Uterine natural killer cells are the most abundant immune cells in deciduate placenta in the early pregnancy of mammals. During pregnancy, uNK cells play the important regulation role in the establishment of uterine immunotolerance environment, the development of placenta and fetus. However, studies on the regulation mechanisms of unique biological activity of uNK cells in pregnancy are limited. According to the related researches, the presentation of the important regulation role of uNK cells in the establishment of maternal-fetal immunotolerance environment and the development of placenta and fetus were well given in this paper, and the regulatory mechanism of hormones, glucose metabolism and DNA methylation on NK cells in other tissues were well summarized in order to provide new research directions and ideas for exploring the molecular regulation mechanism of uNK cells.

Early embryo loss, that is, embryo death before implantation, is the leading factor determining the pregnancy rate in dairy cows. The high mortality of embryo before implantation in dairy cows brings tremendous economic losses to many farms. At present, it is generally recognized that early embryo loss is mainly related with chromosomal abnormalities, reproductive hormone disorders, heat stress, immune factors, twin pregnancy, diseases and vitamin B deficiency. The factors affecting early embryo loss are reviewed in order to provide a comprehensive reference for preventing early embryo loss in dairy cows.

With the development of the global horse industry, the economic value of horses is growing. Assisted reproductive technology is beneficial to dig out the potential value of excellent equine, and in vitro maturation (IVM) of horse oocytes is an important part of assisted reproductive technology. Oocyte recovery is a preparation for in vitro maturation, for the isolated ovary, more oocytes can be obtained by using the scraping method. To obtain oocytes continuously for a long time and to protect the development ability of oocytes, ovum pick up (OPU) is widely used in mare. The maturation rate of expanded oocytes is higher than that of compact oocytes, and the age of the mares affects the quality of their oocytes. Horse oocytes stored in vitro for a long time will not affect their developmental ability, there are mature systems that can keep horse oocytes in vitro for more than 24 hours without affecting their maturation rate. The basic culture medium commonly used in horse oocyte maturation system is M199, and the addition of FBS, FSH, LH, IGF-1 and other substances can significantly improve the maturation rate, and the commonly used culture environment is 38-39℃, 5% CO₂ and saturated humidity incubate for 30 hours. Mature oocytes have dilated cumulus cells and polar body, and the cytoskeleton and microtubule structure of oocytes will be changed after maturation. This review summarized the research on the collection and in vitro maturation of equine oocytes, focusing on the recovery rate of different collection techniques and some key factors affecting the in vitro maturation rate of equine oocytes, in order to provide some knowledge and experience for further research of IVM and in vitro fertilization (IVF) in the future.

α-ketoglutarate (AKG), an important intermediate in the cycle of tricarboxylic acid (TCA), is a precursor of glutamic acid and glutamine, also the best substitate of glutamine. As a new functional feed additive for animal production, AKG is receiving more and more attention recently due to its good stability and unique nutritional physiological functions, which will be of great significance for the development of ecological health breeding industry. This paper reviews the metabolic mechanism, nutritional physiological function and application of AKG in animal production, which would provide a direction for the further study and rational application of AKG.

Feline infectious peritonitis is a systemic fatal viral disease, which is one of the main causes of death in young and young adult cats. In recent years, the disease has been widely prevalent in the world and has shown a certain rising trend. More and more scholars have researched and explored it, but its pathogenesis and immune mechanism have not been completely clear. In this paper, the etiology, pathogenesis, epidemiology, clinical symptoms, diagnosis, and treatment as well as immunity and prevention of feline infectious peritonitis were comprehensively described, providing reference and guidance for the scientific diagnosis, treatment, and prevention of the disease.

Clustered regularly interspaced short palindromic repeat (CRISPR) is an acquired immune system that exists widely in archaea and bacteria, mediated by RNA and assisted by Cas protein. It has been found that the CRISPR/Cas9 was the most widely used CRISPR system. This review mainly summarizes the basic principle and research progress of the CRISPR/Cas9 system, and highlights its application in the prevention and control of important swine virus diseases, including host modification and virus modification. This technology provides a powerful tool for the study of virus pathogenic mechanism, next generation vaccine development and disease resistance breeding. It has a far-reaching impact on the control of the diseases.

The aim of this study was to investigate tissue expression profiles and polymorphism of Fsp27 gene in sheep (Ovis aries) and their association with tail fat deposition of different sheep breeds. The qRT-PCR was applied to detect the expression level of Fsp27 gene in different tissues of Altay sheep in adequate nutrition stage, and its expression levels in tail fat of Altay sheep in adequate and deficient nutrition stages, then the PCR-SSCP combined with sequencing were used to detect polymorphism of Fsp27 gene in 5 sheep breeds with different tail fat deposition ability and their association with tail fat deposition in sheep were analyzed. The results showed that Fsp27 gene was expressed at a high level in tail fat of Altay sheep in adequate nutrition stage and extremely higher than those in the other tissues (P<0.01), and expressed at a relatively high level in perirenal fat and subcutaneous fat which was extremely higher than those in heart, liver, spleen, lung, kidney, stomach, intestine, skin and skeletal muscle tissues (P<0.01), but expressed at a very low level in heart, liver, spleen, lung, kidney, stomach, intestine, skin and skeletal muscle tissues, and there were no significant difference among them (P>0.05). In addition, the expression level of Fsp27 gene in tail fat tissue of Altay sheep in adequate nutrition stage was significantly higher than that in deficient nutrition stage (P<0.01). Twenty-five SNPs were detected in Fsp27 gene sequence of sheep with different tail fat deposition ability, G/A mutation at g. 16771741 site in exon3 and C/T at g. 16774969 site in exon5 were missense mutations, and they were significantly associated with tail fat deposition ability of sheep. G was dominant allele in Altay sheep, Small Tail Han sheep and Hu sheep at g. 16771741 site, the allele frequency was 86.8%, 83.7% individuals and 85.7%, respectively, but only 30.3% and 11.1% individuals of Chinese Merino and Suffolk sheep were G allele at this site. T was dominant allele in Altay sheep at g. 16774969 site, 84.7% were TT genotype, 15.3% individuals were CT genotype, and no CC genotype was detected, and the genotype frequency of TT and CT in Small Tail Han sheep and Hu sheep were 65.9%, 50.4% and 22.7%, 41.1%, respectively, but CC was dominant genotype in Chinese Merino and Suffolk sheep, their frequency were 97.4% and 69.4%, and no TT genotype was detected. These results indicated that Fsp27 gene expressed at a high level in tail fat tissue of Altay sheep, and its expression level was closely related with nutrition state of Altay sheep. The G/A mutation at g. 16771741 site in exon3 and C/T mutation at g. 16774969 site in exon5 of Fsp27 gene were highly correlated with tail fat deposition ability of sheep, so could be used to help selecting low fat sheep breed.

In order to explore the regulatory effects of c-Myc gene on the cell proliferation and the related genes expression, the eukaryotic expression vector pc-Myc-EGFP of the c-Myc-(G₄S)₃-EGFP fusion gene was constructed and its structure and physicochemical characteristics were analyzed by bioinformatics in this experiment. After transfecting into sheep embryonic fibroblasts (SEF), the effects of c-Myc on the cell proliferation and the related genes expression were detected by cell counting and qRT-PCR, respectively, and the corresponding expression regulation mechanism was analyzed by the promoter analysis software. The results of restriction enzyme digestion and sequencing indicated that the vector was successfully constructed. The bioinformatics analysis showed that the sheep c-Myc protein, with a molecular weight of 48 474.78 u, was mainly located in the nucleus and contained a helix-loop-helix (HLH) domain. The fusion protein c-Myc-(G₄S)₃-EGFP was highly hydrophilic and contained 31 phosphorylation sites. (G₄S)₃ had a high degree of flexibility (9.848 5), c-Myc and EGFP on both sides maintained their respective spatial conformation and did not interfere with each other. The further cellular assay results indicated that the recombinant plasmid pc-Myc-EGFP was expressed successfully in SEF. Overexpression of c-Myc promoted the proliferation of SEF and increased the expression level of cyclin D2, Cdk4, Cdk6, cyclin E1, cyclin A2 and cdc25A mRNA up to 5.20, 3.10, 6.54, 6.52, 23.37 and 8.22 times (P<0.01) respectively, while E2F1 mRNA up to 1.83 times (P<0.05). The results of promoter analysis indicated that there were binding elements for c-Myc and E2F1 on the 5'regulatory region of the above genes. In conclusion, sheep c-Myc promoted SEF proliferation by up-regulating the expression of the cell cycle-related genes, which might be through directly acting on E-box elements of the 5'regulatory region of target genes, or through the indirect regulatory role of other transcription factors such as E2F1.

This study was performed to obtain the sequence of Krüppel-like factor 11 (KLF1) gene, clarify its expression pattern in tissues and cells of goats, and further elucidate its effects on the differentiation of goat intramuscular preadipocytes with its possible action pathway. The expression of KLF11 in goat different tissues and cells at different adipogenic differentiation stages was detected by real-time fluorescence quantitative PCR (qPCR). From both morphology and molecular biology, Oil red O staining and qPCR were used to reveal the effect of interfering KLF11 on the differentiation of intramuscular adipocytes of goats with its possible action pathway. The results showed that KLF11 gene was 1 752 bp in length, CDS region was 1 518 bp in length, which encoded 505 amino acids. KLF11 gene was widely expressed in the various tissues of goats. The expression level of KLF11 was higher in liver, longissimus dorsi and abdominal fat. Compared with the preadipocytes, the expression of KLF11 gene extremely significantly increased at 48 h of differentiation(P<0.01).After obtaining the effective interference sequence of KLF11-siRNA1, the expression of KLF11 was significantly decreased by KLF11-siRNA1 to 63.58%, obviously promoted accumulation of lipid droplets were odserved in the intramuscular adipocytes of goats. After interfering KLF11, the mRNA levels of PPARγ and C/EBPβ, the adipocyte differentiation marker genes were extremely significantly up-regulated (P<0.01), while the expression of Pref-1 was extremely significantly down-regulated (P<0.01). Meanwhile, the expression levels of some members of KLFs family (KLF1,KLF2,KLF4,KLF5,KLF8,KLF10,KLF14,KLF16) significantly declined and others increased after interfering KLF11 gene. KLF11 gene could inhibit the differentiation of goat intramuscular preadipocytes through regulating the expression of PPARγ, C/EBPβ and Pref-1, and this effect might be achieved by synergizing with KLF2, KLF4, KLF10 and antagonizing with KLF7, KLF15.

This study aimed to explore the molecular mechanism by which the liver regulates the absorption and conversion of nutrients and affects the body weight of goose. RNA-Seq technology was used to analyze the RNA expression in livers of Sichuan White goose with different average daily gain (high:H; middle:M; low:L) at 70-days-old. The differentially expressed genes were screened, their functional enrichment and cluster correlation analysis were performed. The results showed that the average daily gain of the H, M, and L groups were significantly different (P<0.05), which were (52.24±0.41), (44.44±0.42), and (37.80±0.47) g·d^-1, respectively. There were 14 632, 14 651 and 14 819 expression genes identified, of which 13 776 genes were co-expressed. The expression level of the top 100 genes accounted for 65.38%-70.37% of all gene expression, including 85 co-expression genes and 17 specific expression genes. Four hundred thirty five differentially expressed genes were identified, and mainly enriched in apoptosis, protein refolding, insulin secretion, nutritional response, cell growth, amino acid transport, and cAMP signaling pathway. The 5 gene modules constructed from differentially expressed genes were significantly associated with average daily gain and lipid metabolism index (|r|≥ 0.75, P<0.05). Overall, body weight related potential candidate genes and metabolic pathways were identified. The molecular mechanism by which the liver regulates nutrient absorption and conversion and affects the weight of goose was explored, which make us better understand how liver metabolism affects the weight of goose and can help us to design new selection strategies to improve goose production.

The aim of this study was to elucidate the transcriptional regulation mechanism and expression patterns of bovine IRX3 gene and identify the key transcription factors of promoter. The distribution of bovine IRX3 mRNA in 3 adult bulls tissues was determined through qPCR, including heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi, large intestine, small intestine, reticulum, rumen, abdomen and testicle. In addition, the 1.8 kb promoter sequence of IRX3 gene was cloned and dual luciferase receptor vectors with the 6 different deletion fragments of the promoter were constructed, and then transfected into 3T3-L1 and C2C12 cell lines, respectively. Furthermore, the key transcription factors in the core promoter region were predicted by online software, and the transcriptional regulation of key transcription factors on IRX3 gene was identified in 3T3-L1 cell line by site-directed mutation and siRNA interference. The results showed that IRX3 had a broad tissue distribution in 13 tissues, and it was highly expressed in the lung, kidney, heart, subcutaneous fat and longissimus dorsi (P<0.05). The promoter core region of bovine IRX3 gene was located at -372/-42 bp using the dual luciferase reporter vector. Combined with site-directed mutation and siRNA interference demonstrated that NRF1, KLF4, HOXA5 and CREB1 regulated the transcriptional activity of IRX3 gene. The tissue expression analysis results indicated that the expression level of IRX3 was relatively higher in longissimus dorsi and subcutaneous fat. There were 8 CpG islands in the 1.8 kb region of IRX3 promoter. NRF1, KLF4, HOXA5 and CREB1 were located in CpG islands and regulated the transcriptional activity of IRX3 gene. These results lay an important theoretical foundation for exploring the molecular regulation mechanism of IRX3 gene in bovine fat deposition.

The aim of this study was to investigate the sequence characteristics, tissue expressions of VRTN (vertebrae development homolog) gene in Bama Xiang pig population and the gene frequency of causal mutation site ins291 for vertebral number and its association with teat number and litter size. The tissues of three 0-day-old Bama Xiang pigs were collected, and the full-length sequence of VRTN gene coding region was obtained by cDNA cloning technology and bioinformatics analysis was also conducted. The expression of VRTN in heart, liver, spleen, lung, kidney, longissimus dorsi and subcutaneous fat tissue was detected by fluorescence quantitative PCR. Blood samples from 279 Bama Xiang sows were collected to test the frequency distribution of ins291 site in the population, and its association with teat number and litter size was analyzed. The result showed that the full-length of the VRTN gene coding region in Bama Xiang pig was 2 097 bp, its amino acid sequence had lots of conserved regions among different species; VRTN gene encoded 698 amino acids, and it was predicted to express a hydrophilic protein. VRTN protein was most likely having one helix-turn-helix superfamily structural functional region, two low-complexity regions and two internal repetition structures. Moreover, the protein interacted with nuclear receptor subfamily 6 group A member 1 (NR6A1), Prospero homeobox 2 (PROX2) and leucine rich repeat containing 74A (LRRC74A). VRTN gene was expressed in all tissues of 0-day-old Bama Xiang pigs, with the highest expression in longissimus dorsi. The allele frequency of beneficial mutation site ins291 of VRTN in Bama Xiang pigs from the preserved population was 21.15%. There was no significant difference in average litter size, total teat number, left teat number, right teat number and maximum teat number of one side among individuals with different genotypes (P>0.05), but the teat number of individuals with homozygous mutant genotype(ins/ins) was higher than those with other genotypes. These results indicate that molecular breeding method can be used to increase the favorable allele frequency of VRTN ins291 in the industrialized productions of Bama Xiang pigs, which don't affect litter size.

The study aimed to investigate the development and distribution of blood vessels and the expression of angiogenesis-related factors in fetal placenta of Dazu Black goat during early pregnancy, and to analyze the correlation between the expression of angiogenesis-related factors and vascular development. Fifteen 8-month-old, healthy, young female Dazu Black goats were randomly selected, after natural mating with the same ram (recorded as 0 d on the last breeding day), fetuses and fetal placenta were collected by cesarean operation at 20, 25, and 30 d of pregnancy, and fetuses and fetal placenta were collected after slaughter at 45 and 60 d of pregnancy. Placental vascular development and distribution were investigated by immunohistochemical technology, and the expression level of angiogenesis-related genes was detected by qRT-PCR technique, and the correlation between the expression of angiogenesis-related genes and the placental vascular distribution was analyzed. The results showed that fetal body weight and body length increased gradually with the progress of pregnancy, and the positive rate of vascular endothelial cells was at a high level (>26%). With the progress of pregnancy, goat fetal placenta vascularity area/tissue area(CAD) gradually increased; capillary number/tissue area (CND) increased during 25-30 d and decreased during 30-60 d of pregnancy; Average capillary area (APC) showed an opposite trend to CND; There was no significant change in capillary circumference/tissue area(CSD). The expression of VEGFA in fetal placenta was gradually increased with the progress of pregnancy and correlated with vascular distribution. The expression level of FLT1 and KDR in fetal placenta first increased and then decreased, reaching the peak at 30 and 45 d of pregnancy, respectively. The expression of ANGPT1/2 and its receptor TEK, FGF2 and its receptor FGFR2 were higher at 30 d of pregnancy, which could be related to angiogenesis. In conclusion, before 30 d of pregnancy, fetal placenta mainly formed branches through capillaries to promote the increase of vascular area; at 30-60 d of pregnancy, the increase of vascular area was mainly promoted by the thickening of blood vessels per unit, and the expression of angiogenesis-related genes was higher at 30 d of pregnancy, which might be related to cotyledon formation. VEGFA in goat fetal placenta during early pregnancy could act together with other angiogenesis-related factors to regulate the angiogenesis.

The aim of this study was to investigate the effect of vitrification on the genomic methylation patterns of bovine GV oocytes. Fresh and vitrified GV oocytes were collected. Single-cell whole-genome methylation sequencing(ScWGBS) technology was used to detect the whole-genome methylation levels of the fresh and vitrified bovine GV oocytes, in order to reveal the difference of DNA methylation patterns between the fresh and vitrified bovine GV oocytes. The results showed that vitrification didn't significantly affect the whole-genome methylation level of bovine GV oocytes. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) were used to analyze 140 differentially methylated regions(DMRs). It was found that DMRs were mainly involved in cell development, cytoskeleton organization, and they were mainly enriched in the PI3K-Akt and GnRH signaling pathways, and the partial related genes were screened about oocyte maturation (TSC2), cytoskeleton (NUDC), cell viability (MAFK) etc.. These results can provide the information foundation and research direction for improving the efficiency of vitrified GV oocytes.

The purpose of this study is to explore the influence of different treatments on the microbial activity and live bacteria composition during the preparation of faecal bacteria suspension, so as to provide reference for optimizing the application of faecal microbiota transplantation(FMT) on pig. Seven experimental groups were set up in this study:microbial suspension from fresh feces by static settlement (FS), microbial suspension from fresh feces by static settlement and stored at -80℃ for 1 week (FFS), microbial suspension from fresh feces by centrifugation (FC), microbial suspension from fresh feces by centrifugation and stored at -80℃ for 1 week (FFC), microbial suspension from feces frozen in nitrogen by static settlement (NS), microbial suspension from feces treated with glycerin and frozen in nitrogen for 24 h by static settlement (GNS), and microbial suspension from feces frozen in nitrogen by static settlement and stored at -80℃ for 1 week (NFS). All suspensions were spread on plates and cultivated at 37℃ anaerobic incubator for 48 h. The colonies growing were counted. In addition, 87 single colonies were randomly selected from the four groups of FS, FFS, FC and FFC and purified for full-length sequencing of 16S rRNA gene. The results showed that there were significant differences in bacteria activity when preparing suspensions from fresh feces by static settlement and centrifugation methods, and the activity of faecal bacteria in the centrifugation group was significantly lower than that in the static settlement group (P<0.05). However, there was no significant influence of suspensions from frozen feces on the activity of faecal bacteria regardless of static settlement or centrifugation (P>0.05). Whether the suspensions were prepared by static settlement or centrifugation, no significant impact was found on the activity of faecal bacteria either from fresh or frozen feces (P>0.05),nor did on the live bacteria composition. When microbial suspensions were prepared by static settlement, there were no significant difference in faecal bacteria activity (P>0.05) among FS, NS, GNS and NFS groups. The above results indicate that when preparing faecal microbial suspensions by static settlement, ultra-low temperature cryopreservation has no significant effect on the activity and composition of faecal bacteria. The present research provided a useful suggestion for the application of long distance and large size faecal microbiota transplantation technology in pig production in the future.

The purpose of this study was to accurately evaluate the nutrient production of manure and the biological yield of corn under different fertilizer application rates based on the model of growing-finishing pigs and corn planting farmland, and to establish the manure carrying coefficient of corn planting farmland based on the use of manure. Eight 13-week-old Duroc×Landrace×Yorkshire growing-finishing pigs with similar parity and body weight ((33.2±3.5) kg) were used in the experiment. Digestion trials were conducted at 16 and 25 weeks old, respectively. The excretion of feces and urine and the production of nitrogen and phosphorus were measured. In the corn planting experiment,6 treatment groups were set up in the experimental field with normal production level. The control group was treated with chemical fertilizer, the negative control group treated with no fertilizer, the 4 experimental groups were treated with pig manure according to 100%, 130%, 160% of standard nitrogen requirement and 100% of standard phosphorus requirement of corn planting, respectively. The biomass production and nitrogen and phosphorus contents of corn plants were measured at the late milk-ripe stage and the full-ripe stage, respectively. The demand for nitrogen and phosphorus in corn planting were calculated and fitted with the production of nitrogen and phosphorus in manure of growing-finishing pigs, and the manure carrying parameter of growing-finishing pigs based on corn planting was calculated. According to the application rate of pig manure organic fertilizer in 130% nitrogen demand, if the corn was harvested at the late milk-ripe stage, the requirements of nitrogen and phosphorus for corn planting in one season were 122.1 and 53.0 kg·hm^-2, respectively. If the corn was harvested at the full-ripe stage, the requirements of nitrogen and phosphorus for corn planting in one season were 190.2 and 62.8 kg·hm^-2, respectively. The field stock capacity was calculated based on the application rate of pig manure in 130% nitrogen demand. And the manure carrying parameter of growing-finishing pigs was 61.1 and 95.2 pigs·hm^-2 based on nitrogen and 90.5 and 107.3 pigs·hm^-2 on the basis of phosphorus, which the corn were harvested at the late milk-ripe stage and full-ripe stage, respectively. The application of pig manure could also increase the biomass yield of corn, thus enhancing the amount of nitrogen and phosphorus. The results showed that the manure carrying parameters of growing-finishing pigs were 95.2 pigs·hm^-2 and 107.3 pigs·hm^-2 based on the nitrogen and phosphorus requirement of corn respectively.

This study aimed to construct a TPL2 (MAP3K8/COT) knockout PK-15 cell line PK-15-TPL2^-/-, and evaluate the impact of FMDV and SVA replication before and after the gene knockout. It provides good biological material for research the mechanism of TPL2 in the process of virus infection and points out the direction of increasing the production of FMDV and SVA for vaccine production. Two single guide RNAs (sg RNA) against the TPL2 gene were screened. Sg RNA was synthesized and inserted into a lentiviral expression vector. The lentivirus was packaged and infected with PK-15 cells, and single cells that had been transfected with sg RNA were sorted by flow cytometry. The DNA mutation of TPL2 in the cell line was confirmed by sequencing, and the expression of TPL2 protein in the cell line was detected by Western blot. Replication of FMDV and SVA in PK-15-TPL2^-/- cells were evaluated by IFA, RT-qPCR, Western blot and TCID₅₀. On this basis, the activation of the interferon pathway in FMDV or SVA infected PK-15-TPL2^-/- cells was studied by measuring the mRNA expression level of interferon (IFN) and IFN stimulating gene (ISG). In TPL2 knockout PK-15 cell line, the TPL2 gene had a base insertion and deletion mutation, TPL2 protein expression was not detected in PK-15-TPL2^-/- cell line. Viral copy number was measure and compared between PK-15 and PK-15-TPL2^-/- cells after FMDV and SVA infection. The results showed that the TPL2 gene knockout significantly promoted FMDV and SVA replication. At the same time, RT-qPCR further confirmed that the mRNA expressions of IFN-α, IFN-β, IFN-γ, ISG15, ISG54 and ISG56 in PK-15-TPL2^-/- cells were significantly lower than these in PK-15 cells after FMDV and SVA infection. In summary, the TPL2 knockout PK-15 cell line was constructed successfully. The TPL2 gene has antiviral effects on FMDV and SVA. Compared with control cells, the TPL2 gene knockout is more productive to FMDV and SVA replication. Results indicated that the CRISPR/Cas9 gene-editing technology can be used as an effective tool to improve virus yield for FMDV and SVA vaccine production. It provided good biological material for studying the mechanisms of TPL2 in the process of virus infection.

The objective of this study was to prepare monoclonal antibodies against p62 protein of African swine fever virus (ASFV), and apply it to immunohistochemistry (IHC) detection of ASFV antigen in infected tissue samples. BALB/c mice were immunized with purified recombinant p62 protein expressed by baculovirus system and hybridoma cells were obtained by fusion of spleen cells and myeloma cells. An indirect ELISA based on the purified p62 protein was developed. Eighteen hybridoma cell lines stably secreting MAbs against the p62 protein were harvested after screening and subcloning. The results of indirect immunofluorescence assay (IFA) showed that all of the monoclonal antibodies could react with ASFV and did not react with swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus and porcine circovirus 2 with good specificity. Three MAbs could recognize ASFV p35 protein, and 15 MAbs recognize p15 protein. Among of them, 14 MAbs heavy chains belong to IgG1 and 4 MAbs heavy chains belong to IgG2a. All light chains were κ. The results of immunohistochemistry showed that 5 MAbs had positive reactivity with the ASFV in lung, tonsil, lymph node of pigs infected with ASFV. The monoclonal antibodies against p62 protein obtained from the study laid a foundation for the establishment of immunological detection method of African swine fever virus and could provide important biomaterials for research on the structure and function of p62 protein.

The objective of this study was to screen the adjuvant suitable for an inactivated vaccine against Erysipelothrix rhusiopathiae. The five inactivated vaccines of mineral oil adjuvant (referred as oil vaccine), aluminum hydroxide adjuvant (referred as alh vaccine), ISA201 dual phase oil emulsion adjuvant (referred as ISA201vaccine), GEL02 water-soluble polymer adjuvant (referred as GEL vaccine), and IMS1313 water-soluble nano-sized adjuvant (referred as IMS1313 vaccine) were prepared, which the antigens of that were all inactivated bacteria of E. rhusiopathiae strain HG-1, isolated and identified as serovar 1a. The immunoprotection test results of the mouse showed that after 14 days of second immunization, mice were intraperitoneally challenged with 4LD₅₀ HG-1 solution, giving the protection rates of the five vaccines 100% (7/7), 14% (1/7), 14% (1/7), 71% (5/7) and 14% (1/7), respectively. According to the above test results, the safety test of piglets was carried out by using alh vaccine and GEL vaccine, and the results showed that the side effects of the two adjuvant vaccines were small. Fourteen days after the second immunization, groups of pigs vaccinated with alh vaccine and GEL vaccine were challenged intravenously with 16 LD₁₀₀ of HG-1 solution, and the results showed that alh vaccine and GEL vaccine provide 60% (3/5) and 100% (5/5) protective efficacy, respectively. This study identified GEL adjuvant as the optimal adjuvant for the development of an inactivated vaccine against swine erysipelas.

The purpose of this experiment is to clarify the pathogenicity and molecular epidemic characteristics of Shigella from yak, and to provide new ideas for exploring the epidemic ways of Shigella and formulating reasonable control strategies. In 2017, 1 396 samples of yak anal cotton swabs were collected in Gansu, Qinghai and Tibet, and isolates were systematically identified by selective medium screening, biochemical identification and serum agglutination test. Seven virulence genes (ipaH, ipaBCD, ial, sen, set1A, set1B and stx) were detected by PCR. MLST typing of 15 pairs of housekeeping gene sequences provided by Mc MLST website database was carried out. Referring to the PulseNet experimental method of CDC in the United States, The chromosome of S. flexneri and S. sonnei were digested by restriction endonuclease NotⅠ and XbaⅠ, and PFGE analysis was performed on these isolates. The results showed that 41 strains were consistent with the biochemical characteristics of Shigella. They were divided into 4 biochemical phenotypes, B3 (36.59%) and B4 (32.35%) were the main biochemical phenotypes. Serum agglutination test identified 23 strains of S. flexneri, including four serotypes 1a (n=2), 2a (n=16), 2b (n=3), Xv (n=2); 18 strains were S. sonnei bacteria are divided into phase Ⅰ (n=12) and phase Ⅱ (n=6). A total of six virulence genes ipaH, ipaBCD, ial, sen, set1A, and set1B were detected, and the carrying rates were 100%, 92.68%, 73.17%, 70.73%, 26.83%, and 26.83%, respectively. There are 7 virulence genotypes, of which VT5 and VT7 are the main epidemic types, accounting for 43.9% and 24.39%, respectively, while Shigella carrying two or more virulence genes account for 92.68%. Fourty-one strains of Shigella were divided into 10 types of ST, of which ST100, ST116 and ST155 were the main epidemic types. S. flexneri of NotⅠ enzyme digestion was divided into 13 PTs, while S. sonnei was divided into 14 PTs by XbaⅠ enzyme digestion. The biochemical phenotype, serotype, ST, and PT type of Shigella isolated from yaks are both polymorphic and prevalent. The Shigella isolates carried the same virulence genes as human, and carrying rates of ipaH, ipaBCD, ial, and sen genes were higher, which posing a potential threat to public safety.

An isolate of Listeria monocytogenes CMG47 was obtained from a goat farm in Shanghai. To identify the genotyping and biological characteristics of this isolate, multiplex PCR was performed in lineage identification and serotyping, multi-locus sequence typing (MLST) was used for genotyping. Major virulence factors were examined by PCR, hemolysis and phospholipase activity were analyzed by in vitro observation and real-time fluorescent quantitative PCR. The virulence was determined by intraperitoneal injection in ICR mice and intravenous injection in zebrafish. Study results revealed that the isolate belonged to lineage I, 1/2b for its serotyping, ST619 for its genotyping, and carried major virulence factors such as prfA, inlA, inlB, plcA, plcB, mpl, actA and hly. It showed weak hemolysis but did not exhibit apparent phospholipase activity. The isolate was of high pathogenicity comparable to the reference virulent strain EGDe in mice and zebrafish models (P>0.05). The isolate belongs to the dominant serotyping and genotyping for Listeria infection, which carries major virulence factors and is highly pathogenic. This work provides the molecular basis for further analysis of the prevalence and distribution characteristics of sporadic listeriosis and is very important for the establishment of Listeria monitoring system and risk assessment as well.

In order to prepare a monoclonal antibody (MAb) against Mycoplasma hypneumoniae (Mhp), the splenic cells from BALB/c mice immunized with the P97 C-terminal region of Mhp J strain, which contains R1 region (P97CR1 protein) and was expressed in Escherichia coli (E. coli) BL21(DE3), were fused with SP2/0 myeloma cells, and a hybridoma cell line A3 stably secreting the MAbs against P97 Protein of Mhp were successfully screened. The identification results indicated the A3 MAb was IgG1 subclass with κ light chain. The A3 MAb was able to specifically recognize the P97CR1 protein of Mhp expressed in E. coli BL21(DE3) using Western blot analysis. Flow cytometry analysis showed that the MAb was able to recognize natural P97 protein expressed on the Mhp. The epitope sequence recognized by the A3 MAb was identified as LDDNLQby pepscan technique. The epitope sequence is highly conservative among the P97 proteins of all Mhp strains. The blocking ELISA analysis showed that the binding of the A3 MAb to the P97CR1 protein was blocked by high immune positive serum of Mhp. The preparation of A3 Mab provided foundation for further development of a blocking ELISA assay with high specificity and sensitivity for detecting Mhp infection.

This study aimed to clarify the infection of the pathogenic Bartonella in the ectoparasite Melophagus ovinus in sheep in parts of Tibet. From January to September 2019,298 sheep parasites in Linzhi, Xigaze and Naqu were collected. The morphological identification and PCR amplification of the 18S rRNA gene of the Melophagus ovinus were used to identify the body and the pathogen gltA gane of Bartonella was detected, pMD^TM-18T was connected to some positive PCR products and transferred into DH5α competent cells. The positive results were sequenced and analyzed for genetic and evolutionary analysis. The results showed that the positive rate of female and male were 49.8% (113/227) and 42.3%(30/71), there was no significant difference between male and female(χ²=0.944, P=0.267). The total infection rate of the pathogen Bartonella was 48.0% (143/298). Positive rate of Linzhi was significantly higher than that of Xigaze and Naqu area(χ²=13.801, P<0.01; χ²=17.067,P<0.01), there was no significant difference between Xigaze and Naqu areas(χ²=0.084, P=0.771).The positive rate of Melophagus ovinus in free culture, captivity, and slaughterhouse were 44.3% (102/230), 85.0% (34/40) and 23.3% (7/30), respectively. There were significant differences between captive culture and free host culture (χ²=20.929, P<0.01)or slaughterhouse (χ²=24.38,P<0.01).There was significant differences between host free culture and slaughterhouse(χ²=3.989,P=0.046).The sequencing results were uploaded to GenBank database, and obtained three Bartonella gltA gene accession numbers, MN623006, MN623007 and MN623008. The sequence alignment showed that the homology of Bartonella in Yunnan and Xinjiang was 99.6%-100%. In this study, the pathogen Bartonella of Tibetan Melophagus ovinus was detected for the first time, which provided the basis for understanding the pathogen Bartonella carried by Tibetan sheep parasites in vitro and the prevention and control of the pathogen.

This study aimed to analyze the effects of three exogenous stimulating factors on the species, resistance types, and resistance mechanisms, etc. of antibiotic resistance genes (ARGs) by feeding yak with cefquinome (CEF), difloxacin (DIF), and aflatoxin B₁ (AFB₁), and sequence the rumen microorganisms by metagenomics. It is important to study the characteristics and resistance mechanisms of microbial antibiotic resistome or resistance reservoirs. Fifteen yaks were selected and randomly divided into 5 groups. According to the recommended dosage of the instructions, the Cef group and the Dif group were orally administered with CEF 1 mg·kg^-1 and DIF 1 mL·kg^-1, respectively. E1 groups and E2 groups were fed AFB₁ 20 and 60 μg·kg^-1, respectively. Group C was the control group. The rumen fluid samples of yaks were collected 7 days after treatment and DNA was extracted. The DNA was sequenced by Illumina HiSeq. After standardizing the reads counts, the TPM was obtained. An analysis of variance was performed on the TPM. The results showed that 132 ARGs were obtained in the control group, belonging to 30 resistance types, among which the abundance of tetracycline tetQ and tetW genes was higher. The abundance of tetW gene in Cef group increased (P<0.05). The abundance of tetQ in Dif group increased (P<0.05). The abundance of tetracycline antibiotic and cephalosporin antibiotic resistance genes in Cef group increased (P<0.05). The abundance of tetracycline antibiotic and aminocoumarin antibiotic resistance genes in Dif group increased (P<0.05). The abundance of aminocoumarin antibiotic and penem antibiotic resistance genes in the E1 group increased (P<0.05). The abundances of 9 resistance genes such as penems antibiotic and cephalosporins antibiotic in E2 group increased (P<0.05). The abundance of Erm 23S ribosomal RNA methyltransferase in Dif group increased (P<0.05). The abundance of three resistance mechanisms such as ATP-binding cassette (ABC) antibiotic efflux pump in E2 group increased (P<0.05). All three treatments significantly increased the host species of tetracycline antibiotic resistance genes. In conclusion:The rumen is a rich library of ARGs, in which tetQ and tetW are dominant ARGs. Not only CEF, DIF increase the abundance of some ARGs species, resistance types and enzymes related to resistance mechanisms, thereby increasing rumen microbial resistance, but also AFB₁ has a similar effect. Moreover, the high-dose AFB₁ has a greater range of effects on antibiotic resistance than antibiotics. These three factors also increase types of host microorganisms carrying tetracycline antibiotic resistance gene, thereby enhancing the horizontal gene transfer, accelerating the spread of ARGs, and enhancing the resistance of microorganisms to tetracycline antibiotics.

This study aimed to study the evolution and structural characteristics of β-lactamase produced by Staphylococcus aureus (SA) and its action mode in Ningxia province. Antimicrobial susceptibility test and PCR were used to detect the resistance and drug resistance genes of five β-lactams respectively; the evolution analysis and structure function prediction of β-lactamase BlaZ were made by bioinformatics; the mode of action of BlaZ was analyzed by molecular docking and dynamic simulation. The results showed that the resistance rate of Penicillins was significantly higher than Cephalosporin's; the detection rate of blaZ gene was 82.37% and the detection rate of blaZ_TEM-1 gene, which was rarely reported in SA, was 26.05%. According to evolutionary analysis, BlaZ belongs to A-type β-lactamase and contains 63 important trace residues under evolutionary pressure. Meanwhile, molecular docking showed that BlaZ is easy to combine with Penicillins, Avibatan, what's more, formed stable. The results of molecular dynamics simulation showed that the structural flexibility of BlaZ was less than that of TEM-1 and TEM-52, in which there was a difference in the flexibility of Ω-loop region (P<0.05) and it could affect the antibiotic binding. Thus, there was a positive correlation between the flexibility of the region and the binding activity; the structural stability of BlaZ was significantly different (P<0.05) when it was combined with Ampicillin and Ceftiofur, respectively. The BlaZ produced by SA in Ningxia is a A-type β-lactamase. In addition, blaZ_TEM-1 gene was detected from those strains. The bind-ing activity of BlaZ to Penicillins and Avibactam is higher than that of Cephalosporin, and the structural flexibility of Ω-loop region is an important factor affecting the binding activity.

This study aimed to investigate the effects of ethanol extract of Chinese propolis (EECP) on transcript levels of inflammation-related genes and tight junctional permeability in bovine mammary epithelial cells stimulated by bacterial endotoxin (lipopolysaccharide,LPS). The total content of phenolic acids and flavonoids in the EECP was determined by Folin-phenol method and ALNO₃ colorimetry, respectively. Moreover, the inflammatory model of bovine mammary epithelial cells (MAC-T) was induced by bacterial lipopolysaccharide and the effect of EECP on the relative cell proliferation rate was detected via CCK-8 method. Real-time quantitative PCR (RT-qPCR) was used to evaluate LPS-induced bovine mammary epithelial cells inflammatory factors (IL-6, IL-8, TNF-α and IL-1β) relative mRNA transcription levels. To determine the effect of EECP on LPS-induced MAC-T cell, inflammation tight junction permeability. We used RT-qPCR to detect the relative mRNA transcription levels of tight junction proteins (occludin, ZO-1), and further used immunofluorescence to localize tight junction membrane proteins. The results showed that the content of total phenolic acids and total flavonoids in EECP were 106.35 mg GAE·g^-1 and 320.85 mg RE·g^-1, respectively. CCK-8 results showed that the safe concentration of EECP was 0-15 μg·mL ^-1, and can effectively increase the viability of MAC-T cells under LPS stimulation. LPS stimulation significantly increased the mRNA transcription levels of cellular inflammation-related factors IL-6, IL-8, TNF-α and IL-1β (P<0.001). However, the mRNA transcription of IL-6, IL-8, TNF-α and IL-1β were significantly reduced under pretreatment with 2.5-15.0 μg·mL^-1 EECP. Similarly, the LPS model group significantly inhibited (P<0.01) the mRNA transcription of tight junction proteins (ZO-1 and occludin), while the mRNA transcription of tight junction proteins was increased by EECP pretreatment (P<0.05). Immunofluorescence staining experiments also confirmed that EECP could alleviate LPS-induced mammary epithelial barrier dysfunction by up-regulating the expression of tight junction proteins occludin and ZO-1. The results confirmed that EECP has a good protective effect on bacterial lipopolysaccharide-induced inflammation of bovine mammary epithelial cells, which provides a basis for the use of Chinese propolis to prevent mastitis in dairy cows.

Hemangiopericytoma is a soft tissue sarcoma that originate from the pericytes of the capillary wall. In this study, we described a left radiocarpal joint mass in an 11-year-old female mixed-breed dog. Iconography, cytology, histopathology, and immunohistology were used to determine the nature of the mass. X-ray imaging showed that the tumor began in soft tissue and had clear boundaries; cytology showed that the cells were spindle-shaped, with obvious nucleoli and variably sized nuclei; histopathology revealed the presence of spindle cells surrounding blood vessels; immunohistochemistry was positive for vimentin and α-SMA, and negative for desmin and S-100. The number of PCNA positive tumor cells was more than 25%. Masson trichrome staining showed that the content of collagen fibers in tumor tissue was low. The diagnosis of hemangiopericytoma was confirmed by pathology and immunohistochemistry.

The objective of this study was to express the allograft inflammatory factor-1 (AIF-1) of yak in E.coli BL21(DE3) and investigate its effect on mouse macrophages. The expression levels of AIF-1 gene in 5 tissues of yak were detected by q-PCR. A prokaryotic expression vector was constructed to express and purify AIF-1 protein. The expression levels of 4 inflammatory factors in mouse macrophages were detected by q-PCR. The results showed that the expression level of AIF-1 gene was the highest in the spleen of Maiwa yak, which was extremely significantly higher than those in other tissues (P<0.01). Recombinant protein of approximately 29.47 ku was expressed and purified. The expression levels of IL-1β, IL-6, TNF-α and iNOS in mouse macrophages were promoted by 1.0, 10.0 and 100.0 μg·mL^-1 of AIF-1 protein. The results indicated that AIF-1 played a role in the immune response of macrophages. This article will be benefit for further study on the function of yak AIF-1.