemiological data on E. bieneusi intestinal infections in companion animals (dogs and cats) in China exists. In this study, fecal samples (651 from dogs and 389 from cats) in Guangzhou city, Guangdong Province, China, were collected, and the ribosomal internal transcribed (ITS) spacer region from the DNA extracted from them was Polymerase Chain Reaction (PCR)-amplified and sequenced. Results Based on the sequencing data, E. bieneusi was identified in the fecal samples collected from 149 (22.9%) and 79 (20.3%) dogs and cats. Of the factors investigated, poor living conditions appeared to be the major risk factor for contracting the pathogen. Eleven E. bieneusi genotypes, six known (PtEb IX, GD1, D, CD9, EbpC, I) and five novel (designated here as GD2– GD6), were found in dogs. Eight genotypes, six known (PtEb IX, GD1, D, CD9, EbpC, Type IV) and two novel (GD2 and GC1), were identified in cats. Genotype PtEb IX was most common in both dogs and cats, followed by genotype GD1. Conclusions Although PtEb IX was the most common E. bieneusi genotype in dogs, this is the first report of this genotype dominating in cats. The same genotype distribution of the pathogen between the two different companion animals species in the same geographic area indicates that inter-species transmission is probable. The widespread existence of zoonotic E. bieneusi genotypes (D, EbpC, Type IV) in companion animals indicates that they are potential sources of environmental contamination and infections in humans.

Background Coccidiosis is one of the most frequently reported diseases in chickens, causing a significant economic impact on the poultry industry. However, there have been no previous studies evaluating the prevalence of this disease in broiler farms in Guangdong province. Therefore, this study aims to conduct an epidemiological investigation into the occurrence of Eimeria species and associated risk factors in intensive management conditions across four regions in Guangdong province, China. A total of 394 fecal samples were collected from 89 broiler farms in Guangdong province. The prevalence of Eimeria species infection was determined using PCR, and the occurrence of Clostridium perfringens type A was assessed using quantitative real-time PCR. Results The results showed an overall prevalence of 98.88% (88/89) at the farm level and 87.06% (343/394) at the flock level. All seven Eimeria species were identified, with E. acervulina (72.53%; 64/89), E. tenella (68.54%; 61/89), and E. mitis (66.29%; 59/89) at the farm level, and E. acervulina (36.55%; 144/394), E. mitis (35.28%; 139/394), and E. tenella (34.01%; 134/394) at the flock level. The predominant species combination observed was a co-infection of all seven Eimeria species (6.74%; 6/89), followed by a combination of E. acervulina , E. tenella , E. mitis , E. necatrix , E. brunetti , and E. maxima (5.62%, 5/89). A combination of E. acervulina , E. tenella , E. mitis , E. necatrix , E. brunetti , and E. praecox (4.49%; 4/89) was also observed at the farm level. Furthermore, the study identified several potential risk factors associated with the prevalence of Eimeria species, including farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were identified as potential risk factors associated with prevalence of Eimeria species. Univariate and multivariate analyses revealed a significant association between E. necatrix infection and both grower chickens (OR = 10.86; 95% CI: 1.92–61.36; p  < 0.05) and adult chickens (OR = 24.97; 95% CI: 4.29–145.15; p  < 0.001) compared to starter chickens at the farm level. Additionally, farms that used groundwater (OR = 0.27; 95% CI: 0.08–0.94; p  < 0.05) were less likely to have E. maxima compared to those that used running water. At the flock level, the prevalence of E. tenella was significantly higher in the Pearl River Delta (OR = 2.48; 95% CI: 1.0–6.15; p  = 0.05) compared to eastern Guangdong. Interestingly, flocks with indigenous birds were less likely to have E. brunetti (OR = 0.48; 95% CI: 0.26–0.89; p  < 0.05) compared to flocks with indigenous crossbred birds. Furthermore, flocks that used anticoccidial drugs (OR = 0.09; 95% CI: 0.03–0.31; p  < 0.001) or a combination of vaccines and anticoccidial drugs (OR = 0.06; 95% CI: 0.01–0.25; p  < 0.001) were less likely to be positive for E. tenella compared to flocks that only used vaccines. Finally, flocks with C. perfringens type A infection were significantly more likely to have E. necatrix (OR = 3.26; 95% CI: 1.96–5.43; p  < 0.001), E. tenella (OR = 2.14; 95% CI: 1.36–3.36; p  < 0.001), E. brunetti (OR = 2.48; 95% CI: 1.45–4.23; p  < 0.001), and E. acervulina (OR = 2.62; 95% CI: 1.69–4.06; p  < 0.001) compared to flocks without C. perfringens type A. Conclusions This study conducted an investigation on the prevalence, distribution, and risk factors associated with Eimeria species infection in broiler chickens in Guangdong. The farm-level prevalence of Eimeria species was higher than the previous prevalence figures for other areas and countries. E. brunetti was identified at higher prevalence in Guangdong than previously survived prevalence in different regions in China. Farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were considered as potential risk factors associated with prevalence of Eimeria species. It is imperative to underscore the necessity for further surveys to delve deeper into the occurrence of Eimeria species under intensive management conditions for different flock purposes.

Background 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. Methods 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. Results 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% Procell fetal bovine serum without the use of antibiotics. Subsequent analysis of the isolate’s 18S rRNA gene, ITS1-5.8S rRNA-ITS2 gene, and EF-α gene, through polymerase chain reaction, DNA sequencing, and phylogenetic analysis, revealed its close association to Pentatrichomonas hominis . Light microscopy and scanning electron microscopy demonstrated the presence of various distinct cellular structures, including four anterior flagella, recurrent flagellum, undulating membrane, pelta and axostyle. Additionally, transmission electron microscopy revealed the existence of organelles such as the Golgi complex, rough endoplasmic reticulum, food vacuoles, and hydrogenosomes. This study represents the first successful isolation of monoclonal cells of P. hominis to our knowledge and serves as a valuable baseline for future research focused on the isolation and purification of various other parasites. Additionally, it offers practical guidance for the diagnosis and management of Trichomonas spp. infections in pigs. Conclusions In summary, our findings underscore the efficacy of our novel isolation technique as a valuable tool for the diagnosis and management of Trichomonas spp. infections, which can help mitigate the significant economic losses encountered in the pig breeding industry. Graphical Abstract

Background Necrotic enteritis (NE) is an infectious intestinal disease caused by Clostridium perfringens ( C. perfringens ) that is now re-emerging and causing concern within the poultry industry. Previously, the supplementation of antibiotics in feed was the most popular control strategy against C. perfringens . However, with the ban on supplementing growth-promoting antibiotics in livestock feed, alternatives to antibiotics will be essential in order to control necrotic enteritis. A possible alternative to antibiotics could be the medium or long chain fatty acids (MCFA or LCFA) as these are able to destroy cell membranes which in turn results in the death of bacteria. In this study, the in vitro antimicrobial activity of different combinations with microencapsulated caprylic acid (C8: 0), capric acid (C10: 0), lauric acid (C12: 0) and myristic acid (C14: 0) against C. perfringens and in vivo control the NE-inducing C. perfringens in broiler chicken were analyzed. Results The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay results revealed that three different combinations of medium/long chain fatty acids varied in antimicrobial activities against C. perfringens type A strain (CVCC52, quality control), C. perfringens type A strain (C8-1), C. perfringens type G strain (D25) and C. perfringens type G strain (MZ1). Specifically, combination of C12: 0 and C14: 0 (C12–14) showed the highest antimicrobial activity against the four strains of C. perfringens (MIC ≤ 12.5 μg/mL, MBC = 50 μg/mL), followed by the combination of C10: 0 and C12: 0 (C10–12) (MIC, MBC ≤ 50 μg/mL). The in vivo study, 189 of 818-crossbred chickens that were fed a wheat-based diet and randomly divided into nine groups, with six treatment groups supplemented with either a high dose (1 g/kg) or low dose (0.5 g/kg) of three combinations respectively. The remaining three groups comsisted of a positive group supplement with avilamycin (0.01 g/kg), an infected control and an uninfected control. All chickens were challenged with C. perfringens from day 14 to day 17, except those in the uninfected control group. On day 20, the duodenum and jejunum necrotic lesions scores were calculated and the results showed that there was significant decrease in the C12–C14 high dose group (1.43 ± 0.23, 0.48 ± 0.13) and the C10–12 high dose group (1.52 ± 0.19, 0.48 ± 0.11) compared to the infected group (2.86 ± 0.21, 1.20 ± 0.28). Conclusions This finding indicated that dietary microencapsulated C12–C14 and C10–C12 could inhibit the growth of C. perfringens in chickens, which proves is viability to serve as an alternative to antibiotics used for necrotic enteritis caused by C. perfringens .

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. Pentatrichomonas hominis est un parasite protozoaire qui infecte le tractus gastro-intestinal des humains et des mammifères, provoquant douleurs abdominales et diarrhées. Cependant, sa présence chez les porcs et son potentiel pathogène causant des diarrhées chez les porcelets n’ont pas été bien étudiés. Cette étude visait à évaluer la prévalence de P. hominis chez les porcs et son potentiel de transmission zoonotique. Au total, 406 échantillons fécaux de porcs ont été collectés dans quatre élevages porcins des provinces du Guangdong et de l’Anhui. L’extraction de l’ADN fécal a été réalisée à l’aide d’un kit disponible dans le commerce. La méthode de PCR nichée a été utilisée pour détecter la présence d’une infection à P. hominis . Les échantillons positifs ont ensuite été séquencés et les caractéristiques génétiques du gène de l’ARN ribosomique (ARNr) 18S ont été analysées. Le taux d’infection global de P. hominis était de 34,98 % (142/406), avec 28,47 % (80/281) dans la province du Guangdong et 49,60 % (62/125) dans la province de l’Anhui. Parmi les différents groupes d’âge, les porcelets allaités présentaient le taux d’infection le plus élevé, soit 40,24 % (68/169). L’analyse génétique des isolats de P. hominis a montré que le génotype PH-1 était prédominant et présentait un degré élevé de similitude avec les séquences de P. hominis obtenues chez l’homme, le chat et le porc, indiquant un potentiel de transmission zoonotique. Le taux d’infection élevé et la diversité génétique soulignent la nécessité de mesures de contrôle efficaces dans les élevages porcins pour réduire la transmission des parasites et le risque zoonotique.

Pentatrichomonas hominis , a flagellated parasitic protozoan, predominantly infects the mammalian digestive tract, often causing symptoms such as abdominal pain and diarrhea. However, studies investigating its pathogenicity are limited, and the mechanisms underlying P. hominis -induced diarrhea remain unclear. Establishing an in vitro cell model for P. hominis infection is imperative. This study investigated the interaction between P. hominis and IPEC-J2 cells and its impact on parasite growth, adhesion, morphology, and cell viability. Co-cultivation of P. hominis with IPEC-J2 cells resulted in exponential growth of the parasite, with peak densities reaching approximately 4.8 × 10 5 cells/mL and 1.2 × 10 6 cells/mL at 48 h for initial inoculation concentrations of 10 4 cells/mL and 10 5 cells/mL, respectively. The adhesion rate of P. hominis to IPEC-J2 cells reached a maximum of 93.82% and 86.57% at 24 h for initial inoculation concentrations of 10 4 cells/mL and 10 5 cells/mL, respectively. Morphological changes in IPEC-J2 cells co-cultivated with P. hominis were observed, manifesting as elongated and irregular shapes. The viability of IPEC-J2 cells exhibited a decreasing trend with increasing P. hominis concentration and co-cultivation time. Additionally, the mRNA expression levels of IL-6, IL-8, and TNF-α were upregulated, whereas those of CAT and CuZn-SOD were downregulated. These findings provide quantitative evidence that P. hominis can promote its growth by adhering to IPEC-J2 cells, inducing morphological changes, reducing cell viability, and triggering inflammatory responses. Further in vivo studies are warranted to confirm these results and enhance our understanding of P. hominis infection. Pentatrichomonas hominis , un protozoaire parasite flagellé, infecte principalement le tube digestif des mammifères, provoquant souvent des symptômes tels que des douleurs abdominales et de la diarrhée. Cependant, les études portant sur sa pathogénicité sont limitées et les mécanismes sous-jacents à la diarrhée induite par P. hominis restent flous. L’établissement d’un modèle cellulaire in vitro de l’infection à P. hominis est impératif. Cette étude a examiné l’interaction entre P. hominis et les cellules IPEC-J2 et son impact sur la croissance du parasite, l’adhésion, la morphologie et la viabilité cellulaire. La co-culture de P. hominis avec des cellules IPEC-J2 a entraîné une croissance exponentielle du parasite, avec des densités maximales atteignant environ 4,8 × 10 5 cellules/mL et 1,2 × 10 6 cellules/mL à 48 h pour des concentrations d’inoculation initiales de 10 4 cellules/mL et 10 5 cellules/mL, respectivement. Le taux d’adhésion de P. hominis aux cellules IPEC-J2 a atteint un maximum de 93,82 % et 86,57 % après 24 h pour des concentrations d’inoculation initiales de 10 4 cellules/mL et 10 5 cellules/mL, respectivement. Des changements morphologiques dans les cellules IPEC-J2 co-cultivées avec P. hominis ont été observés, se manifestant par des formes allongées et irrégulières. La viabilité des cellules IPEC-J2 a montré une tendance à la baisse avec l’augmentation de la concentration de P. hominis et de la durée de co-culture. De plus, les niveaux d’expression d’ARNm d’IL-6, d’IL-8 et de TNF-α étaient régulés positivement, tandis que ceux de CAT et de CuZn-SOD étaient régulés négativement. Ces résultats fournissent des preuves quantitatives que P. hominis peut favoriser sa croissance en adhérant aux cellules IPEC-J2, en induisant des changements morphologiques, en réduisant la viabilité cellulaire et en déclenchant des réponses inflammatoires. D’autres études in vivo sont nécessaires pour confirmer ces résultats et améliorer notre compréhension de l’infection à P. hominis .

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.