仔猪腹泻致病性大肠杆菌分型鉴定及耐药性分析

为了解贵州省规模化养猪场腹泻仔猪致病性大肠杆菌流行情况及耐药性变化,本研究运用凝集试验、PCR和药敏纸片琼脂扩散法等方法对分离的78株致病性大肠杆菌进行血清型、毒力基因及耐药性分析。结果显示,78株致病性大肠杆菌以O138、O87血清型为主,占定型菌株的60.8%;其中62株致病性大肠杆菌检出毒力基因,检出率为79.5%,可分为8种毒力基因类型,分属肠致病性大肠杆菌（EPEC）、肠产毒性大肠杆菌（ETEC）和肠聚集性大肠杆菌（EAEC）,毒力基因eaeA、elt和escV检出率较高,分别为38.5%、28.2%和21.8%;分离到的致病性大肠杆菌对β-内酰胺类药物高度耐药,均为多重耐药株,耐药种类可达8种以上。结果表明,当前贵州省规模化养猪场腹泻仔猪致病性大肠杆菌的毒力基因检出率较高且基因型复杂,耐药性严重。本试验结果可为规模化养猪场防控仔猪腹泻提供基础资料及理论依据。     

曲靖地区仔猪腹泻细菌性病原的分离鉴定与耐药性分析

为了解曲靖地区仔猪出现腹泻的细菌性病因,对5个养殖场148份出现仔猪腹泻症状的样品进行细菌分离培养及耐药性分析,结果显示,检出致病性大肠杆菌(E.coli)114份,阳性率为77.03%;沙门氏菌(Salmonella)26份,阳性率为17.57%,混合感染率为17.57%.致病性大肠杆菌和沙门氏菌对多种抗生素产生了耐药性,其中114株致病性大肠杆菌中97株呈多重耐药性,占总菌数的85.09%,26株沙门氏菌中20株呈多重耐药性,占总菌数的76.92%.结果表明,致病性大肠杆菌是导致当地哺乳仔猪腹泻的主要细菌性病原,部分仔猪继发感染沙门氏菌,两种细菌对多种抗生素产生了耐药性,多重耐药现象较严重.     

仔猪腹泻大肠埃希菌的分离鉴定及药敏试验

为了了解甘肃省武威市凉州区部分地区引起仔猪黄白痢的致病性大肠埃希菌的主要血清型及其致病性,从凉州区4家规模化猪场采集了60份仔猪肛门拭子,用细菌分离培养方法分离出20株大肠埃希菌菌株,然后经形态观察、生化试验,确定了该次分离菌株为致病性大肠埃希菌。经血清型鉴定,表明20株分离菌株分布在8个血清型中,其中优势血清型为O8（9株）,占总数的45%。药物敏感性试验结果表明,分离菌株对临床常用药物具有抗药性。该研究能够对凉州区仔猪腹泻病的流行病学研究、诊断和免疫防制提供科学参考。     

新疆某牛场大肠杆菌的分离鉴定及16S rRNA序列分析

为了调查新疆某规模化奶牛场犊牛腹泻的原因并确定病原,无菌采集15份腹泻犊牛粪样进行病原的分离培养,采用形态学观察、生化试验、血清学和致病性分析等方法鉴定分离菌株,利用大肠杆菌16S rRNA通用引物进行基因序列扩增并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的其他菌株序列进行同源性比对,采用Mega 6.0软件依据16S rRNA序列构建分离株系统进化树并进行分析,同时对分离菌株的黏附性基因进行鉴定。结果从15份样品中分离获得2株分离菌株;试验显示,分离株均发酵葡萄糖,MR试验、吲哚和甲基红试验呈阳性,硫化氢、氧化酶、DNA酶、VP试验呈阴性。2株分离菌株血清型均为O78,且均具有致病性。药敏试验显示,分离菌株均对环丙沙星、卡那霉素、氟哌酸、庆大霉素、磺胺甲唑、头孢哌酮、壮观霉素、多黏菌素B、呋喃妥因敏感。分离株16S rRNA基因序列与大肠杆菌菌株NF738(GenBank登录号:MT649856)同源性为≥99.7%,且处于同一大分支。经PCR鉴定,分离菌株具有K88黏附性基因。本研究结果证实了引起新疆某规模化奶牛场犊牛腹泻的病原为致病性产肠毒素型大肠杆菌。     

北京地区仔猪大肠杆菌血清型鉴定及其毒力因子检测

本试验旨在调查北京地区仔猪腹泻的原因及猪源大肠杆菌血清型、毒力因子的分布情况。收集北京京郊地区仔猪腹泻样本400份,应用含2%血清的TSA培养基分离大肠杆菌,通过血清型鉴定试验和毒力因子检测验证O抗原。结果显示,400份样本中分离到64株大肠杆菌,定型42株。其主要为O101型（18.7%）、O64型（12.5%）、O8型（10.9%）、O20型（10.9%）、O45型（4.7%）、O149型（4.7%）、O2型（1.6%）、O89型（1.6%）8种血清型,其携带的毒力因子为STa、Stx2e、astA和eaeA等。结果表明,引起京郊仔猪腹泻的大肠杆菌主要有8个血清型,其中O101型发病比例最高。其携带的毒力因子主要为astA和eaeA,占全部菌株的50%以上,STa和Stx2e因子占全部菌株的30%以上。本试验结果将为今后京郊地区仔猪腹泻病的防控提供有效的数据支持。     

Detection of Serotypes and Virulence Factors of Escherichia coli from Piglets in Beijing

To investigate the cause of piglets diarrhea, and the distribution of the serotype and virulence factors of swine Escherichia coli in Beijing, 400 diarrhea samples were collected. TSA serum agar culture method was used to isolate Escherichia coli, serotype identification test and virulence factor genes test were used to verify the presence of O-antigen. 64 strains of E.coli were isolated from 400 diarrhea samples, among which 42 strains of E.coli were classified as 8 serotypes:O101(18.7%),O64(12.5%),O8(10.9%),O20(10.9%),O45(4.7%),O149(4.7%),O2(1.6%) and O89(1.6%), and the major virulence factors were STa, Stx2e, astA and eaeA. There were 8 mainly serotypes that caused piglets diarrhea in Beijing area, among which O101 serotype accounted for the highest proportion. The major virulence factors were astA and eaeA, accounted for more than 50% of all strains, STa and Stx2e accounted for more than 30% of all strains. These data would provide effective data to support the prevention and control of piglet diarrhea in Beijing.     

Isolation,Identification and Drug Resistance Analysis of Interstinal Pathogenic Escherichia coli and Salmonella Isolated from Diarrhea Piglets

In order to find out the serotype, resistant phenotype and genotype of Escherichia coli (E. coli) and Salmonella in piglets, this study collected 128 samples of diarrhea piglets from seven large-scale pig farms in five cities in Guizhou province, and the E. coli and Salmonella were isolated and identified. The pathogenicity of the strain was identified by animal test. The drug resistance of the main pathogen was tested by drug susceptibility paper. The resistance gene of each pathogen was detected by PCR. The drug resistance and genotype correlation of the bacterial were analyzed. The results showed that 78 strains of pathogenic E. coli and 21 strains of Salmonella were isolated and identified in this study. The serotypes of pathogenic E. coli were predominantly O138 and O87. Salmonella Typhimurium and Salmonella Enteritidis were predominant serotypes. The susceptibility test showed that the resistant strains of 78 strains of E. coli were more than 80% resistant to β-lactams and more than 40% for other antibacterials. The resistance rate of 21 strains of Salmonella to aminoglycosides was more than 50% and more than 20% to other types of antibacterials; 12 and 10 kinds of drug resistance-related genes of E. coli and Salmonella were detected, respectively; The coincidence rate of resistant genotype and phenotype of two kinds of bacteria were above 60%, and both were multiple drug resistance. This study provided a theoretical basis for comprehensive prevention and control of piglets diarrhea.     

腹泻仔猪肠道致病性大肠埃希氏菌和沙门氏菌的分离鉴定与耐药性研究

为探明仔猪细菌性腹泻肠道致病性大肠埃希氏菌和沙门氏菌流行的血清型、耐药表型及耐药基因型,本试验采集了贵州省5个地（州）市7个规模化养猪场的128份腹泻仔猪肠道样本,并对采集的样本进行了大肠埃希氏菌和沙门氏菌分离与鉴定,通过动物试验鉴定菌株的致病性,利用血清学方法鉴定其血清型,并通过药敏纸片法对主要致病菌进行耐药性研究,采用PCR技术检测各致病菌株耐药相关基因,分析细菌耐药表型和耐药基因型相关性。结果显示,本研究共分离鉴定到78株致病性大肠埃希氏菌与21株沙门氏菌,致病性大肠埃希氏菌血清型以O138、O87为主,沙门氏菌血清型以鼠伤寒沙门氏菌、肠炎沙门氏菌居多;药敏试验结果表明,本试验分离到的78株致病性大肠埃希氏菌对β-内酰胺类药物耐药率达80%以上,对其他种类的抗菌药耐药率均超过40%,分离鉴定的21株沙门氏菌对氨基糖苷类药物耐药率达50%以上,对其他种类的抗菌药耐药率均达20%以上;本试验分离鉴定的致病性大肠埃希氏菌共检出12种耐药相关基因,沙门氏菌共检出10种耐药相关基因,两种细菌耐药基因型与耐药表型符合率均达60%以上,且均为多重耐药。本研究为仔猪腹泻的综合防控提供了理论依据。     

腹泻犊牛大肠埃希菌的分离鉴定及药敏试验

对陕西省某奶牛场一起犊牛腹泻病例进行诊断和治疗。无菌采集腹泻犊牛肝脏和脾脏组织进行微生物学检测、小鼠致病性试验和药敏试验。结果显示,被检测病料中,牛病毒性腹泻病毒(BVDV)、牛腺病毒7型(BAdV-7)、牛冠状病毒(BCoV)、牛轮状病毒(BRV)检测结果均为阴性;分别从脾脏和肝脏组织分离培养到2株细菌,其培养特性和生化试验结果与大肠埃希菌高度相似,16S rRNA基因测序结果与大肠埃希菌的同源性为100%;动物致病性试验显示,分离菌能致试验小鼠死亡;药敏试验结果显示,该菌对左氧氟沙星、庆大霉素高度敏感,对头孢呋辛、恩诺沙星不敏感;临床选择敏感药物左氧氟沙星进行治疗,犊牛腹泻很快得到控制。     

Fimbriae in Escherichia coli isolated from the small intestine of piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively.Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated.Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

Thermostable factor(s) in soya producing a net excess of secretion in the ligated gut test in pigs

The ligated gut test (LGT) is the standard method for the examination of Escherichia coli strains for enterotoxin production in pigs. As solid pig feed has been associated with diarrhea, soya products (the main protein source for piglets) were investigated with the same test as E. coli strains. After injection of different soya products into ligated segments of the small intestine fluid accumulation was observed, indicating a net excess of secretion. The factor in soya products responsible for this effect was found to be thermostable, as its effect was unaltered after heating at 120 degrees C during an hour. No indications of a possible allergic phenomenon accounting for the fluid accumulation were found. From the results of this study it is concluded that soyabean products can produce results in the LGT similar to those produced by enterotoxigenic E. coli strains.     

Comparison in gnotobiotic pigs of lesions caused by verotoxigenic and non-verotoxigenic Escherichia coli

To compare the pathogenesis of calf and rabbit strains of E. coli, gnotobiotic pigs were infected with 10(10) colony forming units (cfu) of verotoxigenic strain RDEC-1 or S102-9, or a non-verotoxigenic E. coli (X114/83). Pigs were killed 4 days later, and intestinal tissue was fixed and examined by light, scanning, and transmission electron microscopy. Strains S102-9 and RDEC-1 caused diarrhea, attached to enterocytes, and effaced microvilli, confirming that the calf and rabbit strains possessed similar mechanisms of pathogenicity. Non-verotoxigenic strain X114/83 did not cause diarrhea, but in 5/5 piglets it was detected in histological sections adherent to enterocyte surfaces. Exfoliated enterocytes were seen in 4/5. Bacteria attached to enterocytes by "cups" and "pedestals," with effacement of microvilli, were seen by electron microscopy in 1/5 piglets. It was concluded that strain S102-9 appears to be an animal equivalent of human enterohemorrhagic E. coli, that verotoxin is not essential in the pathogenesis of attaching and effacing lesions, and that the lesions induced by S102-9 are more severe in gnotobiotic pigs than in gnotobiotic or conventional calves.     

猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析

为了解江苏省某规模化猪场猪流行性腹泻病毒(PEDV)遗传变异情况,本试验采集腹泻仔猪小肠组织及肠道内容物,通过RT-PCR进行PEDV鉴定。将检测为阳性的病料组织经20μg/mL胰酶处理后,接种到Vero细胞中进行培养,将细胞培养物进行反复冻融后,收获病毒液,进行RT-PCR鉴定。采用分段重叠策略对其S基因进行全基因序列扩增,并在此基础上分析该毒株S基因的核苷酸同源性及遗传进化关系。结果显示,经RT-PCR鉴定,病料组织呈PEDV阳性,阳性病料接种Vero细胞盲传4代后,出现明显的细胞病变,表现为细胞合胞体病变,空泡化,最终裂解脱落。收获的病毒液经RT-PCR鉴定后,确认分离到1株PEDV,命名为JS2017株。核苷酸序列分析表明,JS2017株S基因序列与美国变异株OH1414、加拿大变异株ON-007、中国变异株PEDV-LYG和CH/PDS/2015位于同一进化分支,其核苷酸同源性高达99.0%以上;其中与中国变异株YC2014亲缘关系最近,核苷酸同源性为100.0%;与经典株CV777、DR13株同源性较低,亲缘关系较远。结果表明,本研究分离获得的JS2017毒株是一株PEDV地方流行变异毒株,与2014年中国江苏分离株(YC2014株)亲缘关系最密切,与当前猪流行性腹泻疫苗株亲缘关系较远。     

一规模化猪场断奶仔猪腹泻大肠杆菌毒力因子的检测

从疑似断奶仔猪腹泻的仔猪中分离、鉴定出15株病原性大肠杆菌。经O血清型鉴定,11株为0131、4株未定型,所有O131血清型菌株呈β溶血。多重PCR检测毒素基因(STa、STb、LT、SLT-2e)和大肠杆菌粘附素单抗(F4、F5、F6、F41、F18)检测菌毛,O131菌株的毒素为STa、STb、SLT-2e,表达F18粘附素,未定型菌株的毒素为STa,共表达F6和F18粘附素。对15株大肠杆菌用16种抗生素进行药敏试验,结果表明:分离株对阿米卡星、痢特灵、新霉素敏感,而对多种抗生素产生了不同程度的耐药性。运用本场大肠杆菌分离株灭活苗免疫猪群,取得良好效果。     

肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。     

Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays.

We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.     

藏香猪源大肠埃希菌致病性和血清型检测及药敏试验

为了解四川省藏香猪源大肠埃希菌致病性、血清型及耐药性情况,从四川省藏香猪养殖场中无菌采集腹泻仔猪肝脏、肛拭子及粪便等组织病料253份中,分离得到155株大肠埃希菌。采用人工感染小鼠致病性试验、玻板凝集试验和KB药敏纸片法分别测定155株大肠埃希菌分离菌株的致病性、血清型及耐药性。结果显示,小鼠致病性试验表明155株分离菌中120株有致病性;玻板凝集试验表明120株致病性大肠埃希菌分离株属于14个血清型,以O111、O147、O109和O119为主要流行的优势血清型;耐药性试验表明120株致病性大肠埃希菌分离株对阿莫西林、氨苄西林、新霉素、磺胺间甲氧嘧啶4种药物耐药较严重,耐药率在94.2%~98.3%之间,对庆大霉素、氟苯尼考、多西环素等6种药物耐药率在44.2%~85.0%之间,对其他药物耐药率在15.0%~37.5%之间。说明该地区藏香猪源致病性大肠埃希菌血清型呈多样性分布,耐药性严重。     

Occurrence of mannose resistant hemagglutinins in Escherichia coli strains isolated from porcine colibacillosis

Three-hundred and fifty-eight E. coli strains isolated from piglets were tested for the presence of hemagglutinins by the use of the active hemagglutination test with or without mannose. Additionally 86 strains from the mentioned number of strains were investigated for the presence of common fimbriae using the same method but growing the strains in media especially suited for the development of this kind of fimbriae. These 358 strains and additionally 202 E. coli strains were tested using antisera for 987P and K88 antigens. It was found, using the active hemagglutination test, that 51.4% of the strains were hemagglutinating. The hemagglutinating strains carried the K88 antigen. All these strains were isolated from new-born and weaned piglets with enterotoxic form of colibacillosis, called also E. coli diarrhea. From cases of this form of colibacillosis originated also 26.7% of the strains in which common fimbriae (type 1) were detected. This result was obtained when the BHI medium was used for cultivation. In case of TSA medium only 2.3% of strains were positive. No specific or common fimbriae were found in strains recovered from septic form of colibacillosis and oedema disease (called also enterotoxaemic form of colibacillosis). No strain of 560 examined showed the presence of fimbrial 987P antigen.     

中兽药防治大肠杆菌性仔猪腹泻研究

仔猪腹泻是严重的仔猪疾病之一,常用抗生素进行治疗,但细菌耐药性导致药物的治疗效果下降。目前,有很多研究报道中草药防治仔猪腹泻取得了良好的疗效。本文从中药抗动物腹泻的作用机理、仔猪大肠杆菌性腹泻的中兽医辨证、中药抗仔猪大肠杆菌性腹泻的作用机理三个方面展开论述中药防治仔猪腹泻的研究进展,为防治仔猪腹泻的中兽药开发提供理论基础。