动物性饲料中沙门氏菌的分离鉴定

通过常规分离培养鉴定技术,对饲料厂成品饲料１０２份,鱼粉９７份、肉骨粉９８份进行了沙门氏菌的分离鉴定,结果从２９７份样品中到沙门氏菌４５株分离率１５．２％（４５／２９７）。成品饲料阳性１８份,阳性率１７．６％（１３／１０２）。鱼粉阳性１４份,阳性率为１４．４％（１４／９７）,肉骨粉１３４份,阳性率为１３．３％（１３／９８）。     

Dot-ELISA检查香肠中沙门氏菌的研究

以硝酸纤维膜为固相载体，利用沙门氏菌多价血清和辣根过氧化物酶（HRP）标记制备的羊抗兔IgG，成功地建立对香肠的Dot-ELISA检查法，同时，采用常规分离培养鉴定技术作对照试验。结果显示，在86份香肠中，用Dot-ELISA检出沙门氏菌阳性为36份，阳性率为41．86％；而常规分离培养鉴定技术检出沙门氏菌阳性为34份，阳性率为39．53％，两种方法的阳性符合率为86．17％，经统计分析，t=0．3111，p〉0．05，两种方法差异不显著。     

2006--2012年广西鸡沙门氏菌感染情况调查

运用分子生物学、血清学以及病原分离鉴定等多种方法，对2006～2012年广西规模养殖场、农村散养鸡以及活禽市场鸡血清和组织样本进行鸡白痢-伤寒沙门氏菌抗体和沙门氏菌病原检测和鉴定。结果发现，血清样本中规模场鸡白痢伤寒抗体阳性率较低，平均为2．16％，其中种公鸡的阳性率平均为1．55％，核心群母鸡阳性率平均为1．66％，生产群母鸡阳性率平均为4．16％；但广西农村散养鸡和活禽市场鸡白痢-伤寒沙门氏菌的污染情况不容乐观，其中农村散养鸡白痢-伤寒沙门氏菌阳性率为21．78％～24％，平均为22．52％（320/1421），活禽市场鸡阳性率为20．81％～33．2％，平均达到27．51％（2800/10177）；发病鸡的组织样品沙门氏菌平均阳性率为4．9％（29/592），阳性株以伤寒沙门氏菌为主，共13株占44．83％（13/29），其次为亚利桑那沙门氏菌8株，占27．59％（8/29），第三种是沙门氏菌的某些亚种（无法定型），最少为猪霍乱沙门氏菌2株，占6．9％（2/29）。     

应用斑点酶联免疫吸附试验快速检测饲料中的沙门氏菌

选用硝酸纤维素（NC）膜作固相载体，建立检测沙门氏菌的斑点酶联免疫吸附试验诊断方法，检测成品饲料100份，对沙门氏菌的最低检出量为400个／mL，沙门氏菌阳性检出率43．00％（43／100），常规分离培养阳性检出率为38．00％（38／100），两者符合率为83．72％，差异不显著（P＞0．05）。试验表明该方法简便、特异、快速、结果直观，便于在基层推广使用。     

饲料原料及浓缩料、配合料中沙门氏菌的分离鉴定与分析

通过饲料中沙门氏菌的国标检测方法,对饲料原料、配合饲料、浓缩饲料及精料补充料进行沙门氏菌的分离鉴定,结果3000多个各类饲料样品中饲料用棉籽粕沙门氏菌阳性3份、大豆粕沙门氏菌阳性3份、鱼粉沙门氏菌阳性5份、骨粉沙门氏菌阳性7份、生物蛋白饲料沙门氏菌阳性2份、鸡配合饲料沙门氏菌阳性8份、鸡浓缩饲料沙门氏菌阳性5个、猪配合饲料沙门氏菌阳性2份、猪浓缩饲料沙门氏菌阳性2份、草鱼配合饲料沙门氏菌阳性1份,牛羊精料补充料沙门氏菌阳性6份。     

Dot-ELISA检测猪胴体中沙门氏菌的研究

应用Dot-ELISA法对西宁某猪屠宰点 85份猪胴体进行了沙门氏菌的检测 ,同时采用常规分离培养鉴定技术作对照实验。结果在 85份肉样中 ,Dot-ELISA检出沙门氏菌阳性 6 5份 ,阳性率为 76 .4 7% (6 5 /85 ) ;而常规分离培养鉴定技术检出沙门氏菌阳性 6 7份 ,阳性率为 78.82 % (6 7/85 ) ,此两种方法的阳性符合率为 86 .5 7%。经统计分析 ,两种方法差异不显著 (P >0 .0 5 )。     

禽源沙门氏菌的血清型分布与耐药性分析

对89株禽源沙门氏菌临床分离菌株进行了血清型鉴定,鉴定出肠炎沙门氏菌（S．enteritidis）17株,纽波特沙门氏菌（Snewport）1株,鼠伤寒沙门氏菌（S．typhimurium）6株和鸡白痢沙门氏菌（S．pullorum）65株。结果表明,89株禽源沙门氏菌中有24株（27．0％）为能够引起人的沙门氏菌感染的血清型,具有重要的公共卫生学意义,而且这24株沙门氏菌中多重耐药菌株有12株（50．0％）。用微量稀释法检测了89株禽源沙门氏菌对18种抗菌药物的耐药性。检测结果表明,所有分离菌株均对阿米卡星和头孢曲松敏感;在使用的其他16种抗菌药物中,89株禽源沙门氏菌分离菌株对链霉素耐药率最高,为43．8％,其次为四环素、头孢噻吩、头孢噻呋、氟苯尼考、奥比沙星、萘啶酸和氨苄西林,耐药率分别为30．3％、22．5％、22．5％、20.2％、20.2％、19．1％和19．1％;对氯霉素、环丙沙星、沙拉沙星、恩诺沙星、左氟沙星、庆大霉素、头孢噻肟和卡那霉素呈现轻度的耐药性,耐药率分别为10．1％、9．0％、9．0％、7．9％、7．9％、6．7％、6.7％和2．2％。     

饲料及其原料中沙门氏菌的分离鉴定和耐药分析

为了解饲料及其原料中沙门氏菌污染情况及耐药情况,为沙门氏菌病的防控工作提供理论依据,特针对饲料及其原料开展沙门氏菌污染情况及耐药情况筛查。试验共收集饲料及原料样品707份,参照GB/T 13091－2002饲料中沙门氏菌检测方法进行沙门氏菌的分离和鉴定;分离株以API 2.0E鉴定试剂盒确认;后参照GB 4789.4－2010进行血清分型,并采用K-B纸片琼脂扩散法测定分离株耐药情况。结果表明,共检出阳性样品56份,阳性率为7.9%,其中52份为动物性原料;经血清分型得到阿贡纳沙门氏菌（Agona）5株,阿贡纳沙门氏菌Ⅱ（AgonaⅡ）1株,博内茅斯（Bournemouth）1株和摩科拉（Mokola）1株,其余菌株因血清种类有限,仅分型至群,未能进一步分型;药敏试验结果显示,饲料中沙门氏菌耐药率比人源和动物源性沙门氏菌低,但仍分离到13重耐药株1株,4重耐药株2株,3重耐药株9株,2重耐药株12株,且各菌株耐药谱不同。饲料及其原料中沙门氏菌检出率和耐药性相对较低,但仍存在个别菌株耐药情况严重的现象,有必要继续加大、加强针对抗生素使用的监管力度。     

鲫鱼鳃中沙门氏菌的分离与鉴定

应用常规细菌分离对西宁市某市场60份鲫鱼鳃样品进行了沙门氏菌的分离与鉴定。结果显示,在60份鲫鱼鳃样品中共检出沙门氏菌阳性菌株13份,阳性率21．7％（13／60）。表明：此批鲫鱼鳃样品污染沙门氏菌的情况比较严重,应该引起有关部门和消费者的重视。     

Dot-ELISA检测火腿肠中沙门氏菌的研究

以硝酸纤维膜为固相载体 ,利用沙门氏菌多价血清和辣根过氧化物酶 (HRP)标记制备的羊抗兔IgG ,成功地建立对火腿肠中沙门氏菌的Dot ELISA检测法 ,同时 ,采用常规分离培养鉴定技术作为对照试验 ,对 80份火腿肠进行了沙门氏菌的检测。结果显示 ,在 80份火腿肠中 ,用Dot ELISA检出沙门氏菌阳性为 2 2份 ,阳性率为 2 7 3 % (2 2 80 ) ;而常规分离培养鉴定技术检出沙门氏菌阳性为 2 0份 ,阳性率 2 5 0 0 % (2 0 80 ) ,两种方法的阳性符合率为 86 3 6% (P >0 0 5 )。     

江苏部分地区羊群边界病流行情况调查

本实验室从安徽某羊场发生腹泻的山羊病料中检测分离到边界病病毒（borderdiseasevirus,BDV）,证明BDV在我国羊群中存在。为了进一步了解BDV在江苏羊群的流行情况,本研究收集江苏部分地区发生腹泻和健康羊群的血清和组织样品,采用RT—PCR方法进行检测,并对阳性样品进行病毒的分离鉴定,测定分离毒株5’-UTR基因片段,与其他已报道的毒株进行同源性比较并绘制进化树。结果表明有27．4％（29／106）样品呈阳性,不N羊场BDV阳性率为0～67％,共分离到4株不同的BDV毒株,它们之间的同源性为73．9％～95．6％,而与其他BDV毒株的同源性为66．2％～91．6％。进化分析表明AH12-02与其他各毒株均较远,形成单独的分支,另3个毒株与BDV3型毒株关系最近。采用ELISA试剂盒对血清样品BDV抗体进行检测发现不同羊场抗体阳性率存在较大差异（0～100%）,还有抗体阴性持续感染个体的存在。以上结果丰富了我国BDV分子流行病学数据,为进一步探索BDV在我国羊群中的流行情况奠定了基础。     

湖南省奶牛源金黄色葡萄球菌分离鉴定与耐药性分析

为了掌握湖南省内奶牛源金黄色葡萄球菌的耐药情况,指导临床合理用药,抽取牛乳样品,通过对细菌的分离培养和纯化,采用基质辅助激光电离飞行时间质谱仪和全自动生化鉴定仪进行鉴定,并对分离菌株进行18种抗菌药物耐药性检测;结合耐药表型和mecA基因检测判定MRSA菌株。结果显示,2020-2021年从湖南地区257份牛乳样品中分离出金黄色葡萄球菌39株,89.74%的临床分离菌株表现为不同程度耐药,其中对青霉素耐药最为严重,耐药率达82.05%;对红霉素、克林霉素、替米考星、庆大霉素、头孢西丁耐药率在20%-35%之间;对多西环素及万古霉素绝对敏感,耐药率为0。38.46%分离菌株表现为多重耐药,以7重耐药最多。鉴定出19株MRSA,其中包含15株OS-MRSA和4株OR-MRSA。本研究可为湖南省内奶牛企业制定合理用药措施提供依据。     

Occurrence of different serotypes of Erysipelothrix rhusiopathiae in retail pork and fish.

Retail pork (38 samples), cod (10 samples) and herring (10 samples) were obtained from 12 stores in the area of Lund in southern Sweden during September and October 1990. Erysipelothrix rhusiopathiae was isolated from 50% of the pork samples, 60% of the cod samples and from 30% of the samples from herring. Serotype 2 dominated on retail pork as well as on fish samples constituting 53% of the pork isolates (10 strains) and 33% of the cod isolates (2 strains). All E. rhusiopathiae isolates originating from herring were serotype 2 (3 strains). Serotypes 1b, 6, and 8 were isolated from retail pork only (6, 2 and 1 strains, respectively). Serotype 5 was isolated from cod only (3 strains) and so was serotype 9 (1 strain). The public health hazards with the occurrence of virulent strains of Erysipelothrix rhusiopathiae in retail pork and fish are discussed.     

Ribotyping of Streptococcus uberis from a dairy's environment, bovine feces and milk

Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.     

我国部分地区乳房炎奶样中大肠杆菌的分离鉴定及耐药性分析

为了解我国部分地区奶牛乳房炎奶样中大肠杆菌的耐药情况,以指导牛场合理使用抗生素,提高治疗效果和畜产品质量,从全国六个省市自治区采取158例奶牛乳房炎的奶样,对奶样中的大肠杆菌进行分离纯化与鉴定,并进行药敏试验。结果显示,158份奶样中,共分离出38株大肠杆菌,其中黑龙江18株(分离率为36.0%)、上海3株(分离率6.0%)、北京3株(分离率27.2%)、新疆10株(分离率33.3%)、山东4株(分离率26.7%)、陕西0株(分离率0)。药敏试验显示,磺胺异噁唑耐药率最高,为73.7%;环丙沙星、诺氟沙星、四环素、链霉素药物的敏感率最高,为100%。分离菌株最多耐13种药物,最少耐4种药物,呈现多重耐药性。不同种类的抗生素中,上海和山东对β-内酰胺类药物和氯霉素类药物以及黑龙江、上海和新疆对磺胺类药物的耐药情况比较严重,耐药率高达50%以上。结果表明,各地区分离株对多种抗菌药物产生了不同程度耐药性和多重耐药,临床兽医在治疗时应注意合理用药,提高疗效和畜产品质量,减少耐药性的产生。     

广西猪瘟病毒E0和E2基因的克隆及序列分析

通过分析目前广西猪瘟病毒(CSFV)的E0和E2基因特征,为了解广西地区CSFV的分子流行病学、遗传变异及综合防控提供科学依据。试验采用RT-PCR方法,对阳性猪瘟样品进行CSFV的E0及E2基因的扩增,经克隆、测序后,利用DNAStar软件对序列进行比对分析,同时绘制系统遗传进化树。结果表明,从阳性猪瘟样品中成功扩增CSFV的E0及E2基因。序列比对分析发现,GX2毒株与参考毒株的E0基因核苷酸同源性在83.1%~94.1%,其推导氨基酸同源性在85.9%~99.6%;与参考毒株的E2基因核苷酸同源性为81.7%~93.7%,其推导氨基酸同源性为89.0%~97.0%;E0与E2基因均属于基因Ⅱ群。氨基酸变异位点分析表明,E0蛋白的RNase活性区域氨基酸基序位点没有发生变异;E2蛋白中15个位点上的半胱氨酸均未发生变异,但单抗识别位点S734R发生变异。遗传进化分析显示测定的GX2毒株与近年来广西CSFV流行毒株的变异趋势相似,与中国传统疫苗株HCLV、经典强毒株Shimen的同源性较低,亲缘关系较远,与广西近年来的流行毒株GXWZ02株的同源性较高,亲缘关系较近。     

Prevalence of Treponema hyodysenteriae in healthy pigs.

The prevalence of Treponema hyodysenteriae in faecal samples from healthy pigs of various ages in different farrowing units was investigated.Samples from herds designated as Category I were processed within 2 hrs. of sampling. Samples from herds designated as Category II were transported 2 to 3 days before cultivation procedures started. T. hyodysenteriae was demonstrated in 53.7 % to 93 % of the samples collected from Category I herds. No marked difference in the frequency of positive samples from the various age groups of pigs was found. In Category II herds, the organism was demonstrated in 10 % of the samples.The degree of beta-haemolysis shown by isolated strains was grouped into 3 groups: weak, moderate and strong. Strongly betahaemolytic strains, supposedly enteropathogenic, were demonstrated in all Category I herds. Such strains were found in 4.6 % to 25 % of the positive samples in these herds. In Category II herds, 2 out of 17 positive samples harboured strongly beta-haemolytic strains of T. hyodysenteriae.The amount of growth of T. hyodysenteriae on primary plates inoculated with sample material originating from the 2 categories of herds was mostly moderate or abundant. Strongly beta-haemolytic isolates originating from Category I herds produced abundant growth on primary plates in approx. 60 % of samples harbouring such strains. In samples from Category I herds with strains producing weak or moderate beta-haemolysis sparse and moderate amount of growth of the organism was predominant.     

Evaluation of the presence of porcine reproductive and respiratory syndrome virus in pig meat and experimental transmission following oral exposure

A study was performed to evaluate the presence of porcine reproductive and respiratory syndrome virus (PRRSV) in pig meat collected at slaughterhouses and its potential transmission to pigs via pig meat. A total of 1039 blood samples were collected from pigs upon their arrival at the abattoir. The following day, meat samples (n = 1027) were collected from the carcasses of these same pigs. Samples originated from 2 Canadian slaughterhouses, 1 situated in the province of Quebec and the other situated in the province of Manitoba. Serum samples were tested for antibodies to PRRSV and both serum and meat samples were also tested for PRRSV nucleic acid by polymerase chain reaction (PCR). Seropositivity to PRRSV for all serum samples was 74.3%. Furthermore 45 (4.3%) of the total serum samples and 19 (1.9%) of the 1027 meat samples were positive for PRRSV by PCR. Sequence analysis of open reading frame (ORF) 5 performed on 15 of the 19 PRRSV strains identified in pig meat indicated that 9 were field strains and 6 were vaccine-like (98% to 99.7% nucleotide homology with the Ingelvac RespPRRS/Repro vaccine). One of these 6 strains presented an intermediate 2-6-2 restriction fragment length polymorphism (RFLP) cut pattern and the others showed the characteristic 2-5-2 RFLP pattern of the vaccine strain. All strains sequenced were determined to be North American strains. In only 1 of the 19 PRRSV-positive meat samples could PRRSV be isolated. To test the potential infectivity of meat samples containing residual PRRSV, 11 of the PCR-positive meat samples (weighing 1.05 to 1.8 kg) were each used in feeding experiments of 2 PRRSV antibody-negative specific pathogen-free pigs of 9 wk of age. Samples were cut into several pieces and fed to each pair of pigs on 2 consecutive days. Each pig pair was housed in a separate cubicle and serum samples were collected at -7, 0, 7, 14, and 20 to 21 days post exposure. Seven pig pairs were found to be infected by PRRSV following ingestion of meat samples, including meat samples containing vaccine-like virus, as judged by the demonstration of PRRSV antibodies and/or PRRSV nucleic acid in the serum. In summary, the present study indicated that low residual quantities of PRRSV may be found in a small percentage of pig meat collected at slaugtherhouses. Furthermore, when this meat was fed raw to pigs in the experimental setting designed, pigs could be infected by PRRSV.     

Antimicrobial Resistance and Virulence Genes of Campylobacter spp. Isolated from Chicken and Swine Farms in Jiangsu Province

The aim of this study was to investigate the antimicrobial resistance, virulence genes of Campylobacter spp. isolated from chicken and swine farms in Jiangsu Province. Campylobacter spp. were isolated and identified from two hundred and fifty fecal samples collected from twenty-five animal farms in Jiangsu Province. Campylobacter strains were tested for the antimicrobial susceptibility against to 9 kinds of antimicrobial agents by using an agar dilution method. Eight virulence genes (cdtB, cadF, htrB, clpP, csrA, wlaN, cstⅡ, and cgtB) were detected by polymerase chain reaction. Ninety-three Campylobacter strains were isolated and identified from 250 samples, including 45 C. jejuni strains and 48 C. coli strains. The highest percentage of antimicrobial resistance was found for nalidixic acid (80.0%), tetracycline (71.1%) and ciprofloxacin (66.7%) in C. jejuni isolates. The C. coli isolates were most frequently resistant to erythromycin (87.5%), nalidixic acid (79.2%) and azithromycin (72.9%). Moreover, the multi-drug resistance (resistance to three or more antimicrobial classes) was common among Campylobacter isolates with a rate of 67.7%. On the other hand, the 93 Campylobacter strains showed a wide variation for the presence of the 8 virulence genes, cdtB and cadF were positive for all isolates,while htrB, clpP, csrA, wlaN, cstⅡ and cgtB was 97.8%, 76.3%, 18.3%, 5.4%, 2.2%, and 0%, respectively. The results indicated that the multiple drug resistance of Campylobacter strains from animal origin was relatively serious. In addition, the virulence-associated genes were detected widely among Campylobacter strains.     

江苏部分地区鸡源与猪源弯曲菌耐药性分析与毒力基因检测

旨在了解江苏省部分地区鸡源与猪源弯曲菌的耐药及毒力基因携带情况。从江苏省25个规模养殖场采集250份粪便样品进行弯曲菌的分离鉴定,采用琼脂平板稀释法测定9种抗菌药物的最小抑菌浓度（minimal inhibitory concentrations,MICs）,PCR方法扩增弯曲菌8种与致病力相关的毒力基因。结果显示：共分离得到93株弯曲菌,包括空肠弯曲菌45株,结肠弯曲菌48株;空肠弯曲菌对萘啶酸（80.0%）、四环素（71.1%）和环丙沙星（66.7%）的耐药率较高,而结肠弯曲菌对红霉素（87.5%）、萘啶酸（79.2%）和阿奇霉素（72.9%）产生较强的耐药性,分离株多重耐药现象严重,多重耐药率达67.7%;8个毒力基因在弯曲菌分离株中的携带率不同,cdtB和cadF携带率为100%,htrB为97.8%,clpP为76.3%,csrA为18.3%,wlaN为5.4%,cstⅡ为2.2%,cgtB为0%。结果提示,江苏省畜禽养殖场弯曲菌分离株多重耐药情况严重,毒力相关基因在弯曲菌中分布广泛。