Recombinant linear multiple epitopes of σB protein protect Muscovy ducks against novel duck reovirus infection.

Infection by the novel duck reovirus (NDRV) in ducklings causes high mortality, which leads to substantial economic losses in the duck industry in China. To date, no commercial vaccine is available for this disease. In this study, linear B cell epitopes of the σB protein of the NDRV were predicted and recombinant multiple linear B cell epitopes (MLBEs) were constructed through linkers. The recombinant MLBEs were then expressed and purified. One-day-old Muscovy ducklings were immunized with different doses of MLBEs and challenged with 5 × 104 ELD50 of the virulent CHY strain of NDRV 14 days after immunization. The ducklings vaccinated with 20 and 40 µg of MLBE performed no clinical signs or gross or histopathological lesions, indicating 100% protection against infection. The viral load in the liver and spleens of these birds was significantly lower than that in the control group. Additionally, these ducklings exhibited positive seroconversion at 7 days after vaccination on enzyme-linked immunosorbent assay. These results indicate that MLBE of σB could be used as a candidate for developing vaccines against NDRV infection.

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.

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.

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 rapid quantitative method to differentiate MS1 vaccine strain from wild-type Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an economically important pathogen in the poultry industry. Vaccination is an effective method to prevent and control MS infections. Currently two live attenuated MS vaccines are commercially available, the temperature-sensitive MS-H vaccine strain and the NAD-independent MS1 vaccine strain. Differentiation of vaccine strains from wild-type (WT) strains is crucial for monitoring MS infection, especially after vaccination. In this study, we developed a Taqman duplex real-time polymerase chain reaction (PCR) method to identify MS1 vaccine strains from WT strains. The method was specific and did not cross-react with other avian pathogens. The sensitivity assay indicated that no inhibition occurred between probes or between mixed and pure templates in duplex real-time PCR. Compared with the melt-based mismatch amplification mutation assay (MAMA), our method was more sensitive and rapid. In conclusion, the Taqman duplex real-time PCR method is a useful method for the diagnosis and differentiation of WT-MS and MS1 vaccine strains in a single reaction.

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.

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.

Impact of duck astrovirus on susceptibility to infection across duck ages.

An outbreak of duck astrovirus (DAstV) has occurred in duck farming regions of China, causing substantial economic setbacks in the duck industry. This investigation aimed to examine the variations in DAstV pathogenicity among ducks at different age intervals. Infections were induced in ducks at distinct age groups (1, 7, 14, 21, and 28 d) utilizing the DAstv-1-GDB-2022 strain. The results indicate increased pathogenicity of the DAstv-1-GDB-2022 strain in ducklings aged 21 to 28 d, manifesting as liver and kidney enlargement, severe bleeding, and potential fatalities. Conversely, ducklings aged 1 and 14 d displayed milder symptoms postinfection. Notably, viral shedding continued in ducks of diverse age groups even 21 d postinfection (Dpi). Moreover, DAstV replicates in various tissues, predominantly affecting the liver. Immunohistochemical tests using rabbit anti-DAstV antibodies revealed robust positive signals in both the liver and kidneys, which correlated with the clinical symptom severity observed through macroscopic and microscopic examinations. Serum biochemical assays and indirect ELISA demonstrated a consistent response to DAstV infection across different age groups, with older ducklings exhibiting increased sensitivity. In conclusion, this study successfully replicated clinical symptoms similar to those of natural DAstV infection using the DAstv-1-GDB-2022 strain. Importantly, we systematically delineated the differences in susceptibility to DAstV among ducks at various ages, laying the foundation for further research into the pathogenic mechanisms of DAstV and potential vaccine development.

Epidemiological investigations and multilocus sequence typing of Mycoplasma gallisepticum collected in China.

Mycoplasma gallisepticum (MG) is one of the important pathogens in poultry industry and has led to major economic losses. Understanding the epidemiology is crucial to improve the control and eradication program of MG. This study collected 1,250 chicken samples, including trachea and lung, from China in 2022 to investigate the epidemiology of MG. Among the collected samples, 938 samples were positive for MG infection, resulting in an average positive rate of 75.04%. Additionally, 570 samples were positive for both MG and Mycoplasma synoviae (MS) coinfection, with an average positive rate of 45.60%. A total of 183 MG infection positive samples in this study were selected for genotyping, and the multilocus sequence typing (MLST) method based on 7 housekeeping genes was used. As a result, 183 samples belonged to 11 sequence types (STs), with ST-78 being the most prevalent. After BURST analysis, all 183 sequences were divided into group 3. Besides, 119 reference sequences from database and 183 sequences of this study were selected to construct the phylogenetic tree using the neighbor-joining method. The results revealed that the sequences from China, total 196 sequences, were classified into 4 branches. The findings suggest that the MG strains in China exhibit diverse genotypes, which may be related to international trade and the use of live vaccines. Furthermore, we detected the drug susceptibility of 10 isolated strains randomly, which may be helpful to guide the clinical use of drugs to control MG infection.

Effects of a complex probiotic preparation, Fengqiang Shengtai and coccidiosis vaccine on the performance and intestinal microbiota of broilers challenged with Eimeria spp.

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.

Epidemiological investigations and multilocus sequence typing of Mycoplasma synoviae isolates from chicken farms in China.

Mycoplasma synoviae (M. synoviae) is an important pathogen in poultry industry and has led to major economic losses. Understanding the epidemiology is crucial to improve control and eradication program of M. synoviae. In this study, 487 samples suspected with M. synoviae infection were collected from August 2020 to June 2021 in China. Among 487 samples, 324 samples were MS positive, the positive rate was 66.53%, and 104 strains were isolated from 324 positive samples. The multilocus sequence typing (MLST) method based on seven housekeeping genes was used to conduct genotyping 104 M. synoviae strains isolated, and the 104 isolates belonged to 8 sequence types (STs) after MLST genotyping, and ST-34 had the highest proportion. After BURST analysis, all 104 isolates were divided into group 12 with other 56 strains isolated from China. Phylogenetic tree constructed by neighbor-joining method showed that nearly all of Chinese isolates (160 isolates) clustered together and separated from other reference isolates (217 isolates) in the PubMLST database. In conclusion, this study suggested that the M. synoviae strains in China were highly similar and independent of abroad strains.

Isolation, identification, and pathogenicity analysis of newly emerging gosling astrovirus in South China.

Goose astroviruses (GoAstV) cause fatal gout and decrease product performance in the waterfowl industry across the world. Since no effective vaccines are available, studies on the epidemiology of the virus are necessary for vaccine development. In this study, we collected 94 gout samples from goose farms in the Guangdong Province of South China. Among them, 87 samples (92.6%) tested positive for GoAstV, out of which five GoAstV strains were isolated after four generations of blind transmission through healthy 13-day-old goose embryos. The whole genome of the isolates was sequenced and further analyzed by comparing the sequences with published sequences from China and other parts of the world. The results of the alignment analysis showed that nucleotide sequence similarities among the five GoAstV isolates were around 97.4-98.8%, 98.6-100%, 98.1-99.8%, and 96.7-100% for the whole genome, ORF1a, ORF1b, and ORF2, respectively. These results showed that the GoAstV isolates were highly similar to each other, although they were prevalent in five different regions of the Guangdong Province. The results of the phylogenetic analysis showed that the whole genome, along with the ORF1a, ORF1b, and ORF2 genes of the isolates, were clustered on a single branch, along with the recently published GoAstV-2, and were very distinct from the DNA sequences of the GoAstV-1 virus. In this study, we also reproduced the clinical symptoms of natural infection using the GoAstV-GD2101 isolates, confirming that the gout-causing pathogen in goslings was the goose astrovirus. These findings provided new insights into the pathogenicity and genetic evolution of GoAstV and laid the foundation for effectively controlling the disease.

Study of microencapsulated fatty acid antimicrobial activity in vitro and its prevention ability of Clostridium perfringens induced necrotic enteritis in broiler chicken.

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.

Molecular characterization of chicken infectious anaemia virus (CIAV) in China during 2020-2021.

Chicken infectious anaemia virus (CIAV) has been identified as the causative agent of chicken infectious anaemia (CIA), causing huge economic losses to the poultry industry globally. In this study, a total of 573 clinical samples were collected from 197 broiler farms in 17 provinces of China during 2020-2021. Among them, 375 samples (375/573, 65.4%) were positive for CIAV by real-time PCR. The positive rate of CIAV detection between different regions of China ranged from 46.67% (North China) to 81.25% (Central China). The nucleotide sequences of the VP1 gene were obtained for 91 CIAV strains, whole genome sequencing was successful for 72 out of 91 strains. Phylogenetic analysis based on the VP1 gene revealed that 91 CIAV strains currently circulating in China belong to three genotypes (II, IIIa and IIIb), and most of the CIAV strains belong to genotype IIIa. Phylogenetic analysis of the whole genome showed that 71 CIAV strains belong to genotype IIIa, and one strain belongs to genotype II. Sequence analysis showed several amino acid substitutions in both the VP1, VP2 and VP3 proteins. Our results enhance the understanding of the molecular characterization of CIAV infection in China.RESEARCH HIGHLIGHTS A molecular systematic survey of CIAV in China during 2020-2021.CIAV genotype IIIa is the predominant genotype in China.

Drug resistance patterns and genotype associations of Trichomonas gallinae in meat pigeons (Columba livia): insights from Guangdong Province, China.

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.

Effects of Bacillus subtilis and coccidiosis vaccine on growth indices and intestinal microbiota of broilers.

Avian coccidiosis is the most serious parasitic disease in the poultry industry. Therefore, the aim of the current study was to explore the effects of B. subtilis and live coccidiosis vaccine alone or in combination on the production performance and anticoccidiosis, as well as the dynamic changes of intestinal microbial community. Nine hundred ninty Mahuang chickens were randomized into 4 preimmune groups including control group, coccidiosis vaccine immunization group; B. subtilis administration group and a group that was administrated a combination of live coccidiosis vaccine and B. subtilis group. Intestinal mucosal scraps collected from all these experimental groups at the age of 8 d and 15 d for microbial community 16S rRNA gene sequencing. At the age of 25 d, 30 broilers from each preimmune group were randomly assigned to a subgroup infected with Eimeria spp. and renamed as CI, V-CI BS-CI, and VBS-CI group. The production performance was monitored at the age of 25 d, 35 d, 45 d, and 55 d for the rest broilers from each pre-immune group. Otherwise, in the Eimeria spp. challenge stage, intestinal mucosal scraps collected for microbial community sequencing, while duodenum, jejunum, and cecum collected for pathological examination after sacrifice at the age of 32 d. In addition, the oocysts per gram of feces (OPG) and intestinal lesion score of broilers after Eimeria spp. challenge were also counted. Overall, the probiotics and coccidiosis vaccine resulted in the significantly improvement of the production performance. Otherwise, the intestinal lesion score and OPG after Eimeria spp. infection was significantly decreased in the VBS-CI group (P < 0.05). Moreover, these protective effects may also be closely related to genus such as Romboutsia, Blautia, and Butyricococcus, as well as microbiota functions like the quorum sensing pathway. According to these results, a combination of B. subtilis and coccidiosis vaccines can improve performance and provide additional protection against Eimeria spp. infection.

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.

Corrigendum: A Novel Intranasal Vaccine With PmpGs + MOMP Induces Robust Protections Both in Respiratory Tract and Genital System Post Chlamydia psittaci Infection.

[This corrects the article DOI: 10.3389/fvets.2022.855447.].

Identification and Pathogenicity Analysis of the Pathogen Causing Spotted Spleen in Muscovy Duck.

Since September 2020, the clinical symptoms of Muscovy duck spleen spots have appeared in Guangdong, Guangxi, Jiangxi, Hunan, Hubei, and other provinces, resulting in a large number of Muscovy duck deaths and great economic losses. The absence of the typical clinical symptoms caused by pathogenic microorganisms makes the cause of the spotted spleen a mystery. High-throughput sequencing results suggested that Riemerella anatipestifer (R. anatipestifer) may be the pathogen. Then, R. anatipestifer was regarded as the research target for isolation, identification, and pathogenicity assessment. After biochemical test, PCR amplification, and serotype determination, it was confirmed that the isolated strain CZG-1 was serotype 15 R. anatipestifer. Typical spotted spleen symptoms were observed after CZG-1 infection. Furthermore, drug sensitivity assays showed the similar drug-resistant spectrum of R. anatipestifer serotype 15 to other serotypes; for example, all test strains were resistant to polymyxin, gentamicin, and neomycin. The CZG-1 strain has high pathogenicity, and its lethal dose of 50% (LD50) is 35.122 CFU/ml. Virulence gene determination showed that the CZG-1 strain had at least five virulence genes, bioF, TSS9-1, TSS9-2, PncA, and 0373Right. Above all, this study identified and proved that the pathogen of spotted spleen in ducks was R. anatipestifer serotype 15, which caused death of ducks without the typical symptoms of bacterial infection. The results of this study enriched the knowledge of symptom after R. anatipestifer infection, provided a reference to the identification of the pathogen of spotted spleen, and provided theoretical basis for prevention and control of spotted spleen.