The traditional breeding in the past century has made the performance of poultry close to the limit of phenotypic selection. The development of genomics has become a new way to break through this bottleneck. Since the first chicken genome draft was published in 2004, the reference genome sequences of chicken, duck, goose, and other poultry have been published in recent years, and the quality has been gradually improved. Based on the high-quality genome reference sequences of poultry, researchers have carried out a series of further studies on important traits of poultry, which have accelerated our understanding of important traits of poultry, and screened effective molecular markers for application in genome selection breeding of poultry. In this manuscript, the reference sequence of poultry genome, the genomics of important traits and its application were reviewed.

Chromatin accessibility refers to the openness of proteins such as nucleosomes or transcription factors to the recombination of other proteins after binding to eukaryotic chromatin DNA, which can reflect transcriptional activity. Open chromatin is closely related to animal growth and development, cell differentiation and other processes. The efficient and accurate localization of open chromatin in the genome can provide vital clues for exploring the regulation mechanism of gene expression. In this paper, the methods of chromatin accessibility detection and the factors of chromatin accessibility are introduced, and the effects of chromatin accessibility on animal development and its development and application prospects are discussed so as to provide a reference for related studies on gene expression regulation during animal development.

This study aimed to investigate the relationship between the expression level of porcine m⁶A methylases METTL3 and deoxynivalenol (DON) induced injury of intestinal porcine epithelial cells (IPEC-J2). In this study, IPEC-J2 cell lines with METTL3 knockdown was successfully established. IPEC-J2 cells from the METTL3 interference group and control group were treated with 1 μg·mL^-1 DON for 48 h, respectively. Real-time quantitative PCR was used to detect the mRNA expression differences of apoptosis-related genes, immune-related genes and oxidase-related genes, and cell proliferation activity, cell cycle, cell apoptosis, and reactive oxygen species (ROS) levels were measured. The results showed that, compared with control group, the expression of immune-related genes TNF-α, IL-6 and IL-12 in the interference group changed significantly (P<0.05); The levels of apoptosis-related genes Bax, Caspase3 and Caspase9 were significantly decreased(P<0.05); The levels of oxidase-related genes GPXs, CuZn-SOD and CAT were significantly increased(P<0.05). The cell proliferation activity was significantly increased, and the total cell apoptosis rate was significantly decreased (P<0.05); The level of ROS was extremely significantly decreased (P<0.01). The results indicate that the down-regulation expression of METTL3 gene contributes to the enhancement of IPEC-J2 cells resisting apoptosis and inflammation induced by DON in a certain extent. The down-regulation expression of METTL3 gene is beneficial to alleviate a series of inflammatory responses and cell damage caused by DON, and will provide a theoretical basis for in-depth study of the mechanism of RNA methylation in the regulation of piglet resistance to DON damage in the future.

This experiment was conducted to study the differences of meat quality and muscle fiber characteristics of black Tibetan lambs at different ages under grazing conditions. Five newborn black Tibetan lambs (average weight (2.31±0.49) kg) and five 12-month-old black Tibetan sheep (average weight (36.58±1.26) kg) were selected under natural grazing conditions in Qinghai. They were divided into two groups according to age, namely lamb group and adult sheep group. Via ATPase staining, ELISA and real-time qPCR, the variation of the nutrient composition, antioxidant capacity, muscle fiber type and myosin heavy chain (MyHCs) genes expression in longissimus dorsi were analyzed between lamb and adult sheep groups. The results showed as follows:1) There were no significant differences in nutrient indexes of longissimus dorsi such as moisture, crude protein, ether extract and ash between the two groups (P>0.05). Similarly there were no significant differences in catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) levels (P>0.05). 2) The diameter and cross-sectional area of type Ⅰ, ⅡA and ⅡB muscle fiber in lambs were significantly lower than those of adults (P<0.05), but the muscle fiber density was the opposites (P<0.05). The proportion of type Ⅰ muscle fiber quantity and the proportion of type Ⅰ and type ⅡA muscle fiber area of lambs were significantly higher than those of adult sheep (P<0.05). 3) The mRNA expression levels of MyHC Ⅰ and MyHC ⅡA in lambs were significantly lower than those in adults (P<0.05), but the mRNA expression level of MyHC ⅡB was the opposite (P<0.05). These results indicated that, with the increase of age, the nutrient composition and antioxidant capacity of longissimus dorsi of black Tibetan sheep had no significant changes, but the muscle fiber dramatic changed from oxidized type to glycolysis type, which influenced the body growth and development to a certain extent.

The aim of this study was to clone and analyze the Zfy gene by bioinformatics in Xinong Saanen dairy goats, and to successfully establish goat fibroblasts cell lines with Zfy gene knockout by CRISPR/Cas9 technique, and provide basic data for further exploring the function and sex regulation of Zfy gene. In this study, the samples of testicles were collected from 3 healthy Xinong Saanen male goats during 3-month-old and total RNA were extracted, respectively. Primers were designed according to the predicted mRNA sequence (GenBank accession No.:XM_018044893) of Capra hircus Zfy gene published in NCBI, and the Zfy gene was segmental amplified and cloned by RT-PCR. The nucleotide sequence and protein structure and function of the CDS region of the Zfy gene were analyzed by related bioinformatics softwares. Four pairs of sgRNAs were designed according to the CDS region of the Zfy gene to construct 4 targeting vectors later transfected into goat fetal fibroblasts (GFFs). The knockout efficiency of the targeting vectors was confirmed by T7E1 digestion method. Then the Zfy gene knockout positive monoclonal GFFs cells were isolated through puromycin screening, and subsequently, PCR amplification and sequencing were used to detect the mutation rate. The results showed that the sequence of the CDS region of the Zfy gene of Xinong Saanen dairy goats with a length of 2 406 bp was successfully cloned. The homology and phylogenetic tree analysis showed that the Zfy gene sequence of goat had high homology with Cervus elaphus, Bos taurus and Ovis aries, and their genetic distance was closer, the homology with Mus musculus was the lowest and the genetic distance was the farthest. Bioinformatics softwares analysis showed that the goat Zfy gene encoded 801 amino acids, and the molecular mass was 90.37 ku. The Zfy protein contained 66 phosphorylation sites, had an isoelectric point of 5.66, was highly hydrophilic, had no signal peptide, and was a structurally unstable non-secretory protein. T7E1 digestion revealed that all 4 targeting vectors could knock out the gene effectively and the knockout efficiency was 12.31%, 40.86%, 31.52%, and 26.37%, respectively. The targeting plasmid with the highest knockout efficiency was selected to transfect into GFFs cells, and a GFFs-positive cell line that could stably target the knockout of Zfy gene was sorted out by puromycin screening, and the mutation rate was 23.91% by PCR sequencing. In conclusion, this study successfully cloned the Zfy gene of Xinong Saanen dairy goats and revealed the physicochemical properties of the protein of Zfy gene, successfully constructed a CRISPR/Cas9 knockout vector for the Zfy gene of goats and screened the best knockout site, and also obtained subcloned cells for Zfy gene knockout, which provided the foundation for in-depth research on the function of Zfy gene of goats and the development of sex control technology.

The aim of this study was to construct the expression profile of adipohenin (ADIG) in different tissues of cattle and to identify its core promoter region and key transcription factors in order to elucidate the molecular mechanism of its transcriptional regulation. Heart, liver, spleen, lung, kidney, muscle and subcutaneous adipose tissues were collected from 3 ((24±2) months old) healthy Nanyang bulls, and the relative expression levels of ADIG gene in each tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). The 2 245 bp sequence of the upstream promoter region of ADIG gene was cloned, 6 pGL3-WT were constructed and cotransfected with pRL-TK into 293T cells, primary adipocytes and 3T3-L1 cells, and further bioinformatics was used to predict the key transcription factors of their core promoter region. The results showed that ADIG gene was highly expressed in lung (P<0.05), kidney (P<0.05) and subcutaneous fat (P<0.05) tissues of Nanyang cattle; the promoter sequence of ADIG gene with 2 245 bp and its 6 missing fragment sequences were cloned. pADIG-1 621/+59, pADIG-1 288/+59, pADIG-850/+59, pADIG-589/+59, pADIG-321/+59 and pADIG-79/+59 dual luciferase reporter vectors were successfully constructed, and the core region of bovine ADIG gene promoter was detected at -321/-79. Bioinformatics preliminarily predicted that the core region of bovine ADIG gene contained C/EBPα, PPARδ, CREB, STAT5 and AP-2α and other transcription factor binding sites. In summary, the bovine ADIG gene is most highly expressed in subcutaneous adipose tissue with a promoter core region located at -321/-79. Transcription factors such as C/EBPα, PPARδ, CREB, STAT5 and AP-2α have regulatory roles on the transcriptional activity of the ADIG gene. The results of this study provide a theoretical basis for exploring the improved breeding of ADIG gene at the molecular level of beef cattle.

The study aimed to investigate the effects of extreme environment on the genetic diversity, genetic structure and genetic differentiation of Lepus tibetanus in the eastern Pamir Plateau by molecular genetics method, and to supply research materials for the conservation of species diversity and genetic diversity in the Pamir Plateau. In this study, the sequence of the mitochondrial CO1 and ND4 genes of L. tibetanus from the eastern Pamir Plateau were detected, and the related bioinformatics softwares for data analysis were used. The results showed that 17 haplotypes were detected in the mitochondrial gene concatenate sequences of 37 L. tibetanus from 4 sampling areas in the eastern Pamir Plateau. The total nucleotide diversity was (0.012±0.006), and the haplotype diversity was (0.935±0.021), which were lower than other Lepu, such as Lepus yarkandensis and Lepus europaeus. Moreover, the genetic diversity of L. tibetanus at relatively high altitudes was lower than that of L. tibetanus at relatively low altitudes. All L. tibetanus were divided into 3 Clade A-C by ML tree and median-joining network. Samples in Clade A had the mixed haplotypes from all the sampling area, samples in Clade B had only L. tibetanus from Khunjerab Pass, and samples in Clade C clustered together with L. yarkandensis in the world Lepus phylogenetic tree. In terms of genetic differentiation, the L. tibetanus population in the Khunjerab Pass area with the highest altitude had the highest degree of differentiation from other populations, and the L. tibetanus population in Wuqia County and Akto County had the lowest degree of differentiation and the largest gene flow. Based on previous studies, it is suggested that the genetic diversity of L. tibetanus from the eastern Pamir Plateau is relatively low, and there is no strong systematic geographical distribution pattern. The L. tibetanus at Khunjerab Pass differed from other L. tibetanus at the mitochondrial gene level under the influence of the extreme environment at the plateau and may have formed different ecotypes. In addition, the introgression of mitochondrial DNA in L. tibetanus caused by hybridization with L. yarkandensis was found.

The study aimed to explore the molecular regulation mechanism of donkey meat tenderness based on the joint analysis of transcriptome and metabolome. In this study, 30 female Guangling donkeys with the same growth environment and feeding conditions, aged 33 to 36 months, were used as the research object. Shear force and intramuscular fat content were measured, and 8 donkeys were selected based on the shear force and intramuscular fat content. The donkeys were divided into high tenderness group (HT, n=4) and low tenderness group (LT, n=4). Differentially expressed genes and metabolites were screened by transcriptome and metabolome analysis, and then combined with KEGG pathway enrichment analysis to construct the relevant network interaction map. The results of transcriptome indicated that a total of 1 253 differetially expressed genes were found in HT group and LT group, of which 832 genes were up-regulated and 421 genes were down-regulated. The results of KEGG analysis indicated that the differetially expressed genes were mainly enriched in carbohydrate metabolism, lipid metabolism, endocrine system, signal transduction and cellular processes. The metabolome showed that 225 differential metabolites were identified in HT and LT group, of which 154 were up-regulated and 71 were down-regulated. KEGG pathway analysis showed that differential metabolites were mainly enriched in carbohydrate metabolism, lipid metabolism, amino acid metabolism, nucleotide metabolism and signal transduction. Joint analysis showed that differentially expressed genes and differential metabolites were significantly enriched in glycero-phospholipid metabolism, pentose phosphate pathway, alanine, aspartate and glutamate metabolism, arginine and proline metabolism and glucagon signaling pathway. Significant differentially expressed genes and metabolites played a key regulatory role in the above metabolic pathways, and can be used as potential candidate genes and metabolites for donkey meat tenderness, which lays a theoretical foundation for exploring the molecular regulation mechanism of meat tenderness and molecular breeding of donkey in the future.

The aim of this study was to establish an in vitro study model of subcutaneous and intramuscular preadipocytes in yak, and to detect the expression differences of key genes in the differentiation process of preadipocytes in the two parts, so as to provide experimental materials and theoretical basis for the study of the molecular mechanism of fat deposition in different parts of yaks. The subcutaneous adipose tissue and longissimus dorsi muscle tissue of 5 healthy male Maiawa yaks aged 18 to 22 months were collected, and the subcutaneous and intramuscular preadipocytes were separated by collagenase digestion. The cells were then divided into intramuscular group and subcutaneous group according to the cell sources. Immunofluorescence identification, growth curve mapping, Oil red O identification of adipocytes and real-time fluorescence quantitative technology were used to detect the expression of key differentiation genes PPARγ, C/EBPα, FASN and HSL. The results showed that yak subcutaneous and intramuscular adipocytes on the first day of cultivation were mostly circular, with the increase of incubation time, formed a spindle, the normal "S" growth curve model for CCK-8 detection, and growth rate of subcutaneous preadipocytes on day 4 was significantly higher than that of intramuscular preadipocytes. After identification by immunofluorescence, the expression of PREF-1 was positive, and they were identified as preadipocytes. After differentiation, mature adipocytes with large and round fat droplets were formed, and red fat droplets were detected by Oil red O staining, and the key genes were expressed. The expression levels of PPARγ, FASN and HSL during subcutaneous and intramuscular preadipocyte differentiation were significantly increased with the increase of differentiation time (P<0.05), the expression of C/EBPα in intramuscular and subcutaneous preadipocytes increased gradually at the prophase of differentiation (P<0.05), and the expression level was decreased at the later stage of differentiation. The expression of FASN and HSL in intramuscular preadipocytes showed significant increase on day 3, while in subcutaneous preadipocytes showed significant increase on day 6. In conclusion, subcutaneous and intramuscular preadipocytes of yak were successfully isolated and differentiated into mature adipocytes. Preliminary detection showed that the expression levels of key adipogenic differentiation genes PPARγ, HSL and FASN all showed an increasing trend during the differentiation period for 9 days, while the expression levels of C/EBPα decreased significantly after 6 days of differentiation. This study provided a reference for further studying the molecular rules of fat deposition in subcutaneous and intramuscular adipocytes in yaks.

This study was conducted to analyze the differences of nuclear morphology (including sperm size and shape) between Holstein bull X and Y sperm, clarifying the effects of freezing and flow sorting on the nuclear morphology of bull X and Y sperm. Six types of sperm nuclei images from 15 healthy Holstein bulls with the same age were captured by fluorescence microscopy for fresh and frozen non-sexed sperm, and fresh and frozen X and Y sperm. The Image J plug-in Nuclear Morphology Analysis was used to analyze the 8 indices information related to the morphology of 37 000 sperm nuclei images. The results showed that the proportion of bulls with significant difference in sperm nucleus morphology between X and Y (P<0.05) was less than 50%. After flow sorting, the sperm nucleus area, perimeter and length of 8 (8/11) bulls, the width of 6 (6/11) bulls changed significantly|The ellipticity, elongation and regularity of sperm nuclei in 5 (5/11) bulls and the circularity of sperm nuclei in 3 (3/11) bulls changed significantly. After freezing, there were significant differences in sperm nuclear area and perimeter among 11 (11/14) bulls, the length of sperm nuclei among 10 (10/14) bulls, width of sperm nuclei among 9 (9/14) bulls changed significantly|And there were significant differences in sperm nucleus circularity among 5 (5/14) bulls, regularity, elongation and ellipticity of sperm nuclei among 6 (6/14) bulls changed significantly. Only 4 bulls had significant differences in the size of X and Y sperm nuclei before freezing, but added to 8 bulls after freezing. Based on the analysis of morphological information from a large number of sperm nuclear images, it could be clarified that flow sorting and freezing had the significant impact on the nuclear size of Holstein bull sperm, and the impact of freezing on the nuclear size of Holstein bull Y sperm was higher than that of X sperm. The research will provide a reference for the improvement of sex-controlled sperm sorting and sperm freezing, and are of great significance to the improvement of bull production performance and cow economic benefit.

The purpose of this study was to explore the molecular characteristics of NGF gene and its expression properties in reproductive organs of female yak (Bos grunniens). The heart, liver, spleen, lung and kidney of female yak at luteal phase were collected. In addition, the ovaries, uterus and oviduct of yaks during fetal, follicular, luteal phases and gestation were collected (n=3). NGF gene was cloned by RT-PCR and its sequence was analyzed by bioinformatics method. The mRNA expression was evaluated by RT-qPCR and NGF protein location in the reproductive organs was detected by immunohistochemistry. The results showed that the CDS region of yak NGF gene was 726 bp, which encoded 241 amino acids. NGF protein belongs to alkaline unstable hydrophilic protein. The results of NGF amino acid phylogenetic tree showed that yaks were first grouped with yellow cattle, and the homology with other species was more than 87%, indicating that NGF gene was relatively conserved in the course of animal evolution. The results of RT-qPCR showed that the expression of NGF gene in ovary was extremely significantly higher than that in heart, liver, kidney, spleen, lung, uterus and oviduct (P<0.01). The relative expression of NGF in ovary of luteal phase was the highest, which was significantly higher than that in fetal, follicular stage and pregnancy (P<0.01). In uterus, the relative expression of NGF was significantly higher during pregnancy than that in fetal and follicular stages (P<0.05). There was no significant difference in the expression of NGF in different physiological stages of oviduct. The immunohistochemistry analysis indicated that NGF protein was mainly localized in granulosa cells and ovarian epithelium cells, with the highest expression in granulosa cells. It was also expressed in yak endometrium and epithelial cells of yak oviduct. In conclusion, the sequence of NGF gene in yak is relatively conserved in the course of evolution, and is highly expressed in the ovary. NGF gene may play an important regulatory role in maintaining ovary function, pregnancy and promoting corpus luteam function in female yaks.

The purpose of this study was to analyze the content of glycerolphospholipids in fat globule membrane in dairy cows with low-fat depression induced by conjugated linoleic acid (CLA). Ten Holstein cows with similar body conditions and at mid-lactation were selected for their own before and after control design. The test was carried out for 12 days, cows were fed with feeding basal diet +400 g CLA every day from day 1 to day 6, and then changed to the basal diet from day 7 to day 12. Cow performance, milk composition and milk fat globules(MFG) size parameters were measured daily. The milk samples were analyzed for glycerophospholipid on the first、7th and 12th day by mass spectrometry. The results showed that CLA treatment had no significant effect on the milk production, feed intake, milk protein and lactose content of dairy cows (P>0.05), but significantly reduced the milk fat rate of dairy cows (P<0.05). The size of fat globules average surface area diameter (D_[3,2])、10% particle volume diameter (Dv(10)) and 50% particle volume diameter (Dv(50)) all showed a trend of first decreased and then increased (P<0.05), while the specific surface area (SSA) first increased and then decreased (P<0.05). With the extension of feeding time, the small MFG distribution gradually increased and then decreased, while the large MFG first decreased and then gradually increased (P<0.05). Analysis of glycerophospholipids found that the concentration of phosphatidylcholine (PC 28:0, PC 30:0, PC 32:0, PC 34:1, PC 36:4 and PC 38:5) and phosphatidylethanolamine (PE 28:0, PE 30:0, PE 31:1e, PE 32:0, PE 32:2e, PE 33:2e, PE 34:0, PE 34:1, PE 34:2, PE 34:2e, PE 34:4e, PE 34:5e, PE 35:1, PE 35:3e, PE 35:5e, PE 36:2e, PE 36:4, PE 36:5e, PE 38:3, PE 38:4) showed a trend of first decreasing and then increasing. And content of phosphatidylinositol (PI) increased. These results indicate that CLA affect the size of fat globules and content and composition of phospholipids in milk fat globule membrane of dairy cows.

The objective of this study was to investigate the effects of rumen-protected nicotinic acid (RPN) and rumen-protected choline (RPC) on lactation performance and hepatic lipid metabolism of perinatal dairy cows, and to provide theoretical basis for alleviating fatty liver of dairy cows. Twenty-four healthy Chinese Holstein perinatal dairy cows with similar parity were randomly divided into 4 groups in a 2×2 factor experimental design with 6 cows in each group, including control group (CON, basal diet), RPN group (RPN, basal diet +18.4 g·d^-1 RPN), RPC group (RPC, basal diet +60 g·d^-1 RPC) and both RPN and RPC group (RPN×RPC, basic diet +18.4 g·d^-1 RPN +60 g·d^-1 RPC), respectively. The experiment was conducted from 14 d prenatal to 21 d postpartum. The weekly weight and milk production of the dairy cows after calving, and the birth weight of the calves were recorded. Blood was collected every week, milk was sampled at 14 and 21 d after delivery, liver biopsies were taken on 21 d relative to parturition. The results showed that the supplementation of RPN or RPC had no significant impact on the performance of dairy cows and calves, including dairy cows body weight, calves body weight, milk production and milk composition (P>0.05). Supplementation of RPN and RPC significantly increased the contents of nicotinic acid (NA), choline (CH) and the level of very low density lipoprotein (VLDL) in plasma (P<0.001). RPN group and RPN×RPC group significantly reduced triglycerides (TG) content in liver (P<0.05). In summary, supplementing RPN and RPC to the basic diet of perinatal dairy cows has no significant effect on production performance, whereas they can improve the liver and body health of perinatal dairy cows by increasing the content of VLDL in plasma and reduce lipid deposition in the liver.

To develop a blocking ELISA for the detection of antibodies against African swine fever virus (ASFV), ASFV P54 protein expressed in E. coli was used as the coating antigen, and monoclonal antibody against P54 protein was prepared. The optimum reaction conditions of blocking ELISA were determined by square matrix titration, and the sensitivity, specificity, repeatability and conformity were evaluated. The results showed that the optimum conditions of the assay were as follows:the antigen coating concentration was 2.0 μg·mL^-1, antigen coating temperature and time were 4℃ overnight, the dilution of tested serum was 1:40, the dilution of enzyme-labelling mAb was 1:1 000, the action time of tested serum was 1 h, the action time of enzyme-labelling mAb was 30 min, and the action time of substrate was 10 min. Through the detection of negative and positive samples, as well as specificity and sensitivity tests, it was determined that the criteria for determining the negativity and positivity of the assay were:Results determined to be positive at cut-off blocking rate ≥ 55.2%, negative at blocking rate <43.4%, and suspicious between the two values. The assay had no cross reaction with positive sera of classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, foot-and-mouth disease virus, swine influenza virus and porcine pseudorabies virus. The coefficient of variation (CV) of intra- and inter-batch repeated tests were both within 10%. The coincidence rate between the assay and commercial ELISA kit was 98%. In conclusion, the blocking ELISA has ideal specificity, sensitivity and repeatability, and can be used for the antibody detection of ASFV clinical samples.

In order to investigate the changes of expression levels of duck interferon-induced transmembrane proteins (duIFITMs) and related cytokines in the early stage of duck hepatitis A virus genotype 3 (DHAV-3) infection, and to evaluate the inhibitory effect of duIFITMs against proliferation of DHAV-3, the high-throughput sequencing and real-time PCR were used to evaluate the expression levels of duIFITMs and their related cytokines in the liver of ducklings at the early stage by the infection of DHAV-3. The cell models of DELC with duIFITM1 over-expression or knockdown were constructed by transfection of pEGFP-duIFITM1 and duIFITM1-siRNA, respectively. Then, the effect of duIFITM1 on DHAV-3 proliferation was evaluated. The results showed that duIFITM1 was significantly up-regulated at 24 and 36 h after DHAV-3 infection in ducklings (P<0.01), but duIFITM5 had no change in the early stage of DHAV-3 infection. Then dulIFITM1 over-expressing and knockdown DELC were infected with DHAV-3. Compared with control group and siRNA interference group, the number of DHAV-3 copies and virus titers decreased in the duIFITM1 overexpression group significantly at both 48 and 60 h (P<0.01). Furthermore, a total of 211 molecules and 74 enriched signaling pathways, which related to immune response against DHAV-3 were founded by transcriptome analyses. The results of real-time PCR detection confirmed that the levels of mRNA of RIG-I and MDA5 had no changes in livers at 12 and 24 h after inoculated by DHAV-3, but it increased to 4.23 times (P<0.01) and 3.61 times (P<0.05) in 36 h compared with the control group, these expression changes of RLRs coincided with that of IFN-α/IFN-β in early stage of DHAV-3 infection. Besides, the expression levels of IRF1 and IRF3 were upregulated significantly in early stage. It was the first time to be demonstrated that the duIFITM1 have an inhibitory effect against DHAV-3 proliferation, which could provide theoretical foundations for the development of new anti-DHAV drugs. Systematic study and analysis of the expression levels of a variety of key immune molecules in the early stage of anti-viral infection might be an important reference for the further exploration of the molecular mechanisms of DHAV-3 infection and immunity.

Pseudorabies virus (PRV) is the pathogen of pig pseudorabies. At present, there are more mature commercial vaccines in the clinical application, but the virus variation is frequent, so the occurrence of pseudorabies is still more common in pig breeding. How to clean the wild virus in the host is the key to the prevention and control of the disease. The adeno-associated virus (AAV) carrying CRISPR/Cas9 system toward TK, gE and VP16 genes of PRV were designed to weaken the virulence and replication ability of PRV, so as to achieve the purpose of eliminating PRV in mice model. The results showed that the pX601 recombinant plasmids (pXTK, pXGE, pXVP16) with CRISPR/Cas9 expression system and corresponding sgRNAs were successfully constructed, and these plasmids were further packaged into AAV-gE, AAV-VP16 and AAV-TK viruses with gene editing function, which significantly reduced the PRV load in cells and mice and increased the survival rate in the cell level and mouse infection test. The treatment result is positively correlated with the times of treatments. The results of the mouse model provide a new reference for the clinical treatment of porcine pseudorabies.

Based on the N-terminal gene of LppA protein of Mycoplasma mycoides subsp. capri (Mmc) Guizhou strain, the eukaryotic recombinant expression plasmid pVAX1-LppA was constructed, and the immune effect was analyzed to provide new ideas for the prevention and control of mycoplasmal pneumonia of sheep and goats (MPSG). Mice were immunized with the constructed eukaryotic recombinant expression plasmid pVAX1-LppA (50, 100, 150 μg), pVAX1 empty vector, and PBS, respectively. The levels of antibodies and cytokines (IL-2, IFN-γ and IL-4) in sera of mice were detected by ELISA. The proliferation of splenic lymphocytes was detected by MTT assay and the ratio of CD4⁺, CD8⁺ T lymphocytes to total cells was detected by flow cytometry. The protective efficiency of mice was evaluated by the challenge test. The lungs of mice were collected for preparation of sections to observe the pathological changes. The results showed that compared with the empty vector pVAX1 and PBS control groups, the levels of antibodies and cytokines (IL-2, IFN-γ, IL-4) in mice in the recombinant plasmid pVAX1-LppA group were significantly increased, the proliferation ability of spleen cells was stronger, and the proportion of CD4⁺ and CD8⁺ T lymphocytes in total cells increased significantly. The recombinant plasmid pVAX1-LppA had a certain ability to protect mice from Mmc challenge. The lung pathological damage was significantly reduced, and the number of cases was decreased post immunization. The maximum protection rate of 100 μg recombinant plasmid pVAX1-LppA was 80%. The results showed that LppA protein had good immunogenicity, and the recombinant plasmid pVAX1-LppA could activate strong humoral and cellular immune responses, which could be used as a candidate vaccine for MPSG.

This study aimed to isolate a phage that can lyse bla_NDM-5and mcr-1 positive Escherichia coli, explore the biological characteristics of the phage, and provide a certain theoretical basis for phage therapy. The isolated phage was observed by transmission electron microscope, the optimal multiplicity of infection, one-step growth curve and other biological characteristics experiments and whole genome sequencing analysis. A strain of E. coli phage vB_EcoM-ZQ1 was successfully isolated. The phage belonged to the order Ceratoviridae and the Mycoviridae family. The optimal multiplicity of infection was 0.1, the incubation period was 20 min, the lysis period was 10 min, and the amount of lysis was 92 PFU·cell^-1. Under the conditions of temperature 4-55℃ and pH4-10, the phage had high activity. It had a certain lysis effect on multi-drug resistant E. coli, with a lysis rate of 25.5%. The phage gene had a total length of 149 112 bp and a GC content of 49.1%. Among 184 ORFs, 70 ORFs encoded functional proteins, and 2 ORFs encoded tRNAs, and no drug resistance genes and virulence factors were found; The BLAST comparison found that the phage had 92.37% similarity with Shigella phage phiSboM-AG3. The results of a homologous phylogenetic tree constructed based on the large subunit of the phage terminal enzyme showed that the phage vB_EcoM-ZQ1 was closely related to the Salmonella phage pSal-SNUABM-02. The results indicated that the bacteriophage vB_EcoM-ZQ1 had stable biological characteristics and complete gene structure, which had laid a certain preliminary foundation for the follow-up prevention and control of bla_NDM-5 and mcr-1 positive E. coli.

Listeria monocytogenes(LM)is an important food-borne zoonotic pathogenic microorganism, which can survival under various stress conditions, such as acidic, alkalic, osmotic and oxidative stresses.Two-component systems play important roles in sensing and adaptating to the environments. In this study, we try to explore the roles of the two-component system HssS/HssR in stresses resistance and pathogenicity of Listeria monocytogenes. A hssS/hssR deleted strain was constructed based on Listeria monocytogenes 10403S, and its biological characteristics were investigated. Growth curves showed that deletion of hssS/hssR weakened the growth ability under oxidative stress (20 mmol·L^-1 H₂O₂) condition. Survival assays showed that the growth rate of deletion strain ΔhssS/hssR was significantly lower than that of the parent strain treated with 20 mmol·L^-1 H₂O₂for one hour (P<0.01). Phagocytosis and proliferation assays in macrophage RAW264.7 cells showed that deletion of hssS/hssR significantly enhanced the ability of anti-phagocytosis (P<0.01) and proliferation in macrophages (P<0.05). Virulence assays conducted on chicken embryo showed that the LD₅₀ of the parent strain and deleted strain were 10^2.2and 10^1.4, respectively. And the bacteria load in the liver of chicken embryo infected by the deletion strain was significantly higher than that of parent strain (P<0.01), indicating that deletion of hssS/hssR significantly enhanced the virulence of Listeria monocytogenes. These results indicated that the two-component system HssS/HssR mediates the oxidative stress resistance and virulence of Listeria monocytogenes.

To prepare monoclonal antibodies (McAbs) against the gametocyte antigen EnGAM22 of Eimeria necatrix, BALB/c mice were immunized with recombinant gametocyte antigen rEnGAM22 purified by Ni-NTA affinity chromatography. After three times of immunization, the spleen cells of the immunized mice were fused with myeloma cells SP2/0. Hybridoma culture supernatant were examined by the pre established ELISA assay. Two hybridomas stably secreting specific antibody were obtained after four times of subcloning by limiting dilution method, namely 2F3 and 3D3 respectively. The subclasses and specificities of McAbs were identified and the recognition of McAbs to natural proteins and the localization of McAbs in E. necatrix were studied. The results showed that the subclasses of McAbs 2F3 and 3D3 were identified as IgG2a and IgG2b, respectively. The titer of purified ascites were 1:256 000 (2F3) and 1:64 000 (3D3), respectively. The McAbs 2F3 and 3D3 could recognize rEnGAM22 and the native gametocyte protein of E. necatrix. The indirect immunofluorescence assay with McAbs 2F3 and 3D3 showed that EnGAM22 were located in wall-formating bodies of macrogametocytes and the walls of oocyst, which indicated that EnGAM22 protein was involved in the formation of oocyst wall. The McAbs 2F3 and 3D3 might be used for studying the molecular mechanism of oocyst wall formation in coccidia.

This experiment was conducted to observe the immunoprotective effects of the prokaryotic expression products of surface antigens SAG13 and SAG14 of Eimeria stiedae in rabbits. We analyzed the transcription level of Es-SAGs in unsporulated oocyst stage, sporulated oocyst stage, schizogony stage and gametogony stage by relative fluorescence quantitative PCR, and expressed Es-SAG specifically expressed in the schizogony stage. Then forty 45-day-old coccidia-free rabbits were randomly divided into four groups:blank control group (B), parasite challenge control group (P), rEs-SAG13 immunization group (SAG13) and rEs-SAG14 immunization group (SAG14). Rabbits of group B, group P, group SAG13 and group SAG14 were subcutaneously injected with 1 mL PBS, 1 mL PBS, 1 mL 100 μg·mL^-1 rEs-SAG13 and 1 mL 100 μg·mL^-1 rEs-SAG14, respectively, then the same amount of booster immunity was given after 14 days. Two weeks after the second immunization, 1×10⁴ sporulated oocysts were infected orally in all groups except group B. Three weeks after infection, the differences of clinical symptoms, oocyst excretion, relative weight gain, feed conversion ratio, liver index and liver lesions were observed in each group. Results revealed that the transcription level of Es-SAGs at each stage is significantly different, and SAG13 and SAG14 are members that are specifically expressed in schizogony stage. The rEs-SAG13 and rEs-SAG14 have good reactogenicity. The typical symptoms of rabbit hepatic coccidiosis appeared in the control group, while the symptoms of the other groups were not obvious. The oocyst reduction rates of rEs-SAG13 immunization group and rEs-SAG14 immunization group were 82.8% and 51.9%, respectively. The average relative weight gain of immune group was significantly higher than that of parasite challenge control group. There were significant differences in liver index and liver lesion score between rEs-SAG13 immunization group and parasite challenge control group. However, there was no significant difference in liver index and liver lesion score between rEs-SAG14 immunization group and parasite challenge control group (P<0.05). These results indicated that rEs-SAG13 and rEs-SAG14 can reduce weight gain loss and oocyst output in varying degrees, alleviate liver lesions, but rEs-SAG13 is more effective.

BaeSR is an important two component system in Salmonella Typhimurium, which affects bacterial resistance by regulating the transcription and expression of downstream target genes.The aim of this study was to explore the regulatory mechanism of BaeR overexpression in S. Typhimurium to fluoroquinolones (FQs), and to find the potential target gene of BaeR. In this study, we first expressed, purified and validated the BaeR protein, and screened the drug-related differentially expressed genes (include ompW, sodB, tolC, mdtD, spy and marR) from transcriptomics as potential target genes of BaeR. Combined with LacZ reporter gene fusion assay and electrophoretic mobility shift assay (EMSA), we studied the resistance mechanism of BaeSR to S. Typhimurium. The target band appeared at 28 ku after purification, which indicated that BaeR protein was successfully expressed. LacZ reporter gene fusion assay results show that, BaeR can positively regulate the expression of ompW, sodB, tolC, mdtD, spy and marR after overexpression. EMSA results indicate that BaeR protein can directly bind to the promoter region of marR and regulate the expression of marR gene. In conclusion, BaeSR can positively regulate the transcriptional expression of target genes ompW, sodB, tolC, mdtD, spy and marR in S. Typhimurium, and directly regulate the expression of marR, thus affecting the resistance of S. Typhimurium to FQs.

This study aims to understand the role of TLR4 signal transduction in the pathogenesis of acute lung injury caused by Pseudomonas aeruginosa infection. C3H/HeJ mice lacking of normal TLR4 signaling due to mutations in Tlr4 gene was used to evaluated the role of TLR4 signaling in the regulation of acute lung injury caused by Pseudomonas aeruginosa. A total of twenty-one TLR4-deficient (C3H/HeJ) male mice and twenty-one SPF wild-type (C3H/HeN) male mice, aged 6-8 weeks, were randomly divided into 4 groups:C3H/HeN control group, C3H/HeJ control group, C3H/HeN infection group, and C3H/HeJ infection group. Mice in the two infection groups were nasally infected with a lethal dose of Pseudomonas aeruginosa. After infection, the survival rate, clinical symptoms, pulmonary pathological changes, lung bacterial load, and the level of cytokines in lungs were observed. The results showed that:1) Compared with the wild-type C3H/HeN infection group, the clinical symptoms of mice in the TLR4-deficient C3H/HeJ infection group were significantly aggravated. The median survival time of mice in the TLR4-deficient C3H/HeJ infection group was shorter. The body temperature of mice in the C3H/HeJ infection group was extremely significantly lower than that in the C3H/HeN infection group after 8 h of infection (P<0.01), and the pulmonary pathological changes and the degree of pulmonary edema were more serious than that of the C3H/HeN infection group (P<0.05); 2) The level of TNF-α and IL-1β in lung of mice in the TLR4-deficient C3H/HeJ infection group extremely significantly lower than that in the wild-type C3H/HeN infection group after 8 h of infection (P<0.01); 3) Mice pulmonary bacterial load in TLR4-deficient C3H/HeJ infection group was significantly higher than that in the wild-type C3H/HeN infection group (P<0.05). In summary, the lack of TLR4 protein significantly increased the pathogenicity of Pseudomonas aeruginosa to mice.

This study was carried out to investigate the effect of polysaccharides from ZhuKuQin (ZKQPs) on the intestinal flora and immune function of piglets with dampness-heat diarrhea. We analyzed the monosaccharide composition of ZKQPs by HPLC, and studied the direct effects of ZKQPs on common intestinal pathogens in vitro antibacterial experiments. At the same time, we establish a piglet diarrhea model, and treated with Pulsatilla powder and three doses of ZKQPs (25, 50 and 75 mg·kg^-1). After the treatment, the small intestine tissues and the contents of the cecum were collected. The cecal colony was identified and counted; the changes of intestinal flora were analyzed by 16S rDNA high-throughput sequencing, and the mRNA expression of small intestinal immune-related cytokines was determined. The results showed that the primary structure of ZKQPs is mainly composed of rhamnose, glucose, galactose and xylose. In vitro tests, E. coli, Salmonella, and Clostridium wischii were sensitive to ZKQPs and showed a concentration dependence within a certain range. In animal experiments, ZKQPs effectively promoted the growth of intestinal Lactobacilli in piglets with diarrhea, and had a strong inhibitory effect on the growth of pathogenic E. coli. Among them, 50 mg·kg^-1ZKQPs had the most significant effect. The results of 16S rDNA high-throughput sequencing showed that ZKQPs regulate the Alpha diversity of diarrhea piglets, and had a significant impact on the intestinal flora at the class and genera level. The relative abundance of Bacillus and Lactobacillus decreased significantly and the relative abundance of bacteria increased significantly in diarrhea piglets; after ZKQPs treatment, the relative abundances of Bacillus and Lactobacillus increased significantly and were higher than normal, and the relative abundance of Clostridia significantly decreased, which significantly improved the intestinal flora structure of diarrhea piglets. In piglets with diarrhea, the level of Th1 cytokine IL-2 mRNA in each segment of the small intestine increased (P<0.01), while the Th2 cytokine IL-4 and IL-5 mRNA levels decreased; after treatment with ZKQPs, the levels of IL-2 mRNA in the small intestine decreased, the levels of IL-4 and IL-5 mRNA generally increased, and the mRNA levels of different cytokines vary in different locations in the small intestine. ZKQPs 50 and 75 mg·kg^-1 have the most significant effects on them, comprehensive analysis ZKQPs 50 mg·kg^-1 treatment of diarrhea piglets has the greatest impact on the level of cytokine mRNA. This study shows that ZKQPs have a good inhibitory effect on the growth of intestinal pathogens in vitro, can change the abundance and diversity of the intestinal microbiota of diarrhea piglets, increase the relative abundance of Lactobacillus and improve the structure of the flora. At the same time, ZKQPs regulate the intestinal immune response and effectively enhance the anti-diarrhea ability of piglets.

The study aimed to investigate the effect of Sarcopyramis napalensis Wall extract (SNE) on hepatic lipidosis induced by methionine-choline-deficient (MCD) diet and its underlying mechanisms. Sixty male mice were divided into 6 groups, namely MCS (control diet of MCD diet) group, MCS+400 mg·(kg·bw)^-1 SNE group, MCD group, MCD+100 mg·(kg·bw)^-1 SNE group, MCD + 200 mg·(kg·bw)^-1 SNE group, MCD + 400 mg·(kg·bw)^-1 SNE group. Mice were fed with MCD diet and treated with SNE simultaneously for 6 weeks. The levels of hepatic lipidosis, liver fibrosis and the abilities of anti-oxidation were determined by HE and sirius red staining of liver section, and by the analysis of serum alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA). The activation of adenine monophosphate activated protein kinase (AMPK) was determined by Western blot. The mRNA expression levels of sterol regulatory element binding protein-1C (SREBP-1c), liver-type fatty acid-binding protein (L-FABP), fatty acid transport protein-3 (FATP-3), fatty acid transport protein-4 (FATP-4), intercellular adhesion molecule-l (ICAM-1), vascular cell adhesion molecule-l (VCAM-1), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2) were determined by real-time fluorescence quantitative PCR. Hepatic lipid metabolomics were conducted by LC-MS/MS. The results showed that 200 and 400 mg·(kg·bw)^-1 of SNE could significantly improve hepatic steatosis, reduce liver inflammation and liver fibrosis in MCD diet-fed mice. 100, 200, 400 mg·(kg·bw)^-1 of SNE could significantly reduce the activities of serum ALT and AKP and the levels of hepatic MDA, and significantly increase the activities of hepatic GSH-Px in MCD diet-fed mice. The results of Western blot and real-time fluorescence quantitative PCR showed that SNE significantly activated AMPK in liver tissue of MCD diet-fed mice, and significantly inhibited the mRNA expression levels of SREBP-1c, L-FABP, FATP-3, FATP-4, ICAM-1, VCAM-1, MMP-2, and TIMP-2. The results of hepatic lipid metabolomics showed that SNE significantly decreased the contents of triglyceride (TG), phosphatidylglycerol (PG) and ceramide (Cer), and increased the contents of sphingomyelin (SM) and coenzyme Q9 in MCD diet-fed mice. These results indicate that SNE can reduce the synthesis of hepatic TG by modulating AMPK/SREBP-1c pathway, and can also reduce the intake of fatty acids in the liver by inhibiting the expression of FATP and L-FABP mRNA, thus improve hepatic steatosis of MCD-diet fed mice. Moreover, SNE can attenuate the oxidative stress through increasing the content of SM and coenzymes Q9 and decreasing the content of Cer in liver tissue of MCD-diet fed mice, thus improves liver inflammation and fibrosis. Taken together, SNE therefore exerts the protective effect on hepatic lipidosis.

This study aimed to assessment the detoxification of flumazenil combined with epinephrine on Gelsemium elegans Benth (G. elegans) poisoning rats, providing reference for G. elegans toxic mechanism and poisoning cases. G. elegans poisoning rat model was constructed, and 60 rats were randomly divided into 4 groups:control group, flumazenil rescue group, epinephrine rescue group and combined drug (flumazenil + epinephrine) rescue group. The poisoning and death of the groups' rats were recorded; the blood pressure and heart rate were monitored. Then evaluate the rescue effect of drugs in each group. Molecular docking and whole cell patch clamp technique were used to predict the target sites of gelsenicine (main toxic substance of G. elegans) and rescue drugs, and elucidate their possible detoxification mechanisms. We found that the LD₅₀ of G. elegans was 0.25 g·kg^-1 by gavage in rats; flumazenil combined with epinephrine had a significant detoxification effect on G. elegans poisoning rats; the survival rate increased from 25% to 92% (P<0.01), and the mean time to poisoning symptoms was extended from 16.56 to 25.98 min (P<0.01); the mean time to death was prolonged from 40.13 to 58.05 min (P<0.01); the rescue effect was significantly higher than that of the flumazenil and epinephrine alone rescue group. The combination of flumazenil and epinephrine significantly alleviated the clinical manifestations of decreased blood pressure and heart rate in poisoning rats. The results of molecular docking computer simulation showed that both flumazenil and gelsenicine had better binding effect to the same active site of GABA_A receptor. The binding energy of flumazenil was stronger than gelsenicine (-85.47 vs -77.15 kJ·mol^-1), which could reverse the toxicity effect of G. elegans. The whole cell patch clamp results showed that gelsenicine significantly prolonged the opening time of GABA_A receptor ion channels, and this effect was antagonized by flumazenil. In conclusion, the combination of flumazenil and epinephrine had a significant detoxification effect in G. elegans poisoning rats. The target of G. elegans neurotoxicity may relate to the GABA_A receptor that flumazenil action in the CNS; epinephrine was essential to prevent the decrease of blood pressure and heart rate caused by poisoning. Combination of drugs could use a therapy for both the causes and symptoms.

This study aims at exploring the effect of electric acupuncture at Baihui acupoint on rats with spleen-deficiency diarrhea. Sixty-four Wistar rats were selected and divided into four groups:sham operation group (Sham), model group (Model), electric acupuncture Baihui group (Baihui), positive control group (PC). Except for the Sham group, the rest groups were established spleen-deficiency diarrhea model by gavage of Folium Sennae decoction. The rats of Baihui group were electro-acupunctured at the Baihui acupoint and the rats of PC group were gavaged with montmorillonite powder for 2 weeks after successful modeling. During the whole experiment, the symptoms and signs, body mass and diarrhea index of all rats were observed and recorded. After the experiment, the serum levels of D-xylose, amylase (AMS), and inflammatory factors such as interleukin-2 (IL-2), IL-12, and tumor necrosis factor-α (TNF-α) of all rats were detected. The spleen and pancreas were taken out, and the indexes of spleen and pancreas were measured. The results showed that the rats in the Model group suffered from weight loss, sluggish spirit, messy hair, decreased activity, watery stools, and perianal and tail adhering to loose stools. Electric acupuncture at Baihui acupoint could gradually restore the general behavioral state of rats with spleen-deficiency diarrhea, and significantly increase the body mass of male rats (P<0.05), decrease the diarrhea index (P<0.05), increase the content of serum D-xylose and amylase (P<0.05), decrease the serum content of IL-2, IL-12, and TNF-α (P<0.05). The results demonstrated that electric acupuncture the Baihui acupoint could improve the symptoms and signs of rats with spleen-deficiency diarrhea, increase body mass, adjust organ index, improve digestion and absorption, and relieve inflammation. Therefore, it has a certain therapeutic effect. This study provides a quick and convenient treatment method for clinical treatment of spleen-deficiency diarrhea, and could also provide a theoretical basis for the study of the efficacy and mechanism of Baihui acupoint.

Oct4 and Cdx2 play important roles in the development of sheep pre-implantation embryos, which regulate the development of fetus and placenta. Oct4 and Cdx2 also regulate the expression of interferon-tau(IFNT), which plays a key role in embryo implantation and pregnancy recognization.In this study, embryos of different development stages were obtained by in vitro maturation,fertilization and cultured. Expression of Oct4 in the ovine embryos were analyzed using immunofluorescence staining. The results showed that expression of Oct4 protein was not detected in the zygotes and the early cleaved embryos. At the 16~32 morula cell stage, only a part of cells expressed Oct4 protein. Co-expression of Oct4 and Cdx2 was existed in the trophoblast of ovine blastocyst.

To explore the ultrastructure changes of dairy goat spermatozoa during liquid storage at 4℃, and provide a morphological basis for elucidating the relevant mechanisms that affect the quality of dairy goat spermatozoa during liquid storage. The semen of Guanzhong dairy goats was collected and stored in a liquid state at 4℃, sperm viability at 0, 24, 48, 72, 96, 120 and 144 h of storage was detected by eosin staining, the ultrastructure of spermatozoa at 0, 48, and 96 h was observed by scanning electron microscope and transmission electron microscope. The results of eosin staining showed that sperm viability gradually decreased with the prolongation of liquid storage time. The ultrastructure showed that at 0 h, the sperm morphology and structure were normal, the acrosome was intact and smooth, the mitochondrial sheath of sperm tail were intact, and the mitochondria were arranged regularly, and the morphology and size were normal; at 48 h, some spermatozoa showed structural damage such as tail breakage and curling, nuclear membrane expansion and folding, and mitochondrial swelling; at 96 h, some spermatozoa suffered damages such as acrosome damage, main segment fracture, nuclear disintegration and fragmentation, mitochondrial deletion and disordered arrangement. At 48 and 96 h, some spermatozoa exhibited typical apoptotic morphological features, such as needle-like protrusions on the nuclear surface, cystic vacuoles and apoptotic bodies. The above results showed that during the liquid storage at 4℃, the sperm of dairy goats suffered structural damage, some spermatozoa had typical apoptotic morphological characteristics, sperm viability decreased gradually, suggesting that the sperm structure damage of dairy goats led to the decrease of viability.

A detection method is urgently needed, which not only can improve the detection and diagnostic ability for the newly discovered bovine hepacivirus strain GDZJ, BovHepV-GDZJ, but also contribute to the preliminary understanding of tick carrying BovHepV in Guangdong province. In this study, the specific primers of Nested PCR for BovHepV-GDZJ strain were designed using the gene regions with high abundance in high-throughput sequencing data as templates through macrotranscriptomics, high-throughput sequencing, and PCR. The results showed that the minimum detection value of the method for the standard plasmid containing the gene fragment of BovHepV-GDZJ strain was 6.8 copies·μL^-1, and it had no response to common pathogens carried by ticks such as arenavirus. Moreover, the positive rate was 46.08% (53/115) in the 115 tick samples collected in Zhanjiang, Guangdong. In this study, BovHepV was first detected from ticks, which revealing the possible transmission route of BovHepV. What's more, a nested PCR method for the detection of BovHepV-GDZJ strain was established to provide technical help for the detection and prevention of BovHepV in Guangdong. It also provides some reference value for the control of the HNV epidemic.

This study was conducted to develop the duplex PCR assay for detection simultaneously and discrimination of canine bocavirus (CBoV) and canine circovirus (CCV). The published gene sequences of CBoV and CCV in GenBank were analyzed and compared with each other, respectively. Two pairs of specific primers were designed with the Primer Primier 5 software for PCR amplification. The results showed that two specific fragments of the two viruses were simultaneously amplified and the length of amplified fragments were as follows:170 bp for CBoV and 239 bp for CCV. The sensitivity and specificity tests showed that the assay could detect at least 3.0 pg of target DNA, and there was no cross-reaction with those clinically relevant pathogens, ie. canine parvovirus type 2, canine distemper virus and canine parainfluenza virus. The detection results for 30 clinical samples with the duplex PCR showed that 4 samples were positive for CBoV and 3 for CCV. These results indicated that the method could provide technical support for the detection of CBoV and CCV, and epidemiological investigation of canine viral diarrhea.

This study aimed to investigate the effect of cofilin2 gene expression on the proliferation of duck enteritis virus (DEV). The complete gene sequence of cofilin2 from duck (GenBank:MF434775),four pairs of shRNA sequences were designed,inserted into vectors and transfected into duck embryo fibroblasts. shRNA sequences with high interference efficiency were screened by FQ-PCR and fluorescence microscope observation. The shRNA sequences with high interference efficiency were transfected into duck embryo fibroblasts to inhibit cofilin2 gene expression. Then check how cofilin2 is expressed during DEV replication. Result were as follows:Four pairs of shRNA interfering plasmids were successfully transfected into duck embryo fibroblasts and expressed by fluorescence microscopy. The silences were 4.96%±0.78%, 41.12%±2.94%, 17.55%±2.07%, 54.02%±1.73%, respectively, and cofilin2-86 was the most efficient. After cofilin2-86 transfection into duck embryo fibroblasts and DEV infection, the expression of DEV nucleic acid decreased significantly at different time. In conclusion, RNA interference targeting cofilin2 gene plays an important role in DEV replication and proliferation.

The objective of this experiment was to analyze the effects of cyst of fallopian tube on slaughter performance, organ index, serum biochemistry and histopathology of Hy-Line Grey laying hens. Sixteen 54-week-old Hy-Line Grey laying hens from the same feeding environment were selected and divided into control group (CTR, n=9) and cyst of fallopian tube group (CFT, n=7). Hens were slaughtered and serum samples were collected to determine slaughter performance, organ indices and serum biochemical indexes. The results showed as follows:1) Live weight and dressed weight were significantly higher in cyst of fallopian tube group hens compared to the control group (P<0.01), and dressed percentage was significantly higher (P<0.05); but the percentage of carcass was significantly lower (P<0.01). 2) In the CFT group, the cecum index, weight and organ index of duodenum, jejunum and ileum were all highly significantly lower than that in the CTR control group (P<0.01), while the cecum weight and the index of ovary were significantly lower than CTR group (P<0.05). 3) The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (P<0.01), and the activity of serum alkaline phosphatase (ALP) were extremely significantly lower in cyst of fallopian tube group compared to the control group (P<0.01). 4) Cyst of fallopian tube caused different degrees of pathological damage to the lungs, ovaries and oviducts of laying hens, such as extensive hemorrhage in the lungs and the alveolar cavity of the lungs filled with large amounts of red exudate; capillary congestion and inflammatory cell infiltration in the cortical area of the ovaries; chorionic villus breakage and inflammatory cell infiltration in the plasma membrane layer of the oviducts. In conclusion, cyst of fallopian tube affected the slaughter performance and organ index of laying hens, increased serum ALT and AST activity, decreased serum ALP activity; it also caused different degrees of pathological damage to the lungs, ovaries and oviducts of laying hens.