Amino acid (AA) balance is of importance to the health and growth of mammals, and GCN2 and mTORC1 signaling pathways play crucial roles in maintaining AA balance in body. GCN2 signaling pathway can effectively sense intracellular AA starvation, mTORC1 signaling pathway can respond to the change of extracellular AA level. Combining with the recent studies regarding GCN2 and mTORC1, this review summarized characteristics of the GCN2 and mTORC1 signaling pathways and the related response mechanism under the condition of AA starvation and sufficiency, e.g. protein synthesis, refusing to feed, an increase of AA transporter, AA synthase expressions and autophagy initiation, etc.. Understanding the regulatory mechanism by which cellular AA balance is kept under different AA nutrition status contributes to the nutritional regulating of nitrogen for animals, thus improving animal gut health.

Mycoplasma is a critical pathogen of humans and animals. Due to lacking of highly effective drugs and vaccines, these diseases cause serious economic losses. The unknown pathogenesis and immunologic response restrict the development of novel drugs and vaccines. Secretory proteins are involved in direct communication between pathogens and hosts and usually considered as the key factors of virulence and immunity. Therefore they are potential targets of novel drugs and vaccines. But the study of secretory proteins of Mycoplasma has just started and only a few of Mycoplasma secretory proteins have been identified. In addition, the functions of secretory proteins in pathogenesis and immunity are mostly unknown. This paper reviewed the identified secretory proteins of Mycoplasma, their secretion pattern, and the methods used in related research, in order to provide some support to future study of Mycoplasma secretory proteins.

The objective of this study was to clone the coding sequence (CDS) of porcine ADAM10 gene and its splicing variant, analyze the gene structure and functions using bioinformatics methods, and investigate the mRNA levels of ADAM10 gene and its splicing variant in different tissues in porcine. According to the sequence of porcine ADAM10 gene in GenBank, the specific primers were designed for cloning the CDS of ADAM10 gene and its splicing variant using reverse transcription-PCR (RT-PCR) and T/A cloning methods. The protein structure of ADAM10 and its splicing variant were analyzed through bioinformatics methods. The mRNA expression patterns of porcine ADAM10 gene and its splicing variant in different tissues in porcine were analyzed by semi-quantitative PCR. The results showed that the complete CDS of ADAM10 gene was 2 247 bp, encoding 748 amino acids. The CDS of splicing variant, missing the sequence of exon 2-7 and partial exon 8, had a length of 1 344 bp which encoded 447 amino acids. Compared with the intact protein, the splicing variant protein missed the leading peptide sequence. However, the splicing variant protein contained signal peptide, transmembrane domain and metalloproteinase and depolymerized domains. Futhermore, the tertiary structures of the 2 proteins were identical. Sequence alignment and phylogenetic analysis showed that the intact ADAM10 protein was highly conserved among species. Semi-quantitative PCR analysis showed that the intact mRNA of ADAM10 gene was highly expressed in spleen and lowly in kidney, mammary gland, leg muscle, fallopian tube, ovary, uterus and small intestine; the mRNA of splicing variant was highly expressed in lung and lowly in spleen and stomach. Our study provides the basic materials for further studying on the structure and biological function of porcine ADAM10 gene.

This study aimed to investigate the genetic connectedness of 3 dominant pig breeds (Duroc, Landrace and Yorkshire pigs) in China, and to explore the plausibility of joint genetic evaluation in China pig breeding. The connectedness ratings (CR) from 2011 to 2016 were calculated to assess the genetic connectedness among 95 national nucleus pig breeding farms. In total, 514 226, 924 717 and 2 031 688 records of days to 100 kg for Duroc, Landrace and Yorkshire pigs from National Pig Database were analyzed and several joint genetic evaluation groups were established through clustering analysis. The results indicated that the average connectedness ratings among Duroc, Landrace and Yorkshire in China increased steadily from 0.04%, 0.06%, 0.06% in 2011 to 0.32%, 0.43% and 0.44% in 2016, respectively. In general, the connectedness rating in China was low, and even no connections among some breeding farms. However, 3, 2 and 4 groups, in which relative high genetic connectedness was observed among breeding farms, were clustered in Duroc, Landrace and Yorkshire, respectively. The joint genetic evaluation could be carried out in these groups. For Duroc pigs, the 3 joint genetic evaluation groups including 16, 7 and 8 breeding farms, correspondingly, the average connectedness ratings of 3 groups were 1.89%, 1.05%, 1.58%, respectively. Likewise, the 2 groups of Landrace pigs included 14, 9 herds and the corresponding average connectedness rating were 3.53% and 1.56%. The 4 groups of Yorkshire pigs included 15, 6, 5, 4 herds and the corresponding average connectedness rating were 2.79%, 1.01%, 1.37% and 1.23%, respectively. The joint genetic evaluation groups of 3 breeds accounted for 35.1%, 24.5% and 31.6% of total nucleus farms in Duroc, Landrace and Yorkshire pigs, respectively. Nevertheless national-wide joint genetic evaluation for pig breeding in China is not practical, the regional joint genetic evaluation groups cross farms is feasible. Meanwhile, genetic connectedness among breeding farms need be further strengthened through enhancing genetic exchange, constructing regional AI stations, utilizing central performance test stations and standardizing pig registration etc.

The aim of the present study was to clone the full-length cDNA of TMSB15A, analyse the structure and function of TMSB15A gene and determine its expression profile in Rongchang pig. Using the RCAE (Rapid-amplification of cDNA ends)-PCR and RT-PCR(Real-time quantitative PCR), we cloned the full-length of TMSB15A mRNA and detected the expression profile of TMSB15A gene in Rongchang pig. The results showed that the TMSB15A mRNA of Rongchang pig was 654 bp in length. This sequence consisted of 138 bp of CDS, 86 bp of 5' UTR and 430 bp of 3' UTR. The amino acid sequence deduced from TMSB15A encoded a putative protein consisting of 45 amino acids. Bioinformatics analysis indicated that TMSB15A protein had 10 negatively charged amino acids and 9 positively charged amino acids. The theoretical molecular weight of TMSB15A protein was 5.2 ku and the theoretical isoelectric point was 5.3. It was predicted that no hydrophobic amino acids, signal peptides or transmembrane domain were found in TMSB15A protein. TMSB15A protein might play physiological function in the cytoplasm. The tertiary structure of TMSB15A comprised α-helices and random coils. Rongchang pig TMSB15A had the greatest genetic relationship with TMSB15A of Homo sapiens, Nomascus leucogenys and Pan troglodytes (97.8%), followed by Camelus ferus and Oryctolagus cuniculus (93.3%), and the lowest relationship with Equus caballus(11.1%). The highest expression level of TMSB15A was in the spleen and thymus (P<0.01). Medium expression levels were observed in lymph node and lung. The expression of TMSB15A in heart, liver, kidney and muscle was the lowest. These data provide a basis for investigating the functions of TMSB15A and will inform further research in Rongchang pig resistance breeding.

This study aimed to obtain the RNA editing sites information in adipose tissue in chicken lines divergently selected for abdominal fat content, and provide the foundation for fat study of broiler. Ten individuals from the 19th generation of fat and lean broiler lines divergently selected for abdominal fat content from the Northeast Agricultural University were used. The transcriptome and genome sequencing datasets were used for identifying RNA editing sites in abdominal fat tissues. Bioinformatics method was used to construct the candidate set of RNA editing sites, after stringent data filtering and screening. The result showed that we classified and annotated RNA editing sites in the candidate set.In the adipose tissue of lean line broilers, 28 transition, 101 transversion and 71 insertion-deletion (InDel) RNA editing sites types were found; while in the adipose tissue of fat line broilers, 30 transition, 84 transversion and 77 InDel RNA editing sites types were found. Most of RNA editing sites were located in the regulatory and intergenic regions of genes (intron 41.91%; intergenic 33.08%; downstream 20.59%; upstream 2.94% and exon 1.47%, respectively). We validated 4 RNA editing sites, 3 of which located in genes (ATP1B3, SLC6A6 and FLNB) related to chicken adipogenesis. In the current study, identification and validation of RNA editing sites were performed in chicken abdominal fat tissues, which lays the foundation for further investigating the molecular mechanism of RNA editing in adipose tissue growth and development.

The objective of this study was to analyze the changes of the liver metabolic function and its effects on nutrients conversion and utility based on differentially expressed gene analysis in the liver. Full-sib White Leghorn layer hens for one week before and after first egg laying were chose for liver sampling. For analyzing and screening the important genes involved in the nutrient metabolic pathway in the liver, the transcriptome data was obtained by RNA-seq technology and bioinformatics analysis, and the gene expression differences was compared before and after the first egg laying. Two hundred and twenty-two differentially expressed genes with more than 2-fold changed were obtained by the transcriptomics sequencing (P<0.05). GO enrichment analysis and KEGG pathway analysis of differentially expressed genes showed that differentially expressed genes obtained in this study mainly involved in lipid metabolism and tryptophan metabolism.In this study, there were some function-unknown differentially expressed genes were found, such as Ring finger protein 186 (RNF186). We obtained the differentially expressed genes in liver from White Leghorn hens before and after first eggs laying and analyzed the changes of metabolism pathway of nutrients in the liver. These results proved that the liver metabolism pathway of nutrients were changed by these differentially expressed genes and the changes might improve the nutrients utilization of the feed after first egg laying.

This study aimed to identify and analyze the expression patterns and mechanism of buffalo mammary gland miRNAs in the lactation and non-lactation periods. Buffalo mammary gland tissues in lactation and non-lactation periods were collected, their miRNAs expression profiles were analyzed by Solexa sequencing technology, pre-miRNAs, mature miRNAs and novel miRNAs were identified, respectively, the first preferred nucleotide and chromosome distribution were analyzed. The highly and differentially expressed miRNAs in the lactation and non-lactation periods were detected. The results showed that 2 miRNAs expression profiles from buffalo lactation and non-lactation mammary glands were constructed. 12 768 110 and 12 569 467 high-quality reads between 18 and 31 nt were obtained seperately. Three hundred fifty-nine mature miRNAs and 363 pre-miRNAs belonged to 259 miRNAs families, and 5 buffalo-specific miRNAs were confirmed by sequencing. U was the most common nucleotide at the 5'end of 19 and 25 nt miRNAs. The bbu-let-7b, bbu-let-7a, miR-26a and miR-21 showed high expression in both periods. bbu-miR-148a, 143, 200c, 200a and bbu-let-7f were specificially highly expressed in non-lactation period, the bbu-miR-125b, 29a and bbu-let-7c were specifically highly expressed in lactation period. The bbu-miR-148a, 143, 200a, 141 and 30a-5p decreased the expression in the lactation period to less than 1/2 of the non-lactation period, bbu-miR-26a, 29a, 125b, 99a and bbu-let-7c were highly expressed in the lactation period more than or equal to 2 times of the lactation period. The result indicate that 2 miRNAs profiles from buffalo lactation and non-lactation mammary gland tissues were constructed. 359 mature miRNAs and 363 pre-miRNAs belonging to 259 buffalo miRNAs families were identified. 5 buffalo genome-specific miRNAs and 10 highly differentially expressed miRNAs were obtained, which lay the foundation for further elucidating the mechanism of key miRNAs in lactating buffalo.

The aim of this study was to investigate the effects of silencing the inhibin α-subunit (INHα) gene or treatment with inhibinA on the secretion of estrogen (E2), progesterone (P) and expressions of related genes in sheep granulosa cells (GCs), explore the role of inhibin in the secretion of E2 and P. GCs were isolated from antral follicles (3-7 mm) obtained from the ovaries of small-tailed Han sheep (1.0-1.5 years old). GCs were transfected with siRNA or treated with inhibinA (200 ng·mL^-1). The concentrations of E2 and P were tested by ELISA. The mRNA levels of related-genes (CYP11,3β-HSD and CYP19) were analyzed by quantitative real-time PCR (qRT-PCR). The results showed that siRNA was transfected into GCs at 48 h and the silencing efficiency of INHα was 87%. Knockdown of INHα by siRNA decreased the concentrations of E2 and P at 48 h compared with the control (P<0.05). Secretion of E2 and P were increased in the group treated with inhibinA (P<0.05). Knockdown of INHα by siRNA decreased the mRNA expressions of 3β-HSD and CYP19, and increased the mRNA expression of CYP11 (P<0.05). The mRNA expressions of 3β-HSD, CYP11 and CYP19 were increased in the group treated with inhibinA (P<0.05). These results indicated that inhibin was a key regulator and involved in the process of follicular growth and ovulation by regulating the secretion of steroid hormone in sheep GCs.

This study aims to investigate whether GPNMB differentially expresses in the skin of mice with various hair colors and to determine its effect on the melanin synthesis. Three approximately 12-day-old healthy C57BL/6 mice were randomly selected from strains with black, brown and gray coat colors, respectively. Three pieces of dorsal skin tissue were collected from each mouse using a skin collector after shaving. Immunohistochemistry assay revealed that GPNMB was mainly located in the hair follicle matrix, the inner and outer root sheath, and the dermal papilla in the skin with various hair colors. The Western blot analysis and qRT-PCR assay showed that the expression level of GPNMB was the lowest in the skin of gray mice. Compared with the control group, relative levels of MC1R, β-catenin and SLC45A2 significantly increased in mouse melanocytes overexpressing GPNMB.we detected the levels of GPNMB and MC1R in mouse melanocytes which had been radiated by UVB. The results showed that UVB had an impact on the GPNMB and MC1R expression, and the level of GPNMB was various with different UVB radiation doses.The above results suggested that GPNMB was differentially expressed in the skin of mice with different hair colors, and it was involved in the formation of the hair color by regulating the melanin synthesis.

This study aimed to investigate the effects of S. aureus on boar sperm motility during storage at 17℃ from the perspectives of sperm metabolism and protein phosphorylation modification levels, and provide a theoretical basis for the molecular mechanisms underlying the study of pathogens on sperm quality. Sperm motility parameters were determined using computer assisted sperm analysis (CASA) and the varying of intracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, cyclic adenosine monophosphate (cAMP) content and totally ATP level were measured by reagent kits. Protein phosphorylation levels and the localization of phosphorylated targets were analyzed by Western blot and immunofluorescence technique, respectively. The results of CASA showed that 10³ CFU·mL^-1 S. aureus-treated could significantly inhibit sperm motility during boar semen storage at 17℃ (P<0.05). With the increase of S.aureus, sperm motility parameters were significantly decreased after 3 days of inoculation (P<0.01). Similarly, GAPDH activity and ATP level in the treatment groups were also markedly decreased compared to that in the control groups after 3 days of inoculation (P<0.05). The results of Western blot showed that there were significant differences in sperm protein phosphorylation levels between the treatment groups and the control groups (P<0.05). The changing trends of phosphorylation levels were consistent with that of intracellular cAMP level, which suggested that S.aureus affecting protein phosphorylation levels might following the sAC-cAMP-PKA signaling pathway. Taken together, S.aureus reduced intracellular ATP content through lowering glycolytic GAPDH activity, meanwhile, S.aureus inhibited the phosphorylation levels of motility-related proteins located in the posterior nucleus, equatorial piece and flagellar regions of boar sperm following the sAC-cAMP-PKA signaling pathway. Both the reduction of ATP and the inhibition of protein phosphorylation levels led to the decrease of boar sperm motility.

In order to discover new molecular marker related to piglet diarrhea, growth and boar reproduction traits, the polymorphism of porcine IL6 gene was investigated and the association analysis was performed in different pig breeds. The DNA fragment of IL6 gene was amplified and sequenced. A SNP (g.1704674C>T), which could alter the recognition site of HpaⅡ, was detected in intron 3 of IL6. The SNP was genotyped by PCR-RFLP in 281 Min piglets, 182 Landrace piglets, 215 Duroc boars and 69 Yorkshire boars. Its association with piglet diarrhea, growth and boar reproduction traits was analyzed. The expression of IL6 was detected in PBMC stimulated with LPS by real-time PCR. The results showed that Min piglets with CC genotype had significantly higher diarrhea index than TT genotype piglets (P<0.05). The 14, 21, 28, 35-day body weight and ADG of Min piglets with CC genotype were significantly lower than that of TC genotype (P<0.05), and the 21-day body weight of Min piglets was also significantly lower than that of piglets with TT genotype (P<0.05). In Landrace piglets, birth weight of piglets with TT genotype was significantly higher than that of piglets with CC genotype (P<0.05), and the 7-day body weight was significantly higher than that of individuals with TC genotype (P<0.05). In Duroc boars, the ejaculate volume of boars with TT and TC genotype was significantly higher than that of boars with CC genotype (P<0.05). In Yorkshire boar, the sperm density of boars with CC genotype was significantly higher than that of boars with TC genotype (P<0.05). At 12 and 24 h after LPS stimulation, the expression of IL6 gene was significantly increased in PBMC (P<0.01 or P<0.05). Therefore, it can be concluded IL6 g.1704674C> T had effect on piglet diarrhea, growth and boar reproduction traits, and which may be a potential molecular marker in pig breeding.

The objective of present study was to investigate the effect of dietary wine grape pomace (WGP) supplementation on metabolism and development of rumen in lambs. A total of 24 Dorper (♂)×Small Tail Han sheep (♀) F1 male lambs ((25±1) kg of body weight, 5 month old) were randomly selected and divided into 4 groups equally. Lambs in 4 groups were fed with diets supplemented with WGP at 0%, 5%, 10%, 20% on a dry weight basis (group 0%, group 5%, group 10%, group 20%), respectively. The experiment lasted for 80 d. At the end of experiment, all lambs were slaughtered, and stomaches were weighted. The rumen fluid was collected for analyzing rumen fermentation and enzymes activity of microorganism, and rumen tissues were sampled for epithelial development measurements. The results showed that:1) No significant effect of dietary WGP supplementation on rumen pH and total volatile fatty acid (TVFA) concentration was observed (P>0.05). Propionic acid molar ratio was significantly increased, whereas acetic acid molar ratio and acetic acid/propionic acid ratio were decreased when lambs were fed with WGP diet (P<0.05); meanwhile, decreased concentrations of lactic acid, ammonia nitrogen and urea nitrogen were observed (P<0.05). 2) Amylase, protease and cellulases activity (except for pectase) were decreased with the increasing WGP supplementation levels (P<0.05), while pectase activity was exhibited as group 10% > 0% > 5% > 20% (P<0.05), the total protein concentration in rumen fluid had no significant difference among different groups(P>0.05). 3) Among factors related to VFA absorption and metabolism, relative mRNA expression of MCT1 and AE2 were significantly decreased when lambs were fed with WGP diet (P<0.05), whereas no significant difference at MCT4 and Na⁺-K⁺-ATPase mRNA expression levels among different groups were observed (P>0.05). 4) Both absolute weight and relative weight of rumen were not significantly altered among the groups (P>0.05). The cuticle thickness of lambs in WGP-added groups was significantly higher than that in group 0% WGP, and ruminal papillae length in group 20% WGP was significantly lower than that in other groups (P<0.05). In conclusion, WGP could effectively change rumen metabolism, and benefit to rumen development. Moreover, WGP supplementation amount could not exceed 10% considering the rumen epithelial tissue development.

The present study aimed to investigate the effect of melatonin (MT) on lipid metabolism and its effect on lipid metabolism enzymes and factors, and to reveal the mechanism of lipid metabolism in male rats. Twenty SD male rats with similar age and body weight were randomly divided into 2 groups (10 rats per group),which were control group (normal diet, ND) and MT treated group (normal diet with oral melatonin solution, ND+MT). The MT was administrated at 10 mg·kg^-1 BW per day.The experiment lasted for 60 days. At the end of the experiment, blood sample were collected to extract plasma to detect biochemical, hormonal and lipid metabolism related indicators; The rats were sacrificed, the liver and the adipose mass in the abdomen, such as omentum, perirenal fat and epididymal fat was separated and weighed. In additon, liver tissue was sampled for Gene chip and related molecules analysis. The result showed that:1) The liver weight, liver index, omental weight, omental index and epididymal fat weight in ND + MT group were decreased significantly (P<0.05). Low density lipoprotein in serum was significantly decreased (P<0.05), high density lipoprotein and total cholesterol in serum were decreased, while very low density lipoprotein increased.The contents of hepatic long chain fatty acids (LCFA) was significantly decreased (P<0.05). 2) Gene chip and fluorescent quantitative PCR result showed that the mRNA expression of lipolysis related factors and enzymes, such as CPT1(P<0.05) and ALDH3a2 (0.05 < P < 0.1) in liver tissue were increased, while the mRNA expression of fat synthesis related enzyme, such as ACSL3 and FAS was decreased significantly (P<0.05). MT can greatly affect the lipid metabolism with the reduction of visceral fat, its mechanism is connected with the response of hormones and lipid metabolism related enzymes or factors to MT.

A droplet digital polymerase chain (ddPCR) detection method, based on gB gene of pseudorabies virus (PRV) was developed to improve the detection and quantification of PRV. The concentration of primer, probe concentration and annealing temperature in ddPCR reaction were optimized. The specificity and sensitivity of the established ddPCR method were also determined and applied to the detection of field samples. In results,the optimal primer concentration was 0.9 μmol·L^-1, the probe concentration was 0.25 μmol·L^-1, and the annealing temperature was 60℃ for ddPCR.The R² value of ddPCR standard curve was 0.998,showed ddPCR had a good linear response. The Limit of Detection of ddPCR was 6.1 copies·μL^-1, lower than that of fluorescence quantitative PCR (qPCR). The coefficient of variation was 2.81%,the ddPCR was specific for detecting PRV. Field samples were detected by ddPCR, qPCR and virus isolation. Compared with the virus isolation, the sensitivity of ddPCR method was 87.5%, specificity was 96.2%, and the coincidence rate was 97%. The results indicate that the new ddPCR assay provides a specific, sensitive tool for quantitative detection of PRV.

Risk-based epidemiological survey was conducted in Yunan Province during 2011-2016 in order to better understand the epidemiological relations and their genetic diversity among the circulating Newcastle disease virus (NDV) in this region. A total of 2 565 swab samples were collected from 48 sampling units in Kunming, Yuxi, Dali, Lijiang, Pu'er and Qujing city. Results were as follows:Virus isolation and sequencing were used to determine the molecular characterization of the isolates. Fifty-two NDVs were isolated from samples collected from 17 live bird markets. The individual positive proportion of NDV was 2.03%, and the population positive proportion was 35.41%. Among 52 NDV isolates, 17 NDVs isolated from 7 retail markets were proved to be virulent strains based on the sequence analysis of the Fusion gene. The individual positive proportion of virulent NDV was 0.66%, the population positive proportion of virulent NDV was 14.58%. Phylogenetic analysis revealed that all 52 NDV isolates were clustered into two classes, namely ClassⅠ and ClassⅡ. All 17 Class Ⅰ NDV isolates were belonged to genotype 3. Thirty-five Class Ⅱ NDV isolates comprised 4 different genotypes, namely genotype Ⅰ (2 isolates),Ⅱ (16 isolates),Ⅵ (13 isolates),Ⅶ (4 isolates). The genetic diversity of circulating NDVs in Yunnan province was proved, all Ⅵ NDV isolates were obtained from pigeon, and 4 genotype Ⅶ NDVs were isolates from chicken and goose. Two genotype Ⅶh NDVs were first isolated from Yunnan in 2013 and 2016 respectively. Genotype Ⅶh NDVs has been endemic in some south-east Asian countries, such as Vietnam, Malaysia, Indonesia, etc. Wild birds may play the role for introduction from neighboring countries. So more active surveillance should be conducted to determine the distribution of the new virus and some control measures also need to be used to prevent the transmission of the new NDVs.

In order to understand the epidemic situation of exogenous avian leukemia virus (ALV) in broiler flocks in Shandong province, blood plasma were collected for virus isolation and identification by ELISA (detection of P27 antigen) and PCR. A strain of new ALV subgroup was isolated and identified from commercial broiler flocks in a farm of Shandong province, named FC1505. The env gene of the isolate was sequenced and indicated that the amino acid sequence of gp85 had the highest gp85 identity of more than 95% to the strains of suspected subgroup K of ALV from Chinese native breed chickens. Its gp85 identity to other known chicken ALV subgroups A-E and J was lower than 90%. In order to further analyze the molecular properties of the isolate, the genome of the virus was sequenced and compared with those from other ALV reference strain of different subgroups. The results showed that the gag, pol and gp37 genes of the isolate were relatively conservative, with the identity more than 90%, and showed the highest identity to suspected ALV-K isolates. Whole genome sequence analysis supported that the FC1505 isolate belongs to ALV-K. Following the reports of K subgroup ALV in Jiangsu and Southern China provinces of China, this is the first report of an ALV-K isolate from broilers of Shandong province and its whole genome analysis.

To analyze genetic variation of canine parvovirus (CPV), a new virus strain was isolated from the feces of a dog infected by CPV. The virus strain, named as CPV-YH, was identified by red cell agglutination assay, morphology, artificial infection of Beagle dogs and molecular biology. The results showed that the isolate induced specific CPE in A-72 cells, and it could agglutinate porcine red blood cells. The virus were round or hexagonal by the electron microscope, and there was no capsule, with a diameter of about 20 nm. Animal regression test showed that the infected dogs had typical symptoms of CPV disease such as hemorrhagic enteritis, liver edema, pulmonary hyperaemia. The complete genome of CPV-YH was 4 921 nt and it shared the highest nucleotide identity (99%) with CPV-LZ2 isolates isolated from Lanzhou in 2011. The amino acid phylogenetic tree of VP2 showed that CPV-YH isolate, 2013-BJ-P27 (China) and UY306 (Uruguay) isolates were on the same small branch. Compared with the CPV strains on GenBank, the homology of VP2 gene nucleotide and amino acid showed 98%-99.4% and 97.1%-99.5% identities, respectively. In conclusion, one CPV mutant strain was successfully isolated, and these data would provide valuable theoretical basis for the epidemic situation and prevention and control of CPV.

To indicate the genetic variability of Enterocytozoon bieneusi in goats, the multi-locus sequence genotypes (MLGs) of 122 E. bieneusi isolates from different production categories were studied using the multi-locus sequence genotyping (MLST) technique based on micro-(MS1, MS3, MS7) and mini-satellite (MS4) loci. The results showed that, the respective amplification efficiencies in loci MS1, MS4 and MS7 were 27.9%(34/122), 18.0%(22/122), 50.8%(62/122), while no amplifications were detected in the locus MS3. The nucleotide sequences analysis indicated that, 16, 9 and 18 genotypes were found in all positive samples for loci MS1, MS4 and MS7, respectively, forming fourteen MLGs. Among them, the amplification efficiencies of three loci among 50 positive samples of cashmere goats were 10.0% (5/50), 14.0% (7/50) and 90.0% (45/50), respectively, belonging to three, three and ten genotypes, and five MLGs. The amplification efficiencies of 56 positive samples of dairy goats in three loci were 28.6% (16/56), 19.6% (11/56) and 8.9% (5/56), grouping into four, four, one genotypes, and seven different MLGs. The amplification efficiencies of three loci of 16 positive samples of black goats were 81.3% (13/16) and 25.0% (4/16), 75.0% (12/16), clustering into 9, 2, 7 genotypes, and two MLGs.

The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. The aim of the present study was to establish an indirect ELISA for practical use. According to the specific sequence of EMA-1 genes of Theileria equi, a pair of primers was designed and synthesized. The EMA-1 gene of Xinjiang strain was cloned, and then was inserted into the prokaryotic vector pGEX-4T-1. The recombinant plasmid (pGEX-4T-1/EMA1) were transformed into Escherichia coli BL12(DE3), and the GST-EMA1 protein was obtained by induction of IPTG.The fusion protein was purified by extracting the inclusion bodies with gel slices,and the purified protein was used as coated antigen for the establishment of indirect ELISA method to detect the antibody to Theileria equi.The results indicated that the expressed EMA-1 had an apparent molecular mass of 56 kDa which was largely consistent with its theoretical value, and expression of protein was identified by Western blot, which confirmed that the protein had highly specificity and reactionogenicity; the purified recombinant GST-EMA1 protein was tested in an ELISA for the detection of antibodies anti-T. equi in horses, and the indirect ELISA could clearly differentiate the T. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera; The intra-and inter-assay demonstrated that the coefficient of maximum variation was 14.79% and 11.06% respectively; 96 serum samples collected from horses in the state of Yili, Xinjiang were used to compare the indirect ELISA and cELISA commercial kit, the resules showed that their positive rates of T. equi infection were 27.1% (26/96) and 25.0% (24/96) respectively, and the total coincidence rate was 95.8%. These results suggest that the GST-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for indirect ELISA test and that provided the means of effective detecting and monitoring for T. equi (especially in recessive infection) in Xinjiang.

This experiment was conducted to investigate the regulation mechanism of death receptor pathway involved in the aflatoxins (AF) B₁-induced thymocytes apoptosis. Ninety healthy chickens were randomly divided into two groups, and fed on control diet and AFB₁ containing diet (formulated by adding 0.6 mg·kg^-1 AFB₁ into the control diet) respectively for 21 days. Thymus was sampled for detecting the relative weight, T-cell subsets, percentage of apoptotic cells and relative mRNA expression of genes in the death receptor pathway at 7, 14 and 21 days of age. Our results showed that in the AFB₁ group, the relative weight of thymus was decreased; the percentages of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ T-cells were decreased, and the percentage of apoptotic thymocytes was increased, when compared with those in the control group. Also, the relative mRNA transcription of RIP1, Caspase-8, Caspase-9, Caspase-3, Fas, FasL, ASK1, IKIP and JNK were up-regulated, and those of Bcl-2, NF-κB1 and Bid were down-regulated. It was speculated that the mechanisms of the excessive apoptosis of thymocytes could be related to the following three downstream death receptor pathways:①AFB₁ could directly induce the apoptosis through the Fas-FasL-FADD-Caspase-8-Caspase-3 pathway; ②Fas could take part in the excessive apoptosis through Fas-ASK1-JNK-Bcl-2 pathway which was mediated via mitochondria; ③The constitutive activation of the TNF-α-RIP1-IKIP-NF-κB1-caspase-3 could be a pathway contributing to the apoptosis of thymocytes induced by AFB₁.

This study was conducted to explore the effect of ciprofloxacin on the transcription of several active efflux system related genes, acrA, acrB, acrD, acrE, acrF, mdtA, marA, robA and soxS,in clinical E. coli strains Y35 and J45.The minimal inhibitory concentrations (MICs) of ciprofloxacin to E. coli strains Y35 and J45 induced by ciprofloxacin were determined using the standard broth microdilution method. A real-time fluorescent quantitative PCR assay was established to observe the difference of active efflux pumps genes transcription level between parental strains and the 10^th, 20^th, 30^th generation of ciprofloxacin induced strains. Results were as follows:The results showed that, the 30^th generation of ciprofloxacin induced strains exhibited increased MICs of ciprofloxacin (2-fold), which was 256 μg·mL^-1. For Y35, 8 of 9 genes transcription level of the 10^th and 30^th of ciprofloxacin induced strains were increased compared with the parental strain, except gene mdtA, and the values of relative transcription level were between 1.20 and 96.07 (the differences were statistically significant, P<0.05 or P<0.01). The transcription level of acrD gene increased most markedly. The expression level of acrA, acrB and marA in the 20^th generation of ciprofloxacin induced strains were decreased compared with the 10^th generation strain, however,these genes showed increased transcription level compared with the parental strain. The relative transcription level of acrE, robA and soxS were between 1.40 and 3.81 (P<0.05 or P<0.01). For J45, only acrB and acrF gene transcription level were increased in the 10^th generation of ciprofloxacin induced strain, the relative value of transcription level were 2.76 and 1.73, however, the transcription level of other genes were decreased. acrA, acrB, acrE and mdtA gene transcription level were increased in the 20^th generation of ciprofloxacin induced strain, the value of relative transcription level were 15.35, 58.89, 31.56 36.50, respectively (P<0.01). All the genes transcription were increased to a high level in the 30^th generation of ciprofloxacin induced strain,the transcription level of acrF gene increased most markedly, it was 102.54 times of the parental strain. The results of this study suggest that continuous induction of the clinical resistance E. coli strains by subinhibitary concentration of ciprofloxacin might result in stronger resistance to ciprofloxacin, and increased level of transcription of active efflux pump genes. The increased transcription level of efflux pump genes might contribute to the stronger resistance of the induced strains to ciprofloxacin.

This experiment was conducted to investigate the effects of recombinant escherichia coli expressing heat-stable enterotoxin (STa) on intestinal immune and barrier function and antioxidant capacity of 7 days old piglets. Twenty-four 7 days old piglets were allotted to four treatments:control group, STa group (2×10⁹ CFU E. coli LMG194-STa), LMG194 group (2×10⁹ CFU E. coli LMG194), and K88 group (2×10⁹ CFU E. coli K88). The pigs were fed with artificial milk and slaughtered 7 days later. The result showed that, compared with the control group, STa group significantly reduced IFN-γ and IL-4 gene transcription levels (P<0.05), and increased VNN1 transcription level in ileum (P<0.05). STa group also significantly reduced gene transcription levels of Villin and MMP3 in ileum (P<0.05) and protein expression levels of Occludin and Claudin-1 in jejunum (P<0.05). STa group significantly reduced activity of glutathione peroxidase (GSH-Px) in duodenum and ileum (P<0.05), and significantly reduced activity of myeloperoxidase (MPO) in ileum (P<0.05), and had a higher production of malondialdehyde (MDA) in duodenum (P<0.05). These results suggest that recombinant Escherichia coli expressing STa can reduce intestinal immune and barrier function and antioxidant capacity of 7 days old piglets.

The objective of this study was to evaluate the protective effect of Hericium Erinaceus polysaccharide (HEPs) on intestinal epithelial cell injury induced by H₂O₂. In this study, IPEC-J2 cells were divided into five groups:the control group, the model group, the HEPs low dose treated group, the HEPs medium dose treated group, the HEPs high dose treated group. MTT method was used to select the optimal dose of H₂O₂ and HEPs, respectively; intracellular ROS was observed and determined by DCFH-DA staining; the content of malondialdehyde (MDA) in the supernatant of cultured cells and the activities of superoxide dismutase (SOD), catalase (CAT) and the release rate of lactate dehydrogenase (LDH) in cells were detected by colorimetric method; the transcription level of ZO-1 gene was detected by quantitative real-time PCR (qRT-PCR). The results showed that:(1) The 0.4 mmol·L^-1 was the most suitable H₂O₂ concentration for oxidative stress modeling. (2) The data indicated that H₂O₂ increased the intracellular ROS and induced cell apoptosis significantly, and decreased the vitalities of SOD and CAT significantly; H₂O₂ significantly increased the content of MDA and the release rate of LDH. But, compared with the model group, after HEPs pretreatment, the content of ROS in cells decreased significantly (P<0.01); at the same time, the activities of SOD and CAT were improved significantly (P<0.01), MDA content and LDH release rate were significantly decreased. (3) The results of quantitative real-time PCR displayed that H₂O₂ could significantly reduce the transcription of ZO-1 gene (P<0.01). Compared with H₂O₂ group, HEPs could significantly improve the transcription of ZO-1 genes after HEPs pretreatment (P<0.01). The results of Western blot showed that the expression of ZO-1 protein was similar to that of ZO-1 gene. Results showed that HEPs had protective effect on IPEC-J2 cells in H₂O₂ induced oxidative damage state.

To investigate the toxicity and mechanism of action of fumonisin B₁ (FB₁) on human umbilical vein endothelial cells (HUVEC), HUVEC cells were treated with FB₁ in different concentration. The cell vitality, cell cycle, cell oxidative stress, apoptosis, mitochondrial membrane potential change and transcription changes of apoptosis-related genes were detected by Cell Counting Kit-8, flow cytometry, fluorescence microscope, Hoechst33342 staining and real-time quantitative PCR. In result, the vitality of FB₁ treated cells (FB₁ dose exceed 5 μg·mL^-1 and process time exceed 12 h) were significantly decrease (P<0.01 or P<0.05). And the fluorescence intensity of reactive oxygen active (ROS) were enhanced when being treated with 10, 20 μg·mL^-1 of FB₁. FB₁ could prevent HUVEC from G₀/G₁ phase to S phase and reduce the mitochondrial membrane potential. The transcription of apoptosis gene Caspase-3 were increased, Bcl-2 were significantly declined (P<0.05). Caspase-9 and Bax genes transcriptions were significantly increased (P<0.01). The results showed that FB₁ has cytotoxic effect on HUVEC cells, which could inhibit cell proliferation, arrest cell cycle at G₀/G₁ phase and induce apoptosis via mitochondrial pathway. Results in the present works provided the foundations for further research of toxicity and mechanism of FB₁ on human cells and indications for some relevant diseases treatment.