This study utilized Holstein bovine lung resident mesenchymal stem cells (LR-MSCs) and observed their osteogenic differentiation ability in vitro under different conditions, with the hope of providing new seed cells for clinical application and tissue engineering. The Holstein calf (healthy Holstein fetal calf, 0 days old, male) was used to obtain lung tissue via sterile methods and to isolate LR-MSCs through techniques like tissue adherence. The cells were primarily cultured. The growth curves were drew, proliferation timings, and potency for the 3rd, 9th, and 15th generations were measured. The expression of stem cell surface markers (CD29, CD44, CD73, CD90, CD106, CD166, CD34 and CD45) was detected using RT-PCR. Chromosomal karyotype analysis was performed on these genes, and their pluripotency was observed after genetic transformation. The results indicate that LR-MSCs could be obtained through both techniques, exhibiting spindle and spiral shapes in vitro and showing a classic "S" growth curve. The 9th generation had a shorter doubling time than the 15th generation, but longer than the 3rd (P<0.01); colony-forming rates for the 9th generation LR-MSCs were significantly decreased compared to the 3rd generation, but higher for the 15th generation, showing statistical significance (P<0.05). RT-PCR revealed the specific expression of CD29, CD44, CD73, CD90 in LR-MSCs. The preliminary studies found that LR-MSCs could specifically express CD29, CD44, CD73, CD90, CD106, CD166, but not CD34, CD45. Chromosome sequencing showed LR-MSCs to be of a normal diploid karyotype (2n=60, XY). Immunofluorescence staining and RT-PCR analysis proved the multilineage differentiation potential of Holstein bovine LR-MSCs into adipocytes, osteocytes, and chondrocytes, which may offer a new seed cell type for upcoming tissue engineering research.

The purpose of this study was to investigate the effect of lysophosphatidic acid(LPA) with different concentrations on the expression of hyaluronate synthase 2(HAS2), prostaglandin-endoperoxide synthase 2(PTGS2) and pentraxin 3(PTX3) in Yak cumulus cells (YCCs). The YCCs of healthy adult (3-4 years old) female yaks were taken as the research object. YCCs in logarithmic growth phase were taken, and different concentrations of LPA (blank control, negative control, 5, 15, 30, and 50 μmol·L^-1) were applied to the cultured YCCs in vitro. After 12, 24, 36, and 48 hours of cultivation, CCK-8 was used to detect the cellular viability of YCCs. The relative expression levels of HAS2, PTGS2, and PTX3 mRNA and protein in YCCs were detected by RT-qPCR and Western-blot assays. Immunofluorescence staining was used to detect the distribution of cumulus dilatation factors HAS2, PTGS2, and PTX3 in YCCs. In this experiment, each treatment group had 3 replicates. The results showed that, LPA incubation for 12, 24, 36, and 48 h had a significant promoting effect on the viability of YCCs. When the incubation time was 24 h, LPA had the most significant promoting effect on the viability of YCCs. Compared to the control group, when the LPA concentration was 15 μmol·L^-1, the viability of YCCs increased most significantly at different incubation times(P<0.05). Compared with the blank control group, the highest relative expression levels of HAS2, PTGS2, and PTX3 mRNA and protein were observed(P<0.05) when the LPA concentration was 15 μmol·L^-1, and the fluorescence intensity of HAS2, PTGS2, and PTX3 in YCCs was significantly enhanced. However, when the LPA concentration exceeded 15 μmol·L^-1, the relative expression levels of HAS2, PTGS2, and PTX3 mRNA and protein gradually decreased. The study results suggests that LPA has a promoting effect on the viability of YCCs. When the incubation time is 24 hours and the LPA concentration is 15 μmol·L^-1, the viability improvement is the most significant(P<0.05). LPA can enhance the expression of the cumulus expansion factors HAS2, PTGS2, and PTX3 in YCCs, and its effect is dose-dependent, with the optimal concentration being 15 μmol·L^-1. The research results provide a theoretical basis for elucidating the molecular mechanism of LPA promoting the expansion of yak cumulus cells, and for further improving the quality of yak oocytes and the success rate of in vitro fertilization (IVF).

This study aimed to obtain the gene sequence and expression pattern of bone morphogenetic protein 15 (BMP15) in tissues of New Zealand white rabbits, construct its eukaryotic expression vector, and predict the biological function of BMP15. In this study, 180-day-old healthy New Zealand white rabbits were selected as the study subjects. The CDS region of BMP15 gene was amplified using RT-PCR, the target fragment was ligated to linearized Pmcherry-N1 and pCMV-Myc empty vectors through T4 ligation method, and the eukaryotic expression vector was constructed. The properties and structure of the encoded protein were analyzed by bioinformatics. The overexpression of pCMV-Myc-BMP15 was detected by real-time fluorescence quantitative PCR ( qRT-PCR ) and Western blotting. Subsequently, qRT-PCR was employed to detect the expression level of the BMP15 gene in different tissues. The subcellular localization of BMP15 gene was detected by laser confocal method. The localization of endogenous BMP15 in ovarian tissue of New Zealand white rabbits was determined using immunofluorescence technique. The results revealed that the CDS sequence of BMP15 gene in New Zealand white rabbit was 1 182 bp. PCR and sequencing results confirmed the successful construction of the pCMV-Myc-BMP15 and Pmcherry-N1-BMP15 eukaryotic expression vectors. Bioinformatics analysis indicated that BMP15 gene encoded 393 amino acids. The protein exhibited an instability coefficient of 55.32, with an isoelectric point of 9.69, suggesting stability as a basic protein. BMP15 possessed 26 phosphorylation sites, 15 glycosylation sites, a signal peptides, and lacked transmembrane domains. Phylogenetic analysis revealed that New Zealand white rabbit had a close relationship with Sus Scrofa and a distant relationship with Gallus. Secondary and tertiary structure analysis indicated that BMP15 was a hybrid protein, primarily composed of α-helices, irregular coils, extended strands, and β-turns. Protein interaction prediction suggested that BMP15 protein had interactions with FSHR, FIGLA, BMPR1B, AMHR2, and NOBOX proteins related to ovarian growth and development. Tissue expression analysis demonstrated specific expression in ovarian tissues. Confocal laser scanning revealed cytoplasmic expression of BMP15. Transfection of pCMV-Myc-BMP15 into 293T cells resulted in significant upregulation at both mRNA and protein levels. Immunofluorescence detection of ovarian tissue confirmed cytoplasmic localization of BMP15 in granulosa cells. In this study, the eukaryotic expression vector of BMP15 was successfully constructed, and the physical and chemical properties, as well as biological characteristics of BMP15 gene and its encoded protein, were predicted and analyzed. BMP15 gene overexpression was successfully achieved in HEK293T cells, and information regarding subcellular localization, tissue expression, and distribution in ovarian tissue was obtained. These findings provide a theoretical basis for the subsequent studies on the function and mechanism of BMP15 gene in ovarian growth and development.

The aim of this study was to analyze the effect of follicular fluid exosomes on the genes of ovarian granulosa cells, to understand the regulation mechanism of follicular fluid exosomes during follicular development, and to provide a theoretical basis for the study of sow reproduction. In this study, 6-month-old binary sows with good health status and similar body weight were selected as experimental materials, and 150 ovaries were collected after slaughter, and follicular fluid exosomes and porcine ovarian granulosa cells (POGCs) were obtained using gradient centrifugation, and follicular fluid exosomes and porcine ovarian granulosa cells was co-cultured in vitro. Porcine ovarian granulosa cells (granulosa cell samples, GC, n=3) and porcine ovarian granulosa cells co-cultured with exosomes (granulosa-exosome co-culture samples, GCE, n=3) were sequenced by RNA-seq. The results showed that an average of 5.54×10⁷ clean reads were obtained per sample in the GC and GCE groups, and the Q20 and Q30 quality scores were greater than 92%. A total of 1 310 979 SNP mutations and 104 498 InDel mutations were obtained in 6 samples in the GC and GCE groups, among which the number of homozygous SNP/Indel mutations was 170 426 and the number of heterozygous SNP/Indel mutations was 1 245 052, and the number of mutant heterozygotes was significantly higher than the number of homozygotes and with a high incidence of SNPs. In addition, among the mutation types, there were 973 003 transformations and 339 974 transversion, and the number of transformations was significantly higher than the number of transversion. After gene annotation, the mutations mainly occurred in the 3'UTR, 5'UTR, and intron regions of the genes, followed by exons and intergenic regions. In addition, after comparison with the differential genes, the 1 583 candidate genes were screened, and it was found by GO and KEGG functional enrichment that the candidate genes were mainly related to cell cycle and cell proliferation/apoptosis processes. In addition, a total of 14 key candidate genes related to cell cycle and proliferation/apoptosis pathways were identified, of which 11 genes had interaction relationships. These SNP/Indel information obtained in this study can provide a scientific basis for subsequent studies on the regulation of exosomes on sow reproduction.

The aim of this study was to analyze the regulatory role of arginine and its metabolites in the induction of apoptosis in piglet sertoli cells under heat stress conditions. In this study, 25 3-week-old healthy male piglets were selected, testes were collected and sertoli cells were isolated, which were randomly divided into 3 groups with 3 replicates. The cultured sertoli cells were treated at 44 ℃ for 30 min as a heat stress model. Mass spectrometry multiple reaction monitoring (MRM) was used to analyze the metabolites of amino acid. The apoptosis rate of cells was detected by flow cytometry. Western blotting was used to detect the expression of iNOS, BAX, FAS, ARG1, ODC and SSAT. Quantitative real-time PCR was used to detect the mRNA levels of Arg1, Arg2 and Odc. The contents of spermine were analyzed by the kit.The results showed that heat stress significantly decreased the levels of arginine (P<0.01) and citrulline (P<0.05), increased the levels of putrine (P<0.01) and ornithine (P<0.01), but did not affect the level of spermine (P>0.05). Heat stress also significantly increased the mRNA expression of Inos (P<0.01), Arg1 (P<0.01), Arg2 (P<0.01) and Odc(P<0.05) and the content of NO (P<0.01), leading to an increase in the apoptosis rate (P<0.01). Addition of 0.05 mmol·L^-1 exogenous arginine increased the viability of SCs (P<0.05) and arginine content (P<0.01) under heat stress, and decreased BAX (P<0.05) and FAS (P<0.05) protein levels and relative apoptosis rate (P<0.01), significantly reduced the protein level of iNOS (P<0.01) and the content of NO (P<0.01), increased the content of spermine (P<0.01), and alleviated the apoptosis of SCs under heat stress (P<0.01). Similar results were obtained with the addition of exogenous spermine. These results indicate that heat treatment induces apoptosis of SCs by enhancing Arg-NO metabolic pathway. Addition of exogenous arginine and spermine enhances Arg-spermine metabolism and reduces NO production, thereby inhibiting heat stress-induced apoptosis of SCs.

The aim of this study was to investigate the effect of alternative splicing factor heterogeneous nuclear ribonucleoprotein F (hnRNPF) on spermatogenesis in Mongolian horses. Testicular sertoli cells were isolated and cultured from 3-year-old Mongolian horse. The hnRNPF overexpression lentiviral vector was constructed and transfected into the sertoli cells of Mongolian horse testis. CCK-8 was used to detect the proliferation of cells at 0 h, 24 h, 48 h and 72 h after transfection, and the optimal infection time was determined with 3 duplication per group. The RNA of transfected and untransfected Mongolian horse sertoli cells was extracted, and the primers of genes known to be related to spermatogenesis were designed for qRT-PCR to detect the expression of spermatogenesis-related genes in the two types of cells. The results of agarose gel electrophoresis showed that the hnRNPF lentivirus overexpression vector was successfully constructed. CCK-8 assay showed that the cells transfected with the expression lentiviral vector had the highest activity at 72 hours, and the activity of transfected cells was higher than that of untransfected cells, indicating that the overexpression of hnRNPF could promote the proliferation of Mongolian horse sertoli cells.qRT-PCR results showed that the expression levels of spermatogenesis-related genes (PKMYT1, CDC25C, YWHAZ, BUB1, BTRC, CCNE1, CALM1, PLK1, REC8, MAPK3, and ADCY7) were significantly different between the two types of cells. All were highly expressed in cells transfected with hnRNPF overexpression vector. The results suggest that hnRNPF may directly or indirectly promote spermatogenesis in Mongolian horses, which provides a new idea for improving the sperm count and semen quality of Mongolian horses.

The study aimed to explore the molecular mechanism of exogenous melatonin (MLT) on mink ovarian development. In this study,200 female white and healthy 3 months old minks with similar body weight were selected. They were divided into experimental groups and control groups. The individuals in experimental group was implanted with an implant containing 18 mg·grain^-1 MLT, while the individuals in control group was implanted with an empty implant without MLT. When the minks were in preparation for breeding, the ovaries of 4 minks in the experimental group and 4 minks in the control group were randomly collected.Then the total RNA was extracted by the Trizol method, and the cDNA library was constructed. The Illumina platform was used for sequencing and bioinformatics analysis and differentially expressed genes screening, and GO functional annotation analysis and KEGG analysis of differentially expressed genes were performed to find out the candidate genes and metabolic pathways related to MLT regulating ovarian development.To further verify the reliability of sequencing data, 6 differentially expressed genes were randomly selected for qPCR verification. The transcriptome results showed that both the experimental and the control groups had more than 120 000 SNP sites, and the SNP sites both were predominantly found in the gene region. The SNP mutation types of conversion were more than transversion and heterozygous in both groups. The number of SE events in the experimental group was the largest. A total of 9 631 new genes was obtained in this study, of which 1 323 and 1 317 new genes were annotated respectively in TrEMBL and NR databases, the annotation ratio was significantly higher than that of other data. The annotation ratio was significantly higher than that of other data. A total of 206 differentially expressed genes were found between the experimental group and the control group, including 36 unannotated genes, 69 up-regulated genes, and 137 down-regulated genes. GO functional enrichment showed that a total of 7 differentially expressed genes were enriched in the reproductive process. KEGG pathway enrichment results showed that a total of 7 differential genes was enriched in 5 pathways related to reproduction. Based on the results of GO functional classification and KEGG pathway annotation analysis, a total of 4 regulatory genes related to MLT affecting ovarian development were screened, all of which were up-regulated genes, including KRT19, GSTT1,ALAS1 and NR4A1. In this study, we performed transcriptome analysis of mink ovaries with or without MLT implantation during the pre-mating stage and found that MLT may promote ovarian development by regulating KRT19, GSTT1, ALAS1, and NR4A1.The results provide a theoretical basis for mink breeding work.

The objective of this study was to optimize the loading sample size and filter medium of The American Oil Chemists' Society (AOCS) standard method for the determination of acid insoluble ash (AIA) to simplify the assay procedure and improve the accuracy. The repeatability, additivity and accuracy of AIA determined with AOCS modified method and the AOCS standard method were compared to obtain a reliable technique for accurately evaluating the dietary apparent metabolizable energy (AME) for broilers. Two experiments were conducted in the current study. In experiment 1, three batches determination of AIA content for each of 6 samples (3 diets, 3 excreta) using two methods (AOCS modified method, AOCS standard method) in a completely randomized design was to compare the reproducibility of the 2 methods. Then, the actual AIA content of 6 mixed diets and 6 mixed excreta samples were determined by the 2 methods. Meanwhile, the AIA value was calculated according to the composition of the mixed diet and mixed excreta and their AIA content. The difference between the determined and the calculated values was compared to test the additivity of the 2 methods by a paired experimental design. In experiment 2, a paired experimental design was used to compare the difference between the calculated AME of 13 diets using two AIA determination methods and the AME measured by the total collection method to test the accuracy of the 2 methods to evaluate the dietary AME. The results showed that the total coefficient of variation (TCV) of AIA determined by the AOCS modified method in each of 3 diets and 3 excreta samples ranged from 2.13% to 5.83% and 1.71% to 2.83%, respectively, while the TCV of AIA determined by the AOCS standard method ranged from 4.69% to 15.37% and 1.50% to 5.79%, respectively. The difference between the determined and calculated AIA values of 6 mixed diets and 6 mixed excreta by the AOCS modified method ranged from 0.004% to 0.017% and 0.002% to 0.029%, respectively. The difference between determined and calculated values of those samples by AOCS standard method ranged from 0.037% to 0.071% and 0.015% to 0.072%, respectively. The differences of 0.021 to 0.251 MJ·kg^-1 DM were observed between the AME evaluated by AIA determined with the AOCS modified method and AME determined by total collection. Whereas the differences of 0.025 to 1.799 MJ·kg^-1 DM were observed between AME evaluated by AIA determined with the AOCS standard method and AME determined by total collection. In conclusion, the AOCS modified method is superior to the AOCS standard method in the reproducibility and additivity of AIA determination and in the accuracy of evaluating dietary AME.

This study was conducted to investigate the effects of dietary supplemented with sodium humate (HNa) on the liver tissue inflammation and antioxidant capacity of Salmonella Typhimurium (S. Typhimurium) infected broilers. A total of 320 healthy 1-day-old Arbor Acres broilers were randomly assigned into 5 treatment groups with 8 replicates of 8 birds each, named CON group (basal diet), ST group (basal diet), L-HNa group (basal diet+0.2 g·kg^-1 HNa), M-HNa (basal diet+0.4 g·kg^-1 HNa), and H-HNa group (basal diet+0.6 g·kg^-1 HNa), respectively. On day 22-24, the broilers in the CON group were gavaged with 1 mL of PBS, while the broilers in the ST, L-HNa, M-HNa and H-HNa groups were gavaged with 1 mL of 3×10⁹ CFU·mL^-1 S. Typhimurium, daily, respectively. The trial lasted for 24 days. The results showed that: 1) Compared with the ST group, dietary supplemented with 0.6 g·kg^-1 HNa significantly increased the average daily gain (ADG) (P<0.05) of broilers. 2) Compared with the CON group, livers of broilers in the ST group exhibited lots of inflammatory cell infiltrations and liver index increased, while dietary supplemented with different concentrations of HNa relieved hepatic histopathological damage (P<0.05). 3) The activities of serum alkaline phosphatase (AKP), alanine aminotransferase (ALT), and aminotransferase (AST) were significantly higher in the ST group broilers compared with the CON group (P<0.05). 4) Compared with the CON group, the broilers in the ST group had lower activities of liver catalase (CAT), superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC) and higher concentration of MDA (P<0.05). Importantly, dietary supplemented with HNa alleviates the damage of S. Typhimurium infection on the antioxidant function of broilers liver. 5) Compared with the ST group, dietary supplemented with different concentrations of HNa significantly decreased the mRNA expression of liver interleukin-1β (IL-1β), IL-18, tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and cluster of differentiation 68 (CD68) (P<0.05), while significantly increased the mRNA expression of IL-10, arginase-1 (ARG1), and cluster of differentiation 163 (CD163)(P<0.05). 6) Compared with the CON group, S. Typhimurium infection significantly elevated the protein expression of nuclear factor kappa beta p65 (NF-κB p65), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and Caspase-1 (P<0.05), significantly decreased the protein expression of NF-kappa-B inhibitor alpha (IκBα)(P<0.05). However, dietary administration of HNa inhibited the activation of NF-κB and NLRP3 signaling pathway. In summary, dietary supplementation of HNa alleviated liver damage of broilers caused by S. Typhimurium infection by decreasing liver inflammation response, increasing liver antioxidant function and suppressing activation of NF-κB and NLRP3 signaling pathway in the liver.

This study aimed to express the S protein of Bovine coronavirus (BCoV) and evaluate its immunogenicity in mice. The S protein of BCoV/SWUN/HXD-4/2021 strain was seclected as a template for codon optimization in insect cells. The complete S gene was synthesized and the recombinant plasmid pFastBac-Dual-S and Bacmid-S were constructed. The recombinant baculovirus rpFastBac-S was harvested by transfection of insect cells and identified by PCR, Western blot and indirect immunofluorescence assay. Baculovirus titers were determined. The effects of different infection doses (MOI=0.005, 0.05, 0.5, 1, 2) on protein expression were optimized, and the best infection condition was selected to express protein. The protein was purified by ultracentrifugation and immunized mice, and the immune effect was evaluated by indirect ELISA and neutralization test. After optimization, the S gene CAI (codon adaptation index) was adjusted from 0.39 to 0.87, and the average GC content was adjusted from 35.8% to 50.6%. Double enzyme digestion and PCR confirmed that the recombinant plasmid and the recombinant bacmid were successfully constructed. Sf9 cells were transfected with 5 μg Bacmid-S and the cytopathic effect tended to be steady after the fourth cell passage. Typical cytopathic effect appeared 20 h after baculovirus infection. The recombinant bacmid-S was harvested and identified. The results of PCR electrophoresis showed that the recombinant bacmid carried the S target protein gene (size 4 108 bp). IFA showed that specific green fluorescence was observed in the experimental group. Western blot results showed that the recombinant baculovirus rpFastBac-S protein was the S target protein, and the target size was about 250 ku. When the MOI was 0.5, the protein expression level was the highest. The mice were immunized with 50 μg protein, 20 μg CpG adjuvant and equal volume of MF59 adjuvant by intramuscular injection. Indirect ELISA results showed that the mice immunized with the protein produced IgG specific antibody, and the antibody titer was higher than that of the control group (P<0.001), and the highest antibody titer was 1∶12 800. The results of neutralization test showed that the highest neutralization titer of mouse serum was 1∶224, and the average neutralization titer of experimental group was higher than that of inactivated BCoV group, but the difference was not statistically significant (P>0.05). In conclusion, the S protein of BCoV was expressed by baculovirus expression system, and the immune protective effect of S protein was evaluated, which provided a theoretical foundation for the subsequent research of bovine coronavirus vaccine.

Cattle viral diarrhea pathogens have adverse effects on animal husbandry. BVDV, BoAstV, BCoV, MRV, BKoV, BoNeV, BNoV, BRV, BToV were well known as the cause of diarrhea. On the other hand, BVDV, BCoV and BToV also caused respiratory system symptoms. Among them, BVDV not only causes alimentary tract symptoms and respiratory symptoms, but also leads to abnormal reproductive function of cows causing great economic losses. In order to understand the prevalence of the main viral diarrhea pathogens in cattle, 323 fecal samples from diarrheal cow in Hebei province of China were detected by PCR to detect nine common viral diarrhea pathogens. Prevalence of 1.85% (6/323), 14.24% (46/323), 57.89% (187/323), 0.31 (1/323), 10.84% (35/323), 4.33% (14/323), 2.48% (8/323), 4.64% (15/323), 11.76% (38/323), was determined for BVDV, BCoV, BRV, BoAstV, BoNeV, BNoV, MRV, BToV, BKoV, respectively. There were 76 co-infection samples accounting for 23.53% (76/323). Homology and genetic evolution analysis of the complete genomic sequence, S, HE, N, M and E genes of HBLF2302 strain showed that HBLF2302 gene had high homology with BCoV/NMG1/2022 and was GIIb subtype. Based on the amino acid sequence alignment and the tertiary structure prediction of HBLF2302 S protein, it was found that the 157 site was mutated to 157T and the 854 site was mutated to 854I in the S protein. 318V is a unique mutation, which changes the force with the side chain residue and forms a hydrogen bond connection with 631C. It was found that the Chinese strain was different from the GⅡb subtype in other regions, and there were three unique sites in the ORF1a region. This study enriched the epidemiological data about bovine diarrhea pathogens for laying the foundation of bovine pathogens prevention and control.

The species-specific acidic nuclear phosphoprotein 32A (ANP32A) regulates RNA polymerase activity of influenza A virus from different hosts. To analyze species-specific difference of canine ANP32A (canine ANP32A, caANP32A) and its role in the cross-species infection of influenza virus, RT-PCR was used to amplify and clone caANP32A from Madin-Darby canine kidney (MDCK) cells and different tissues of canine lung, spleen and intestine. The interaction of caANP32A with influenza A virus RNA polymerase was analyzed by Laser confocal assay. The effect of caANP32A on RNA polymerase activity of influenza A virus was detected in 293T cells by double luciferase reporter gene experiment. The results showed that the new caANP32A was amplified from MDCK cells, which had four more amino acids insertion than the previously reported caANP32A. The new caANP32A was amplified from the lung, spleen and intestine of dogs; After sequencing and analyzing the ANP32A gene, it was found that the new caANP32A was not formed by alternative splicing of mRNA; Laser confocal assay showed that the co-location of the new caANP32A with the RNA polymerase of H3N2 CIV in the nucleus. Polymerase activity experiments showed that overexpression of novel caANP32A in mammalian cells did not promote the RNA polymerase activity of H9N2 avian influenza virus (AIV) and H3N2 canine influenza virus (CIV), but chANP32A could. The new caANP32A cloned in this study, compared to previous reports, has four amino acids LSLV insertion at positions 176 to 179, but the new caANP32A still has species restriction on the polymerase activity of AIV and does not enhance the RNA polymerase activity of CIV. This study provides a basis for further elucidating the role of dogs in the cross-species infection of influenza virus.

The aim of this paper was to construct a lipoteichoic acid synthase ltaS gene deletion strain to investigate the effect of the ltaS gene on the pathogenicity of Listeria monocytogenes. In this study, we used homologous recombination to construct a strain of Listeria monocytogenes with a deletion of the ltaS gene. The effect of the ltaS gene on the growth and bacterial morphology of Listeria monocytogenes was explored by growth curves assay and observation of bacterial morphology. The anchoring of InlA and InlB on the bacterial surface of wild strain EGDe-prfA* and deletion strain ΔltaS were investigated by Western blot assay. The effect of ltaS gene on the ability of Listeria monocytogenes to infest using DF-1 for cell adhesion invasion assay and mice RAW264.7 for phagocytosis assay with multiplication assay was determined. Effect of ltaS gene deletion on pathogenicity of Listeria monocytogenes as assessed by mice organ bacterial load test and survival assay. The results showed: A strain of Listeria monocytogenes with a deletion of the ltaS gene was successfully constructed by homologous recombination technology. Growth curve assay and light microscopic observation revealed that the ltaS gene did not affect normal growth in BHI and bacterial morphology of Listeria monocytogenes. Deletion of the ltaS gene resulted in a significant reduction of the bacterial surface virulence factors InlA and InlB anchor quantification, as confirmed by Western blot analysis. The results of the cell adhesion rate and invasion rate assay showed that the adhesion rate and the invasion rate of ΔltaS to DF-1 was highly significantly lower than that of the parental strain EGDe-prfA* (P<0.01). Phagocytosis assay revealed that the phagocytosis of ΔltaS by RAW264.7 was not significantly different from that of the parental strain EGDe-prfA* (P>0.05), the amount of ΔltaS multiply in RAW264.7 was significantly different from that of the EGDe-prfA* (P<0.05).The results of the mice pathogenicity virulence test showed that the bacterial load in the liver and spleen of mice infected by ΔltaS was significantly lower than that of EGDe-prfA* (P<0.01). And all mice infected with EGDe-prfA* died within 96 h, while the survival rate of mice infected with the ΔltaS was 80%. The LD₅₀ of parental strain EGDe-prfA* was 1.7×10⁴ CFU and the LD₅₀ of deletion strain ΔltaS was 7.49×10⁶ CFU. In summary, ltaS gene deletion significantly reduced the anchoring quantities of surface InlA and InlB and diminished the ability of infection ability, pathogenicity of Listeria monocytogenes.

To clarify the infection and biological characteristics of Corynebacterium pseudotuberculosis (Cp) in surface lymph node abscesses of goats in Chongqing and Guizhou, Cp isolation and identification, pathogenicity tests, antimicrobial susceptibility tests, virulence or antibiotics resistance gene detection and phylogenetic analysis were carried out in this study. The results showed that 23 strains of Cp were isolated from 40 samples, with a separation rate of 57.5%. All the Cp isolates harbored pld, FagA, FagB, OppB, OppD, OppF, SodC, SpaC, pknG, NanH, sigE, and cp40 genes. The results of antimicrobial susceptibility tests showed that 95.65% (22/23) of Cp was sensitive to cefradine, amikacin and doxycycline, followed by penicillin, ampicillin, tetracycline, vancomycin and ciprofloxacin, with a sensitivity rate of 91.30% (21/23), while 100% of Cp isolates were resistant to furazolidone and 82.61% (19/23) resistant to neomycin and kanamycin. The detection rate of aminoglycoside O-phosphotransferase gene aph(3')-IIa and broad-spectrum class A beta-lactamase gene bla_TEM-116 were 0 and 4.35% respectively. The fusA gene of Cp was sequenced and submitted to GenBank with login numbers ON969140-ON969162, and phylogenetic analysis of fusA showed that all the Cp isolates were clustered into Cp biotype ovis. This study verified that Cp was the major pathogen responsible for surface lymph node abscesses of goats in Chongqing and Guizhou, and clarified its biological characteristics which provided information for this pathogen prevention.

This study aimed to obtain rHfFer1 and rHfAV422 of Haemaphysalis flava and evaluate their anti-tick vaccine efficacy. The rHfFer1 and rHfAV422 recombinant proteins of H. flava were obtained by prokaryotic expression and twenty-two rabbits were randomly divided into three groups (Quil-A saponin control group and two immunization groups), among them, there were six rabbits in the Quil-A saponin control group and eight rabbits in each of the two immunized groups. Fourteen days after the first immunization, the same dose was given for the second immunization, and the larvae infestation were conducted one week after the second immunization. Immune efficacy was evaluated according to the engorged and molting rate. The results showed that the engorged and molting rate of the rHfFer1 and rHfAV422 immunized groups were significantly different from those of the control group (P<0.05), the calculated anti-tick vaccine efficacy of rHfFer1 and rHfAV422 were 68.06% and 50.00%, respectively. The result indicated that rHfFer1 and rHfAV422 could be used as candidate antigens for the development of anti-tick vaccines.

An infectious clone was constructed by mutating the WH303 site of CSFV C strain to provide candidate strains for the study of marker vaccine of CSFV C strain. In this study, CSFV C strain was used as the research material, and WH303 site was mutated by overlapping PCR method, and then cs-full and CS-full-flag were cloned into the full-length infectious clones of CSFV C strain. In vitro transcription RNA, and then transfected into PK15 cells, continuous passage of cells to complete the cell rescue and proliferation. The results showed that the specific restriction endonuclease digestion reaction verified the successful construction of infectious clone. RT-PCR and peroxidase monolayer cell test detection indicated that the virus had been rescued successfully. The virus can still be detected in the 17th generation of virus cell, indicating that the virus can be stably passaged. In summary, MC and MC-flag of C strain WH303 mutated infectious clones were constructed successfully in this study, MC strain was saved successfully, and the stable virus strain was obtained.

The aim of this research was to systematically evaluate and analyze the infection status of bluetongue virus (BTV) in sheep flocks in China from 2012 to 2022, and to provide evidence for the prevention and control of BTV in sheep flocks in China. A Meta-analysis was conducted after searching the databases of CNKI, Wanfang, VIP, PubMed and ScienceDirect for articles on the sheep BTV prevalence in China published from 2012 to 2022. After searching and screening, a total of 28 studies were brought into the Meta-analysis, including 38 226 sheep of which 9 109 sheep were BTV positive. The overall prevalence of BTV was 23.8% (95%CI: 23.4,24.3) in sheep and goats, 29.4% (95%CI: 14.7,44.1) in sheep and 25.1% (95%CI: 18.6,31.7) in goats. Among provinces/cities in China, the prevalence was highest in Chongqing 34.6% (95%CI: 32.0,37.1), followed by Yunnan province and Inner Mongolia Autonomous Region. Subgroup analysis showed that there were significant regional differences in the prevalence of BTV (P=0.011). The prevalence of BTV in eastern, southern, western, northern and central areas of China were 6.1% (95%CI: 4.3,7.9), 33.8% (95%CI: 29.5,38.1) and 21.7% (95%CI: 17.1,26.2), 21.0% (95%CI: 8.3,33.6) and 26.6% (95%CI: 24.3,28.8), respectively. At the same time, detection methods and climate types were also risk factors for BTV prevalence in sheep in China(P<0.05). The results showed that the prevalence of BTV was high and the distribution was wide among sheep in China. It is necessary to continue the surveillance and prevention of BTV infection in China.

There are few reports on PVL⁺ ST22 Staphylococcus aureus (S. aureus) isolated from equine milk in China. This study was conducted to evaluate the pathogenicity of an S. aureus (PVL⁺ ST22) isolated from equine milk in Xinjiang, hoping to provide basic data for the study of S. aureus from equine milk in China. S. aureus were isolated from apparently healthy equine milk in Xinjiang using the selective medium and then identified by biochemical and molecular identification methods. The obtained S. aureus were subjected to multilocus sequence typing (MLST), spa typing, antimicrobial susceptibility testing, and biofilm-forming ability, hemolytic activity, virulence gene detection, along with mouse skin abscess and pathogenicity assay. The isolated strain was then subjected to mouse macrophage adhesion, invasion, and intracellular proliferation assays to assess their pathogenicity. The results indicated that a PVL⁺ ST22 S. aureus (1/65, 1.5%) strain was isolated from equine milk, with spa typing t304. It was resistant to penicillin only, had no biofilm-forming ability, but carried 9 virulence genes (sec, seg, seh, tsst-1, hla, hlb, clfA, clfB, and cna ) and has hemolytic activity. Mouse skin abscess and pathogenicity tests also showed that it caused skin abscess formation and death in mice at high doses of infection. ST22 S. aureus has adhered (4.26%), invaded (6.45%), and proliferated intracellularly in mouse macrophages and caused apoptosis. In conclusion, the PVL⁺ ST22 S. aureus isolated from equine milk is highly pathogenic and may pose potential risks in terms of host pathogenicity and public health safety.

This study aimed to provide different methods for solving drug-resistant Pseudomonas aeruginosa infections. P. aeruginosa phage was isolated and purified from hospital wastewater using chicken-derived P. aeruginosa as host bacteria, and biological characterization and genomic analysis were performed, while its effect was tested using a checkerboard method in combination with three antibiotics. The results showed that a strain of P. aeruginosa phage PaVOB was isolated from hospital wastewater, TEM showed that virus particles had an icosahedral head with an average diameter of 73.02 nm and a long tail with an average length of 155.57 nm, it belongs to the family Siphoviridae in the order Caudovirales, had a double-stranded DNA genome, had a narrow host spectrum, was resistant to low temperatures (-20 ℃) and reached the optimal temperature at 37 ℃, had good acid-base tolerance, and maintained activity at pH 3-12, the optimal MOI is 0.001, the one-step growth curve shows that the latent period is 10 min, the outbreak amount is 50 PFU·cell^-1, and the adsorption test shows that the adsorption rate of PaVOB at 10 min is 99.1%. The full length of PaVOB genome was 72 179 bp, (G+C)% content was 54.81%, with VB PaeP FBPa1 The affinity was less than 0.1. ORF prediction showed that PaVOB included 74 ORFs, and none of the software predictions revealed the presence of virulence genes, drug resistance genes and tRNAs. In addition, the results of antibiotic combination showed that PaVOB had synergistic effect with ceftriaxone, had synergistic effect with enrofloxacin at potency of 10⁷ PFU·mL^-1, and had unrelated effect with gentamicin. P. aeruginosa phage PaVOB is highly adaptable to the environment and has clear genomic information, which can be selectively applied in combination with different antibiotics to exert synergistic effects and has the possibility of treating clinically resistant Pseudomonas aeruginosa.

To investigated the probiotic function of Eurotium cristatum(EC) in black tea and reveal the preliminary mechanism of inhibit Salmonella Typhimurium,we used dilution coating method to isolate EC, and analyzed its taxonomic status by morphology and ITS rRNA gene sequence. And then the agar diffusion inhibition test, thermal acid-base and UV stability test, electron microscopy and mouse animal test were used to evaluate the preliminary mechanism of inhibition of S. Typhimurium bacteria by this Ec-12 supernatant. A strain of Ec-12 was identified as E. cristatum based on morphology, growth characteristics and ITS rRNA gene sequence analysis, and named strain Ec-12. The supernatant of the crude isolate Ec-12 produced good inhibition at pH near neutral conditions with good stability. The supernatant of tEc-12 showed good bacterial inhibition at pH near neutral and good stability of inhibition. The inhibition of S. Typhimurium was observed by electron microscopy, which showed that the supernatant of the crude isolate Ec-12 caused deformation of the body and roughness of the surface layer of the bacterium. It also significantly reduced the rate of diarrhea and mortality caused by S. Typhimurium infection in mice, improved the integrity of mouse villi and increased the secretion of mucin (P<0.05), reduced the apoptosis of colonic cells (P<0.05), and inhibited the harm of S. Typhimurium to mice. The supernatant of Ec-12 strain acted on S. Typhimurium in the murine gastrointestinal tract, disrupting the structural integrity of S. Typhimurium, while acting on the murine intestine, improving the integrity of intestinal villi, promoting the secretion of colonic mucin, reducing the apoptosis of colonic cells, and thus reducing the diarrhea and death caused by S. Typhimurium. It has a probiotic potential.

In the process of modern large-scale pig breeding, whether it is the stress response caused by weaning, replacement and replacement, or bacterial and viral infections, it is very easy to affect the health of the digestive tract and cause intestinal inflammation with diarrhea as the main symptom. The expression of renin-angiotensin system (RAS) in jejunum tissues of pigs with clinical diarrhea was studied to explore the relationship between RAS and intestinal inflammation. The study was conducted on nursery pigs with clinical symptoms of diarrhea, and the pathological changes of jejunum tissues of diarrhea pigs were observed by histopathology, Western blot and RT-qPCR. To clarify the expression changes of RAS components, especially angiotensin-converting enzyme 2(ACE2), and explore the effects of RAS on intestinal inflammation injury in pigs. The results showed that the pro-inflammatory factors TNF-α and IL-1β were significantly increased in jejunum tissues of pigs with clinical diarrhoea piglets, while the anti-inflammatory factor IL-10 was not significantly changed. Histopathology showed that jejunum epithelial villi exfoliated, glandular hyperplasia and hemorrhagic sites. The mRNA and protein levels of ACE2 and MasR and the content of Ang (1-7) were decreased, while the protein levels of ACE and AT1R and the content of Ang II were increased. The results suggest that local jejunum RAS is activated in the jejunal tissue of pigs with clinical diarrhoea piglets. The activity of ACE-Ang II-AT1R axis was much higher than that of ACE2-ang (1-7)-MasR axis, and ACE2 was inferior in anti-inflammatory damage.

The study aimed to detect the presence of microsatellite instability (MSI) in Marek's disease (MD) tumors by high-resolution melting (HRM) method. Clinical samples were collected and diagnosed as Marek's disease by clinical signs, pathological anatomy, histopathology, PCR and sequencing. Muscle, liver, spleen, blood and tumor samples were collected from five affected chickens. DNA was extracted from the samples, and HRM assay was performed by using 15 pairs of primers for amplifying microsatellite DNA sequences. The difference melting curves of the normalization processing were obtained. The differences in the melting curves among samples were compared and analyzed for the presence of MSI in 15 microsatellite markers. The various clinical symptoms and pathological findings of the affected chickens were consistent with MD. The sequence of the viral meq gene had high homology with the virulent strain of MDV prevalent in China in recent years. HRM analysis showed that melting curves of PCR production were significantly different in muscle, liver, spleen, blood, and tumor samples. That is MSI phenomenon and MSI frequencies were different in chickens and microsatellites markers. There were 14,7,5,3 and one microsatellite markers presented MSI in five chickens respectively. The markers MCW0200 and MCW0220 presented MSI in all chickens' samples, however the markers ADL0158 had no MSI phenomenon in all chickens' samples. These microsatellite PCR amplification fragments differ in size were further demonstrated by nondenaturing polyacrylamide gel electrophoresis and sequencing analysis. There was an increase and decrease of microsatellite repeat units, and insertions or deletions of gene fragments in the sequences. The results suggest that the MSI phenomenon in MD tumors can be detected by HRM analysis, and HRM was a sensitive, accurate, simple and high-throughput method.

In this study, three fusion proteins were constructed by linking Fc fragment, membrane penetrating peptide R11 and membrane penetrating peptide TAT to cFGF-21 respectively, aiming to investigate the long-lasting hypoglycemic effect of cFGF-21 on type 1 diabetes. A mouse model of type 1 diabetes was established with streptozotocin (STZ) and subsequently treated with cFGF-21, R11-cFGF-21, Fc-cFGF-21 and TAT-cFGF-21, respectively. Treatment was divided into three phases: 1) daily dosing (1-14 days); 2) every 3 days (15-29 days); and 3) every 5 days (30-44 days). During the treatment, mice were tested for blood glucose every three days, and at the end of each phase, experimental mice were tested for blood glucose fluctuations over 12 h. Before the end of the experiment, oral glucose tolerance test was performed on all mice. The expression levels of inflammation-related factors IL-10, IL-1β, IL-17A and anti-inflammatory factor IL-10 in mice serum were measured by ELISA. The transcript levels of glucose metabolism-related factors in liver, fat, intestine and kidney tissues were detected by Real-time PCR. At the end of the experiment, all mice were tested for glycosylated hemoglobin (HbA1c), lipid quadruple and liver function, serum insulin and HbA1c and pathological analysis of mouse pancreatic tissues were performed. In the first phase of treatment, the hypoglycemic effect of TAT-cFGF-21 was significantly lower compared with the cFGF-21 group, and there was no significant difference between the R11-cFGF-21 and Fc-cFGF-21 groups; in the second and third phases of treatment, compared with the cFGF-21 group, the blood glucose in the TAT-cFGF-21 group increased significantly, and the R11-cFGF-21 and Fc-cFGF-21 groups could control blood glucose at a lower level within 3-5 days of dosing. After 8 weeks of treatment, oral glucose tolerance (OGTT) and dyslipidemia were significantly improved in the R11-cFGF-21 and Fc-cFGF-21 groups compared to the cFGF-21 group, glycosylated hemoglobin (HbA1c) was near normal levels, and liver damage was significantly repaired, while there was no significant difference between the TAT-cFGF-21 and cFGF-21 groups. In addition, ELISA and HE staining results showed that oxidative stress and inflammatory response were significantly improved in the R11-cFGF-21 and Fc-cFGF-21 groups compared with the cFGF-21 and TAT-cFGF-21 groups, and the islet β-cell repair effect was obvious. The significant increase of long-term hypoglycemic capacity of cFGF-21 modified by R11 and Fc, while the long-term hypoglycemic capacity of TAT-cFGF-21 was weaker. In addition, cFGF21 modified by R11 and Fc significantly improved oxidative stress, pancreatic inflammation and lipid metabolism, and repaired liver injury and pancreatic β-cells, with significantly better therapeutic effects than cFGF21.

Trichomonas gallinae is one kinds of highly transmissible parasitic disease, which seriously affects the production performance of pigeons, even leading to the death of pigeons. These bring huge economic losses to the pigeon industry. However, in clinical practice, the therapeutic drugs that used to prevent and control of Trichomonas gallinae are mostly chemical drugs, the use cycle of these chemical drug is short and the side effects are large. In order to develop the natural anti-parasitic drugs in clinic, this paper studied the clinical efficacy of Gentiana scabra against Trichomonas gallinae, providing the new medication strategy for the treatment of Trichomonas gallinae in the pigeon industry. By isolating, identifying, and purifying of Trichomonas gallinae, the stable strains were obtained for subsequent experiments. The relative growth inhibition rate and minimum effective concentration of Gentian water extract against Trichomonas pigeon were determined in vitro. In order to further observe the efficacy, different doses and concentrations of Gentian water extract were added to the basic diet, and the effects of Gentian water extract on body weight, survival rate, activity of Trichomonas pigeon, blood biochemical indexes and contents of inflammatory factors were measured in vivo, so as to evaluate the clinical efficacy of Chinese herbal medicine Gentian against Trichomonas pigeon in vivo and in vitro. The results of in vitro test showed that the minimum effective concentration (MEC) of Gentian water extract was 50 mg·mL^-1, which showed strong anti-Trichomonas pigeon activity in vitro. The results of in vivo experiments showed that the water extract of Gentian herb had the effect of restoring the body weight and improving the survival rate of the sick pigeons. The insecticidal effect of Gentian water extract was significant compared with control group (P<0.05), but had no significant difference compared with metronidazole group (P>0.05), and the duration of efficacy of Gentian 500 mg·mL^-1 per group was longer than that of metronidazole group; The contents of alanine aminotransferase (ALT), AST and ALT/AST in serum were lower than those in control group. Serum levels of three inflammatory factors, interleukin-6 (IL-6), interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β), were higher than those in the control group, similar to metronidazole group. Gentian water extract has significant clinical efficacy in the treatment of Trichomoniasis of pigeon, and its efficacy reaches the therapeutic effect of metronidazole, and has no significant toxic side effects. Therefore, Gentian can be used as a potential natural drug development against trichomoniasis of pigeon.

The aim of this study was to evaluate the effect and mechanism of Xiangsha Liujunzi decoction on ETEC-induced diarrhea in weaned piglets. Twenty-four 21-day-old weaned piglets were randomly divided into three groups: the control group (CON), the model group (MOD), and the Xiangsha Liujunzi decoction group (XS). The XS group was given Xiangsha Liujunzi decoction (1 mL·kg^-1, 1 g·mL^-1) for 14 consecutive days, and the other groups were given the same amount of sterile water. On the 15th day, piglets in groups MOD and XS were given 10¹¹ CFU·mL^-1 ETEC bacterial solution at a dose of 1 mL·kg^-1 for 3 consecutive days, and piglets in group XS were given Xiangsha Liujunzi decoction. The diarrheal degree of the piglets in each group was recorded by fecal score. Pathological changes of the ileum were observed by HE staining. By means of qRT-PCR, the mRNA expression levels of IL-1β, IL-6, and IL-8 in ileum tissue were detected. Transcriptomics and metagenomics were used to analyze the differential expression of RNA in intestinal tissues and changes in the intestinal flora, respectively. The protein levels of p-p38/p38, p-ERK/ERK, and p-JNK/JNK in ileum tissue were detected by Western blot. The results showed that, after challenging ETEC, the diarrheal score was significantly higher (P<0.01) in the MOD group compared to the CON group. The structure of ileum tissue was destroyed in the MOD group. The V/C value was significantly decreased (P<0.01), and the mRNA expression of IL-1β, IL-6, and IL-8 were significantly increased (P<0.01) in the MOD group compared to the CON group. At the phylum level, Proteobacteria was significantly increased (P<0.01), and Firmicutes was significantly decreased (P<0.01) in the MOD group compared to the CON group. At the genus level, Lactobacillus was decreased, and Shigella was increased in the MOD group compared to the CON group. The ratios of p-p38/p38 and p-JNK/JNK in ileum tissues were significantly increased (P<0.05, P<0.01) in the MOD group compared to the CON group. In comparison with the MOD group, the diarrhea score in the XS group was significantly lower (P<0.01). The structure of the ileum tissue was intact in the XS group. The V/C value was significantly increased (P<0.01), and the mRNA expression of IL-1β, IL-6, and IL-8 in the ileum tissue was significantly decreased (P<0.05, P<0.01) in the XS group compared to the MOD group. At the phylum level, Proteobacteria was significantly decreased (P<0.01), and Firmicutes was significantly increased (P<0.01) in the XS group compared to the MOD group. At the genus level, Lactobacillus was increased, and Shigella was decreased in the XS group compared to the MOD group. The ratios of p-p38/p38 and p-ERK/ERK in ileum tissues were significantly decreased (P<0.01) in the XS group compared to the MOD group. Ten genes with significant differences related to inflammatory immunity were screened by the intestinal tissue gene transcriptome, which were TNFAIP8L2, TRIM67, CXCL2, EGF, NOX1, CCL28, FABP2, FABP6, IL1RAP, and CEBPB. The possible marker species for each group were Lactobacillaceae in the CON group, Shigella in the MOD group, and Deinococcus and Eubacterium in the XS group. In conclusion, Xiangsha Liujunzi decoction can effectively alleviate ETEC-induced diarrhea in weanling piglets. Treatment with Xiangsha Liujunzi decoction increases the abundance of beneficial bacteria in the intestinal flora and reverses the changes in the structure of the intestinal flora induced by ETEC. Moreover, Xiangsha Liujunzi decoction inhibits the activation of the MAPK signaling pathway, thereby alleviating ETEC-induced intestinal inflammatory injury.

This study aimed to investigate the effect of Asiatic acid (AA) on lipopolysaccharide (LPS)-induced apoptosis and autophagy in the kidneys of broilers. Forty broilers (one day old) were fed adaptively until seven days old and then were randomly divided into the control group (Con), LPS group (LPS), low dose AA group (LPS+AA 15 mg·kg^-1) and high dose AA group (LPS+AA 30 mg·kg^-1). The broilers in the AA pretreatment group were gavaged with corresponding doses of AA daily for 14 consecutive days. Except for the control group, broilers in other groups were intraperitoneally injected with 0.5 mg·kg^-1 LPS at the age of 16, 18 and 20 days to establish an acute kidney injury (AKI) model. All broilers were sacrificed at the age of 21 days, and kidney samples were collected. Hematoxylin-eosin (HE) staining was used to observe the histopathological changes in the kidney tissue. The kidney coefficient was calculated and the levels of antioxidant enzymes in kidney tissue were detected. Real-time fluorescence quantitative (qRT-PCR) and Western blot were used to detect the expression of apoptosis-related genes and proteins as well as the expression of autophagy-related genes and proteins. Immunohistochemistry and immunofluorescence were used to detect the expression and distribution of cytochrome c (Cytc) and microtubule-associated protein 1A/1B-light chain 3 (LC3) in kidney tissue. The TUNEL staining was used to detect the apoptosis rate of the kidneys. The results showed that AA could alleviate LPS-induced kidney pathological damage of broilers, and significantly reduce the kidney coefficient and malondialdehyde (MDA) content as well as significantly increase the activities of glutathione peroxidase (GSH-Px), superoxide enzyme (SOD) and catalase (CAT) (P<0.05). AA significantly reduced the mRNA expression of P53, BAX and Caspase3 in LPS-induced AKI of broilers (P<0.05), and significantly promoted the mRNA expression of BCl2, Beclin1, ATG5, LC3-I and LC3-II in LPS-induced AKI of broilers (P<0.05). Moreover, AA pretreatment significantly reduced the protein expression of P53, Bak1, BAX and Cleaved-Caspase3 in LPS-induced AKI of broilers (P<0.05), and significantly promoted the protein expression of Beclin1, ATG5, LC3 II/I in LPS-induced AKI of broilers (P<0.05). Immunohistochemical and immunofluorescence results also showed that AA pretreatment significantly reduced the protein expression and distribution of Cytc in LPS-induced AKI (P<0.001), and significantly promoted the protein expression and distribution of LC3 in LPS-induced AKI (P<0.001). The TUNEL results showed that AA pretreatment significantly reduced the LPS-induced apoptosis rate in the kidney of broilers (P<0.001). In summary, AA alleviates LPS-induced AKI of broilers by inhibiting oxidative stress, promoting renal cell autophagy and reducing apoptosis. This result provides a theoretical basis for AA becoming a potential feed additive and preventing LPS-induced AKI of broilers.

This experiment was conducted to study the mechanism of Escherichia coli (E. coli) infection on mitochondrial damage of mammary epithelial cells and mouse mammary gland. In this study, bovine mammary epithelial cells and mouse mammary gland were used as the research object. Bovine mammary epithelial cells and mouse mammary gland were randomly divided into three groups, including Control group, E.coli group and lipopolysaccharide (LPS) group, respectively. No treatment was conduceted in the Control group, boving mammary epithelial cells and mouse mammary gland tissue in the E.coli group were infected with E.coli (MOI=5) for 6 h or 10⁶ CFU E. coli for 24 h, respectively. In the LPS group they were treated with 1 μg·mL^-1 lipopolysaccharide (LPS) for 6 h or 20 mg·kg^-1 body weight LPS for 24 h, respectively. with three replicates in each group, 6 mice in each group. The results showed that: 1) Cytokeratin 18 in bovine mammary epithelial cells was dyed green and evenly distributed in the cytoplasm, and the nucleus was dyed blue by DAPI. 2) The mitochondria in untreated-bovine mammary epithelial cells were intact and the cell connections were tight. In E.coli group, the space between bovine mammary epithelial cells was increased, and mitochondria were swollen with disappeared mitochondrial cristae and blurred partial mitochondria cristae. 3) E. coli infection or LPS treatment caused the appearance of neutrophils in the mammary glands of mice. E.coli infection or LPS significantly decreased (P<0.05 or P<0.01) mitochondrial energy metabolism (decreased D(520 nm) absorption value, ATP concentration, and the activities of mitochondrial electron transport chain complex Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ), mitochondrial fusion and division (reduced Drp1, Fis1, Mfn1, Mfn2, and OPA1 mRNA expressions) and mitochondrial biogenesis (inhibited PGC-1α, NRF1, TFAM, and D-Loop gene expression). These results indicated that E.coli infection caused energy metabolism disorder of mitochondria, inhibited mitochondrial division and fusion, and reduced mitochondrial biogenesis mainly through LPS in bovine mammary epithelial cells and mouse mammary gland, thus causing mitochondrial damage and eventually leading to mastitis. Therefore, it can be inferred that E.coli infection could cause bovine mastitis through inducing mitochondrial damage, which play an important role in mammary gland inflammation.

The aim of this study was to explore the effect and mechanism of Atractylodes Macrocephala-Cistanche Deserticola on constipation through network pharmacology and animal experiments. The constituents and corresponding target proteins of Atractylodes Macrocephala-Cistanche Deserticola were obtained from TCMSP database, and constipation related targets were obtained from GeneCards, OMIM and other databases. After the genes were sorted into UniProt database, softwares including Venny2.1.0, Cytoscape3.7.2 and STRING were used to draw Venn diagram and protein-protein interaction (PPI) network, and then CentiScaPe 2.2 plug-in was used for analysis. Bioinformatics GO analysis and KEGG signaling pathway enrichment analysis were conducted through DAVID database. Some components with more potential targets and core targets were verified by molecular docking with AutodockTools and PyMOL. Forty mice were randomly divided into blank group (6 mice) and loperamide hydrochloride group (34 mice), which were gavaged with distilled water and 10.0 mg·kg^-1 loperamide hydrochloride, respectively. After successful modeling, 30 mice with constipation were evenly distributed into model group, positive control group and high-, medium- and low-dose drug groups. Blank group and model group were intragastric with distilled water, positive control group was intragastric with 10.0 mg·kg^-1 moxapride citrate, and high-, medium- and low-dose groups were intragastric with 4.8, 2.4 and 1.2 g·kg^-1 Atractylodes Macrocephala-Cistanche Deserticola suspension, respectively, once a day for 7 days. Seven days later, the bowel movement and small bowel movement tests were performed. The results showed that PTGS2 was the most highly connected target gene. A total of 19 core targets for the treatment of constipation including AKT1, TNF and IL-6 were screened out. KEGG enrichment analysis showed that lipid and atherosclerosis and hepatitis B pathways were more critical. Molecular docking results showed that the components with more potential targets in Atractylodes Macrocephala-Cistanche Deserticola had a good combination with the core targets of constipation. Animal experiments showed that compared with the model group, the time of first melena was significantly shortened and the small bowel propulsion rate was significantly increased in the medium- and high-dose drug groups. Atractylodes Macrocephala-Cistanche Deserticola can improve slow transit constipation. A variety of active ingredients may regulate multiple signaling pathways to treat constipation through key targets such as PTGS2, AKT1,TNF and IL-6.

The aim of this paper was to study the antigenicity of glutathione peroxidase EnGPX and its subcellular localization in Eimeria necatrix. Total RNA was extracted from the gametophyte of E. necatrix (Yangzhou strain), and the ORF coding sequence of EnGPX was amplified using RT-PCR. The prokaryotic expression plasmid pET-28a(+)-EnGPX was constructed and transformed into BL21(DE3) for in vitro expression, additionally, a mouse anti-rEnGPX polyclonal antibody was prepared and used to analyze the recombinant protein through Western blotting, and laser confocal immunofluorescence localization analysis. The study found that the coding region of the EnGPX gene was 753 base pairs in length and encoded a protein consisting of 250 amino acids. The resulting recombinant protein had a molecular weight of around 30 ku and was predominantly present in the form of inclusion bodies. The recombinant protein exhibited favorable reactivity and cross-reactivity, as it was recognized by a 6×HIS-tagged monoclonal antibody, a mouse anti-rEnGPX polyclonal antibody, and convalescent serum from E. necatrix, E. maxima, and E. tenella. EnGPX was detected in natural gametophyte proteins, with the encoded protein primarily localized to the type II wall-forming body (WFBII) of the gametophyte and oocyst wall. This study successfully cloned and expressed the glutathione peroxidase (EnGPX) of E. necatrix. The recombinant protein exhibited good reactivity, and natural EnGPX protein was found to be localized on the gametophyte and oocyst wall. The results shed light on the molecular mechanism of EnGPX involvement in oocyst wall formation and identify potential targets for the development of novel subunit vaccines against coccidia.

The aim was to investigate the regulatory effect of a monoclonal antibody against D1133L protein of African swine fever virus (ASFV) during virus replication. Based on the preparation of monoclonal antibody against D1133L protein, ASFV and different concentrations of monoclonal antibodies were added simultaneously to PAMs, and the effect of monoclonal antibody against D1133L on virus replication was analyzed by 50% Hemadsorbing dose (HAD₅₀), real-time quantitative PCR (RT-qPCR), Western blot, and observation of fluorescence. Monoclonal antibodies at different concentrations significantly inhibited the viral titer (P<0.01), the expression level of proteins, the gene transcription level (P<0.01), and the expression level of ASFV-GFP green fluorescent protein (P<0.05) at different infection times in a dose-dependent manner. In summary, monoclonal antibody against D1133L significantly inhibited ASFV replication, the results of the trial are informative in the selection of drug targets for ASFV.

The present study aimed to establish a novel real-time fluorescent RT-PCR assay for detection of pigeon megrivirus (PiMeV). Specific primers were designed based on PiMeVs sequences download from the GenBank, and positive fragments (designated as PiMeV-CHN001 strain) were amplified from racing pigeon feces. Then the 3C gene of PiMeV-CHN001 strain was obtained and analyzed. Based on the 3C molecular characterization, a real-time fluorescent RT-PCR method (RT-qPCR) was developed using the specific primers. The results demonstrated the 3C gene of PiMeV-CHN001 strain had 591 bp (coding 197 amino acids), with the nucleic acid sequence homology with other wild urban pigeons (Columba livia) (MeV-B1 strain and MeV-B2 strain) was 89.5% and 92.0%, respectively. The RT-qPCR assay standard curve had the axial intercept of standard curve was 37.93 and the slope was -3.335, with a linear correlation (R2) of 1.00 and efficiency of 99.4%. The methods were specificity, no-cross amplification signal was found from other pigeon viruses (such as avian influenza virus, pigeon paramyxovirus type I, pigeon torque teno virus, pigeon adenovirus, and pigeon circovirus). Only one specific peaked with a melting temperature (Tm) was (81.69±0.22) ℃ form PiMeV-CHN001, with no primer-dimers peak represent. The lowest limit of detection concentration was 54.0 copies/μL. The intra-and inter-assay were less than 1.5% according to the repeatability test. The developed qPCR was used for PiMeV detection of 42 racing pigeon feces. 2 positive (positive rate was 4.76%) signals were found. In conclusion, we firstly confirmed the presence of PiMeV in racing pigeons in Mainland China, and the data can enrich Megrivirus host spectrum. Moreover, the developed RT-qPCR assay also lays good foundation for further PiMeV epidemiological investigation.