Rumen microbes play a vital role in the production efficiency, health status of ruminants and greenhouse gas emissions. It is hot spots and highlights that the different research methods are used to reveal the structural composition and metabolic function of microbial communities in rumen ecosystems. This article reviews recent advances in rumen microbial composition, genomic function and metabolism in ruminants using omics technologies (metagenome, metatranscriptome, metaproteome and metabolomics) combined with evolving instrumental analysis and bioinformatics technologies. It aims to provide new technical methods and theoretical basis for further regulating rumen microbiome to improve ruminant production and reduce environmental pollution.

Avian necrotic enteritis (NE) is one of the most prominent intestinal diseases in broiler modern intensive production, which has brought huge economic losses to the poultry industry. With the worldwide restrictions and the bans on antibiotic growth promoters, the incidence of chicken necrotic enteritis is on the rise globally, which poses a serious threat to poultry production and health. Therefore, there is an urgent need for new technologies and products to replace antibiotics to prevent and control avian necrotic enteritis. This article reviews the advances in biological measurements for the prevention and control of chicken necrotic enteritis. It provides a technical reference for the biological prevention and control of chicken necrotic enteritis, and provides theoretical considerations for its in-depth research.

This experiment was conducted to clone the full-length cDNA of porcine ADAR1 gene, and explore its expression in different tissues and backfat of pigs with different ages.The cDNA full-length sequence of ADAR1 gene was cloned using rapid-amplification of cDNA ends (RACE) and analyzed by bioinformatics. The real-time PCR was used to detect ADAR1 mRNA expression in different tissues and backfat of Large White pigs with different ages. A 6 259 bp cDNA sequence of porcine ADAR1 was cloned, which encoded 1 145 amino acids. Its amino acid sequence shared an identity equal or more than 85% with other mammals including human, chimpanzee, macaque, gibbon, cow, goat and sheep. The predicted ADAR1 had 2 Zα binding domains, 3 double-stranded RNA binding domains and a deaminase domain. The real-time PCR result showed that ADAR1 was expressed in heart, liver, spleen, lung, kidney, brain, muscle, small intestine and backfat. Furthermore, it was expressed in backfat of individuals with the ages of 7, 60, 120 and 180 days, showing an overall trend of going down firstly and going up then with the individual development. In this study, the full-length cDNA sequence of ADAR1 gene was successfully cloned in pigs, which was widely expressed in pigs and its expression levels were different in backfat of pigs with different ages. These findings provided a theoretical foundation for further function study of ADAR1.

This experiment was conducted to study population relationship of Tibetan sheep at the molecular level. A total of 384 individuals from 20 Tibetan sheep populations from 5 regions including Tibet, Qinghai, Gansu, Yunnan and Sichuan were selected for Affymetrix Ovine 600K chip scanning. The genetic diversity of Tibetan sheep populations was analyzed based on heterozygosity and inbreeding coefficient, and the population clustering of Tibetan sheep was analyzed by PCA, NJ-tree and STRUCTURE. The results showed that the observed heterozygosity of Tibet Langkazi sheep was the lowest (0.235 0) and the inbreeding coefficient was the highest (0.279 6). The observed heterozygosity of Qinghai valley type Tibetan sheep was the highest (0.323 4) and the inbreeding coefficient was the lowest (0.009 0). The results of genetic structure analysis showed that Tibetan sheep in Tibet and Yunnan could be gathered together alone, while grassland sheep in Qinghai, Gansu and Sichuan were clustered together in confusion. The results indicate that the genetic diversity of Tibetan sheep in Tibet and Yunnan is generally lower than that of Tibetan sheep in Qinghai, Gansu and Sichuan. Tibetan sheep in Tibet and Yunnan can maintain regional specificity, while grassland sheep in Qinghai, Gansu and Sichuan regions have little difference in genetic distance and population structure.

The aim of this study was to investigate the effects of overexpressing inflammatory factor interleukin-6 (IL-6) on the proliferation and migration of goat hair follicle stem cells (HFSCs) and elucidate the role of MAPK/ERK signaling pathway in the proliferation and migration of goat HFSCs. The pXJ40-myc-IL-6 overexpression vector was constructed and transiently transfected into the goat HFSCs, and the empty vector cells were used as the blank control group. Western blot was used to quantitatively detect the expression of IL-6 protein, and MTT assay was used to detect the proliferation of goat HFSCs. The migration ability of HFSCs was detected by wound healing assay. Finally, Western blot was used to detect the expression of key kinases in MAPK/ERK and P38 signaling pathway in goat HFSCs. The results showed that after transfecting the IL-6 overexpression vector into goat HFSCs for 3 days, compared with the control group, the proliferation activity of the HFSCs was inhibited and the sustained inhibition effect was observed until the 7th day(P<0.05). After 24 h of transfection of IL-6 overexpression vector into goat HFSCs, the healing rate of wounding site in the treatment group reached 84.2%, which was significantly lower than that in the control group (98.5%, P<0.01). Compared with the control group, the phosphorylation level of the key kinase ERK1/2 (p-ERK1/2) in the MAPK/ERK signaling pathway was significantly down-regulated (P<0.01), the phosphorylation level of the key kinase p38 (p-p38) in the P38 signaling pathway was up-regulated, however, the change was not statistically significant (P>0.05). In summary, the overexpression of IL-6 gene has an inhibitory effect on the proliferation and migration ability of goat HFSCs, and its negative regulatory role on the proliferation and migration of goat HFSCs maybe through the MAPK/REK signaling pathway. The results provide basis for revealing the mechanism of migration and tissue repair of goat HFSCs.

The objective of this study was to estimate the genetic parameters of productive life of Chinese Holsteins, and to compare the prediction reliability and stability of different evaluation models. The genetic parameters of productive life records of 90 049 Chinese Holstein cows from 29 herds in Beijing were estimated by single-trait animal model (STAM), single-trait sire model (STSM) and multiple-trait animal models (MTAM). All models used for genetic parameter estimation took into account the fixed effects of herd, birth year-season and group of age at first calving, the random effect of individual additive genetic effect (animal model) or sire genetic effect (sire model), as well as the residual effect. The heritability of productive life derived from the single-trait animal model and single-trait sire model were 0.052 and 0.047, respectively. Multiple-trait animal models were relatively stable, with estimated heritability between 0.057 and 0.069, and the genetic correlations among these traits ranged from 0.779 to 0.998. For the data with large amount of censored records, multiple-trait animal models based on the first 3 lactations are more appropriate, meanwhile these model are more stable. It is the first genetic evaluation for productive life of Chinese Holstein, and these results provide theoretical basis for improving economic efficiency of farms and facilitating balanced breeding in the future.

The objective of this study was to investigate the effects of the S100A10 gene, which was identified as a candidate gene regulating Simmental marbling score by genome-wide association study (GWAS), on adipogenic differentiation of mice preadipocytes. In this study, white preadipocytes were isolated from inguinal white adipose tissue of C57BL/6 mice and induced white/brown adipogenic differentiation in vitro. The expression of the S100a10 gene was interfered using siRNA. The differentiation of preadipocytes were detected by Oil Red O staining. Gene and protein expression levels were measured by qRT-PCR, Western blot and immunofluorescence. The results showed that lipid droplets decreased significantly after S100a10 knocking down. And qRT-PCR assay showed that the expression level of white adipocyte-specific genes Pparg, C/ebpa, Fabp4, Glut4, Adiponectin and Leptin were significantly down regulated in white adipogenic differentiation(P<0.01). The expression level of brown adipocyte-specific genes Ucp1, Pgc1a, Fabp4, Elovl3 and Cox7a were significantly decreased in brown adipogenic differentiation(P<0.01). Western blot result showed that the expression of FABP4 and PPARG were significantly decreased in white adipogenic differentiation(P<0.01), the immunofluorescence assay showed that the expression of FABP4 was decreased in white adipogenic differentiation. The protein expression of brown adipogenic differentiation specific genes FABP4 and UCP1 were obviously decreased in brown adipogenic differentiation(P<0.01). The results showed that knocking down S100A10 inhibited the white/brown adipogenic differentiation in mice preadipocytes. This study provides scientific basis for the further research of the effect of S100A10 on intramuscular fat deposition in beef cattle.

The aim of this study was to analyze the genomic methylation patterns of in vitro fertilized(IVF) blastocysts from fresh and vitrified bovine oocytes. In this study, the whole-genome methylation level and differential methylation regions of in vitro fertilized(IVF) blastocysts from fresh and vitrified bovine oocytes were detected by single-cell whole-genome methylation sequencing technique(scWGMS). The difference of DNA methylation level between in vitro fertilized(IVF) blastocysts from fresh and vitrified bovine oocytes was investigated. The results showed that the whole methylation level of in vitro fertilized(IVF) blastocysts from fresh oocytes was significantly higher than that of in vitro fertilized(IVF) blastocysts from vitrified oocytes in bovine(P<0.05). GO and KEGG were used to analyze 143 DMRs. It was found that biological process of these DMRs were mainly involved in metabolism, growth and development, cell localization and response to cell stimulation. The pathways were mainly enriched in growth and development, nucleic acid binding and histone acetylation, and the partial related candidate genes were screened(FARP2, PI4KA, FAM3D, NCOR2, ZNF827, etc.). In this study, we found that the whole genome methylation level of in vitro fertilized(IVF) blastocysts from vitrified bovine oocytes was significantly decreased, and the DMR region was mainly involved in ATP binding, growth and development and histone acetylation, which provided information reference for improving the quality of IVF blastocysts from vitrified bovine oocytes.

The aim of this study was to identify molecular characteristic and three-dimensional structure of TEDDM1 in bovine follicle, its function was analyzed by combining expression characteristic of TEDDM1 in different physiology states of follicles. DF and SF in first follicular development wave during estrous cycle were collected, GCs were isolated, total RNA was extracted and the reverse transcription was performed by RT-PCR, the specific primers were designed to amplify TEDDM1 and the sequence was sequenced, bioinformatics method was used to analyze CDS region sequence structure; Using RPLP0 as reference gene, qRT-PCR was performed to detect the experssion of TEDDM1 in DF and SF; The rabbit anti-TEDDM1 antibody was used to dectet expression level and localization of TEDDM1 in bovine follicles. The results showed that full length of TEDDM1 CDS region was 903 bp, encoding 300 amino acids, contained 7 parallel alpha helical structures across the cell membrane, which was a typical G protein-coupled receptor, sequence of TEDDM1 had the highest similarity with Bison (99.4%); The functional domain analysis showed that DUF716 existed in TEDDM1. The results of qRT-PCR analysis showed that expression level of TEDDM1 mRNA in SF was significantly higher than that in DF (P<0.05); The immunohistochemical analysis showed that TEDDM1 was expressed in GCs and membrane cells layer of DF and SF, and specific color intensity result showed that expression of TEDDM1 in SF GCs and membrane cells was higher than that in DF. The study provides a basis for further study on regulatory effect of TEDDM1 in bovine follicular development, signal transduction and hormone regulation, and lays a foundation for further research on mechanism of bovine follicular development.

The aim of this study was to investigate the effects of tumor necrosis factor-α (TNF-α) on yak oocytes maturation in vitro, the expression of epidermal growth factor (HIF-1α) and heat shock protein receptor (HSP70) in yak oocytes and the subsequent embryo development. In this experiment, TNF-α with different concentrations (0, 10, 25, 50 ng·mL^-1) were added to in vitro maturation medium of yak oocytes. After the cumulus-oocyte complexes (COCs) matured, the oocyte maturation rate and the development rate of early embryos after in vitro fertilization were counted. The HIF-1α and HSP70 genes of yak were amplified by RT-PCR. The mRNA and protein expression of HIF-1α and HSP70 were detected by real-time PCR (qRT-RCR), Western blotting and immunofluorescence staining. The results showed that:1) The addition of TNF-α to the oocyte maturation medium promoted the maturation rate of oocytes. With the increase of TNF-α concentration, the maturation rate, cleavage rate and blastocyst rate of oocytes increased gradually. When the concentration of TNF-α was 25 ng·mL^-1, the maturation rate and cleavage rate of oocytes reached the highest, 84.98% and 66.85%, respectively, and the blastocyst rate also reached 21.48%, which were significantly higher than that in the control group(P<0.05). 2) With the increase of TNF-α concentration, the expression level of HIF-1α decreased gradually. When the concentration of TNF-α was 0 ng·mL^-1, the expression level of HIF-1α was the highest, which was significantly higher than that in other groups (P<0.01). The expression level of HIF-1α was the lowest when the concentration of TNF-α was 50 ng·mL^-1, which was sig-nificantly lower than that in other groups (P<0.01). Immunofluorescence assay showed that HIF-1α was expressed in both cumulus cells and oocytes. 3) When the concentration of TNF-α was 25 ng·mL^-1, the expression level of HSP70 was the highest, which was significantly higher than that in other groups (P<0.01), and when the concentration of TNF-α reached 50 ng·mL^-1, the expression level of HSP70 was the lowest. The results showed that TNF-α significantly increased the developmental capacity of oocytes during maturation of yak oocytes in vitro, and also induced the expression of HSP70 and inhibited the expression of HIF-1α. These would provide a theoretical basis for exploring the role of TNF-α, HIF-1α and HSP70 in the reproductive process of yak and the impact on embryo development.

The purpose of this experiment was to study the effects of oregano essential oil on the fatty acid composition in muscles of Gansu Alpine Fine-wool sheep. Thirty 60-day-old healthy Gansu Alpine Fine-wool sheep with the similar body condition were selected and randomly divided into 3 groups:control group, adding 2 and 4 g·d^-1 oregano essential oil groups (group I and group Ⅱ). There were 10 sheep per group. Six sheep in each group were slaughtered randomly after 3 month, the samples of longissimus dorsi (LD) muscle and semitendinosus (SE) muscle were collected, and the fatty acid composition and conjugated linoleic acid (CLA) content were measured. The results showed that:1) The concentration of blood HDL-C of ewes in group I and rams in group Ⅱ was significantly lower than that of ewes in control group (P<0.05), and there was no significant difference in other blood biochemical parameters (P>0.05) among the 3 groups. 2) Oleic acid (C18:1n9c) and palmitic acid (C16:0) were the main fatty acids in muscle of different parts. In LD muscle, the content of MUFA and UFA of the ewes in control group were significantly higher than that of rams in control group(P<0.05), while the content of n-6PUFA of ewes in control group was significantly lower than that of rams in control group (P<0.05), the SFA content of rams in group I was significantly higher than that of rams in group Ⅱ(P<0.05); The content of MUFA of rams in group Ⅱ was significantly higher than that of ewes in groupⅡ, the rams in control group and group I(P<0.05); The content of PUFA of ewes in group Ⅱ was significantly higher than that of rams in group Ⅱ, ewes in control group and group I(P<0.05), the UFA content of rams in group I was significantly lower than that of rams in group Ⅱ(P<0.05). In SE muscle, the contents of PUFA, n-6PUFA and n-3PUFA of rams in control group were significantly higher than that of ewes in control group (P<0.05), the MUFA content of rams in group Ⅱ was significantly higher than that of rams in control group and group I(P<0.05), n-6/n-3 content of rams in group I was significantly higher than that of ewes in group I (P<0.05). 3) The content of c9t11-CLA in muscles of ewes was higher than that of rams in the 3 groups, and the content of c9t11-CLA of ewes in group I was significantly higher than that of rams in group I (P<0.05); The c9t11-CLA content in LD muscle of rams and ewes in group Ⅱ was significantly higher than that in control group (P<0.05), and there was no significant difference between group I and group Ⅱ (P>0.05). In summary, the fatty acids composition in muscle of Gansu Alpine Fine-wool sheep are different with animal sex, tissue location and additive dosage, adding oregano essential oil in diet can increase CLA content in muscle, and the supplementation effect of 4 g·d^-1is better than 2 g·d^-1.

This experiment was conducted to explore the effects of acute heat stress on antioxidant capacity, immune function and expression of HSP70 family genes in blood lymphocytes in goats. Five (12±0.5)-month-old healthy female goats (F₁ of Boer goat×Guanzhong dairy goat) were selected and raised separately in cage in an environment controlled chamber (temperature:20℃, relative humidity:60%), adaptation period lasted for 5 days. At the 6th day, five goats were treated with acute heat stress at 38℃ for 12 h. Blood samples were collected before heat stress (0 h, 20℃) and after 2, 4, 8 and 12 h of heat stress. Serum antioxidant indexes (total antioxidant capacity, superoxide dismutase, glutathione peroxidase activity and malondialdehyde content), serum immune indexes (serum immunoglobulin and cytokine contents) and the expression of HSP70 family member genes (HSPA1A, HSPA6 and HSPA8) in blood lymphocytes were measured by colorimetric method, ELISA and RT-PCR, respectively. The results showed as follows:1) Heat stress time had significant effects on serum antioxidant indexes. Compared with 0 h before heat stress, serum T-AOC (P<0.05), SOD (P<0.05) and GSH-Px (P<0.01) activities decreased significantly after 8 h of heat stress, and MDA content increased significantly after 4 h of heat stress (P<0.05). 2) Heat stress time had also significant influence on serum immune indexes. The contents of serum TNF-α and IL-2 increased significantly after 4 h of heat stress (P<0.05), while the contents of IL-1β and IFN-γ increased significantly after 8 h of heat stress (P<0.05). The contents of IgG (P<0.01), IgM (P<0.01) and IgA (P<0.05) decreased significantly after 4 h of heat stress, while the content of IL-4 decreased significantly after 12 h of heat stress(P<0.01). 3) Heat stress time significantly increased the expression levels of HSP70 family genes (HSPA1A, HSPA6 and HSPA8) in blood lymphocytes. The mRNA expression of HSPA1A rose firstly, then declined, and then it reached the peak at 4 h after heat stress. In addition, the mRNA expression of HSPA1A was significantly higher at 2, 4, 8 and 12 h after heat stress than at 0 h (P<0.01). The mRNA expression of HSPA6 increased significantly at 2 h after heat stress (P<0.01), and returned to pre-stress level after 4 h (P>0.05). The mRNA expression of HSPA8 was significantly higher at 4 (P<0.05), 8 (P<0.01), 12 h (P<0.01) after heat stress than that at 0 h. In this experiment, acute heat stress at 38℃ could inhibit the immune and antioxidant functions and increased the mRNA expression of HSPA1A, HSPA6 and HSPA8 genes in blood lymphocytes. HSPA1A was more sensitive to temperature and time of heat stress, and could be used as a molecular marker of early heat stress in goats.

This experiment was conducted to understand the prevalence and variation of H9 subtype avian influenza virus (AIV) isolated in East China. Diseased samples from clinical suspected cases and swab samples from poultry in live poultry markets were collected and subjected to inoculate SPF chicken embryos. H9 AIVs were identified by HA and HI test. HA genes of partial H9 AIVs were amplified by RT-PCR, and PCR productions were subjected to sequence. Antisera from SPF chickens vaccinated with four selected strains were performed cross-reactivity by HI test to analyze the antigen variation. Results were as follows:62 isolates of H9 subtype AIV were identified, and the isolation rate was 2.02% (62/3 074). Sequence analysis of 25 H9 subtype AIVs revealed that the HA genes were belonged to the h9.4.2.5 lineage, and further were divided into Group A and Group B. The amino acid at the HA gene cleavage site was PSRSSR↓G, which is consistent with the characteristics of low pathogenic avian influenza virus. Compared with the amino acid of the HA gene of the Y280-like strain, the left arm of the receptor binding site (RBS) was changed to NGLMGR. There was a change of R (64%)/K (36%) at antigenic residue 92. The cross-reactivity of antisera showed that the HI antibody titers were differed by 2-8 log2 between the isolates and the isolates could be categorized to two antigenic phenotypes. Therefore, antigen drift is appeared in the prevalent H9N2 AIVs, and at least two antigenic phenotypes are existed simultaneously.

TANK-binding kinase 1(TBK1) is a key enzyme responsible for IRF3, IRF7 phos-phorylation and type Ⅰ interferons expression during viral infection. Furthermore, TBK1 is an important element in antiviral natural and acquired immune response. To study the effect of TBK1 gene knockout on porcine pseudorabies virus (PRV) replication, in this study, TBK1 knockout PK-15 cell line was constructed by CRISPR/Cas9 technology. Next, the cell viability of PK-15-TBK1^-/- cells was monitored by CCK-8 assay. Then, the following indexes were used to comprehensively evaluate the effect of TBK1 gene knockout on PRV replication:fluorescence intensity of PRV-GFP was assayed by flow cytometry; mRNA levels of PRV-gB, PRV-gE, PRV-TK, IL-1β, IFN-β and ISG15 were measured by RT-qPCR; protein expression levels of PRV-gB and PRV-gE were evaluated by Western Blot; infectivity of progeny virus was determined by titer determination. Results were as follows:Firstly, T7E1 assay results showed that target bands were identified from 3 sgRNA target sites in exon 2 regions of TBK1 gene. TBK1-sgRNA1 cells with highest editing efficiency were selected with limiting dilution method for monoclonal cultivation by inoculating into a 96-pore plate. Then, No.4 cell strain from 6 independent TBK1 stable knockout monoclonal cells was chosen for CCK-8 assay. The results showed that knockout of TBK1 gene had no effect on cell viability. In addition, flow cytometry results showed that positive cells infected with PRV-GFP account for 56.89% of the total PK-15 cells, and those were 77.95% of the total PK-15-TBK1^-/- cells, indicating that PK-15-TBK1^-/- cells could enhance PRV replication. Moreover, RT-qPCR and WB results showed that PK-15-TBK1^-/- cells could up-regulate PRV mRNA transcription and protein translation. Titer determination showed that the TCID₅₀ of new progeny virions in PK-15 cells and PK-15-TBK1^-/- cells were 10^6.8 TCID₅₀·0.1 mL^-1 and 10^8.5 TCID₅₀·0.1 mL^-1, respectively. Besides, RT-qPCR results showed that up-regulation transcription of IL-1β, IFN-β and ISG15 induced by PRV infection were resisted in PK-15-TBK1^-/- cells. In conclusion, the above results indicate that knockout of TBK1 gene promotes PRV replication in PK-15 cells, which might be linked to the inhibition of transcription of IL-1β, IFN-β and ISG15.

In order to investigate the molecular epidemiological characteristics and genomic changes of porcine reproductive and respiratory syndrome virus (PRRSV) epidemic strains from Shandong province and its neighboring areas in China since 2017, in this study, 70 samples of suspected PRRS symptoms were collected from some pig farms in Shandong, Henan and Jiangsu provinces, and PRRSV was detected by RT-PCR. The genetic recombination of virus in PRRSV positive samples, the 561st, 586th and 592nd amino acids of Nsp9 which determine the pathogenicity and replication of Chinese highly pathogenic PRRSV (HP-PRRSV) were analyzed to explore the genetic evolution of PRRSV isolates. The results showed that 11 strains of PRRSV isolates were screened from the above samples, and the detection rate of PRRSV was 55.7%. Nsp2 sequence analysis showed that compared with the classical American strain VR-2332, 30 amino acids were discontinuously deleted at 481 and 533-561 sites in seven isolates, which were identical to the deletion characteristics of HP-PRRSV prevalent in China since 2006. Two PRRSV isolates had total deletions of 33 discontinuous amino acids in three fragments at 481, 533-561 and 595-597, respectively; and two PRRSV isolates showed a total of 73 discontinuous amino acid deletions in two fragments at 475-518 and 533-561, respectively. The Nsp2 genes of PRRSV mutant strains have undergone significant new deletions. The results of gene recombination analysis by the software RDP4 showed that the last four new type of deleted virus strains had large gene recombination, and the number of recombinant sites and recombinant fragments were different. The four strains all used the highly pathogenic strain JXA1 as the main parental strain for the recombinant and most of the recombination changes were concentrated on Nsp2 protein region, and partial recombination changes were also observed in the non-structural and minor protein regions. In addition, the amino acid variation analysis of positions 561, 586 and 592 of Nsp9 showed that the three critical amino acid sites of the four virus isolates were consistent with Chinese HP-PRRSV. This study accumulated data for further exploration of the genetic variation of PRRSV and related biological characteristics.

This experiment was conducted to develop a TaqMan based real-time fluorescent RT-PCR for the detection of PDCoV. Based on the M gene sequence of PDCoV in GenBank, a pair of primers was designed, and M gene of PDCoV was amplified and cloned into pMD18-T vector to get the recombinant plasmid. Then another pair of primers and a TaqMan probe was designed, and a TaqMan based real-time fluorescent RT-PCR assay was developed by optimizing the conditions and systems of reaction. Sensitivity, specificity and reproducibility assay were determined. The results showed that this assay could detect the PDCoV between 1.0×10¹-1.0×10⁹ copies·μL^-1 with a good linear relation, and the sensitivity limit was 1.0×10¹ copies·μL^-1. There were no cross-reaction with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), pseudorabies virus (PRV) and other porcine viruses. The CV (Coefficient of Variation) of intra-assay and inter-assay were no more than 1%. The TaqMan based real-time RT-PCR assay was then used to detect 100 diarrhea clinical samples of porcine that collected from 2017 to 2018, and the PDCoV positive ratio was 23%, and the coincidence rate with SYBR Green Ⅰ real-time PCR assay was 100%. Piglets were inoculated with PDCoV, and fecal samples were collected and detected by TaqMan real-time RT-PCR and common RT-PCR assay, the results showed that the sensitivity of the former was much better than the latter. This assay provides a high sensitivity, specificity and reproducibility method for detection of PDCoV.

In order to establish a rapid and sensitive nanoparticle-assisted PCR (nano PCR) assay for detection of canine parvovirus (CPV), a pair of primers was designed based on the conserved region of the VP2 gene to amplify 574 bp fragment. The annealing temperature, primer concentration and other reaction conditions of nano PCR were optimized, and its specificity and sensitivity were evaluated. The results showed that the established nano PCR had good specificity and sensitivity, with a minimum nucleic acid detection amount of 2 copies·μL^-1, and its sensitivity was 1 000 times higher than that of conventional PCR. Seventy-five clinical samples collected from Beijing, Guangxi and Jilin were tested, the positive rate of CPV was 89.3% (67/75) and 70.7% (53/75) by nano PCR and conventional PCR, respectively. The results showed that the method had higher sensitivity and was suitable for the detection of low amount of CPV in clinical samples. The established nano PCR in this study provided a new method for rapid, sensitive and accurate diagnosis of CPV infection in the early stage,which laid a foundation for the prevention and control of CPV and had significant value in clinical application.

Enterococcus faecium has been reported as a conditional pathogen in clinical practice, mainly causing endocarditis and sepsis in the host. At present, the research on drug resistance of E. faecium has become increasingly prominent, mainly because the bacteria are natural resistant to β-lactam and its resistant ability to glycopeptide antibiotics (such as vancomycin) are increasing, but the drug resistance transmission mechanism of this strain is still under study. The purpose of this study was to describe the characteristics of a multidrug resistance genomic island from E. faecium. Highlight the spread of the genomic island in different genera of bacteria. At the same time, it provides data that support for the spread of bacterial resistance in wild animals. Single-molecule sequencing technology (de nove) was used in this E. faecium, and the genomic island was analyzed using all kinds of molecular biology tools. Results were as follows:This strain of E. faecium carries multiple resistance genes on its chromosome; The Tn554 transposase carrying ANT(9) and ErmA and the pair of intercalating elements IS256 carrying AAC(6')-le-APH(2″)-la together constitute a multidrug resistance genomic island from Staphylococcus aureus. The genomic island is resistant to macrolides, lincosamides and aminoglycoside antibiotics. In conclusion, a strain of E. faecium was isolated from the visceral tissue of dead forest carp, which carries a multi-drug resistant genomic island PRI1. This genomic island is currently not reported in E. faecium. At the same time, this strain is derived from wild animals, so the resistance of wild animals has become more and more serious.

To investigate the mechanisms of CpxR regulate the colistin susceptibility of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). The cpxR-pmrB or cpxR-phoQ co-expressed genes were acquired by overlapping PCR. The pmrB and phoQ single overexpressed strains and cpxR-pmrB or cpxR-phoQ co-expressed strains were constructed in this paper. The minimal inhibitory concentrations (MICs) of colistin for the above strains were measured by the 2-fold serial broth microdilution method. Growth curves were generated by OD_{600 nm} of strains in LB medium and colistin time-kill assay were carried out with survival rate of strains after incubated with different concentrations of colistin. The results showed that the MIC of cpxR overexpressed strain JSΔΔ/pR was dramatically decreased by 93.75% compared with JS which was consistent with the previous study. The MICs for pmrB and phoQ single overexpressed strains JSΔΔ/pB and JSΔΔ/pQ were increased by 300% compared with JS. However, the MICs for cpxR-pmrB or cpxR-phoQ co-expressed strains JSΔΔ/pRB and JSΔΔ/pRQ were decreased by 50% and 75%, respectively. Furthermore, the growth curves showed that unlike the low growth rate of JSΔΔ/pR, the growth rate of JSΔΔ/pB, JSΔΔ/pQ, JSΔΔ/pRB and JSΔΔ/pRQ were slightly lower than JSΔΔ/pHisA. Colistin time-kill assay showed that JSΔΔ/pB and JSΔΔ/pQ was more susceptible than JSΔΔ/pRB and JSΔΔ/pRQ when incubated with increasing concentrations of colistin. In conclusion, the results indicated that CpxR was able to regulate the colistin susceptibility of Salmonella Typhimurium through colistin resistance-related genes pmrB and phoQ.

Toll-like receptor 3 (TLR3) is a pattern recognition receptor that stimulates innate immunity. In order to prepare a monoclonal antibody that can bind to chicken TLR3 (chTLR3) protein with natural structure, BALB/c mice were immunized with recombinant plasmid pVAX1-Igk-chTLR3, which could be expressed in cells, and monoclonal antibodies against chTLR3 were prepared by hybridoma technique. The expression of chTLR3 expression of chTLR3 in peripheral blood lymphocytes and spleen of cadmium poisoned chickens were detected by Western blot, flow cytometry and indirect immunofluorescence. The results showed that a positive hybridoma cell line was obtained. The number of chromosomes of the cell line was 94±2, the titer of the cell supernatant was 1:2⁵, and the antibody subclass was identified as IgG3. The cell supernatant not only specifically bound to the prokaryotic expression protein of chTLR3, but also bound to chTLR3 protein in chicken peripheral blood lymphocytes. Western blot, flow cytometry and indirect immunofluorescence were used to detect the expression of chTLR3 in peripheral blood lymphocyte and spleen of chickens in control group and cadmium-exposed group. The results indicated that the monoclonal antibody against chTLR3 with natural activity was successfully prepared. The antibody can be used to study the distribution, localization and expression of chTLR3 in chicken tissues and cells by Western blot, indirect immunofluorescence and flow cytometry. It provides an important material basis for tracing the distribution and expression of chTLR3 protein and its role in the pathogenesis of chicken diseases.

To investigate the effect of Polysaccharides from ZhuKuQin (ZKQPs) on the recovery function in damp-heat diarrhea Piglet intestine, 60 piglets were divided into blank control group (n=10) and damp-heat diarrhea model group (high temperature, humidity environment combined with high fat compound feed), and 50 model piglets were randomly divided into model group, positive drug group (Pulsatilla powder), ZKQPs high, middle and low dose group, with 10 piglets in each group, drugs were mixed with feed, for 7 days. Small intestinal goblet cells (GC) were stained with PAS and observed by Microscope, RT-PCR and ELISA were used to detect the transcriptional quantity and content of mucoprotein-2 (MUC-2) and intestinal trilobular factor (ITF-3) in intestinal mucus of damp-heat diarrhea piglets. The results were as follows:1)The number of GC in duodenum, jejunum and ileum of piglets in model group decreased significantly when compared with that in blank group (P<0.05); The number of GC in duodenum, jejunum and ileum of piglets in ZKQPs high, middle dose group and positive drug group increased. The number of GC in duodenum, jejunum and ileum of piglets in the ZKQPs high dose group and the positive drug group were significantly different when compared with the model group (P<0.05), and the number of GC in duodenum and ileum of piglets in ZKQPs middle dose group was significantly different (P<0.05). 2)The transcriptional quantity and content of MUC-2 in duodenum, jejunum and ileum of piglets were decreased in model group, and the difference was significant when compared with that of blank group (P<0.05).The transcriptional quantity and content of MUC-2 in duodenum, jejunum and ileum of piglets in each ZKQPs dose groups and positive drug groups increased. The transcriptional quantity and content of MUC-2 in duodenum, jejunum and ileum of piglets in ZKQPs high dose group and positive drug group were significantly different from those in model group (P<0.01). 3)The transcriptional quantity and content of ITF-3 in duodenum, jejunum and ileum of piglets were decreased in model group, and the difference was significant when compared with that of blank group (P<0.05). The transcriptional quantity and content of ITF-3 in duodenum, jejunum and ileum of piglets in ZKQPs high, middle dose group and positive drug group increased.The transcriptional quantity and content of ITF-3 in jejunum and ileum of piglets in ZKQPs high, middle dose group and positive drug group were significantly different from those in model group (P<0.01). ZKQPs can increase the number of GC and the transcription of MUC-2 and ITF-3 mRNA in small intestine of damp-heat diarrhea Piglets, and promote the secretion of MUC-2 and ITF-3 in small intestine of damp-heat diarrhea Piglets.

Senecavirus A(SVA) is a nonenveloped, single-stranded RNA virus, which can cause vesicular lesions in sows and newborn piglet death. In order to establish a rapid assay for detection of SVA, we compared the genetic sequences of 40 SVA genes published on NCBI, and the best primers was selected from three pairs of primers which designed based on the conserved regions of sequences. The RT-PCR detection method of SVA was established successfully after the optimization of primer concentration, annealing temperature, extension time and cycle number. SVA, Encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV) were detected by RT-PCR, which showed a good specificity with no cross-reaction. The sensitivity test was carried out as well, and sensitivity of the detection could reach up to 1 TCID₅₀, which shows it is more sensitive than the methods reported in foreign literatures. One hundred samples of pig tissue were detected using this method, and the positive rate was 2%. The establishment of this method, with its great specificity, high sensitivity and ideal reliability, could provide an effective technical means for SVA detection and epidemiological investigation.