The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor- mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN- was upregulated slightly at 5 and 9dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

中国兽医药品监察所国家猪瘟参考实验室是由农业部2002年公布的第一批国家参考实验室,2017年6月1日,世界动物卫生组织(OIE)确认中国兽医药品监察所猪瘟实验室成为新的OIE猪瘟参考实验室,该实验室是国内最早开展猪瘟(Classical Swine Fever,CSF)研究的权威实验室之一.按照国家重大动物疫病防控的需求以及OIE 参考实验室职能的要求,该实验室近年来承担了近22项国家和省部级的研究课题,研究经费达2000万元.

对四川省2009-2014年共2 954株猪、鸡源大肠杆菌进行13种抗生素耐药性测定,同时对耐头孢噻呋大肠杆菌及O157菌株耐药基因进行检测.结果表明:大肠杆菌对抗生素耐药谱广,多重耐药严重.大肠杆菌对抗生素耐药率呈逐年波动下降趋势,猪源大肠杆菌对抗生素耐药率总体耐药率为40.40％～ 92.32％,抗生素耐药率依次为:TET(92.32％)＞SIZ(91.96％)＞ AMP(91.25％)＞SXT(87.56％)＞DO(77.67％)＞FFC(73.97％)＞SH(69.91％)＞EN(66.00％)＞AMC(61.10％)＞ OFX (59.39％)＞CN (57.82％)＞ PME (42.53％)＞CEF(40.40％).鸡源大肠杆菌对抗生素的总体耐药率为9.17％-89.41％,抗生素耐药率依次为:SIZ(89.41％) ＞AMP (85.01％) ＞SXT(83.40％) ＞TET(83.01％) ＞EN(72.03％)＞OFX(63.37％) ＞AMC(62.14％)＞DO(58.90％) ＞FFC (55.10％) ＞SH (50.52％) ＞CEF (46.9％) ＞CN (33.91％)＞PME(9.17％).2009年-2014年猪源大肠杆菌耐药率总体高于鸡源大肠杆菌.研究表明,监测大肠杆菌的抗生素耐药性对动物的养殖、疾病的预防及耐药性的控制有着重要意义.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an epidemic etiology in pigs of all ages causing reproductive failure and respiratory manifestation. PRRSV has been circulating in Chinese pig farms for almost 20 years. The aim of the present study was to fully understand the extent of the genetic diversity and molecular characteristics of PRRSVs in Central China. A strain of PRRSV isolated from a recent outbreak farm in Hunan province in Central China, designated HUN-2014, was sequenced and analyzed with 39 other PRRSVs from 1998 to 2014 in Central China. Comparative results of genomic sequences revealed that all 40 PRRSVs belonged to the North American genotype (NA genotype) and shared 88.8-99.0% homology. Phylogenetic analysis showed three subgenotypes, namely conventional PRRSV (C-PRRSV), specially mutant PRRSV (S-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), in all 40 PRRSVs. Moreover, comparative analysis of amino acid (AA) sequences of NSP2, GP3, GP5 and ORF5a revealed the main evolution trend of PRRSVs in Central China from 1998 to 2014, which was from C-PRRSV to HP-PRRSV, accompanied by different evolving directions to S-PRRSV. In conclusion, both the major evolutionary trend and special features of genetic variation should be emphasized as theoretical basis for development of new vaccines and control strategies for PRRS.

10 lesioned lung tissues from typical swine mycoplasmosis were grinded,cleaned,and subjected to isolation,and a mycoplasma strain was obtained.The mycoplasma was proved to be Mycoplasma hyopneumoniae (Mhp)by culture character,serological identification,biochemical identification,PCR test and sequencing.The purefied isolated strain was named Mhp strain S.Afterwards,Homology comparison was performed on genetic sequence and amino acid sequence of P46 and P97 R1 region with the available sequence of other strains,the results show that the amino acid sequence of P46 gene is more than 99% homology with other strains,the number of P97 R1 region amino acid repeat sequence of the Mhp strain S is 11,different from other strains.The immunogenicity test results showed that the isolation strain had good immunogenicity.

选用猪、兔、鸡、小鼠和豚鼠5种试验动物分别制备猪肺炎支原体高免阳性血清,并测定了其对猪肺炎支原体的代谢抑制效价.再从兔高免血清中纯化特异性抗体,比较抗体纯化前后的代谢抑制效价是否发生变化.结果表明,5种试验动物高免血清对猪肺炎支原体的代谢抑制价分别为0、104、103、105、102,纯化后的抗体仍可抑制猪肺炎支原体的生长,代谢抑制价没有明显变化.由此可见,不同动物来源的高免血清对猪肺炎支原体的代谢抑制价不同,代谢抑制作用的主要作用物质为特异性抗体;首次证明病原靶动物——猪的高免抗血清对猪肺炎支原体没有代谢抑制作用,其他支原体是否也表现为对靶动物(或人)抗血清的耐受性还有待进一步研究.

为了比较两种猪瘟病毒抗体检测试剂盒对猪瘟疫苗免疫抗体的检测结果,本试验将27头猪瘟抗体阴性仔猪免疫猪瘟活疫苗后7、10、14、17、20、23、27、30、34 d共9个时间点采血,分别用两种猪瘟病毒ELISA抗体检测试剂盒进行抗体检测,同时采用OIE指定方法-荧光抗体病毒中和试验对检测结果进行验证.结果表明,虽然两种试剂盒均可在免疫后30 d 100％检测到免疫猪体内的猪瘟抗体,但国产试剂盒在疫苗免疫后第10天便可在6/21猪体内检测到猪瘟抗体,20 d时抗体检测全为阳性,而美国IDEXX公司的试剂盒在疫苗免疫后27 d才在11/21猪体内检测到猪瘟抗体,30 d时才全部变为阳性,而两种试剂盒对6头阴性对照猪血清连续跟踪检测34 d均为阴性.由此可以得出结论:国产试剂盒在疫苗免疫后的早期抗体检测中敏感性明显离子IDEXX公司的试剂盒.

为再评价猪瘟兔化弱毒疫苗的免疫效力,采用近年在我国流行的不同临床致病力和不同基因亚型的9株猪瘟病毒流行毒株,进行免疫保护效力研究.结果表明,以C-株疫苗种毒生产的猪瘟活疫苗(传代细胞源)对我国目前流行的猪瘟病毒高、中、低致病力毒株及不同基因亚型(1.1、2.1、2.2)流行毒株均具有坚强的保护力,且免疫猪接种不同流行毒株毒后不排毒.研究结果为我国继续使用猪瘟兔化弱毒疫苗进行全面免疫提供了重要科学依据.

Objective]In order to make researches on the RNA location and distribution of Classical Swine Fever Virus (CSFV)in infected PK15 cells, a rapid, accurate and sensitivein situ hybridization (ISH) technology was established.[Method]After comparison with the complete sequences of CSFV, BVDV and BDV to avoid the homology regions, a set of specific probes of CSFV RNA andβ-actin were designed and synthesized. A reference strain CSFV (HeBHH1/95) was used to optimize the ISH technology through comparison several parameters such as fluorescence intensity, repeatability, protease K concentration and formalin fixation time. After these parameters were determined, an ISH technology was established. Fluorescent antibody test (FAT) was used to compare the sensitivity with the ISH technology. All the sub genotypes of CSFV (sub genotype 1.1, 2.1,2.2 and 2.3) which are present in China and other normal pig infectious virus (BVDV, PPV, PRV,PCV-2) were used to detect specificity of this ISH technology. A high virulent strain of CSFV (SM) was inoculated in PK15 cells. Infected cells were sampled at 0.5 hours post inoculation (hpi)、1, 3, 6, 8, 10, 14, 18, 24, 36, 48, 72, and 96hpi. Then FAT was performed in parallel to detect the expression and location of CSFV E2 protein.[Result]CSFV RNA were detected in infected PK15 cells by using the ISH technology. The optimal concentration of protease K was 1:1 000 and the optimal time of formalin fixation was 30 minutes. The minimum of detection is 10-8/200μL which is 3.5 orders of magnitude higher than FAT. The specific tests showed that the ISH technology could react with sub genotypes 1.1, 2.1,2.2 and 2.3 of CSFV in China and has no cross reaction with BVDV, PPV, PRV,PCV-2 viruses. CSFV RNA ISH test results showed that CSFV RNA were firstly detected in nucleus at 0.5 hpi and gathered in nucleus from 0.5hpi to 6hpi; at 10hpi, there were more CSFV RNA gathered in cytolymph than before and less CSFV RNA were detected than before in nucleus;at 24hpi, CSFV RNA mainly gathered in cytolymph around nucleus; at 36hpi, more and more CSFV RNA gathered in cytolymph and got maximum at 72hpi; at 96hpi, the amount of CSFV RNA declined. The results of FAT showed that little E2 protein was detected in cytolymph at 8hpi. From 10hpi to 24 hpi, only few cells were detected positive. After 36hpi, the expression of E2 protein increased gradually and got to the maximum at 72hpi. But after 96hpi, the amount of E2 protein declined. The trend of E2 protein expression was in accord with CSFV RNA.[Conclusion] A visualizationin situ hybridization technology was firstly established to detect CSFV RNA. The location and distribution of CSFV RNA was studied by using this technology. The results proved that CSFV RNA enters cell early than 0.5hpi and CSFV RNA has ever existed in nucleus.

Here, we report the complete genome sequence of strain GD-2011, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that was isolated from a stillborn fetus. The GD-2011 strain is characterized by a discontinuous 30-amino acid deletion in the nonstructural protein 2. In addition, GD-2011 had a 1-amino acid insertion in glycoprotein 5, which does not exist in any other HP-PRRSV strains.

Alphaviruses belong to the family of Togaviridae, which are encapsulated by a capsid protein and possess single strand plus RNA. In recent years, the domestic and foreign research on alphavirus-based vectors is active and the alphavirus-based vectors have been used for vaccine research, gene therapy and molecular biology research. Based on the structural principle, the alphavirus vectors are classified into three main types:replication-competent vector, replication-deficient vectors and DNA-layered vector. In this review, the transformation, characteristics and application of three different alphavirus-based vectors are discussed in order to provide references for the development of safer, more effective alphavirus-based vectors.

对获得的91株鸡源奇异变形杆菌进行了11种抗生素敏感性测定,从中筛选出5株耐药性较高的耐药奇异变形杆菌.以奇异变形杆菌标准菌株为模型,进行了31种中药水溶性粗提物抑菌活性研究.以庆大霉素和青霉素为参照,采用管碟法对6种中药水溶性粗提物的抑菌活性进行效价估算.最终将6种中药水溶性粗提物作用于筛选出的耐药奇异变形杆菌,验证其抑菌活性.结果表明,31种中药中地榆、虎杖、大黄、黄芩、大青叶和绵马贯众对奇异变形杆菌标准菌株的抑菌效果最佳,对耐药奇异变形杆菌抑菌效果明显优于试验的11种抗生素.

为了从细胞水平探讨PRRSV GD强毒株和PRRSV弱毒疫苗接种仔猪后对其外周血淋巴细胞亚群的影响,运用血常规技术和流式细胞技术分析了猪体外周血淋巴细胞亚群的动态变化.将30日龄20头SPF猪分成3组,分别感染HP-PRRSV GD第5代强毒株、GDr180弱毒疫苗株及留作对照.于感染后第0、3、7、10、14、21、28及35天采血进行外周血淋巴细胞亚群测定,分析淋巴细胞亚群的变化.结果表明:HP-PRRSV GD强毒株感染可导致白细胞、淋巴细胞数量、单核细胞、粒细胞、B细胞、Tc细胞、Th细胞、Tm细胞、和γ6T细胞数量的下降;而弱毒疫苗GDr180株免疫则表现为白细胞、淋巴细胞、单核细胞、引起粒细胞、B细胞、Tc细胞、Th细胞和Tm细胞的上升,对γ6T细胞的影响不大;阴性对照组在整个检测期间各种细胞基本上保持稳定状态.从实验结果可以看出:与HP-PRRSV GD强毒株破坏免疫细胞不同,PRRSV弱毒疫苗GDr180株免疫接种能刺激猪免疫细胞增殖,给试验猪提供良好的免疫反应.

The potency detection method of C-strain vaccine based on classical swine fever virus rabbit infection dose( RID) is widely used in China, however, this method is greatly influenced by bread,individual difference and housing environment. In order to establish an alternative potency test strategy avoid using experimental animals, the permissive cell was inoculated with serial diluted vaccine, and then the antigen-capture ELISA, RT-nestPCR and traditional RT-PCR were utilized to detect the progeny virus. The highest positive dilution was termed as the indicator of the vaccine’ s potency. The method of cell infection dose( CID) was established. The results showed that the higher live virus particles in vaccine, the higher dose of cell infection virus. Results from the potency tests of different types of C-strain vaccines indicated that the alternative strategy could be applied to potency test of ST cell-line produced C-strain vaccine and bovine testicular cell produced C-strain vaccine. Potency tests of 12 batches of ST cell-line produced C-strain vaccines and 3 batches of bovine testicular cell produced C-strain vaccines were conducted with both the alternative strategy CID and the RID test parallel. The results showed, the positive dilution of all 12 batches of ST cell-line produced C-strain vaccine were 105 CID/dose, and 2.6×104~3.0×104RID/dose. And the positive dilution of 3 batches of bovine testicular cell produced C-strain vaccine were 104 CID/dose and 7.0×103~8.0×103RID/dose. The potency test results of the alternative strategy CID were at certain extent corresponding to that of RID test, and it could reflect the concentration of infectious virus particles in vaccine. The alternative strategy CID is of potentiality to replace the RID test.

In order to prepare antibody for detection of classical swine fever virus( CSFV) , the E2 gene of vaccine strain hog cholera lapinised virus ( HCLV ) was cloned into vector pFastBac1, through thransposon and transfection, a recombinant baculovirus carried HCLV E2 gene was constructed and recombinant E2 protein was expressed. After immuning rabbits with recombinant E2 protein expressed by baculovirus, the antibodies against CSFV was prepared. The results showed that the recombinant baculovirus could secret E2 protein, and the antibodies had good specificity and could be used to prepare detection reagent for CSFV.

The objective of this study was to investigate the prevalence of antimicrobial resistance in Salmonella from pig in Sichuan province. A total of 2660 samples were collected from the anus of pig in the pig farms in Sichuan during 2009 to2014. Totally,151 Salmonella strains were isolated. The results showed that the isolation rate was 5. 68% and the dominant serotype was Derby( 60. 26%). Antimicrobial susceptibility of Salmonella against 13 antimicrobials was performed using microdilution method. The highest resistance rate was observed in ampicillin( 94. 7%),amoxycillin / clavulanic acid( 92. 7%),spectinomycin( 93. 4%),tetracycline( 95. 4%),sulfagan( 91. 4%),trimethoprim and sulphame-thoxazole( 90. 1%),enrofloxacin( 86. 8%),while ceftiofur and polymyxin E were the most susceptible antimicrobials,followed by the other 4 antimicrobials. The frequency of resistance showed a high trend during 2009 to 2014 including ampicillin,amoxycillin / clavulanic acid,spectinomycin,tetracyclin,sulfagan,trimethoprim and sulphame-thoxazole,and showed a rising trend including ceftiofur,gentamicin,doxycycline,florfenicol,enrofloxacin,ofloxacin,polymyxin E,espeshially ceftiofur,doxycycline,florfenico and ofloxacin kept fastly rising trend. Besides,the multidrug-resistance strains reached a high level of more than 60% during 2009 to 2014. In conclusion,the results indicated that the drug-resistance of Salmonella strains was serious in Sichuan during 2009 to 2014. It was urgent that the domestic industry reasonably applied the antimicrobial drugs to control the development of antimicrobial resistance.

The outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) in 2006 devastated the Chinese swine industry. HP-PRRS virus is still the predominant strain in mainland China, rather than the classical PRRSV strain, and the attenuated live vaccine remains the preferred choice for protecting piglets against HP-PRRSV infection. To fully evaluate the safety of strain GDr180, the 180th attenuated virus of the HP-PRRSV strain GD, we used clinicopathological, microscopical, ultrastructural, serological and molecular biological methods to assess the different clinical manifestations and respiratory characteristics of piglets inoculated with HP-PRRSV strain GD or strain GDr180. The 5-week-old piglets inoculated with strain GD displayed marked clinical signs, including fever, anorexia, dyspnoea and tachypnoea. Significant interstitial pneumonia was present, characterized by thickened alveolar septa infiltrated with mononuclear cells and cell debris. However, the piglets inoculated with strain GDr180 and the negative control piglets showed neither clinical signs nor microscopical or ultrastructural lesions. Ultrastructural observation of the piglets' tracheas and examination of the dynamic tissue distributions of PRRSV strain GD and attenuated strain GDr180, by immunohistochemistry and fluorescence quantitative reverse transcription-polymerase chain reaction, confirmed significant differences in their pathogenicity and distribution in the respiratory systems of piglets. The differences in pathogenicity are attributable to the different severity of the pathological changes in the pigs inoculated with the two strains. Thus, the HP-PRRSV GDr180 strain is practically harmless to the respiratory systems of piglets and may be a safe candidate for inducing immunity against HP-PRRS.

To investigate the drug resistance of Proteus mirabilis,1 829 cotton swabs of cloacal samples were collected from the broilers in the appointed poultry farms.67 strains of Proteus mirabilis were obtained through isolation,purification,and identification,and the isolation rate was 3.66%.The 67 strains of Proteus mirabilis were tested for drug resistance using eleven kinds of antibiotics.The results showed that the Proteus mirabilis from the region generally possessed higher levels of drug resistance against the eleven kinds of antibiotics.The average resistance rates reached more than 80.00% in the nine kinds of drugs including colistin E,florfenicol,bacteresulf,enrofloxacin,cotrimoxazole,ampicillin,doxycycline,spectinomycin and tetracycline.The drug resistance against gentamicin was 62.69% and the drug resistance against ceftiofur was zero.The results indicate that Proteus mirabilis has a higher isolation rate in the broiler farms,and is one of the pathogens that need to be focused on monitoring in the clinic by veterinarian.