Prevalence, geographic distribution and risk factors of Eimeria species on commercial broiler farms in Guangdong, China.

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.

Metabolic Blockade-Based Genome Mining of Sea Anemone-Associated Streptomyces sp. S1502 Identifies Atypical Angucyclines WS-5995 A-E: Isolation, Identification, Biosynthetic Investigation, and Bioactivities.

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A-E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A-E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A-E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 µM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A-E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.

Mechanisms of intestinal epithelial cell damage by Clostridiumperfringens.

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.

Prevalence and diversity of Trichomonas gallinae in meat pigeons (Columba livia) in Guangdong Province, People's Republic of China.

Pigeon farming for meat has developed into an important economic industry in most countries, especially in China. Trichomoniasis, caused by the protozoan parasite Trichomonas gallinae, is a worldwide disease in pigeons. However, studies of the prevalence and distribution of T. gallinae lineages in domestic pigeons in southern China are limited. In this study, a total of 636 pigeon throat swabs samples from four regions in Guangdong Province were screened for T. gallinae by in vitro culture assays and microscopy. The results revealed an overall prevalence of T. gallinae infection in southern China of 26.6% (169/636). There were significant differences in the infection rate of T. gallinae between the four regions (χ2 = 117.948, df = 4, P = 0.000), with up to 44.6% in the Pearl River Delta region. The infection rate of young pigeons was as high as 70.8%. The rDNA sequences (18S rRNA/ITS1-5.8S rRNA-ITS2) of 153 positive samples were amplified and sequenced. Results identified 58.2% (89/153) overall as ITS-A (18S-VI) (also known as ITS-OBT-Tg-1) and 41.8% (64/153) as ITS-B (18S-IV) (also known as ITS-OBT-Tg-2). Thus, ITS-A (18S-VI) was the dominant T. gallinae genotype in southern China, especially in young pigeon (97.0%, 32/33). In conclusion, a high prevalence of T. gallinae infection in domestic pigeons was identified in southern China, particularly in the Pearl River Delta region. The ITS-A (18S-VI) was the dominant genotype highly pathogenic, which may weaken the immune system of pigeons, and cause a negative impact on the development of the pigeon industry in China.

Occurrence, risk factors and genotypes of Enterocytozoon bieneusi in dogs and cats in Guangzhou, southern China: high genotype diversity and zoonotic concern.

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.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

Molecular and biochemical characterization of Eimeria tenella hexokinase.

Hexokinase (HK) is one of the key enzymes in the glycolytic pathway that catalyzes the phosphorylation of glucose. In the present study, we cloned the HK gene from the coccidian Eimeria tenella (EtHK), expressed EtHK as a His-tagged fusion protein, and characterized its primary biochemical features. Mutagenesis confirmed that residues S159, N216, and D217 are essential or important to the EtHK catalytic activity. EtHK exhibited high affinity for D-glucose (Km = 0.67 to 0.79 mM), but was also able to utilize 2-deoxy-D-glucose (Km = 5.66 mM), D-fructose (Km = 13.76 mM), and D-mannose (Km = 25.41 mM). We also observed that quercetin and mangiferin could inhibit the EtHK enzyme activity (IC50 values = 6.52 and 85.82 µM, respectively). Among the two inhibitors, mangiferin also inhibited the growth of E. tenella in vitro (MIC50 = 0.12 µM). These observations suggest that EtHK may be explored as potential drug target, and mangiferin and its analogs may be explored for developing anti-coccidial therapeutics.

Molecular characterization of Cryptosporidium spp. in domestic pigeons (Columba livia domestica) in Guangdong Province, Southern China.

To investigate the prevalence and assess the zoonotic transmission burden of Cryptosporidium species in domestic pigeons in Guangdong Province, Southern China, 244 fecal samples were collected from four pigeon breeding farms between June 2012 and March 2013. Cryptosporidium oocysts were purified by Sheather's sugar flotation technique and characterized by DNA sequencing of small subunit ribosomal RNA (SSU rRNA) gene. Cryptosporidium species were determined by comparison of sequences with corresponding Cryptosporidium sequences in GenBank and phylogenetic analysis using neighbor-joining (NJ) in MEGA5.2. The overall prevalence of Cryptosporidium infection in domestic pigeons in Guangdong Province was 0.82% (2/244). Two Cryptosporidium species, namely Cryptosporidium baileyi and Cryptosporidium meleagridis, were identified in Huizhou and Chaozhou farm, respectively. These findings confirmed the existence of C. meleagridis infection in domestic pigeons in China for the first time and provided base-line information for further studies to evaluate the public health risk from pigeon to human.

A novel thermophilic strain of Bacillus subtilis with antimicrobial activity and its potential application in solid-state fermentation of soybean meal.

Soybean meal (SBM) is the most important source of plant protein in animal feeds, containing around 41%-48% crude protein. Nevertheless, 70%-80% of these proteins is allergenic antigens that can have adverse implications for the gastrointestinal well-being of animals, especially to young animals. Microbial fermentation is one of the most cost-effective strategies used to reduce allergenic antigens from plant sources. In this study, we report the isolation and characterization of a novel probiotic Bacillus subtilis "L5" strain from lake mud. L5 demonstrated remarkable temperature tolerance across a broad temperature spectrum, thriving at 25°C, 37°C, and 50°C. In addition, antimicrobial assay revealed that L5 exhibits strong antimicrobial activity against Escherichia coli, effectively reducing or eliminating the growth of Gram-negative bacteria in SBM when fermented with L5. When applied to SBM fermentation, L5 efficiently reduced SBM antinutritional factors such as glycinin, ß-conglycinin, trypsin inhibitor, phytic acid, neutral detergent fiber, and acid detergent fiber, which in turn results in an increase in crude protein content and the free amino acid concentration. Our findings on the probiotic and fermentation capabilities of L5 suggest that this novel bacterium has dual functions that make it a strong candidate for improving the nutrient values of feed via its role in fermentation.IMPORTANCESoybean meal (SBM), containing 41%-48% crude protein, is the most important source of plant protein in animal feeds. Unfortunately, 70%-80% of the proteins in SBM is allergenic antigens including trypsin inhibition, ß-conglycinin, and conglycinin, which negatively affect intestine health and function. Microbial solid-state fermentation methods have been applied to animal feeds for decades, to eliminate antinutritional factors. Here, a novel potential probiotic Bacillus subtilis "L5" strain with high enzymatic activity and antimicrobial activity will be a great help to improve the quality and reproducibility of SBM fermentation.

MiR-29b inhibits COC expansion and oocyte in vitro maturation via induction of ROS by targeting CYCS.

Cumulus-oocyte complex (COC) expansion and oocyte maturation are crucial processes for embryo development and fertility across species. Although miR-29b has been detected in porcine ovarian granulosa cells, its specific role in regulating oocyte maturation remains largely unknown. In this study, using the pig as a model, we report that over-expression of miR-29b lead to a decrease of COC expansion area and inhibits oocyte maturation (P<0.05). This suppression correlated with a decrease expression of COC-expansion-associated genes, including SHAS2, ADAMTS1, ADAMTS2, ADAMTS17 and PTX 3 in both mural granulosa cells (mGCs) and cumulus granulosa cells (cGCs). Further investigation revealed that miR-29b over-expression induces reactive oxygen species (ROS) accumulation in both mGCs and cGCs, conversely, knock-down of miR-29b reverses all these effects. Treatment with the antioxidant ß-mercaptoethanol alleviates ROS accumulation, rescues COC expansion and restores oocyte polar body formation impaired by miR-29b mimics. Computational analysis predicted CYCS, the gene encoding cytochrome C, as a potential target of miR-29b. Subsequent examination demonstrated that miR-29b downregulates CYCS at both mRNA and protein levels. Dual-luciferase reporter assays further confirmed that miR-29b interacts with the 3'-untranslated region (3'UTR) of CYCS. Over-expression of CYCS decreases ROS accumulation and promotes COC expansion (P<0.05). These results indicate that miR-29b regulates COC expansion and oocyte maturation in vitro by inducing ROS, likely through targeting of CYCS. This study sheds light on the role of miR-29b in oocyte maturation and provides insight into the regulatory function of miRNAs in ovarian physiology.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

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.

Selection of a suitable reference gene for gene-expression studies in Trichomonas gallinae under various biotic and abiotic stress conditions.

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.

Alleviating Pentatrichomonas hominis-induced damage in IPEC-J2 cells: the beneficial influence of porcine-derived lactobacilli.

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.

Epidemiological investigation of coccidiosis and associated risk factors in broiler chickens immunized with live anticoccidial vaccines in China.

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.

Development of a TaqMan polymerase chain reaction detection method for the precise identification and quantification of an attenuated Eimeria maxima vaccine strain in poultry.

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.

Unraveling the pathogenic potential of the Pentatrichomonas hominis PHGD strain: impact on IPEC-J2 cell growth, adhesion, and gene expression.

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 × 105 cells/mL and 1.2 × 106 cells/mL at 48 h for initial inoculation concentrations of 104 cells/mL and 105 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 104 cells/mL and 105 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.

Title: Découvrir le potentiel pathogène de la souche PHGD de Pentatrichomonas hominis : impact sur la croissance, l'adhésion et l'expression des gènes des cellules IPEC-J2. Abstract: 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 × 105 cellules/mL et 1,2 × 106 cellules/mL à 48 h pour des concentrations d'inoculation initiales de 104 cellules/mL et 105 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 104 cellules/mL et 105 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.

Immunoprotective effects of DNA vaccine against Eimeria tenella based on EtAMA3 and EtRON2L2.

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 µg) of pcDNA3.1(+)-AR to evaluate its immunoprotective effects in vivo. The chickens in the 50 µg and 100 µg 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.

Genotypic diversity and epidemiology of Trichomonas gallinae in Columbidae: Insights from a comprehensive analysis.

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.

Isolation and characterization of a novel hydrolase-producing probiotic Bacillus licheniformis and its application in the fermentation of soybean meal.

Soybean meal (SBM) is one of the most important sources of plant-based protein in the livestock and poultry industry. However, SBM contains anti-nutritional factors (ANFs) such as glycinin, ß-conglycinin, trypsin inhibitor and phytic acid that can damage the intestinal health of animals, inevitably reducing growth performance. Fermentation using microorganisms with probiotic potential is a viable strategy to reduce ANFs and enhance the nutritional value of SBM. In this study, a novel potential probiotic Bacillus licheniformis (B4) with phytase, protease, cellulase and xylanase activity was isolated from camel feces. The ability of B4 to tolerate different pH, bile salts concentrations and temperatures were tested using metabolic activity assay. It was found that B4 can survive at pH 3.0, or 1.0% bile salts for 5 h, and displayed high proliferative activity when cultured at 50°C. Furthermore, B4 was capable of degrading glycinin, ß-conglycinin and trypsin inhibitor which in turn resulted in significant increases of the degree of protein hydrolysis from 15.9% to 25.5% (p < 0.01) and crude protein from 44.8% to 54.3% (p < 0.001). After fermentation with B4 for 24 h, phytic acid in SBM was reduced by 73.3% (p < 0.001), the neutral detergent fiber (NDF) and the acid detergent fiber of the fermented SBM were significantly decreased by 38.40% (p < 0.001) and 30.20% (p < 0.05), compared to the unfermented SBM sample. Our results suggested that the effect of solid-state fermented SBM using this novel B. licheniformis (B4) strain, could significantly reduce phytic acid concentrations whilst improving the nutritional value of SBM, presenting itself as a promising alternative to phytase additives.

Choline supplementation regulates gut microbiome diversity, gut epithelial activity, and the cytokine gene expression in gilts.

Choline is an essential nutrient that is necessary for both fetal development and maintenance of neural function, while its effect on female ovarian development is largely unexplored. Our previous study demonstrated that choline supplementation promotes ovarian follicular development and ovulation, although its underlying mechanism was unclear. To uncover the potential regulation pathway, eighteen female Yorkshire × Landrace gilts were fed with either standard commercial diet (Control group, n = 9) or choline supplemented diet (Choline group, additional 500 mg/kg of control diet, n = 9) from day 90 of age to day 186. At day 186, feces samples were analyzed for effects on the gut microbiome using 16S ribosomal RNA gene V3-V4 region sequencing with Illumina MiSeq, serum samples were analyzed for trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) using HILIC method, and jejunum tissues were analyzed for immune related gene expression using qRT-PCR. Our results show that choline supplementation did not alter the circulating level of TMA and TMAO (P > 0.05), but rather increased gut microbiome alpha diversity (P < 0.05). Beta diversity analysis results showed that the choline diet mainly increased the abundance of Firmicutes, Proteobacteria, and Actinobacteria, but decreased the abundance of Bacteroidetes, Spirochaetes, and Euryarchaeota at the phyla level. Meta-genomic analysis revealed that choline supplementation activated pathways in the gut microbiota associated with steroid hormone biosynthesis and degradation of infertility-causing environmental pollutants (bisphenol, xylene, and dioxins). To further verify the effect of choline on intestinal activity, a porcine intestine cell line (IPEC-J2) was treated with serial concentrations of choline chloride in vitro. Our data demonstrated that choline promoted the proliferation of IPEC-J2 while inhibiting the apoptotic activity. qRT-PCR results showed that choline significantly increased the expression level of Bcl2 in both IPEC-J2 cells and jejunum tissues. The expression of IL-22, a cytokine that has been shown to impact ovarian function, was increased by choline treatment in vitro. Our findings reveal the beneficial effect of choline supplementation on enhancing the gut microbiome composition and intestinal epithelial activity, and offer insights into how these changes may have contributed to the ovarian development-promoting effect we reported in our previous study.