Listeria monocytogenes being a common foodborne pathogen. Food contaminated with this bacterial can cause listeriosis, with clinical manifestations of sepsis and meningitis, and mortality be up to 30%. Listeria monocytogenes can survive under extreme condition such as high salinity, high acidity, and low temperature, so posing a significant threat to the human and animal health. At present, the main method of preventing and treating this disease is the use of antibiotics. However, the multiple drug resistance making the prevention and treatment of listeriosis extraordinarily difficult. The development of phage and related biological agents provides feasible solutions to solve the above problem. This article reviews the applications of Listeria phages in food preservation, biological detection, genetic engineering, and the interaction between phages and hosts, aiming to provide a theoretical basis for Listeria phages in food pollution prevention and control.

The application of genetic evaluation software in the animal field has greatly improved the efficiency of breeding work. With the continuous improvement of genome sequencing technology and the rising of artificial intelligence technology, animal genetic evaluation software also experienced rapid development. This paper firstly introduced the application of conventional breeding and genomic breeding in the field of animal breeding, then focused on reviewing the characteristics and development history of genome-wide genetic evaluation software of GBLUP method, Bayesian method, machine learning and deep learning method, and finally discussed the future development trend of computer software in animal genetic evaluation and breeding, which is intended to provide relevant genetic evaluation software references for researchers in the field of animal breeding.

With the development of sequencing technology, a large number of omics sequencing technologies continue to emerge and promote, resulting in a large number of omics data including genome, epigenome, transcriptome, proteome, metabolome, microbiome and so on. These data are of great significance for in-depth study and revelation of the complex regulatory process of important economic traits (growth traits, reproductive traits, meat traits, disease resistance traits, etc.) of livestock and poultry. The complexity of important economic traits of livestock and poultry can not be revealed through a single level of omics alone, while multi-omics technology can systematically analyze the mechanism and phenotype of important economic traits of livestock and poultry, thus gradually become the main method for studying important economic traits of livestock and poultry. This article reviews the methods, advantages and application of multi-omics techniques in the study of important economic traits of livestock and poultry, aiming to provide references and ideas for the study of important economic traits of livestock and poultry.

Heritability is a measure of the proportion of phenotypic variance explained by genetic factors. In actual heritability evaluation, only additive genetic effects are considered. With the development of genomics, genome-wide single nucleotide polymorphisms (SNPs) have been widely used to evaluate heritability because of the difficulty in achieving the integrity and accuracy of genealogy information that traditional heritability estimation relies on. However, accurate evaluation of SNP heritability is helpful in understanding the extent of genetic variation’s effect on phenotype. Currently, many SNP heritability evaluation models have been developed and utilized. In this paper, the concept, common evaluation methods and influencing factors of SNP heritability are reviewed, and a comparison was made between SNP heritability and the traditional heritability estimation, in order to explore the potential application of SNP heritability in livestock breeding.

Complement regulatory protein 46 (CD46) is widely expressed in most mammalian cells. In addition to its various physiological functions such as complement inactivation, regulation of T cell activation and fertility, CD46 can also serve as a receptor for various bacteria and viruses. It is especially important for the attachment of bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV). The use of gene editing technology to specifically modify CD46 has successfully produced live calve that can resist BVDV infections, proving that this gene can be used as a candidate gene for disease resistance breeding. This article mainly reviews the role of CD46 protein in livestock virus infection and its application in disease resistance breeding.

The puberty of female animals is the period when the estrus appears and ovulation occurs for the first time. The mechanism of puberty onset is complex and closely related to genetic, neuroendocrine, metabolic, nutritional, and environmental factors and so on. However, the specific mechanism of animal puberty onset is still unclear. Recent evidence suggests that epigenetic mechanisms are important coordinators in the process of animal puberty onset. The paper reviews the effects of epigenetic mechanisms such as DNA methylation, histone modification, non-coding RNA, chromatin remodeling, genome imprinting, transcription factor binding, and X chromosome inactivation on the onset of puberty, which provides a reference and basis for the further study of the mechanism of puberty onset in female animals.

Feather color plays a crucial role in a chicken’s appearance, and a thorough understanding of the molecular mechanisms governing feather color formation is essential not only for developing distinct color varieties through breeding but also for identifying and distinguishing local breeds. Chicken feather colors exhibit diversity, with barring being a notable pattern that includes sex-linked barring and autosomal barring. The mechanisms responsible for the formation of these two barring patterns is different. In sex-linked barring, the non-black sections of the barred pattern result from the absence of melanocytes capable of producing melanin. Conversely, in autosomal barring, the non-black portions of the barred pattern arise due to the inhibition of melanin production. The principles governing the formation of light and dark colors in both types of barring are similar, primarily involving the modulation of key genes in the melanin deposition process to achieve varying degrees of pigment deposition. This article offers a comprehensive review of the genetic regulatory mechanisms underlying the formation of barring patterns and color intensity. The goal is to serve as a valuable reference for molecular marker-assisted selection in the breeding of meat and egg chickens based on feather color.

Quorum sensing (QS) is a communication mechanism utilized by bacteria to facilitate intra-species or inter-species communication through the signaling molecules. Through this communication mechanism, microorganisms can collectively conduct physiological functions or regulate behaviors that cannot be achieved by microbial individual. These functions include biofilm formation, toxin secretion, bioluminescence, and other adaptive responses to enhance the adaptability of microorganisms against external stress environment. Recently, multi-omics studies have revealed that LuxS/AI-2 QS, mediated by the autoinducer-2 (AI-2) signaling molecule and regulated by the LuxS protein, serves as the primary communication mechanism among rumen microbe, influencing their efficiency in feed utilization. This review provides a summary of the LuxS/AI-2 QS in rumen microbe, focusing on the chemical structure of AI-2 and its transduction pathways. The roles of LuxS/AI-2 QS in rumen microbial colonization and feed digestion in ruminants were reviewed, as well as the potential roles of LuxS/AI-2 QS in feed digestion and stress regulation. This review may provide a theoretical reference for production strategy by regulating the digestive metabolism and homeostasis of rumen microbe through LuxS/AI-2 QS.

Bile acids are cholesterol derivatives that have been shown to be remarkably effective in improving the digestion and absorption of fats in the daily diet. In the liver, cells utilize cholesterol to form primary bile acids, while under the influence of proteases secreted by intestinal flora, primary bile acids turned to secondary bile acids, which greatly expanding the molecular diversity of the intestinal environment. Currently, the most common secondary bile acid receptors are transmembrane G protein bile acid-coupled receptor-5 (GPBAR1, also known as TGR5) and the nuclear receptor farnesoid X receptor (FXR), which play pleiotropic roles in the regulation of organismal health, in particular to maintain intestinal flora homeostasis and mucosal immune system homeostasis. This review discusses the relationship between bile acids and intestinal flora, the metabolism of secondary bile acids, and the different mechanisms of action of secondary bile acids and their receptors in the intestinal immune system, which may provide a theoretical basis for the prevention and control of intestinal diseases in animals as well as related research.

Embryonic lethal abnormal vision like 1 (ELAVL1), also called human antigen R (HuR).It is a typical RNA binding protein (RBP) that binds to the AU-rich element (ARE) in the 3'untranslated element (3'UTR) and plays an important role in the process of mRNA transcription and translation. ELAVL1 has a wide range of functions and can regulate the occurence and development, apoptosis, migration, and invasion of tumors, making it the key target for many cancer treatments. It can also participate in the regulation of protein and mRNA levels encoding organ development and tissue homeostasis. Many studies have also shown that ELAVL1 regulates viral replication through post-transcriptional regulation and post-translational modification. This review summarizes the biological characteristics, functions, regulatory mechanisms of ELAVL1 and its regulatory role in the process of virus replication, aiming to provide a theoretical reference for further studies on the interaction between ELAVL1 and virus replication in livestock and poultry.

Postbiotics are the preparation of inanimate microorganisms and/or their components/metabolites that confer a health benefit on the host. Effective postbiotics must contain inactivated microbial cells or cell components, and cell components mainly include cell walls, cell membranes, lipoteichoic acid, lipopolysaccharides, extracellular vesicles, pilus and cell surface protein. In this paper, the mechanism of action of postbiotics is reviewed from five aspects of probiotic functions such as regulation of intestinal flora, enhancement of intestinal barrier function, regulation of immune response, regulation of body metabolism and regulation of neurotransmission, etc., and the application of different sources of postbiotics such as Lactobacillus source and yeast source in animals is also summarised, intending to provide a reference for the subsequent classification, development and utilization of postbiotics.

The aim of this study was to estimate the genetic parameters of growth, reproduction traits and body measurements of French Large White pigs, and provide a basis for the selection of target traits and the setting of weight coefficient of index model in breeding process. In this study, 8 751,12 762,7 407 records for growth trait, reproductive trait and body measurements were collected, respectively from a pig breeding farm in Heilongjiang Province from 2020 to 2023. DMU software was used for statistical analyses of all data. Genetic parameters was estimated by using animal models for the following traits: growth traits including the age (D100), average daily gain (DG100), backfat thickness (BF100), eye muscle area at 100 kg (LMA100), reproductive traits including total number born (TNB), number born alive (NBA), number healthy born (NHB), litter birth weight (LBW), body measurements including body length (BL), body height (BH) and tube circumference (CBC) in French Large White pigs. The results showed that the heritability estimates in French Large White pigs were 0.232, 0.220, 0.334 and 0.390 for growth traits (D100, DG100, BF100 and LMA100), respectively, which belonged to the medium heritability traits. The heritability estimates were 0.081, 0.065, 0.060, 0.090 for reproductive traits (TNB, NBA, NHB, LBW), respectively, all belonging to low heritability traits. The heritability estimates were 0.677, 0.877 and 0.227 for body measurements traits (BL, BH and CBC), respectively. BL and BH belonged to high heritability traits, while CBC belonged to medium heritability traits. The genetic and phenotypic correlations between D100 and DG100 in French Large White pigs were -0.999 and -0.994, respectively, showing strong negative correlations. There were positive genetic correlation and phenotypic correlation among reproductive traits, and the genetic and phenotypic correlation between NBA and NHB were the highest, which were 0.995 and 0.977, respectively. The genetic and phenotypic correlations among body size traits ranged from -0.345 to 0.246 and from -0.057 to 0.125, respectively. A negative but not high genetic and phenotypic correlation was estimated between BL and BH. The above results indicate that it is necessary to formulate a reasonable breeding program according to the different characteristics of the genetic parameters for above growth, reproductive traits and body measurements in breeding process. The estimated genetic parameters in French Large White pigs in this study could provide some reference for formulating breeding programs and improving breeding efficiency.

To gain a deeper understanding of the genetic basis of meat quality traits, this study performed GWAS analysis using genotype-based populated sequencing data and co-localisation analysis with previously published EWAS results. The study identified 515 genome-wide significant SNP loci and 4 134 SNP loci at the proposed significance threshold, which involved 406 genes, including 262 protein-coding genes, and resulted in the identification of 6 important candidate genes that may be associated with meat quality traits. Meanwhile, comparison with the results of EWAS revealed that 12 significant CpG loci overlapped with significant SNP loci. The study results can provide a more solid theoretical foundation and data support for breeding efforts to improve pork quality.

This study, using Yorkshire pigs as controls, aimed to explore the differences in blood physiological indicators and the expression profiles of immune-related organ tissues between Tibetan pigs and Yorkshire pigs. Additionally, the expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in Tibetan pig peripheral blood lymphocytes stimulated with LPS was investigated, to understand their impact on the immune characteristics of Tibetan pigs. Forty Tibetan pigs and forty Yorkshire pigs aged 180 days were divided into two experimental groups based on breed. Fresh blood samples were collected for detecting blood physiological indicators, and peripheral blood lymphocytes were cultured for further analysis. Moreover, 8 Tibetan pigs and 8 Yorkshire pigs from each breed were randomly selected and divided into two experimental groups. Fresh blood was collected from the anterior vena cava, and liver, spleen, and submandibular lymph node tissues were collected post-slaughter for RNA extraction using the Trizol method. Each individual sample was subjected to 3 replicates for RT-qPCR and next-generation sequencing analysis to assess the expression levels and genetic polymorphisms of IL-1β, IL-6, and TNF-α genes. Peripheral blood lymphocytes were stimulated with 4 concentrations of LPS (0, 1, 10, 100 μg·mL^-1), with 3 replicates per group. Cells were collected at 0, 24, 36, 48, and 72 h, and RT-qPCR was performed to assess the expression of pro-inflammatory cytokine mRNA. Results indicate that: 1) Tibetan pigs exhibited significantly higher levels of WBC, RBC, HGB, HCT, and PLT compared to Yorkshire pigs (P<0.01). Additionally, the average MCV and PCT were significantly higher in Tibetan pigs than those in Yorkshire pigs (P<0.05). 2) RT-qPCR results revealed significantly higher mRNA expression levels of IL-1β, IL-6, and TNF-α genes in the blood, spleen, and liver tissues of Tibetan pigs compared to Yorkshire pigs (P<0.01). In the submandibular lymph nodes of Tibetan pigs, the expression levels of IL-1β and TNF-α genes were significantly higher (P<0.05), while the mRNA expression level of the IL-6 gene was extremely significantly higher (P<0.01). 3) SNP site screening identified 5 mutation sites in the 3' flanking region of the IL-1β gene. Among them, G⁶⁹⁰T, C¹³⁸³G, C¹⁴⁸⁰T, and A¹⁴⁹⁷G sites showed extremely significant differences between Tibetan pigs and Yorkshire pigs (P<0.01), and the C¹⁴⁵⁴T site exhibited significant differences (P<0.05). The IL-6 gene’s 3' flanking region contained a non-synonymous mutation site C²⁶⁵T, which showed significant differences (P<0.05), and a non-synonymous mutation site A-72G in the 5' flanking region exhibited highly significant differences (P<0.01). The TNF-α gene had a non-synonymous mutation site in the 5' flanking region, but the difference between the two breeds was not significant (P>0.05). 4) Different LPS concentrations stimulated Tibetan peripheral blood lymphocytes, with 10 μg·mL^-1 LPS showing the most significant proliferative effect. At concentrations of 1 and 10 μg·mL^-1 LPS stimulation, the IL-1β gene responded significantly, and at a concentration of 100 μg·mL^-1 LPS, the IL-6 gene exhibited a strong response. In summary, compared to Yorkshire pigs, Tibetan pigs demonstrated stronger resistance in blood physiological indicators, immune organ cytokine expression, and genetic polymorphism. The A-72G site in the IL-6 gene may be closely related to immunological and disease resistance capabilities. The significant responses of IL-1β and IL-6 genes to LPS in Tibetan pig peripheral blood lymphocyte culture experiments highlight their critical role in immune regulation.

The objective of this study was to investigate the impact of birth season on milk performance in dairy cows. The 128 346 DHI measurements were collected from 9 515 dairy cows in Tianjin between 2020 and 2023, these data included birth date, daily milk yield, milk fat percentage, milk protein percentage, and 305-day milk yield. The effects of different birth seasons, birth orders, fields and birth years on lactation performance were analyzed using the general linear model of SPSS22.0 software. Wood models were employed to fit lactation curves for different birth seasons and parities, taking the birth season as the independent variable, the parameters obtained from the lactation curve fitting were used to calculate the secondary parameters: the peak day of lactation, peak milk yield, and lactation persistence as the dependent variables, while single-factor analysis of variance was used to assess the impact of birth season on milk performance. Birth seasons, different parity, birth season×parity interaction had significant effects on lactation performance (P<0.01).Calves born during winter exhibited significantly higher milk yield at one and two parities compared to calves born during other seasons(P<0.05). Calves born during summer showed higher average milk fat percentage and average milk protein percentage than those born in other seasons at various parity levels, these differences were significant at second parity onwards (P<0.05). Furthermore, calves born during winter had significantly higher 305-day milk yield and adult equivalent compared to other seasons (P<0.05), while autumn-born calves at second and third parities demonstrated significantly higher values for these parameters compared to corresponding parities in other seasons (P<0.05). Autumn cows displayed an earlier peak lactation day across different parities with the highest peak volume observed among those with two or more parities. Birth season, parity level, birth field, the interaction effect between birth season and parity level had a highly significant influence on milk performance of Holstein cattle in Tianjin area. The 305 days milk yield of autumn-born calves after 2,3 births were the highest, their milk peak day came earliest and the peak milk yield were highest.

The aim of this study was to identify candidate genes for heart development in goose embryos by analyzing the gene expression differences in embryonic hearts tissue under green light stimulation. The 512 goose eggs were randomly divided equally into green light and dark incubators, with 4 layers and 64 eggs per layer placed in each incubator. During the entire incubation period, the green light incubator provided intermittent light regimen of 15 minutes on and 15 minutes off. On the 13, 16, 20, 24, 28, and 30 day of incubation, 8 eggs were randomly selected from each group, the embryo weight was measured, and the embryonic heart tissue was separated and weighed. The embryonic heart tissues were collected at day13 of incubation, and transcriptome sequencing (RNA-Seq) were performed. Differentially expressed genes (DEGs) were screened, and subjected to functional enrichment analysis to identify candidate genes related to green light promoting heart development. And the reliability of the sequencing results was validated using fluorescence quantitative PCR (qRT-PCR). The green light significantly increased the proportion of embryo weight at 16 and 30 embryonic ages (embryo weight/hatched egg weight×100) (P<0.05), and the heart index (heart weight/embryo weight×100) at 13 and 16 embryonic ages in the green light group were significantly higher than individuals in the dark group (P<0.05). A total of 1 643 DEGs were screened from the green light group and the dark group, 7 DEGs were randomly selected for RT-qPCR validation, and the expression trend was consistent with the sequencing results. Enrichment analysis showed that DEGs were mainly enriched in the PI3K-Akt signaling pathway, AMPK signaling pathway, and other pathways. Four candidate genes: GATA4, GATA5, Smad4 and GHR were ultimately screened out. Their expression in heart tissues at different embryonic ages were analyzed to further validated their regulatory role in heart development. Through the analysis of transcriptomic data of heart tissue, we identified GATA4, GATA5, Smad4 and GHR as candidate genes for goose embryonic heart development, which provide clues to the molecular regulatory mechanism of green light on embryonic heart development, and provide a theoretical basis for the application of green light in goose egg hatching.

This study aimed to analyze the population genetic structure and runs of homozygosity (ROH) of Fuling buffalo and other swamp buffalo breeds in southwest China, and provide the theoretical basis for conservation and genetic improvement of Fuling buffalo. In this study, 26 Fuling buffaloes and one hybrid buffalo (FM_HY) were sequenced using whole-genome resequencing technology. The genome resequencing data of 68 swamp buffalo individuals in southwest China and 7 river buffalo individuals were downloaded from the NCBI database. All the genome data were used to obtain high-quality single nucleotide polymorphisms (SNPs) and to analyze the population genetic structure, ROH and inbreeding coefficient. The whole-genome resequencing results showed that the average sequencing depth of Fuling buffalo was 10×, and a total of 14 326 437 SNPs were identified after quality control. The population genetic structure analysis showed that there were significant differences between the Fuling buffalo and the neighboring areas swamp buffalo breeds including Yibin buffalo, Guizhou buffalo, Guizhou White buffalo and Yanjin buffalo. Dehong buffalo and Diandongnan buffalo were significantly different from the above 5 breeds. When the number of clusters was K=6, Fuling buffalo was divided into two groups, which had a unique population structure compared to the neighboring areas buffalo breeds. A total of 2 722 ROHs were detected in Fuling buffalo, Yibin buffalo and Guizhou buffalo. The 1 593 ROHs were identified in Fuling buffalo, and the 78.15% proportion of ROH was the short ROH with 1-2 Mb. Eight long ROH fragments>16 Mb were only detected in Fuling buffalo population, the longest being 24.56 Mb. Based on ROH, the average inbreeding coefficient of Fuling buffalo was 0.056 8, of which 7 individuals had higher inbreeding coefficient in 0.062 5-0.125 0. The average inbreeding coefficients of Guizhou buffalo and Yibin buffalo were 0.034 6 and 0.041 0, respectively. In conclusion, the population of Fuling buffalo in the conservation farm can be divided into two groups, which have a unique population structure compared to the other neighboring areas buffalo breeds, and 7 Fuling buffalo individuals in the conservation farm have inbreeding risk, so it is urgent to strengthen the protection and utilization of Fuling buffalo resources.

The purpose of this study was to reveal the interrelationships and effects of cytoskeleton and cortical granule migration during in vitro maturation of frozen porcine GV-stage oocytes. Pig ovary samples were collected from healthy multiparous sows.For each sampling, about 300 Pudong White sows weighing 70-90 kg with good production performance were used to take ovarian samples, the number of ovaries collected was 120-150 per time, and the GV stage oocytes with homogeneous and compact cytoplasmic distribution were selected and randomly divided into 3 groups according to the experimental requirements, and 3 replicates were set up in each group, with at least 30 cells in each group, and cytoskeleton stabilizers, cytochalasin B (CB) were used to treat the frozen porcine oocytes before freezing. Porcine GV oocytes were treated with cytoskeleton stabilizer CB before freezing, and then matured in vitro after freezing and thawing. Oocyte survival, fluorescence co-localization of microfilaments and cortical granules at each time point, degree of spreading of the oval mound, rate of discharge of the first polar body, level of glutathione (GSH), and developmental ability were examined. The result showed that treatment with CB before freezing effectively increased the survival rate of GV stage oocytes after thawing (44.11% vs. 27.91%, P<0.01). During the maturation of fresh oocytes, co-migration of cortical granules with microfilaments was observed. Freezing caused abnormal cortical granule-microfilament migration, and the addition of CB prior to freezing alleviated the freezing-induced migration obstacles, as evidenced by a higher proportion of both microfilaments and cortical granules successfully migrating to the plasma membrane in the CB-treated group, and a better homogeneous distribution of migration to the plasma membrane. After in vitro maturation of frozen oocytes, CB pretreatment was associated with a higher rate of discharge of first polar body ((26.79±2.37)% vs. (8.13±0.30)%, P<0.01), GSH levels (23.12±2.65 vs. 7.27±0.79, P<0.01), cleavage rate after parthenogenetic activation ((20.91±2.84)% vs. (5.64±0.37)%, P<0.05) and blastocyst rate ((5.00±0.03)% vs. (0.41±0.01)%, P<0.05) were significantly increased. The present study showed that the microfilament cytoskeleton has a certain regulatory effect on the migration of intracellular cortical particles from the interior to the plasma membrane during oocyte maturation in vitro, and there exists a certain co-localization relationship between the two, and that the addition of CB incubation before freezing can effectively affect the degree of migration of the cellular microfilament cytoskeleton and cortical particles, mitigate the abnormal distribution of the cytoskeleton induced by freezing, and ease the migration obstruction of cortical particles and microfilaments induced by freezing, thereby enhancing cellular resistance, which is manifested as promoting cytoplasmic maturation, improving the survival rate of frozen oocytes, nuclear maturation rate and embryo development potential in vitro.

The previous studies have found that mitochondrial tRNA-Lys(T7719G) variation was associated with litter size, which decreased the apoptosis of granulosa cells in Small-tailed Han sheep. Therefore, the physiological mechanism of mitochondrial tRNA-Lys(T7719G) variation on granulosus cell apoptosis was explored. Three sheep were selected with G and T genotypes respectively, and slaughtered for ovarian tissue culture of granulosa cells. The detection experiment of granule cells physiological function were divided into two groups with 3 replicates in each group. Mitochondrial genome copy number, mitochondrial respiratory enzyme complex Ⅰ, complex Ⅱ, complex Ⅲ, complex Ⅳ and complex Ⅴ activities were detected, respectively. Mitochondrial oxygen consumption and extracellular acid production capacity, ROS production, ATP level and membrane potential, as well as total tRNA-Lys and aminoacylation level, expression level of 13 mtDNA-encoded polypeptide proteins were detected. The results showed that the mitochondrial copy number of G genotype granulosa cells was significantly different from that of T genotype (P<0.01), which was 1.78 times that of T genotype. The activity of mitochondrial complex III in G genotype cells was 1.25 times higher than that in T type cells (P<0.05), indicating that the oxidative respiratory function of G genotype cells was enhanced. The mitochondrial oxygen consumption and ATP-coupled oxygen consumption of G genotype granulosa cells were 6.84% (P<0.05) and 15.46% (P<0.01) higher than those of T genotype cells, respectively. The glycolysis ability of G genotype cells was significantly decreased (P<0.01), the level of reactive oxygen species was significantly decreased by 34%, the ATP content was significantly increased, which was 1.25 times that of T genotype (P<0.05), and the mitochondrial membrane potential was significantly increased, which was 1.24 times that of T genotype (P<0.01). Total tRNA-Lys was significantly increased by 50% (P<0.01) in G genotype cells, and the proportion of amylated tRNA-Lys was increased by 33.4% (P<0.01) compared to T genotype cells. The expression levels of all 13 polypeptides encoded by the mitochondrial genome (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, CtyB, COⅠ, COⅡ, COⅢ, ATP6, ATP8) were significantly increased (P<0.05). The physiological mechanism of mitochondrial tRNA-Lys(T7719G) variation affecting ovine granulosa cell apoptosis was initially analyzed, that is, tRNA-Lys(T7719G) variation affected tRNA-Lys homeostasis and aminoacylation level, affected the translation efficiency of mtDNA-encoded peptides, and caused changes in copy number, ATP level, ROS content, and some enzyme activities, and then the energy metabolism function of mitochondria and apoptosis were changed.

The aim of this study was to investigate the effects of androgen receptor (AR) on goat granulosa cell proliferation and apoptosis using enzalutamide. Firstly, granulosa cells from goats were isolated and subjected to in vitro culture. The suitable concentration of enzalutamide for cell culture was determined by microscopic observation and CCK8 assay. The experiment was then divided into 3 groups: blank control group (no treatment), negative control group (treated with DMSO), and experimental group (treated with enzalutamide). Each group was set up with 3 replicates. Cell proliferation was detected using the EDU assay, while cell apoptosis was assessed using Caspase3 activity and apoptosis detection kits. The expression of genes and proteins related to cell proliferation and apoptosis was evaluated using quantitative real-time PCR (qRT-PCR) and Western blot analysis. The results showed that the AR inhibitor, enzalutamide, significantly inhibited cell proliferation and reduced the mRNA levels of CCND1, PCNA, and MKI67 in granulosa cells. It also suppressed the protein expression of CCND1, PCNA, and β-catenin. Additionally, enzalutamide promoted apoptosis in goat granulosa cells, as evidenced by increased mRNA levels of BAX and BCL2, without affecting their protein expression. Furthermore, enzalutamide, while not affecting the mRNA level of CASP3, enhanced Caspase3 activity. These findings suggest that enzalutamide, by inhibiting AR activity, can modulate the expression of β-catenin, PCNA, CCND1, Caspase 3, and other genes involved in the regulation of granulosa cell proliferation and apoptosis.

The aim of this experiment was to eliminate the inflammatory response of bovine ovarian follicular granulosa cells (GCs) exposed to lipopolysaccharide (LPS) in vitro and to attenuate LPS-induced functional disorders of follicular GCs using D₁₉, a derivative of 2-methoxy-1,4-naphthoquinone (MNQ) from the extract of Impatiens balsamina L. Ovarian tissues were collected from sexually mature and healthy Holstein cattle, and follicular GCs were isolated and cultured. The effect of D₁₉ and LPS on the survival of follicular GCs was determined by MTT assay. The experiment was divided into 3 groups: control, LPS-treated and D₁₉-combined LPS-treated groups, with 3 replicates in each group. The relative expression of inflammatory factors and genes related to steroid synthesis was determined by qRT-PCR. TEM was performed to observe the protective effect of D₁₉ against cellular inflammatory injury. ELISA was performed to measure the levels of estradiol (E₂) and progesterone (P₄) in the supernatant of the culture fluid. The results showed that the maximum no-cytotoxic concentration of D₁₉ acting on follicular GCs for 12 h was 64 μmol·L^-1. LPS concentration of 10 μg·mL^-1 had little effect on the survival of follicular GCs after 12 h of action, but the relative expression of the inflammatory factors IL-6, IL-1β, and TNF-α was significantly higher (P<0.01), whereas the relative expression of the inflammatory factors in the D₁₉+L group was significantly lower compared with the LPS group (P<0.01). The protective effect of D₁₉ against LPS-induced structural damage to organelles was demonstrated by TEM observation. ELISA results showed that the levels of E₂ and P₄ in the culture broth were significantly reduced after LPS treatment (P<0.01), and the relative expression of steroid synthesis-related genes CYP19A1, CYP11A1, 3β-HSD, and STAR as detected by qRT-PCR was also significantly reduced (P<0.01). However, the amount of E₂ and P₄ secretion as well as the relative expression of steroid synthesis-related genes were significantly higher in the D₁₉ combined with LPS treatment than that in the LPS group (P<0.01). In the present study, we demonstrated that D₁₉, a derivative of MNQ, has the function of eliminating the inflammatory response of follicular GCs induced by LPS in vitro, and it can alleviate the functional disorders of follicular GCs caused by LPS to a certain extent.

This study was conducted to investigate the effects of dietary lysine supplementation on fecal fermentation parameters, microbial diversity and microflora structure of beef cattle. Four healthy Jinjiang cattle with similar body weight were randomly divided into two groups with two cattle in each group. A repeated 2×2 Latin square design was adopted with the control group fed the basal diet and the treatment group fed the basal diet +0.20% of lysine. The experiment lasted for 30 days and was divided into 2 phases, in which the first 10 days of each phase designated as the adaptation period and the last 5 days being the sample collection period. Fecal samples were collected and then analyzed for fermentation parameters, microbial diversity and microflora structure. The results showed as follows: 1) Dietary supplementation of lysine significantly increased the concentrations of isobutyrate, isovalerate and branched-chain volatile fatty acids in feces (P<0.05); 2) Lysine supplementation had no significant effect on fecal microbial richness and evenness (P>0.05); 3) Taxonomic annotation found that the supplementation of lysine significantly increased the relative abundance of Lachnospiraceae NK4A136 group in feces, whilst the relative abundance of Christensenellaceae R-7 group was decreased as compared to the control group (P<0.05); 4) Both principal coordinate analysis (PCoA) and analysis of similarity (ANOSIM) showed that lysine supplementation had no significant effect on fecal microbial community structure. In conclusion, dietary supplementation of 0.20% lysine could improve the concentrations of isobutyrate, isovalerate and branched-chain volatile fatty acids in feces, as well as alterations in the relative abundances of Lachnospiraceae NK4A136 group and Christensenellaceae R-7 group, but has no significant effect on fecal microbial diversity and microflora structure.

In order to establish a specific indirect ELISA method that can replace the detection of neutralizing antibodies against bovine coronavirus (BCoV), the recombinant S1 protein of BCoV was prepared by expression of insect baculovirus expression system. The purified recombinant S1 protein was used as coating antigen to establish an indirect ELISA method for detection of BCoV S1 protein antibody. The results showed that the recombinant S1 protein was about 100 ku and could be expressed in the supernatant of insect High5 cell culture medium, and the antigenicity of S1 protein was identified by Western blot. After the reaction conditions were optimized, the antigen coating concentration was 0.125 μg·mL^-1, and the dilution of serum was 1∶200. The type of sealant is 1% gelatin and the sealing time is 3 h at 37 ℃. The action time of serum is incubating 30 min at 37 ℃. The dilution of enzyme-labeled second antibody was 1∶25 000, and the time was incubated at 37 ℃ for 45 min. The time for substrate coloration was 13 min at 37 ℃. The critical value was 0.297. This method had a good specificity, the maximum coefficients of variation of inter-assay and intra-assay repeatability tests were less than 10%. Taking IFA as the standard, the results showed that when the critical value OD_{450 nm} was 0.297, the sensitivity of IFA was 96.88% (31/32) and the specificity was 87.50% (7/8), which was highly consistent with the serum neutralization test (κ=0.844, P<0.01). Taking the neutralization test as the standard, the results showed that when the critical value OD_{450 nm} was 0.297, the sensitivity of ELISA was 100.00% (31/31) and the specificity was 88.89% (8/9), which was highly consistent with the serum neutralization test (κ=0.925, P<0.01). Three hundred and three samples of bovine serum were detected by this method, and the positive rate was 74.91%. Therefore, the established ELISA method has strong specificity, high sensitivity and good repeatability, which can be used as an alternative to neutralization test to detect BCoV antibody levels after immunization.

Rep protein plays an important role in porcine circovirus replication. This study aimed to analyze the endonuclease activity，ATPase activity and helicase activity of porcine circovirus type 3(PCV3)Rep protein. The recombinant proteins were purified by Ni²⁺ affinity column chromatography. We established an enzymatic activity assay of Rep protein by optimizing the reaction conditions，and the key active site of Rep protein was explored. Our results confirmed that Rep has endonuclease activity、ATPase activity and helicase activity in vitro. Rep protein exhibited optimal ATPase activity in reaction with 1.6 μmol·L^-1 Rep and 1 mmol·L^-1 Mg²⁺ at 37 ℃ for 75 min. It exhibited optimal ATPase activity in reaction with 7 μmol·L^-1 Rep and 12.5 mmol·L^-1 Mg²⁺ at 37 ℃ for 75 min. Y89 of PCV3 Rep was essential for its endonuclease activity. And K173, D210, N250 sites were essential for its ATPase and helicase activities. In this paper, we developed a method for analysis of the enzyme activity and preliminarily revealed the main functional sites of PCV3 Rep protein，which laid a foundation for the functional study of PCV3 Rep protein and the development of antiviral drugs based on the activity of Rep protein.

The present study aimed to develop an indirect ELISA to detect antibodies against the Cap protein of porcine circovirus type 4 (PCV4), and to detect PCV4 antibodies in pigs, then compare the results with previous investigations on PCV4 seroepidemiology, so as to understand the prevalence and infection of PCV4 in swine populations more comprehensively. PCV4 Cap protein was successfully expressed in a prokaryotic expression system. By optimizing the reaction conditions, an indirect ELISA method based on PCV4 Cap protein was established. To investigate the serological prevalence of PCV4 in Chinese pig herds, 2 298 serum samples from pigs at different growth stages from 18 provinces in China were analyzed using the established ELISA method. Concurrently, the Cap-ELISA method was compared with Rep-ELISA, and 845 serum samples were tested in parallel. The results showed that PCV4 antibodies were detected in serum samples from 17 out of the 18 provinces. The total seroprevalence of PCV4 was 34.94%, and the positive rates of sows and finishing pigs were 59.95% and 31.64%, respectively. The positive rate of piglets was 21.14%, while nursery pigs had the lowest positive rate at 4.20%. A total of 845 serum samples were tested using both methods, and the coincidence rate was 82.84%. The Cap-ELISA test yielded an overall positive rate of 31.83%, while the Rep-ELISA test had an overall positive rate of 35.03%. Studies have demonstrated the widespread prevalence of PCV4 across the country and its ability to infect pigs of all ages. The Cap-ELISA and Rep-ELISA techniques exhibited a high degree of concordance, and based on the antibody growth and decline trends, both methods indicated that PCV4 infection primarily occurs in finishing pigs and sows. The present study provides the most recent data on the seroepidemiological characteristics of PCV4 in China and PCV4 infection prevalence in pigs at different growth stages, which could facilitate further research on PCV4 and its management.

African swine fever (ASF) is a highly contagious and deadly disease caused by the African swine fever virus (ASFV), which has dealt a heavy blow to the healthy development of China’s pig industry in recent years. The large genome of ASFV makes it difficult to obtain whole-genome sequence about epidemic strains in a timely manner. This study aims to establish a simple and reliable ASFV whole-genome sequencing method using Nanopore third-generation sequencing technology. Thirty-one primer pairs covering the entire ASFV genome were designed and divided into 4 primer pools to amplify the sample. The amplified product was sequenced by Nanopore sequencing technology, and the relevant bioinformatics analysis methods were further optimized, and finally the ASFV whole-genome sequencing method was successfully established. Whole-genome sequence of ASFV with a total length of 189 416 bp was successfully obtained from an environmental swab sample by this method. Validation through first-generation sequencing has shown that the result of this method is 100% consistent with first-generation sequencing results in key genes and certain variant positions, including B646L, EP402R, E183L, MGF_360-12L, MGF_505-3R, and I177L gene. At the whole-genome level, the consistency between the result of this method and next-generation sequencing(NGS)result is 99.94%. In addition, the utilization of Nanopore sequencing technology in this study revealed a 56 bp repeat sequence insertion within the intergenic region flanked by the NP1450L and NP419L genes. This insertion was subsequently confirmed via first-generation sequencing techniques. Intriguingly, NGS methods failed to detect this distinct variant feature. This study successfully established an ASFV whole-genome sequencing method based on Nanopore technology. This method demonstrates excellent simplicity and reliability, providing an essential tool for the current prevention and control and molecular epidemiological research of ASF.

The aim was to compare the strength of Lacticaseibacillus paracasei of porcine origin, Limosilactobacillus reuteri of porcine origin, and Lactobacillus johnsonii of porcine origin as oral vaccine vectors to express exogenous proteins to stimulate immunity production in piglets with a view to selecting suitable lactic acid bacteria as receptor vectors.In this study, the acid and bile salt tolerance of recombinant porcine Lacticaseibacillus paracasei (pPG-T7g10-S1/L. paracasei 27-2), Limosilactobacillus reuteri (pPG-T7g10-S1/L. reuteri J31) and Lactobacillus johnsonii (pPG-T7g10-S1/L. johnsonii 6332) were measured in vitro to investigate the resistance of the three strains expressing PEDV S1 protein.The results showed that all the three recombinant strains could tolerate acid and bile salt environment, and there was no significant difference with their wild type strains. Then to compare the immune effects, the newborn piglets were orally immunized with the three recombinant strains, separately. Indirect ELISA and neutralization test were used to detect the specific antibodies and neutralizing activity of the piglets, and the levels of each cytokine in serum and intestinal mucosa of the immunized piglets were measured. The results showed that after oral immunization, compared with the control group, the levels of serum IgG antibody and SIgA antibody in nasal swabs, anal swabs and intestinal mucus of piglets in the three recombinant strains groups were significantly increased, lasted to the 28th day. The antibody level induced by pPG-T7g10-S1/L. paracasei 27-2 group was significantly higher than that of the other two groups (P<0.05). Pigley-produced specific IgG and SIgA both showed neutralizing activity against PEDV. The levels of IFN-γ, IL-2, IL-4 and IL-10 in the serum of piglets were significantly higher than those in the control group (P<0.05), but there was no significant difference in the levels of cytokines among the three recombinant strains. The levels of cytokines IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-17, IL-21, TGF-β, APRIL and BALL in the jejunal mucosa of piglets were higher than those in the control group. The levels of IL-4, IL-5, IL-6, TGF-β, IL-17, IL-21 and BALL in pPG-T7g10-S1/L. paracasei 27-2 group were significantly higher than those in other groups (P<0.05). In conclusion, in this study, oral immunization of piglets with recombinant L. paracasei, L. reuteri and L. johnsonii, constitutively expressing the major protective antigen S1 of PEDV, showed that they could stimulate mucosal immunity, humoral immunity and cellular immunity against PEDV. Compared with the other two recombinant strains, the recombinant porcine pPG-T7g10-S1/L. paracasei 27-2 had the best oral immunization effect. The results of this experiment provide scientific data for the construction of a more effective oral vaccine for lactic acid bacteria.

C8orf4, also known as thyroid carcinoma 1 (TC1), was originally cloned from thyroid carcinoma and its surrounding normal thyroid tissue. C8orf4 is universally expressed in vertebrates with sequence conservation across species. Previous studies have shown that C8orf4 is highly expressed in tumors and is involved in various cancer cell communication. It is a novel regulator of cancer and inflammation, and enhances inflammation by regulating NF-κB transcription. However, little is known about the effects of C8orf4 on viruses. This experiment was designed to study the effect of C8orf4 encoding protein on the replication of porcine epidemic diarrhea virus (PEDV). PEDV was inoculated into Vero cells, and the effect of PEDV on C8orf4 expression was detected by qRT-PCR and Western blot at different time points. The eukaryotic expression vector pcDNA3.1-C8orf4-His and specific siRNA of C8orf4 gene were designed and synthesized. The effect of C8orf4 on PEDV replication was detected by qRT-PCR and Western blot through overexpression and inhibition of C8orf4 expression. Furthermore, the effect of C8orf4 encoding protein on the proliferation of PEDV at each stage of the replication cycle was determined by overexpression of C8orf4 encoding protein. With the extension of virus infection time, the expression of C8orf4 in cells showed an upward trend. Overexpression of C8orf4 significantly inhibited PEDV replication, while interfered with C8orf4 expression to promote PEDV replication, and C8orf4 encoding protein mainly played a role in the biosynthetic phase of PEDV replication cycle. This study proved that C8orf4 encoding protein can inhibit PEDV replication, which provides a reference for the study of the function of C8orf4 and the basis for the study of anti-PEDV replication mechanism.

The genetic evolution and pathogenicity of isolated infectious bronchitis virus (IBV) strains in the laboratory were studied to investigate the prevalence of IBV and the pathogenic characteristics of dominant strains, and to provide reference for the development of new vaccines. In this study, the complete S1 nucleotide sequences of 60 strains IBV were analyzed, and three dominant genotypes were selected for whole genome sequencing analysis, named MH20, KC and JS96 strains. JS96 strain with higher virulence was selected for SPF chicken pathogenicity experiment. The results of genetic evolution analysis show that GI-19 is the main epidemic strain in China, accounting for about 53.57% of cases. At the same time, variants were emerging, and GVI (new genotype) was obviously prevalent. The whole genome sequencing showed that the complete genome of the three isolated strains had the highest similarity with QX-like strains (more than 97%), but the sequence homology with vaccine strains and classical strains was low, only about 77%. Epitope analysis also showed that there were differences in the number and sequence of B cell epitopes among the isolates, vaccine strains and classical strains. The pathogenicity of JS96 in 1-day-old SPF chickens was higher than that in 15-day-old SPF chickens, and the mortality rate of 1-day-old SPF chickens was 100%. Significant growth retardation and obvious symptoms were observed in some 15-day-old SPF chickens. This study shows that QX-like IBV strain is the main epidemic strain of IBV at present, which has strong pathogenicity to young chickens, is prone to I gene recombination, has low similarity with S protein of vaccine strain and classical strain, and has poor adaptability, so it is urgent to select appropriate vaccines and develop new vaccines in order to control the current epidemic of IBV and reduce losses in the poultry industry.

The purpose of this study was to construct a recombinant genotype Ⅶ Newcastle disease virus (NDV) that can efficiently and stably express H9N2 subtype avian influenza virus (AIV) HA protein. It is expected to develop a bivalent vaccine against NDV and H9N2 AIV to provide a new defense idea for the control of H9N2 AIV and NDV, and has important reference significance for the construction and optimization of NDV vaccine vector. A reverse genetic technique was used to insert the full-length membrane-bound HA and another soluble HA protein with truncated transmembrane domain gene into the non-coding region between the P and M genes of NDV using the genotype Ⅶ NDV-weakening strain rDHN3-mF as the backbone. The recombinant virus rDHN3mF-HA and rDHN3mF-2HA of gene type VII NDV attenuated virus which can express HA protein stably and efficiently was obtained. The stability and biological characteristics of recombinant virus were detected by chicken embryo inoculation, Western blot, HA, MDT and other experiments. Recombinant virus was immunized with 7 d SPF chickens and serum HI assay were measured. And the group was subjected to challenge protection experiments 28 d after immunization. The recombinant viruses were passed through fifteen generations in chicken embryos without mutations.Western blot results demonstrated that recombinant viruses can express specific HA proteins stably and efficiently; The growth curves illustrates the recombinant viruses have similar replication and low virulence characteristics to parental strains; Groups of SPF chickens immunized with recombinant virus produce specific HI antibodies against NDV or H9N2 AIV.The challenge protection test showed that the recombinant viruses can produce 100% protection against chickens after challenge, and the results of throat and swab detection indicate that recombinant virus can significantly reduce the amount of NDV and H9N2 AIV shedding. The results of tracheal and lung qPCR showed that rDHN3mF-2HA significantly reduced the shedding of H9N2 AIV in organs than rDHN3mF-HA 3 dpc. The results indicated that the recombinant NDV of genotype Ⅶ coexpressing membrane-bound and soluble H9N2 subtype avian influenza HA protein was successfully constructed, which laid a foundation for the development and application of a new type of recombinant genetic engineering live vector vaccine to prevent H9N2 AIV and NDV.

The Brucella type IV secretion system (T4SS) effector BtpA plays a significant role as an immunosuppressive molecule in Brucella. This study aims to gain a deeper understanding of the impact of BtpA on the biological properties of Brucella abortus strain A19 and to explore the potential effect of BtpA gene deletion on the immunogenicity of strain A19. The BtpA gene was deleted on a bovine Brucella vaccine strain (strain A19). qRT-PCR was performed to detect the BtpA mRNA expression level in the Brucella A19ΔBtpA deletion strain and to investigate the differences between Brucella A19 and A19ΔBtpA deletion strains with respect to growth curves, intracellular survival, adherence invasion and in vitro stress. In addition, mice were immunized with the A19 and A19ΔBtpA deletion strains, and Brucella-specific antibody levels were measured using iELISA at the 7th, 14th, 21st, 28th and 35th day post-immunization; the expression level of IFN-γ in mouse splenic lymphocytes at day 21 of immunization was detected by ELISpot. IFN-γ-positive CD4⁺ and CD8⁺ T lymphocytes and the classification of CD4⁺ and CD8⁺ T lymphocytes were determined by flow cytometry. The A19ΔBtpA deletion strain was successfully constructed, and the transcript level of BtpA in the A19ΔBtpA deletion strain was significantly lower than that of the A19 strain, and the A19ΔBtpA deletion strain and the parental strain showed a similar trend in terms of the growth curve, intracellular survival, and adhesion invasion. In contrast, the in vitro stress test showed that the surviving bacterial population of the A19ΔBtpA deletion strain was significantly lower than that of the A19 strain under hypertonic stress conditions (P<0.05). In the immunogenicity study, both A19 and A19ΔBtpA groups induced the production of Brucella-specific antibodies in mice very significantly compared with the PBS group (P<0.01); the IFN-γ expression level of lymphocytes in the A19ΔBtpA group was significantly higher compared with that of the A19 group (P<0.05), which resulted in the production of IFN-γ-positive CD4⁺, CD8⁺ T-lymphocytes and CD4⁺/CD8⁺ T-cell ratio was significantly higher (P<0.05). The deletion of the BtpA gene does not impact the in vitro intracellular proliferation of Brucella abortus A19. However, it may be associated with the A19 strain’s ability to withstand hypertonic environments. Furthermore, the deletion strain A19ΔBtpA has shown the ability to induce a more robust Th1-type immune response compared to the A19 strain. Additionally, it stimulates the host to produce Brucella-specific antibody IgG at levels similar to those induced by the A19 strain. These findings suggest that the A19ΔBtpA deletion strain has the potential to be a promising candidate for a Brucella gene deletion vaccine.

The aim of this study was to evaluate the efficacy of the combination of competitive ELISA and indirect ELISA methods in the purification of brucellosis, hoping to provide technical support for the prevention and control of brucellosis. A total of 3 271 bovine serum samples from a cattle farm in the northwest of China were tested using the competitive ELISA (cELISA) antibody detection kit, the indirect ELISA (iELISA) antibody detection kit, and the micro-complement fixation test (mCFT) which developed by the writers team. This study adopted a purification strategy of cELISA for preliminary screening, iELISA for confirm diagnosis. The positive cattle were eliminating, the suspect cattle and negative cattle were respectively quarantined after disinfecting completely. After the previous detection, the group was sampled every month for multiple “Detection-Elimination” cycles. When the individual positive rate was below 2% or all negative, the results were verified using a micro-complement fixation test (mCFT). The test were carried on for 2 months after all the bovine turned negative to ensure the successful of the purification. The results showed that, after the implementation of this purification strategy, the positive rate of the infected group with a positive rate of 35.36% decreased to 25.41% in the first month, 7.16% in the second month, and 1.86% in the third month. By the fourth month, the group had achieved a comprehensive negative conversion, and the negative rate by mCFT verification was 100%. The detection continued for 2 months, and the individual positive rate was 0. So far, the brucellosis purification of the infected group was realized, making nearly half of the cattle in the group free from culling, greatly reducing the economic loss. In conclusion, cELISA is suitable for the preliminary screening, while iELISA is suitable for confirmed diagnosis of brucellosis. The combination of two methods, through multiple consecutive tests, can achieve a comprehensive purification of brucellosis infected within a relatively short period of time.

This study aims to further improve the complete genome database of Mycoplasma bovis, elucidate its biological characteristics and genetic evolution relationship, the whole genome sequencing and sequence analysis of Mycoplasma bovis T10 strain derived from Xizang yaks were conducted. The entire genome of T10 strain was sequenced using the Nanopore and Illumina PE150 sequencing platforms, and the genomic characteristics were analyzed using bioinformatics. Genetic phylogenetic trees were used to compare genetic evolution relationships with domestic and foreign isolates. The gene sequencing results showed that the total genome size of T10 strain was 987 812 bp, with a GC content of 29.27%. It was predicted that 822 coding genes were present, with a total length of 709 129 bp; According to the annotation results of the functional database, 22 genes related to membrane transport proteins, 7 genes related to carbohydrate enzymes, 4 genes related to glycosyltransferases, 43 genes related to secretion proteins, 51 genes related to T3SS effector proteins, 28 genes related to pathogen host interaction, 14 genes related to bacterial virulence, and 10 genes related to resistance were annotated; Through comparative genomic analysis, it was found that T10 and 08M, CQ-W70, Hubei-1, Ningxia-1, and PG45 all exhibit amino acid mutations and gene fragment insertions or deletions, as well as differences in the number of gene families, with a difference in the number of gene families ranging from 8 to 32. The results of genetic evolution analysis show that T10 is in the same branch as HB0801, 16M, Hubei-1, CQ-W70, NM2012, Ningxia-1, 08M, JF4278, KG4397, and PG45, but in different branches from PG1 and PG2. Among them, T10 has the closest genetic relationship with HB0801 and the farther genetic relationship with PG45. This study not only improved the complete genome database of Mycoplasma bovis and elaborated on the genes related to pathogenicity, but also fully elucidated the genetic evolution relationship between T10 strain and other Mycoplasma bovis strains at home and abroad. It clarified the basic genome information of T10 strain and its genetic relationship with domestic and foreign strains, providing a theoretical basis for future research on pathogenic mechanism vaccines.

In order to explore the biological characteristics and immune effect of outer membrane vesicles (OMVs) of capsular type B Pasteurella multocida. Outer membrane vesicles were extracted by ultra-high speed and density gradient centrifuge methods. The shape and size of the outer membrane vesicles were observed by transmission electron microscope (TEM), the proteins of OMVs were analyzed by SDS-PAGE and LC-MS/MS, the protein concentration was determined by the BCA method, and the endotoxin content was determined by limulus lysate test. Antibody titers and immune protection test were proceeded in New Zealand white rabbits. The results showed that OMVs had a regular spherical structure, the average particle size was 108.3 nm. OMVs contained a variety of protein bands, the protein bands were mainly 33，40，53 ku. The endotoxin content was 3.31×10⁶ EU·mL^-1. The serum antibody titer of immunized New Zealand white rabbits was 1∶405 600 when the injection dose was 50 μg per rabbit. The transcription levels of IFN-γ and IL-4 in peripheral blood lymphocytes increased. The immune protection efficiency against capsular tybe B Pasteurella maltocida was 75%. In conclusion, the OMVs of capsular type B Pasteurella multocida have good immunoprotective effcet and has the potential to be an candidate vaccine. This study lay the foundation for the research of OMVs vaccines.

This study aimed to investigate the effect of CD44 (cluster of differentiation 44) on the expression and membrane transfer of Na⁺/H⁺ exchanger 3 (NHE3) in porcine intestinal epithelial cells (IPEC-J2) infected with porcine epidemic diarrhea virus (PEDV). Using IPEC-J2 as the cell model, the expression of NHE3 and PEDV N in IPEC-J2 cells at different time points after infection with PEDV was detected by RT-qPCR and Western blot. After the transfection plasmid regulated the expression of CD44 in IPEC-J2, TCID₅₀ and Western blot were used to detect the changes of PEDV replication level and NHE3 protein expression at different time points after PEDV infection, and the changes of Na⁺ concentration inside and outside IPEC-J2 cells were detected by flame atomic absorption method. Transcriptome data and cell experimental results showed that compared with the control group, the expression level of CD44 protein and mRNA expression in IPEC-J2 cells after PEDV infection showed an upward trend, and increased significantly within 24-48 h (P<0.05), while the expression level of PEDV N protein decreased significantly within 12-48 h (P<0.05). In addition, the results of CD44 recombinant plasmid transfection experiments showed that the viral titer and PEDV N protein expression levels in cells infected with PEDV group after overexpression of CD44 were significantly reduced (P<0.05), while the viral titer and PEDV N protein expression levels in cells infected with PEDV group after interference with CD44 were significantly increased (P<0.05). These results showed that overexpression of CD44 had the effect of inhibiting PEDV replication, and PEDV replication increased after interfering with CD44. At the same time, in order to investigate whether CD44 participated in the regulation of NHE3 expression in IPEC-J2 cells under PEDV infection, Western blot and flame atomic absorption spectrometry were used to detect the expression level of surface NHE3 protein and the concentration of Na⁺ inside and outside the cell after regulating CD44. The results showed that overexpression of CD44 significantly promoted the expression of surface NHE3 protein and enhanced its activity (P<0.05), and the concentration of Na⁺ inside and outside the cell gradually returned to normal levels. In contrast, interference with CD44 significantly reduced the expression and activity of surface NHE3 protein (P<0.05), and the concentration of Na⁺ inside and outside cells was higher than normal, showing an imbalance. The results suggest that CD44 may be a potential therapeutic target for alleviating PEDV-induced piglet diarrhea, and it increases the amount of NHE3 transferred to the plasma membrane by inhibiting the replication of PEDV in IPEC-J2 cells, thereby maintaining the balance of intracellular and extracellular Na⁺.

The experiment aims to investigate the effect of pseudorabies virus (PRV) on the permeability of blood-brain barrier in mice and the correlation between matrix metalloproteinase-9 (MMP-9) and tight junction (TJ). Ninety-six Kunming SPF mice were randomly divided into control group and experimental group. Twenty-four mice of control group received nasal saline and seventy-two mice of experimental group received nasal PRV infection. The experimental groups were divided into 24 h infection group, 48 h infection group and 72 h infection group, each group consisted of 24. The clinical symptoms and the pathological lesions were observed by H&E staining. The relationship between nerve damage and viral load after PRV infection was evaluated by modified neurological score and viral load measurement in brain tissue. The permeability of blood-brain barrier was investigated by wet-dry weight method and Evans blue staining method. The correlation between MMP-9 and TJ was explored through mRNA and protein expression differences. The results showed that the degree of brain lesions increased with the extension of time, and the nerve function injury score reached the highest at the 72nd hour. There were significant differences in viral load in brain tissue 48 hours after challenge. W/D, and Evans blue concentration in brain tissue were time-dependent. The correlation between MMP-9 and TJ showed a positive correlation between MMP-9 and TJ expression. After PRV infection, the expression of MMP-9 increased, and the expression of TJ decreased. The results showed that PRV infection impairs BBB integrity and permeability through an MMP-9-mediated decrease of TJ protein expression. In summary, PRV infection may damage BBB through MMP-9-mediated destruction, which lays a foundation for further elucidation of the PRV invasion mechanism.

Mesomycoplasma (Mycoplasma) hyopneumoniae (Mhp) infection can cause damage and apoptosis of host epithelial cells, and the breakdown of the epithelial barrier often leads to the secondary infection of other bacteria and viruses, causing serious economic losses to the swine industry. However, the pathogenicity and virulence determinants of Mhp are not yet completely elucidated. Previous studies have found that Mhp lactate dehydrogenase (LDH) not only involved in the metabolism of Mhp, but also related to its virulence. Therefore, in this study, recombinant LDH (rLDH) was obtained through prokaryotic expression and affinity chromatography, and different concentrations of rLDH were used to interact with porcine bronchial epithelial cell (PBEC). The effect of rLDH on cellular morphology was observed by microscope, and the effect of rLDH on cellular viability was detected by CCK-8 method. The rLDH concentrations (50 and 100 μg·mL^-1) were selected for further analysis by that there was no significant effect on cellular morphology and viability when it incubated with PBEC, and the apoptosis rate of cells was detected by flow cytometry. The transcriptional and expression levels of apoptosis-related genes caspase3, caspase 8, caspase 9, Bax and BCL-2 were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. The results showed that rLDH induced apoptosis of PBEC in a dose-dependent manner, the function of that was similar with Mhp. The mRNA levels of caspase 3, caspase 9, and Bax of PBEC after incubation with rLDH and Mhp 168 were significantly up-regulated and the mRNA level of BCL-2 was significantly down-regulated, while the mRNA of caspase 8 was not changed significantly. The protein expression levels of caspase 3 and Bax were significantly up-regulated and the protein expression level of BCL-2 was significantly down-regulated. The results showed that Mhp could induce the endogenous apoptosis of PBEC through LDH, in which the apoptosis-related factors caspase 3, caspase 9, Bax and BCL-2 playing important roles.

The objective of this study was to investigate the biological properties of Colletotrichum jasminigenum. The fungi were cultivated in PDA using the three-spot inoculation method, followed by observation of their morphology through lactophenol cotton blue staining and scanning electron microscopy. Fungal DNA was extracted via PCR to amplify the ITS region of the gene and sequenced for comparison of gene homology and genetic evolutionary relationships. The concentration of the spore suspension was adjusted to 5×10⁷ CFU·mL^-1 for mouse infection, and clinical symptoms, serum biochemical indexes, and anatomical lesions were observed. The Kirby-Bauer method was employed to investigate fungal drug resistance phenotypes. The results showed that the isolated conidia were 29.6 μm±0.87 μm in length and crescent-shaped. The mycelium was hyaline, asexual, septate, and branched without bristles. Additionally, the adherent spores were clavate or ovoid in shape. The PCR amplification yielded a sequence length of 586 bp for the ITS region of the fungi, which was subsequently submitted to GenBank (OR472966). Based on both morphology and sequence analysis of the ITS region, it was determined that the isolate belonged to Colletotrichum jasminigenum and was named SLY01. Attacking mice with strain SLY01 for 14 days resulted in a highly significant increase in serum alkaline phosphatase levels (P<0.01) and a significant decrease in liver organ index (P<0.05). Colletotrichum jasminigenum was reisolated from the infected mice. Histopathological results showed edema of the alveolar walls with massive lymphocytic infiltration, extensive granular degeneration with hemorrhage and hepatocellular necrosis in the lobules of hepatocytes, and granular degeneration of the renal tubular epithelium in the kidneys. The results of the drug sensitivity test showed that SLY01 was resistant to caspofungin and voriconazole, sensitive to amphotericin B and moderately sensitive to itraconazole. In this study, Colletotrichum jasminigenum was isolated and characterized. The liver and lungs were identified as the main target organs affected in infected mice, and the fungus showed resistance to various drugs. These findings provide a theoretical basis for further research on fungal infections originating from food sources.

The aim was to study the isolation, purification, characterization and in vitro culture biological properties of luteal cells from cows during pregnancy. In this study, luteal tissues of healthy Holstein cows during pregnancy were collected, and bovine luteal cells (BLCs) were isolated by type II and type IV collagenase digestion. High-purity BLCs were purified by Percoll discontinuous density gradient centrifugation. The purity of BLCs was identified by 3β-HSD live cell staining and oil red O lipid droplet staining. The specific marker proteins of BLCs, oxytocin and synaptophysin, were detected by immunohistochemical staining and immunofluorescence staining. Progesterone in cell culture supernatant was detected by ELISA. G-banding karyotype analysis of BLCs chromosomes, CCK-8 assay of BLC growth curve and serum-dependent flow cytometry to detect cell cycle analysis of BLCs in vitro culture characteristics. The results showed that: 1) A large number of high-purity BLCs could be obtained by one-time separation and purification using collagenase digestion and Percoll discontinuous density gradient centrifugation. 2) The results of 3β-HSD staining and oil red O staining showed that the isolated and purified BLCs had uniform cell types, and their morphology was typical epithelial cell-like cells with irregular polygons. The nucleus was large, the cytoplasm was rich, and small lipid droplets were dispersed. 3) Immunofluorescence results showed that BLCs could express specific markers of luteal cells such as oxytocin and synaptophysin; 4) The concentration of progesterone in the culture supernatant of BLCs was time-dependent. 5) Karyotype analysis showed that luteal cells were composed of 30 pairs of chromosomes, including 29 pairs of autosomes and a pair of XX sex chromosomes. CCK-8 and flow cytometry results showed that BLCs had stable proliferation activity during in vitro culture. In summary, this study successfully isolated and purified high-purity BLCs with typical biological characteristics, and sorted out a set of scientific and perfect methods for the separation, purification, identification and in vitro culture of BLCs, which can provide a stable cell model and technical reference for the study of luteal development, degeneration and functional regulation in vitro.

Suppressor of cytokine signaling 2 (SOCS2) is involved in regulating various physiological and pathological processes in animals, such as cell apoptosis, proliferation, cell cycle, tumor, and inflammatory response. In this study, based on the first cloned SOCS2 gene from goats, the effects of overexpression and interference SOCS2 expression on proliferation, cycle and apoptosis of goat turbinate bone cells were studied. A total of 9 samples of heart, liver, spleen, lung, kidney, rumen, large intestine, small intestine and goat turbinate bone cells of healthy Jianzhou goats (6 animals) were used as samples, and the sequence of the CDS region of the goat SOCS2 gene was cloned by reverse transcription PCR and analyzed the sequence. RT-qPCR was used to detect the expression of SOCS2 gene in different tissues. The cloned SOCS2 gene CDS region ligation vector was constructed as a eukaryotic expression vector pcDNA3.1-SOCS2. Design, synthesis and screen effective siRNAs based on goat SOCS2 gene sequences. After transfecting pcDNA3.1-SOCS2 and siRNAs to turbinate osteocytes with Lipofectamine^TM 3000 reagent, SOCS2 protein expression was detected with Western blot mothed, the MTT method detects the effect of SOCS2 on cell proliferation, flow cytometry to detect the effect of SOCS2 on cell cycle and apoptosis and RT-qPCR was used to detect the transcription levels of cell cycle-related genes p21, CDK2 and apoptosis-related genes Caspase3, Caspase7, PARP1, p53, Bax, BCL2L11 and Bcl-2. The results showed that the total length of goat SOCS2 gene was 1 065 bp, and the CDS zone length was 597 bp, encoding 198 amino acid residues. SOCS2 gene is expressed at the highest levels in the liver tissue; SOCS2 can inhibit cell proliferation. The eukaryotic expression vector pcDNA3.1-SOCS2 was successfully constructed, and the gene was successfully expressed in turbinate bone cells. SOCS2 overexpression can lead to a significant increase in the number of cells in the G2/M+S phase and down-regulation of mRNA expression in CDK2 and p21 genes. It promoted apoptosis and upregulated the mRNA expression of Caspase3, Caspase7, Bax, p53, BCL2L11, and Bcl-2 genes, but there was no significant change in PARP1 expression level. However, after interference the expression of SOCS2, the number of cells in G0/G1 stage increased significantly, and the mRNA expression of CDK2 and p21 genes was upregulated. The apoptosis was inhibited and the mRNA expression of Caspase3, Bax, p53 and PARP1 genes was downregulated, while the expression levels of Caspase7, BCL2L11 and Bcl-2 did not change significantly. In summary, SOCS2 can block cells in the G2/M+S phase, promote apoptosis, and inhibit cell proliferation. The results of this study accumulate experimental data for a comprehensive reveal of the function of goat SOCS2.

The aim of this study was to evaluate the effects of Astragalus polysaccharide (APS), saponins (AS) and probiotics on the resistance to E. coli infection in broilers. A total of 200 1-day-old white-feathered broiler chickens were randomly divided into blank group, test group, positive group and model group, with 50 chickens in each group. The experiment lasted for 42 d. From 1 to 21 days, chicks in the blank group and model group were fed a basic diet and each of them was given 0.2 mL of normal saline every day, while chicks in the test group were fed a basic diet containing APS (1 g·kg^-1) and AS (10 mg·kg^-1), and each of them was given 0.2 mL of probiotic complex bacterial solution (Streptococcus alactolyticus∶Lactobacillus plantae∶Bacillus subtilis=1∶1∶1, the concentration of bacterial liquid was 1×10⁹ CFU·mL^-1), and chicks in the positive group were fed a basal diet containing 50 mg·kg^-1 doxycycline hydrochloride soluble powder every day. At the 21st day, chickens in test group, positive group and model group each were given 0.2 mL Escherichia coli O₇₈ bacterial solution (6×10⁸ CFU·mL^-1). Duodenum and jejunum tissue structure, sIgA, inflammatory factors, and oxidation markers were detected at days 0, 1, 7, 14, and 21 of infection (i.e., days 21, 22, 28, 35, and 42 of the trial period), as well as bacterial count in cecum contents. The results were showed as follows: 1) The number of E. coli in model group (except for the 0 day of infection) was significantly higher than that in blank group, test group and positive group (P<0.05), and the number of chicken lactic acid bacteria in test group was significantly higher than that in blank group, positive group and model group (P<0.05). 2) After 1 and 7 days of infection, the sIgA content in duodenum and jejunum tissues of test group and positive group was significantly higher than that of blank group and model group (P<0.05); After 14 days of infection, the sIgA content of jejunum tissue in test group was significantly higher than that in blank group and model group (P<0.05). 3) When infected for 1 and 7 d, the TNF-α content of duodenum and jejunum in the test group and the positive group was significantly lower than that in the model group (P<0.05); When infected for 7 days, the content of duodenum and jejunum IL-10 in the test group and the positive group was significantly higher than that in the model group (P<0.05); After 14 and 21 days of infection, the content of IL-10 in jejunal tissue in the test group and the positive group was significantly higher than that in the model group (P<0.05). 4) When infected for 1, 7, 14 and 21 days, the MPO activity of duodenal and jejunal tissues in the test group and the positive group was significantly higher than that in the blank group (P<0.05).When infected for 1 d, the SOD of jejunal tissues in the test group and the positive group was significantly higher than that in the model group (P<0.05), and when infected after 7 and 21 days, the T-AOC of duodenal and jejunal tissues in the test group and the positive group was significantly higher than that in the model group (P<0.05). In conclusion, under the condition of the current experiment, the combination of APS, AS and probiotic complex can enhance the ability of chicks to resist E. coli infection, and have the effect of replacing antibiotics.

The aim of this study was to investigate the prevalence and genetic diversity of feline herpesvirus-1 (FHV-1) in Chengdu city. From 2020 to 2023, a total of 178 conjunctiva, pharynx and nose swabs were collected from cats with respiratory symptoms in Chengdu city. The collected samples were subjected to FHV-1 PCR detection, gE gene genetic evolutionary analysis, viral isolation and identification, as well as pathogenicity. The results showed that, the FHV-1 positivity rate in Chengdu city was 11.8% (21/178). In which the FHV-1 positivity rate of veterinary hospitals was 6.4% (7/110) and 20.6% (14/68) in catteries, suggested that the higher rate of FHV-1 infection was in catteries. The genetic evolutionary analysis of five FHV-1 gE genes showed that the nucleotide homology was 99.1%~100% to the vaccine strains,and three unique amino acid mutation sites were found in PX-9 strain (L259Q, C294I, W295F). Five positive samples were isolated and cultured by CRFK cell, only PX-9 strain was successfully isolated and identified from CRFK cells. The results of animal regression experiment by PX-9 strain showed that all cats in the infected group exhibited clinical symptoms, such as increased ocular, nasal secretions and sneezing. The highest viral load was found on the 4th day after inoculation (9.05×10⁷ copies·μL^-1), and the detoxification period is relatively long. The results of FHV-1 viral load were 1.7×10⁷ and 3.5×10⁴ copies·μg^-1 in trachea and lungs of infected group, respectively. The other tissues were negative for FHV-1 DNA. The results of this study indicated that FHV-1 was still prevalent in Chengdu and the prevalent strains had highly pathogenicity.

Enzootic nasal tumor is a viral transmitted infection of goats, which is caused by enzootic nasal tumor virus (ENTV-2) of goats. After infection, it can lead to irreversible canceration of goat nasal meatus ethmoid epithelial cells, which has been widespread in the world, causing serious economic losses to the goat breeding industry. there is an urgent need to establish a fast, accurate, and quantitative detection method for ENTV-2, we designed primers and TaqMan probes based on the conserved region of the ENTV-2 env gene published on GenBank, and developed a TaqMan probe real-time fluorescence quantitative RT-PCR detection method (qRT-PCR) for ENTV-2, and then verified its specificity, sensitivity, reproducibility, and clinical detection effectiveness. The results showed that the concentration of the recombinant plasmid standard template was 6.30×10²-6.30×10⁷ copies·μL^-1, there is a good linear relationship within the range, with a correlation coefficient of R² of 0.996 5, amplification efficiency of 110%, slope of the linear equation of -2.953, and minimum detection limit of 6.30×10¹ copies·μL^-1; The intra group and inter group coefficients of variation are both less than 2%, indicating good reproducibility; There was no cross reaction with Foot-and-mouth disease virus (FMDV), peste des petits ruminants virus (PPRV), Endogenous retroviruses (ERVs), etc., indicating that this method has good specificity. The qRT-PCR established in this study was used to detect 29 clinical samples, and the detection rate of this method is higher than that of ordinary PCR methods. The above results indicate that this study has successfully established a qRT-PCR for ENTV-2, providing a technical means for rapid diagnosis and epidemiological investigation of ENTV-2.

This paper aims to establish a triple fluorescence quantitative PCR method for the simultaneous detection of porcine epidemic diarrhea virus (PEDV), porcine rotavirus type A (PoRVA) and porcine deltacoronavirus (PDCoV). Primers and probes were designed for PEDV-N gene, PoRVA-VP6 gene and PDCoV-M gene, and their performance was evaluated after the conditions were optimized, and compared with commercial kits. The results showed that the triple RT-qPCR method established in this study had good specificity and did not amplify positive nucleic acids such as PRRSV, PCV2 and PRV. The detection limits of PEDV, PoRVA and PDCoV were all 1 copies·μL^-1. The coefficient of variation within and between groups was less than 1%. The sample has wide adaptability, and the coefficient of variation is less than 1% when different sample types are detected. Compared with commercial kits, the detection coincidence rates of PEDV and PoRVA were 92.5% and 97.5%, and the detection range of PDCoV was wider, and the detection effect of PDCoV variant strains was still good. The detection method established in this study has the advantages of good specificity, high sensitivity, strong stability and wide sample adaptability, which provides a good detection method for clinical diarrhea samples.

As no effective treatment for alveolar echinococcosis (AE) is currently available, the therapeutic drugs are needed urgently. Early stage studies have shown that the protease inhibitors of HIV could also be anticancer and anti-parasitic. The purpose of this paper is to study the effect of the inhibitors (HIV PIs) on the activity of DNA damage inducible 1 protein of Echinococcus multilocularis (EmuDdi1). The recombinant vector pFastBac1-EmuDdi1 was constructed and expressed in Sf9 cell, and the soluble protein was successfully purified in P2 generations. Then the enzyme activity of Ddi1 protein was detected with the specific fluorescent substrates, and the inhibition rate of seven HIV PIs including saquinavir (SQV), ritonavir (RTV), amprenavir (APV), atazanavir (ATV), lopinavir(LPV), fosamprenavir (Fos), tipranavir (TPV) and darunavir (DRV) on Ddi1 protein activity was further examined. The results showed that Emu Ddi1had high affinity and activity, and saquinavir showed the highest inhibition rate at 67% to inhibit protease activity of EmuDdi1 with a IC₅₀ at 34. These results suggested that saquinavir is effcetive to inhibit the activity of Ddi1, and could be used to develop a potential targeting drug for AE.