Abstract:2017年４月，“第五届全国人畜共患病学术研讨会”在江苏省南京市召开。此次会议以“同一健康，共同行动”为主题，与会者围绕细菌性、病毒性和寄生虫性人畜共患病的研究现状，共同分享、交流和研讨了各自领域内取得的最新研究进展和成果。会议报道的部分研究成果处于国际前沿，充分展现了我国学者在兽医和医学微生物学研究领域的前沿水平和发展前景。此外，参会专家和学者共同探讨了我国兽医微生物学相关研究领域的重点、难点和热点问题，展现了科研人员对兽医微生物学相关研究的热诚，凸显了兽医病原微生物的控制对人类健康的重要性。《微生物学通报》针对此次研讨会内容，与兽医微生物学专业委员会首次合作，推出了本期“兽医微生物学主题刊”，旨在展示我国兽医微生物学研究领域的新进展和新成果，推动我国兽医微生物学学科的发展和繁荣，践行“同一世界，同一健康”的理念。

Abstract:[Objective] Fowl adenovirus (FAdV) infection in chicken flocks is very common in China currently. Because FAdV transmits both horizontally and vertically in chickens, it is essential and urgent to determine the contamination of attenuated vaccines produced in chicken embryo eggs by FAdV and its risk to induce epidemic of Hepatitis-Hydropericardium syndrome (HHS) caused by FAdV. The objectives were to ascertain the contamination of an attenuated live Newcastle disease virus (NDV) vaccine produced by Chinese veterinary drug company by FAdV and to investigate the possible reasons for current prevalence of HHS caused by FAdV in chicken flocks in China. [Methods] FAdV-specific PCR was employed to detect FAdV from the attenuated live vaccine of NDV La Sota strain. PCR positive samples were inoculated into chicken embryo eggs for the isolation of FAdV. Sequencing, identity analysis and the phylogenetic tree construction were performed to analyze the molecular characteristics, genotyping and serotyping of the isolates. [Results] A FAdV strain was obtained and named L160962 that shared the highest identity with serotype 4 FAdVs in the 52k and hexon genes according to homology analysis. Among the strains investigated, isolate L160962 shared identities at a level of 99.9%?100% with Chinese strains HN/151025 and CH/HNJZ/2015, 99.3% with Austrian strain KR5, and only 79.0%?83.3% with members in species A, B, D and E in the sequences of 52k and hexon. Phylogenetic analysis showed that isolate L160962 was clustered into serotype 4 within the species C with an identity higher than 99.9% and most close to the Chinese strains, suggesting that isolate L160962 is similar to the prevalent strains in China. [Conclusion] Our studies observed contamination of attenuated vaccines produced in chicken embryo eggs by serotype 4 of FAdV, which may be one of the reasons that lead to FAdV infection prevalent in chicken flocks in China.

Abstract:[Objective] Bacteriophages are recognized as the most abundant microorganisms on earth, and have potential to control antibiotic-resistant bacterial infections for their bactericidal activity. Bacteriophages display remarkable genetic diversity and host specificity. To determine the biological characteristics and sequences of an efficient lytic Escherichia coli phage isolated from swine intestines. [Methods] Potential phages in the samples from piglet intestinal contents were isolated and purified, and plaque characteristics of the phage were observed. Morphological analysis was performed by electron microscope. The host spectrum, multiplicity of infection, one-step growth curve, thermal stability, pH stability, sensitivity to ultraviolet and chloroform sensitivity were determined, and genomic evolution was analyzed. [Results] A new lytic Escherichia coli phage, named vB_EcoS_SH2 (SH2), was isolated. The phage plaque was shown as big, circular, transparent and neat. Morphological analysis by electron microscopy revealed that phage SH2 had an icosahedron head with 50 nm in diameter, and a systolic tail fiber with 8 nm width and 120 nm length. Phage SH2 had a short latent period of 10 min and an outbreak period of 60 min, and a burst size of 121 PFU/infected cell. The optimal MOI is 0.1. Genome sequencing and comparison revealed that the nucleic acid type of SH2 is dsDNA, and the DNA genome of SH2 is composed of 49 088 bp with a G+C% content of 45% (GenBank accession number: KY985004). Phage morphology and BLASTp analysis revealed that SH2 belongs to the family of Caudovirales, Siphoviridae. Genomic and phylogenetic analysis suggested that the phage is closely related to phage T1, with 95% homology to T1 phage. [Conclusion] a novel efficient lytic T1-like phage vB_EcoS_SH2 against Escherichia coli was identified. The microbiological properties of SH2 suggest that it may be useful for controlling bacterial populations.

Abstract:[Objective] Litopenaeus vannamei is one of the most important species of shrimp farming in the world, with high output and value and strong market demand. From May to June of 2017, Litopenaeus vannamei was found to have unexplained high morbidity and mortality in one shrimp farm in Shanghai. To determine and identify the cause of death for Litopenaeus vannamei and screen out sensitive drugs, a dominant bacterium strain AVZ01 was isolated from sick L. vannamei. The aim of this study was to provide reference for further prevention and treatment of Aeromonas veronii in China. [Methods] The pathogenic bacteria were isolated from the hepatopancreas and intestinal tract of dead L. vannamei. The LD50 assay was carried out by Bliss method. According to morphological, biochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain was identified. Antibiotic susceptibility test was carried out by K-B methods. [Results] After the artificial infection test with strain AVZ01 from the diseased shrimp, the symptoms of shrimp were similar to those of natural disease, the LD50 was counted to 8.7×105 CFU/mL. According to the morphological characteristics, biochemical characteristics and 16S rRNA gene sequence analysis, the pathogen was identified as A. veronii. The results of antibiotic sensitivity test showed that the AVZ01 strain is highly sensitive to 16 antibiotics, such as metolycins, norfloxacin and gentamicin, etc, and resistant to 9 antibiotics, such as penicillin, oxacillin and cephalexin, etc. [Conclusion] The isolated strain AVZ01 has strong pathogenic to L. vannamei, and the gentamicin and neomycin could be used for the control and treatment in the future.

Abstract:[Objective] In recent years, more and more reported sheep respiratory diseases are partially caused by ExPEC (Extraintestinal pathogenic Escherichia coli), resulting in some economic losses. We determine and characterize the bacterial pathogen from sheep with respiratory tract infection in Shihezi, Xinjiang. [Methods] Bacteria were isolated and identified from the lungs of infected lambs by routine methods combined with 16S rRNA gene sequence analysis. Meanwhile, antimicrobial susceptibility and specific virulence gene of the isolates were tested with K-B and PCR. Pathological examination was carried out from the lung of naturally infected sheep and artificially infected mice. [Results] A pathogenic Escherichia coli was isolated from infected lamb lung, which showed multiple drug resistance and carried 3 virulence genes (iutA, fyuA and ireA). Histology examination showed that the capillary vessel of alveolar wall was congested, bronchial lumen was found hyperemia, infiltration and hyperplasia of lymphocyte could be seen in lung tissue. [Conclusion] The bacterial pathogen isolated from infected lambs was ExPEC.

Abstract:[Objective] H7N9 avian influenza virus can infect chickens, and high pathogenic avian influenza (HPAI) H7N9 strains have appeared after natural mutation. Thus, H7N9 vaccines immunization in chickens would be a tendency, and developing an antibody detection method for immunization is a need. To establish a sensitive, rapid, high-throughput enzyme-linked immunosorbent assay (ELISA) to detect H7N9 subtype avian influenza virus antibodies in chickens. [Methods] Three wild-type hemagglutinin (HA) proteins belonging to W1, W2-A and W2-B clades were expressed by an insect cell-baculovirus expression system, and one recombinant HA (H7-53TM) containing a replaced H3 HA transmembrane domain (TM) was expressed as well. Four HA proteins were purified by ion-exchange chromatography and used as ELISA antigens to detect H7N9 avian influenza virus antibodies. [Results] The results of specificity, sensitivity and repeatability assays showed TM replacement mainly affected the repeatability of the HA antigen. The intra- and inter-coefficient of variables of H7-53TM were less than 10%, showing better repeatability; whereas those of 3 wild-type HA proteins were more than 10%, showing worse repeatability. Therefore, H7-53TM was chosen as ELISA antigen. The results of the Receiver operating characteristic curve (ROC curve) analysis show that the established ELISA could accurately discriminate between H7N9 subtype avian influenza virus-positive and -negative serum specimens. Based on correlation, the established ELISA had significantly strong correlation with HI assay to test 134 chicken serum specimens (r=0.854 6, P<0.000 1), and the established ELISA had significantly correlation with HI assay to test sera collected from chickens vaccinated with vaccine strains belonging to three different clades (r>0.5, P<0.05). [Conclusion] TM replacement can increase the repeatability of the HA protein to detect H7N9 avian influenza virus antibodies, and establishes an indirect ELISA for detecting specific antibodies against H7N9 subtype avian influenza viruses belonging to different clades applied with HA containing a replaced transmembrane domain.

Abstract:[Objective] Brucellosis (Brucellosis) referred to as cloth disease, is caused by Brucella to livestock-based zoonotic infectious diseases, causing serious public health problems. At present, the main method of eliminating the disease around the world is the combination of culling and immunization, so the establishment of rapid and accurate diagnostic methods for the prevention and removal of brucellosis is necessary. We establish a diagnostic method of fluorescence polarization (FPA) for brucellosis and provide a scientific and efficient method for diagnosis of brucellosis. [Methods] In the present study, the conjugate of lipopolysaccharide O-chain (OPS) and fluorescein isothiocyanate (FITC) was purified from purified S2 strain of Brucella spp. As an antigen, and the optimal dilution, dilution concentration, reaction conditions, the results to determine parameters such as the initial establishment of the Brucella fluorescence polarization diagnosis method. The results showed that the sensitivity and specificity of the positive serum of 148 bovine serum (including 70 bovine serum and 78 goat serum) and 155 negative bovine serum (including 82 bovine serum and 73 goat serum) were determined by this method. The intra-assay and inter-assay reproducibility was assessed using the controlled positive and negative serum assay kits. At the same time, 400 samples of bovine serum samples were detected by FPA kit and commercial kit, and the coincidence rate was compared. [Results] The optimal conditions of each component in the kit were: the best sample dilution was 0.5% sucrose phosphate buffer; the concentration of the labeled antigen was 90 μg/mL; the best reaction time was 3 to 5 minutes; δmP value of <20, was negative, δmP value of ≥20, was positive. The sensitivity of the method was 98.6% and the specificity was 98.7%. The comparison of 400 clinical samples showed that the FPA method established in this study coincided with the import commercialization kit 94.0%. [Conclusion] The method of fluorescence polarization diagnosis with good specificity and sensitivity was established.

Abstract:[Objective] Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. PDCoV outbreak was first reported in the United States swine in 2014, and has subsequently been reported in many Asian countries. Nowadays, PDCoV has been becoming a great threat to swine industry worldwide. Using the recombinant nucleocapsid (N) protein expressed in E. coli Rosetta(DE3) as the coated antigen to establish an indirect ELISA for the detection of PDCoV antibody, providing a useful tool for antibody detection and epidemiology surveillance of PDCoV. [Methods] The full-length cDNA of PDCoV N gene was amplified by RT-PCR based on the PDCoV strain CHN-HN-2014. The RT-PCR production was inserted into prokaryotic expressing vector pET-30a to generate a recombinant plasmid pET30a-N. Then pET30a-N was transformed into E. coli Rosetta(DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expressed N protein was purified and used as coated antigen to develop an indirect ELISA for detecting antibodies against PDCoV. After the specificity, sensibility, and repeatability of the developed ELISA method were evaluated, this method was used to test clinical serum samples. [Results] SDS-PAGE and Western blotting analysis showed that the N protein was efficiently expressed in supernatant and was specific reactions with antiserum against PDCoV. The established N-ELISA was highly specific, sensitive and repeatable. A total of 148 serum samples collected from pigs with a known immunization history and 102 serum samples collected from pigs with unknown PDCoV status were detected using both N-ELISA and neutralization test. The results showed that the positive agreement rate was 88.99%, the negative agreement rate was 92.90%, and overall coincidence rate was 91.20%. The positive rate of 267 clinical serum samples examined by N-ELISA was 66.67%. [Conclusion] The established ELISA method can be used as a tool for antibody detection and epidemiology surveillance of PDCoV.

Abstract:[Objective] African swine fever virus (ASFV) can cause high mortality in swine and lead to huge economic losses. Therefore to establish a strict and efficient control system, including the sensitive and accurate diagnosis methods, effective warning mechanism, which avoid the spread of ASF in China. The aim of this study was to develop a novel and high-sensitive droplet digital PCR (ddPCR) method to detect African swine fever virus (ASFV). [Methods] The methods of ASFV real-time quantitative PCR (qPCR) and ddPCR were established and optimal reaction conditions were confirmed based on K205R gene of ASFV. Each method was evaluated for linearity, limit of detection and specificity. The methods were tested in 163 specimens which were collected from domestic or imported clinical sample or serum samples. [Results] The results indicated that the method both had a high degree of linearity (R2≥0.998). The detection limit of ddPCR reached 0.36 copies and was approximately 10 copies/reaction, which was approximately a 10-fold greater sensitivity than qPCR. The cross-reaction was performed with other porcine pathogens, and negative amplification of the cross-reaction assay demonstrated the high specificity of this method. [Conclusion] This high sensitivity and specificity method could be used as an efficient molecular biology tool to diagnose ASFV, which is a reserve technique and very important for prevention of the spread of diseases across borders, and it promotes the development of ddPCR in China.

Abstract:[Objective] With the development of Chinese dairy industry, hay demand is increasing. As an natural forage, hay can be a carrier of livestock pathogens. The aim of this study was to investigate the bacterial community structure and the characteristics of pathogenic bacteria in hay. [Methods] We selected six different dairy farms to take hay samples. The Illumina MiSeq sequencing with 16S rRNA gene V3?V4 variable region was adopted to analyze the surface bacterial community structures of commonly used hay in dairy. [Results] A total number of 15 416 operational taxonomic units (OTUs) were obtained from hay samples under the similarity level of 97%, including 29 phyla, 87 classes, 144 orders, 219 families, and 323 genera. Microbial diversity analysis showed that hay samples had high bacterial diversity, and the diversity corresponding to each samples was specific. The results suggested that the abundance of pathogens in the natural hay was higher than that in the hay from artificial planted grasses. [Conclusion] The diversity, abundance, and characteristics of the main pathogens in hay samples were analyzed in this study, which would be a helpful reference for disease prevention and control in dairy farms.

Abstract:[Objective] Exploring the interaction between porcine reproductive and respiratory syndrome virus (PRRSV) nsp11 and host cellular proteins is important for revealing the function of nsp11 in viral replication. [Methods] The host cellular proteins that interact with nsp11 were screened by immunoprecipitation combined tandem mass spectrometry and analyzed by GO annotation, COG annotation as well as KEGG pathway annotation. The screened host cell protein IRAK1 was selected, and then the interaction between nsp11 and IRAK1 was determined by co-immunoprecipitation and confocal microscopy assays. [Results] Compared with the control group, there were 3 differential bands in PRRSV-infected group and 201 host cellular proteins were identified on these differential bands by further LC-MS/MS analysis. These host cellular proteins are closely related to protein metabolism, transduction of cell signaling pathways and pathogenicity of pathogens. Based on the bioinformatics analysis, host cellular protein IRAK1 was identified to interact with the nsp11. [Conclusion] This study identified the host cellular proteins that can interact with PRRSV nsp11, and bioinformatics analysis showed that these proteins play crucial role in virus replication and pathogenesis. The results indicate the direction of the study of nsp11, and also provide a foundation for elucidating the associated molecular mechanisms of the interaction of host cellular proteins with viral proteins in regulating the viral replication and pathogenesis.

Abstract:[Objective] With the gradual prohibition of the use of antibiotic growth promoters (AGPs) in animal feed, studies on AGPs alternatives are becoming hot topics. As the bile salt hydrolase plays a key role in lipid metabolism, it has become an important direction in AGPs alternatives. This study is aimed to determine the difference in enzymatic properties of bile salt hydrolase from chicken and porcine Lactobacillus based on the prokaryotic expression and purification. [Methods] The encoding genes of chicken bile salt hydrolase (BSHc) and porcine bile salt hydrolase (BSHp) were expressed in E. coli and purified by His-tag affinity column chromatography. The purified products were used to identify the BSH kinetic properties by hydrolyzing the six glycoconjugated and tauroconjugated bile salts. Effects of temperature, pH and metal ion compounds on the BSH activity were also determined respectively. [Results] BSHc and BSHp displayed higher catalytic efficiencies on glycoconjugated bile salts than that of tauroconjugated bile salts, while BSHc had a slightly higher hydrolysis activity on glycoconjugated bile salts than BSHp. The higher enzymatic activity of BSHc and BSHp were observed at the temperature of 45 °C and 42 °C, respectively. The optimal pH for BSHc and BSHp were 6.0 and 5.4, respectively. The metal ion compounds containing Cu2+, Fe3+, Mn2+, and Zn2+ displayed different degrees of inhibition on BSHc and BSHp activity, especially higher inhibition observed in Cu2+ and Fe3+ compounds. The inhibition of the compounds containing Na+, K+, Mg2+, and Ca2+ on BSHc and BSHp activity was relatively weak or no inhibition, but KIO3 had a strong inhibitory effect on BSHc and BSHp activity, KI and CaCl2 also had strong inhibitory effects on BSHp activity. [Conclusion] Based on prokaryotic expression and protein purification, BSHc and BSHp displayed higher catalytic efficiencies on glycoconjugated bile salts than that of tauroconjugated bile salts. The optimal temperature and pH for BSHc was higher than that for BSHp. The metal ion compounds containing Cu2+, Fe3+, Mn2+, and Zn2+ displayed significant inhibition on BSHc and BSHp activity. The results will be helpful for identification of BSH inhibitors and development of AGPs alternatives.

Abstract:[Objective] Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens that severely affect pork industry worldwide. As a reversible post-translational modification, sumoylation plays an important role in regulating viral replication. Protein inhibitor of activated STAT1 (PIAS1), as a member of SUMO E3 ligase PIAS family, can promote sumoylation of target protein, which affects the function of target protein and further participates in the transcriptional regulation process. This study was designed to study the mechanism of interaction between PIAS1 and PRRSV N protein, as well as its effect on N protein sumoylation and viral replication, to provide a scientific basis for further understanding the replication regulation and pathogenesis of PRRSV. [Methods] The interaction between PRRSV N protein and PIAS1 was first verified by yeast two-hybrid, Co-IP and confocal immunofluorescence assay. And then increased dose of PIAS1 were exogenously transfected to observe whether PIAS1 could mediate sumoylation of N protein. The effect of PIAS1 on PRRSV replication was investigated by testing the virus titer in cells with increased/reduced PIASI by using lentiviral transduction or RNA interference technology. [Results] PIAS1 could interact with the PRRSV N protein, and both of them predominantly located in the cytoplasm. Exogenous transfection of PIAS1 did not increase the sumoylation level of N protein. However, the overexpression of PIAS1 could promote PRRSV replication in MARC-145 cells. [Conclusion] PIAS1 has the function of promoting PRRSV replication.

Abstract:[Objective] Avian pathogenic Escherichia coli (APEC) leads to significant economic losses in poultry production, and affects public health. pagP gene plays key roles in pathogenicity and antimicrobial peptide resistance of bacteria. However, the function of pagP in APEC is still unknown. To determine the antibacterial peptide resistance and pathogenicity of pagP mutant strain, APEC pagP mutant strain was constructed. [Methods] We constructed APEC-pagP mutant strain with red recombination system, and the corresponding complementary strain was also constructed. We detected the effects of PagP in cell adhesion and invasion, biofilm-forming ability, outer membrane permeability, antibacterial peptide sensitivity and pathogenicity. [Results] pagP mutant strain and complementary strain were successfully constructed. The antimicrobial peptide resistance tests showed that pagP mutant strain was more sensitive to Polymyxin B and AVBD2 (P<0.01). The pathogenic test showed a significantly reduction of toxicity in pagP mutant strain (P<0.01). [Conclusion] pagP gene in APEC played important roles in resistance to AVBD2 and pathogenicity in APEC.

Abstract:[Objective] It is important to study the pathogenesis and molecular mechanism how Brucella interacts with host cells. Many researches focus on the regulation of endoplasmic reticulum (ER) stress on the infection and inflammation. Brucella species replicate within host cells in the form of ER-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain unclear. To reveal the effect of B. suis S2 on ER stress and the excretion of cytokines in RAW264.7 cells. [Methods] We built an infection model of B. suis S2 using RAW264.7 cells, and detected the expression of GRP78 and CHOP, the makers of endoplasmic reticulum (ER) stress, through real time quantitative PCR (qPCR) and Western blot. We measured cytokines TNF-α, IL-1β and IL-6 via qPCR and ELISA at different intervals after infection. [Results] The optimal MOI used in the infection model was 100:1. Most B. suis S2 were killed during the first 4 to 6 h after they invaded into RAW264.7 cells, then the survived bacteria could reproductive, inducing the apoptosis at 24 h, and necrosis at 48 h. The invasion and reproduction of B. suis S2 increased the expression of GRP78 significantly, causing ER stress of RAW264.7 cells, and decreased the expression of TNF-α, IL-1β and IL-6. [Conclusion] ER stress was involved in the control of B. suis S2 infection in RAW264.7 cells.

Abstract:[Objective] Listeria monocytogenes, an important food-borne pathogenic bacteria, often causes human and animal diseases. The cell wall surface proteins with an LPXTG motif play an important role in the pathogenic process of L. monocytogenes. According to the whole sequence of the reference strain, the function of some proteins among the 41 predicted LPXTG motif proteins is unknown. The aim of this study is to clone and bioinformatics analysis of the gene of LPXTG motif protein Lmo0160 of L. monocytogenes LM90SB2 isolated from diseased sheep in Xinjiang. It could provide the foundation for functional verification. [Methods] A pair of specific primers was designed according to the predicted sequence of L. monocytogenes lmo0160 gene in GenBank. The lmo0160 gene was amplified by PCR, and the amplified products were cloned into pMD19-T vector and verified by PCR, restriction endonuclease digestion and sequencing. In addition, the bioinformatics on nucleotide sequence of lmo0160 gene of LM90SB2 strain as well as putative protein structure had been analyzed. [Results] The sequence length of LM90SB2 lmo0160 gene was 1 708 bp, containing a 1 428 bp open reading frame (ORF) encoding 475 amino acids. The nucleotide sequence similarity of lmo0160 gene of LM90SB2 strain was 99.0%?99.1% with FSAN008100 strain (serotype 4b, USA), CFSAN023463 strain (serotype 4b, USA), J2-064 strain (serotype 4b, USA), F2365 strain (serotype 4b, cheese isolate, USA) and NTSN strain (serotype 4b, sheep brain isolate, Yangzhou, China), 97.2% with M7 strain (serotype 4a, cow’s milk isolated, Zhejiang, China), and 87.2%?91.1% with Finland 1998 strain (serotype 1/2a, USA), N53-1 strain (serotype 1/2a, fish isolate, Denmark), N1546 strain (serotype 1/2a, cooked ham isolate, Switzerland) and EGD-e strain (serotype 1/2a, USA). Its putative amino acid sequence similarity was 91.8%?99.4% with above strains. The phylogenetic tree showed that the lmo0160 gene of LM90SB2 strain was closely relative with CFSAN023463, F2365 and NTSN strains which were in the same branch. However, it was genetically far to the international standard strain EGD-e. The secondary structure prediction showed that LM90SB2 Lmo0160 was hydrophilic protein without signal peptide and transmembrane structure. The protein domain prediction showed that Lmo0160 protein had collagen binding domain and collagen-binding surface protein Cna-like, B-type domain (Cna B). [Conclusion] The lmo0160 gene of LM90SB2 strain was successfully cloned. The cloning and analysis of lmo0160 gene provide an important foundation for further study of lmo0160 gene.

Abstract:[Objective] Fowlpox virus (FPV) is a member of Avipoxvirus. FPV is widely used as a living virus vector in poultry and mammals because of its large genome and containing large numbers of replication non-essential regions. The selection of recombination sites is a prerequisite to construct fowlpox virus vectors. The insertion of foreign genes that do not affect viral replication is a prerequisite for selection of the insertion sites. Therefore, identification of replication non-essential regions for foreign gene insertion would provide additional options to construct recombinant viruses. This study aimed to identify the necessity of thymidine kinase (TK) gene in the replication of FPV NX10. [Methods] The recombinant virus rFPV-ΔTK-EGFP was acquired by homologous recombination using FPV NX10 strain as parental virus and enhanced green fluorescent protein (EGFP) as selection marker. The bromodeoxyuridine (BUdR) was added to the culture of CEF cells to identify the role of TK in FPV replication. [Results] The transfer vector pUC19-TK AB-EGFP was constructed. Although the green fluorescent lesions increased in the cloning and purification of recombinant virus, non-fluorescent lesions can still be observed at the edge of the fluorescent plaque. Western blot analysis of the randomly selected plaque clones after 9 to 15 rounds showed that the EGFP gene inserted in the recombinant virus could be expressed correctly, but PCR results showed that the wild-type viruses accompanied with the recombinant virus. After adding BUdR to the cell culture medium, the recombinant virus could not continue to replicate. [Conclusion] The TK gene of FPV NX10 strain is partly essential for replication.

Abstract:[Objective] Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, leading to great losses for the pig industry worldwide. Adhesion is the first step in the pathogenic process of APP. The newly discovered trimeric autotransporter adhesion (TAA) is an important pathogenic virulence factor of APP, and the 2 464 to 2 574 amino acids of TAA stalk region, named BD3, plays an important role in the bacterial adhesion. However, it is still unclear how APP combines with lung tissues. We express the stalk functional region of TAA, screen and analyze its binding protein in swine lung cells. [Methods] BD3 protein was expressed in prokaryotic expression system, purified, then incubated with swine lung tissue protein. The binding protein of BD3 was picked out by co-immunoprecipitation and analyzed by mass spectrometry. The cDNA library was constructed to get the sequence of the binding protein and then the bioinformatics method was done. [Results] Mass spectrometry analysis revealed that the peptide sequence of the binding protein, and the blast result in the database showed that Keratin 1 was the binding protein. cDNA library was constructed to know the sequence of swine Keratin 1. Bioinformatics analysis showed that low sequence homology of Keratin 1 existed between swine and human and it was a separate evolutionary branch, different from other keratins of swine or human, Keratin 1 had a transmembrane domain at 8 to 100 aa and the secondary structure mainly consisted of α-helix and β-fold. It contained α subunit of G protein at 82 to 362 aa and a TSP1 domain (thrombin-sensitive protein type I repeat domain) at 515 to 552 aa. [Conclusion] The finding of Keratin 1 that is in swine lung cells and binds with BD3 of TAA, lays a foundation for researching the mechanism of TAA adhesion to lung tissue, and has great significance to understand the pathogenic mechanism of APP specially colonized in lung tissues.

Abstract:[Objective] Newcastle disease virus (NDV) is one of the most serious infection problems in China in the last two decades, resulting in huge economic losses for the poultry industry. The genetic evolution of NDV strain is a very important topic for controlling ND. In this study, the evolution of NDV under the immune pressure of vaccine was further assessed by comparing the molecular characteristics and genetic variation frequency of F and HN genes of different genotypes of NDV. [Methods] NDV sequences of F gene and HN gene including 89 NDV isolates in our laboratory, 364 NDV pandemic strains worldwide and 15 classical strains, were collected and downloaded from the GenBank, and further genetically analyzed to determine their evolution, molecular characteristic and substitution rate by using Lasergene 7.1 and MEGA 5.1 software. [Results] Phylogenetic tree analysis suggested that NDV evolved into 15 distinct genotypes from genotype I to XV. Most NDV pandemic isolates during 1997?2015 belonged to sub-genotype VII d, suggesting that this genotype was the dominate isolate in China in the last two decades. Furthermore, comparison based on homologies of the nucleotide and amino acid sequence of F and HN gene of NDV showed that different genotypes had their own distinct characteristics, and significant accumulation of amino acid variation was also found. In addition, in comparing F and HN gene with reference Go/GD/QY/1997, which was the first genotype VII in China, average annual substitution frequencies of NDV pandemic strain nucleotide (amino acid) were 2.31×10?3 (2.26×10?3) and 3.37×10?3 (2.35×10?3), respectively. Substitution rates of F and HN during 1997?2001 were 4.72×10?3 and 8.28×10?3, higher than that during 2002?2015 (1.6×10?3 and 1.84×10?3), when the genotype VII vaccine was initially applied in the field. [Conclusion] Bioinformatics analysis proved that matching NDV vaccine strain with epidemic strains in genotype is an important factor in mitigating NDV variability.

Abstract:[Objective] Infectious bronchitis virus (IBV) that causes respiratory and nephritic diseases in chicken is one of the important viral pathogens in poultry industry. To understand the epidemiology and recombination of IBV in China. [Methods] S1 gene sequences of 92 IBV strains and the genomic sequences of 55 strains isolated during 2002?2016 in China were collected from GenBank and analyzed by bioinformatics. [Results] Thirteen genotypes of IBV, including QX, 4/91, Mass, tl/CH/LDT3/03, CK/CH/LSC/99I, TW-I, TW-II, TC07-2, Ck/CH/LDL/97I, N1/62-associated, Arkansas, New-I and a newly emerging genotype New-II, were found co-circulation in China during 2002?2016 based on phylogenetic analysis of S1 gene. It was notable that ck/CH/LSD/110712 belonging USA branch was found in China in this study. Recombination analysis revealed the S1 gene of New-II genotype IBVs was originated from recombination between tl/CH/LDT3/03- and QX-type IBVs. With analysis of whole genomes of 55 IBV isolates, 52 recombinants were identified through RDP4. The partial sequence of vaccine strains were found in 25 of 52 IBV isolates. The recombination events were further confirmed by SimPlot bioinformatics analysis. [Conclusion] Bioinformatics analysis results demonstrated that multiple genotypes of IBV were co-circulated in China during 2002?2016, and novel IBV genotypic clusters and variants could be emerged through recombination which frequently occurring between field and vaccine strains. It indicates the adverse effects of IBV vaccine on controlling IB in China should be paid more attention.

Abstract:[Objective] To study changes of piglet’s jejunum pathology and jejunum flora caused by porcine circovirus 2 type (PCV2) infections. [Methods] The viremia of three piglets infected with PCV2 by oral and intramuscular injection was determined. Then six weaned pigs, being randomly divided into PCV2 infected group and the control group (n=3), were infected with PCV2 with the same route of infection. The three piglets of each group were slaughtered at 21 d after infection. Jejunum and its contents were collected for microscopic pathology and flora sequencing. [Results] The virus loads of the piglets’ serum achieved a higher level at 21 d after PCV2 infection. The jejunum villous of piglets in the infection group atrophied and fell off. The microbial species and abundance in these jejunums changed, and jejunum flora diversity increased, including the significant decrease of Lactobacillus content and the significant increase of Pseudomonas genus content. [Conclusion] PCV2 infection results in certain changes in piglet’s jejunum flora. A decline in the jejunum beneficial bacteria and the increase of harmful bacteria may further aggravate the piglet jejunum inflammation.

Abstract:[Objective] Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a worldwide important zoonotic pathogen that causes foodborne infections in humans. Type I fimbriae are important bacterial adhesion organelles present in various types of pathogenic E. coli (e.g. Uropathogenic E. coli) that facilitate bacterial colonization. However, EHEC O157:H7 cannot express type I fimbriae because of base deletion in the fim operon. BLAST analysis shows that the open reading frame z3276, a specific genetic marker of EHEC O157:H7, encodes a sequence with high amino acid identity to other E. coli type I fimbriae. It is possible that z3276-encoding protein is a compensatory mechanism for type I fimbriae, but its definitive function in EHEC O157:H7 remains unclear. We explore the biological function of z3276 gene. [Methods] We targeted the reference EHEC O157:H7 86-24 strain to construct a z3276 mutant (?z3276) and its complement strain (C?z3276), and the biological characteristics and pathogenicity of ?z3276 in mice were compared with the parental strain. [Results] Motility and biofilm formation assays revealed a smaller twitching motility zone for ?z3276 on the agar surface and significantly decreased biofilm formation by ?z3276 compared with the parental strain. The adhesion and invasion ability of ?z3276 to HEp-2 cells showed no significant change, but the invasion ability of ?z3276 to IPEC-J2 cells was attenuated. During mouse colonization, we observed shortened and lower fecal shedding for ?z3276 compared with the parental strain. [Conclusion] Thus, z3276 appears to be a crucial virulence factor of EHEC O157:H7.

Abstract:[Objective] Infectious bovine rhinotracheitis (IBR) is caused by BoHV-1 and there is no commercialized marker vaccine against IBR in China. Based on the previously constructed BoHV-1 gG?/tk? gene-deleted vaccine in this lab, the glycoprotein gD extracellular region sequence which served as the best immunogenicity in BoHV-1 and BoHV-5 was further inserted into the site where tk is located and recombinant virus strains BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ were successfully constrcuted. This paper aimed to evaluate the immunogenicity and safety of recombinant virus strains in rabbits and protection against both BoHV-1 and BoHV-5 for development of more effective vaccines against BoHV-1 and BoHV-5 infection. [Methods] The 30 rabbits were divided into 6 groups and vaccinated and challenged by nasal inoculation. After strains were vaccinated, their safety was evaluated by clinical symptoms, temperature, virus shedding. Then the rabbits were challenged with either wt BoHV-1 or wt BoHV-5 at the 28th day after vaccination. The protection was evaluated by clinical signs, temperature, virus shedding, histopathology, isolation and identification of virus. Besides, the immunogenicity was studied by neutralization test, indirect-ELISA and specific PBMC proliferative response. [Results] The rabbits vaccinated with BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ didn’t show clinical signs, nasal virus shedding and viral survival in lung tissues. One rabbit vaccinated with BoHV-1 gG?/tk? had viral survival in lung tissues. After challenge, both strains BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ could diminish the clinical signs and nasal virus shedding and maintain the good structure of lung tissues indicating the protection against BoHV-1 and cross-protection against BoHV-5 were improved. Besides, BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ induced stronger neutralization and ELISA antibodies and PBMC proliferation compared with BoHV-1 gG?/tk?. [Conclusion] BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ are safe and could induce higher levels of immune response than BoHV-1 gG?/tk? strain.

Abstract:[Objective] Salmonella is an important zoonotic pathogen and the main cause of gastrointestinal disease worldwide, causing about 21 million cases of typhoid fever in the world each year, poses a serious threat to the world public health. The current serious problem of antibiotic abuse urges to find a substitute for antibiotics. The antimicrobial peptide JH-3 gains broad-spectrum bactericidal activity after being separated and artificial transformed by our group. In this study, we studied the therapeutic efficacy of antimicrobial peptide JH-3 to treat Chinese multi-drug resistant Salmonella CVCC541 infection in a mouse model. [Methods] In the prevention group, BALB/c mice were continuously intraperitoneally injected with antimicrobial peptide JH-3 or ciprofloxacin (CPFX) for 3 days before (B3d, total 40 mg/kg) Salmonella CVCC541 infection; while the drugs were continuously intraperitoneally injected for 3 days after (P3d, total 40 mg/kg) Salmonella infection in the therapy group. [Results] The ciprofloxacin in prevention group showed the best preventive effect, antimicrobial peptide JH-3 (B3d) also showed effective prevention against Salmonella infection, significantly protected mice from the lethal doses of CVCC541 infection as survival rates was 100% in this group. The clinical score, pathological changes of small intestine and the bacterial burdens in spleen and in other organs were decreased to that of the ciprofloxacin prevention group, suggesting JH-3 has the similar preventive function with ciprofloxacin. The therapeutic effect of JH-3 was poor, though the clinical score, pathological changes of small intestine and the bacterial burden were significantly higher in JH-3 treatment group (P3d) when compared to JH-3 prevention group (B3d), the survival was 70%, which was significantly higher than that of the untreated infection group. [Conclusion] This study systematically evaluated the therapeutic effect of antimicrobial peptide JH-3 on Salmonella infection. Our results showed prophylactic administration of JH-3 gains the best antibacterial effect, which is equivalent to ciprofloxacin treatment, providing a reference for the research and development of novel antibiotics.

Abstract:Exosome is a kind of vesicles secreted by cells. It has been thought to be a new important way of cell and cell communication due to its carrying some molecules such as protein, lipid and nucleic acid. RNA viruses, such as HIV-1, HCV, as a class of important pathogens affect human health. Viruses can promote their replication and transmission using certain relevant functions of the exosomes, however, the relevant study of the interaction of exosome and virus infection has just been started. Many aspects of the exosomes have not been fully recognized and still many studies need to be done. In this paper, we summarize the role of exosome in the promotion of various RNA viruses infection to provide insight into the relationship between RNA viruses and exosomes.

Abstract:Classical swine fever (CSF) caused by classical swine fever virus (CSFV), is a highly contagious disease of swine that is characterized by hemorrhage syndrome and immunosuppression. It is one of the most important swine diseases worldwide and leads to significant economic loss in the pig industry in China and other regions of the world. Studies have shown that infection with CSFV can induce host innate immune response, but this virus can suppress the host innate immunity through influencing the expression of effector molecules of the innate immune system. In this review, we highlight the current understanding of host innate immune response and immune suppression during CSFV infection. In addition, we review the mechanisms by which CSFV infection induces innate immunity and its suppression.

Abstract:Bacteria could sense the signal molecules and regulate their physiological behavior via quorum sensing (QS). QS is involved in the regulation of biofilm formation, virulent genes expression as well as the phages infection. QS could control adaptive immunity through the regulation of CRISPR-Cas systems, which is the latest finding and presumed to be the highlight in the near future. To elucidate its regulating mechanisms and reveal broad prospect for bacterial therapy by phage, we reviewed QS regulating mechanisms on bacterial pathogenicity for the control of pathogenic bacteria.

Abstract:Listeria monocytogenes, an important zoonotic foodborne pathogen, encounters acidic environments such as silage, fermented food, stomach and phagolysosomes. It contains a number of enzyme systems to deal with acid stress: F0F1-ATPase, glutamate decarboxylase, arginine deiminase and agmatine deiminase. The bacterium could maintain its intracellular pH (pHi) homeostasis when exposed to environmental pH (pHex) 4.5, survives well in pHex 3.5. Preexposure of L. monocytogenes cells to mild acid stress (pHex 4.5) could induce acid tolerance response (ATR) that could render them more resistant to fatal acidic stress. SigB (σB) is a positive regulator of ATR, enabling the bacterium to better cope with environmental stresses. Therefore, σB could be a target for development of novel antibacterial drugs. Stringent control of L. monocytogenes contamination in fermented food is of great importance to minimize the risk of listeria infections to consumers.

Abstract:Seneca valley virus (SVV) belongs to Senecavirus genus of family Picornaviridae, and is classified as the unique member of Senecavirus genus. SVV was identified in 2002 while cultivating viral vectors in PER.C6 cell culture and was suggested to be contaminated by fetal bovine serum or porcine trypsin. Many studies about SVV at the initial time have focused on the potential oncolytic activity of SVV in human cancer therapy. It is now identified as a pathogen to infect pigs and cause porcine idiopathic vesicular disease. The clinical signs include: cutaneous vesicular lesions on the snout and coronary bands, lameness, anorexia, lethargy and fever. These clinical signs are similar with that caused by foot-and-mouth disease, swine vesicular disease and vesicular stomatitis. Therefore, these diseases are indistinguishable by clinical signs. In 2015 to 2017, SVV infection occurred in the United States, China, Thailand and many other countries, with increasing spread and outbreaks. Therefore, urgent surveillance and control policies are needed to limit the spread of SVV. Several new diagnostic and control methods have been exploited. In the present review, we summarized the recent spread information, diagnostic and control methods of SVV to provide theoretical foundation for controlling of this disease.

Abstract:Many Gram-negative bacteria adhere to the surface of the host cell by means of the type III secretion system, and then inject the specific protein into the host cell across the membrane, destroying a variety of signal pathways in the host cell, which is beneficial to bacterial infection and colonization. In enteropathogenic Escherichia coli (EPEC), in addition to the intestinal cell shedding site (locus of entericyte effacement, LEE) virulence island encoded type III secretion system (T3SS), in the analysis of intestinal hemorrhagic Escherichia coli O157:H7 of the genome sequence found in a new type III secretion system, E. coli type III secretion system 2 (E. coli type III secretion system 2, ETT2) pathogenicity island. Studies have shown that ETT2 may not have a complete secretion system function in most strains, but it has an important effect on the virulence of bacteria. Therefore, this paper briefly reviews the genetic characteristics of E. coli ETT2, the distribution and prevalence of ETT2, the function and mechanism of ETT2 and other aspects of the main research progress.

Abstract:Classical swine fever (CSF) is a kind of infectious disease which could induce acute, febrile and fatal disease in swine. This disease displays with wild prevalence and high mortality, and could lead to significant damage for the pig industry all over the world. At present, most CSF epidemic countries or areas still prevent the CSF virus (CSFV) infection mainly by the vaccination of CSFV live-attenuated vaccine (LAV). However, the traditional LAV could not differentiate the CSFV antibody of vaccinated pigs from infected ones, so it is very urgent to develop the new tag vaccines to purge and even eliminate CSF. In recent years, many domestic and international researchers continuously modify the genome of CSFV wild strains or attenuated strains by molecular biology and genetic engineering methods to construct new LAVs allow differentiation between infected and vaccinated animals (DIVA) , and most of which are established based on Erns and E2 genes. Among these DIVA vaccines, some candidate vaccines could produce good immune effects and could be used to diffrentiate naturally infected pigs from vaccinated ones, and they are hopeful to replace the traditional attenuated strain vaccines in the future.

Abstract:CRISPR-Cas system is an acquired immune defense system that is found in some bacteria and most of the ancient bacteria. It makes the bacteria immune defense against the invasion of exogenous genes. In addition, CRISPR-Cas system plays a regulatory role in the formation of bacterial biofilm, drug resistance, virulence and other physiological functions, as well as in scientific research. According to this, we discussed the bacterial CRISPR-Cas system and its related research on the immune defense. Moreover, we described the regulatory function of this system in physiological function of bacterial and its future application, to provide a new idea for further study on bacterial resistance and pathogenicity.

Abstract:Microbes have the characteristic of high diversity in natural environment, but 99% of microorganisms remain “unculturable” by using traditional methods. Besides, the culturable fraction can’t represent the diversity of micro-ecology. Therefore, the present tasks are to identify species and functions of new microorganisms. This article reviews some success in the isolation and cultivation of unculturable bacteria from complex bacterial communities and their functions researches, including modifying medium, microbial interactions, capture, isolation and cultivation and high-throughput cultivation.

Abstract:African swine fever (ASF) is caused by ASF virus (ASFV), and it is a highly contagious and often fatal diseases of domestic and wild swine that can occur in peracute, acute, subacute and chronic forms. Following infection of swine, ASF is characterized by fever, high viremic titers, hemorrhagic lesions and, depending on the strain, high morbidity and mortality. ASF was introduced into Georgia in 2007, causing enormous economic losses and it has now been speard to many regions in Russia. On 27 March 2017, an outbreak took place in Irkutskaya Oblast, a region close to the largest land port in the North of China about 1 000 kilometers, which poses China a high risk of disease introduction. Here, in order to provide a reference to the prevention and control of ASF in China, the authors present a review on the epidemic situation and research progress of ASF in Russia.