牦牛致病性大肠杆菌多价蜂胶灭活苗的研制

牦牛大肠杆菌病是严重影响牦牛生产的细菌性传染病,该病既是一种条件性疾病,又是其他诸多疾病的致病因素,因而是继主要的病毒性传染病得到控制以后,引起牦牛生产重大经济损失的重要传染病。抗生素对该病的防治效果良好,但其易产生耐药性,且常呈多重耐药,同时对肠道的微生态具有不良影响,因此对牦牛大肠杆菌病疫苗的研制日益受到广泛的重视。     

Preliminary results of an atiepizootic field test using EDTA-Na extract vaccine from Escherichia coli strains pathogenic to swine in swine production industrial units]

An anti-epizootic field test was applied to industrialised pig farms in the region of Wroc?aw, Poland, to test the effectiveness of a pentavalent EDTA (calcium disodium edetate)--sodium vaccine extracted from Escherichia coli strains with pathogenicity to swine. The vaccine had been received from a centre in Berlin-Buch, GDR. The vaccine failed to provide any protection, when orally applied to nursed piglets. However, both morbidity and mortality were reduced and, thus, an anti-epizootic effect on nursed piglets produced, when the vaccine had been injected intramuscularly to the pregnant mother animals, prior to farrowing. In weaned piglets morbidity was sucessfully reduced by both oral as well as intramuscular administration.     

鸭致病性大肠杆菌的分离与鉴定

对成都华阳地区两个种鸭场的鸭大肠杆菌病流行病学进行调查,并分析其发病特点。从这两个种鸭场的粪便和其中一个鸭场死亡鸭胚中共分离出19株大肠杆菌,我们对其培养特性、形态特征、生化特性、致病性及药物敏感性进行了研究,同时对分离的19株大肠杆菌的0群抗原进行了鉴定,其结果为O141 4株、O138 4株、O101 2株、O154 1株和O78 4株,还有4株未定型,有待于进一步的血清型鉴定。动物实验表明,已鉴定出血清型的15株大肠杆菌均有不同程度的致病性,药物敏感性实验表明,不同菌株对药物敏感性差异较大,但对多数抗菌药还没有产生抗药性。最后针对这两个种鸭场对该病的防治提出了对策。     

Pathotyping avian pathogenic Escherichia coli strains in Korea

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.     

原场鸡大肠杆菌多价苗的研究

用从豫北地区五家大型鸡场发病鸡群分离到5个大肠埃希氏菌优势血清型菌株O1、O2、O78、O86和O111的两次培养物,以蜂胶为佐剂,经甲醛灭活,蜂胶用酒精纯化,按一定比例配制成每毫升含100亿个大肠杆茵的多价蜂胶佐剂灭活苗。经试验证明,该疫苗安全、有效。免疫接种0．5ml／只,临床无不良反应。对同型菌株攻毒,保护率达100％,对地方性鸡大肠杆菌病有较强的预防作用。     

鸡大肠杆菌中草药佐剂多价灭活疫苗最小免疫量试验

取10日龄SPF雏鸡，分为两组。通过免疫攻毒试验检测了3个批次的鸡大肠杆菌中草药佐剂多价灭活疫苗的最小免疫量。结果证明，该灭活疫苗的最小免疫量为1／10个预设剂量。     

鸡致病性埃希氏大肠杆菌不同菌株间同源性抗原的研究

通过兔制备了３株引起鸡败血症的埃希氏大肠杆菌（Ｅ．ｃｏｌｉ）高免血清,对分离自新疆不同地区的３０余株鸡致病性Ｅ．ｃｏｌｉ进行了玻板凝集试验和双向免疫扩散试验。结果证明,在琼扩试验中,不同的Ｅ．ｃｏｌｉ菌株间存在着同源性抗原成份,这种同源性抗原在绝大多数Ｅ．ｃｏｌｉ间都有数种之多。但是,这种相互间的同源抗原在玻板凝集试验中有时并不能表现,甚至有沉淀线出现的血清与抗原之间在玻板凝集试验中也不能检测出来。     

湖南猪致病性大肠杆菌的分离及药物敏感性监测

从湖南47个规模化猪场发生典型仔猪黄、白痢猪只的63份腹泻病猪分离出63株大肠杆菌，其中12株作致病力试验均有致病性，对25种抗菌药物药敏试验，大肠杆菌对胺卡那霉素和头孢唑啉敏感，敏感菌株分别占98．41％和92．06％；多种抗生素产生耐药性，且为多重耐药。     

Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan

The virulence factors of avian pathogenic Escherichia coli (APEC) isolated in Japan were investigated. Serogroups O, serotypes K1 and K5, and genes cva C, iss, iutA, papA, tsh, and usp, which have been thought to be related to virulence, were examined for their association with E. coli strains isolated from diseased and healthy chickens. The frequently recognized serogroups O1, O2, and O78 were found in 56 of 125 (44.8%) strains of diseased chickens (APEC) versus 13 of 100 (13.0%) strains of healthy chickens (commensal E. coli), a significant difference at risk ratio < 0.01. Although iss, iutA, and tsh were widely distributed in the APEC irrespective of O serogroup, papA, usp, and the K1 serotype were detected in serogroup O2 of APEC. The kfiD gene related to the K5 capsule and VT, LT, and ST genes related to exotoxins were not detected in any strains examined.     

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh_s) and a 33 kDa β-domain (Tsh_β). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh_s (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh_β (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37℃ or 42℃. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26℃, 37℃, or 42℃, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37℃ in mucin agar, and it was not detected when the bacteria were grown at 26℃ in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.     

断奶仔猪源产Vero细胞毒素大肠杆菌eaeA基因的检测

根据文献合成了一对引物,利用PCR方法检测了84株来自断奶仔猪水肿病例的产Vero细胞毒素大肠杆菌(VerotoxigenicEscherichiacoli,VTEC)的LEE(locusofenterocyteeffacement)毒力岛的eaeA基因,以了解携带LEE毒力岛的大肠埃希菌在江苏省断奶仔猪群VTEC中的流行状况。结果发现:从其中17株中可扩增到预期的DNA片断,证实它们携带LEE毒力岛,携带率为20.24%。数据分析提示:LEE毒力岛的分布不仅限于EPEC和EHEC,在断奶仔猪源VTEC中也有一定的分布,并可能成为重要的毒力因子。     

鸡致病性E.coli地方株血清型的鉴定、毒力基因及致病性检测

为了研究河北秦皇岛地区鸡致病性大肠杆菌(Escherichia coli,E.coli)地方流行株的优势血清型、毒力基因和致病性,本研究采用常规鉴定方法和16SrRNA PCR方法,从采集自河北秦皇岛地区不同养鸡场的83份病鸡组织中分离鉴定出56株E.coli。人工感染1日龄雏鸡试验表明,46株分离株为致病性E.coli。采用玻片凝集试验、PCR方法分别测定致病性E.coli分离株的血清型分布和16种毒力基因携带情况。结果显示,定型的42株致病性E.coli分离株包括11种血清型,以O7 8、O89、O142及O1为优势血清型;46株致病性E.coli分离株中以irp2、fuyA、ompT、Iss a、iutA、iroN和hlyF毒力基因检出率较高,在89.1%~100.0%,其他毒力基因检出率为22.0%~72.0%。选择优势血清型代表株QH1(O78)、QH1(O89)、QH1(O142)和QH1(O1)人工感染1日龄雏鸡,计算半数致死量(LD50),确定其致病性。结果表明,QH1(O78)株致病性最强,对1日龄雏鸡的LD50为3.16×106 CFU/mL;对14,49日龄雏鸡的LD50分别为1.07×107,5×108 CFU/mL。本研究为河北秦皇岛地区鸡致病性E.coli地方流行株防控提供试验依据。     

猪源大肠埃希氏菌对较新的16种抗生素的敏感性

对从北京某猪场分离的 2 0株猪源大肠埃希氏菌所做的对 1 6种较新抗生素的药敏试验结果表明 ,除对头孢三嗪 ( CRO)、头孢噻甲羧肟 ( CAZ)、头孢噻肟 ( CTX)未产生耐药性外 ,分离菌株对其余 1 3种药物均显示程度不同的耐药。多西环 ( DO)、乙酰螺旋霉素 ( ASP)、杆菌肽 ( B)、新生霉素 ( NB)、氯洁霉素对其无抑制作用。 2 0株耐药菌株中 ,有 1 5种耐药谱型 ,多数为 5耐以上多重联合耐药     

利用MLST技术探讨不同地区致病性耐药鸡源大肠杆菌的遗传进化关系

选取来自山东等10省698株鸡源大肠杆菌为研究对象,分别利用PCR、微量肉汤稀释法、MLST技术方法进行系统进化群分群、耐药表型检测、分子分型,并采用Bio Numerics v4.0软件和SPSS20.0软件进行数据处理分析。结果显示,低致病群B_1群为48.85%(341/698)和非致病群A群24.64%(172/698)分布较多,高致病群D群20.20%(141/698)和B_2群为6.31%(44/698)。鸡源大肠杆菌耐药严重,12种药物的耐药率约在50%以上。B_2和D群菌株对8种抗菌药的耐药率高于A和B_1群菌株,其中对庆大霉素、大观霉素、头孢噻呋、氨苄西林和奥格门丁5种药物极显著性差异(P<0.01),对氟苯尼考和复方新诺明显著性差异(P<0.05)。135株高致病菌株经MLST分型得到48个ST型,ST117(12株)和ST354(12株)最多,其次是ST115(8株),ST2309(8株),ST10型(7株),ST69(6株),ST1011(5株),ST1158(5株)。有29个ST型各1株,18株菌株未分出序列型。不同地区大肠杆菌ST型分布存在差异,ST69和ST354型分布最为广泛(4个省),其次是ST115、ST117和ST2309型(3个省),ST10、ST48、ST57、ST362、ST371、ST1011和ST1158(2个省),其余36个ST型菌株仅在1个省市有检出。135株菌株分为35个聚类簇,其中23个聚类簇的遗传相似性在40%以下,但有6个聚类簇菌株间的遗传相似性均在80%以上。48个ST型菌株共获得了76种耐药谱。我国各地健康鸡群隐性携带相对较高比例的高致病菌株,且耐药程度相对严重,耐药谱较广。鸡源大肠杆菌菌株分布具有多态性和地域性,ST型与耐药谱之间无相关性。     

The occurrence and the importance of enterotoxins from strains of Escherichia coli from bovine mastitis]

Baby mouse and Y1 cell culturing tests were conducted into 117 Escherichia (E.) coli strains from mastitis of cattle to clear up enterotoxin formation. The rate of the latter was found to be relatively low, with enterotoxins recorded from only 7 strains (6%). No correlations were established between enterotoxin formation, on the one hand, and antibiotic resistance as well as biochemical activities of E. coli isolates, on the other. The question still remains open, if enterotoxin-forming strains derive their udder pathogenicity from endotoxins or with support by enterotoxins.     

黑龙江省部分地区鹅致病性大肠杆菌血清型鉴定及药敏试验

收集来自黑龙江省部分地区的疑似鹅大肠杆菌致死鹅的肝脏、心脏、肺脏、肾脏等组织样本,用于进行大肠杆菌分离培养.经形态观察,生化试验,共鉴定出61株大肠杆菌.经血清型鉴定,表明O9、O18、O64、O93这4种血清型在黑龙江省内分布广泛,为优势血清型.药敏试验表明,分离菌株呈现出不同程度的耐药性,临床常用的庆大霉素、氨苄青霉素、氟哌酸抑菌作用很弱,而妥布霉素,四环素,阿米卡星类对分离菌株几乎无抑制作用.耐药率低于33％的仅有3种,分别是先锋噻肟,复达欣,菌必治,以上3种药物可以作为临床用药的候选药物.     

Differentiation of pathogenic and nonpathogenic Escherichia coli isolated from poultry

Twenty-one isolates of Escherichia coli recovered from chickens and turkeys were evaluated for pathogenicity in 1-week-old chicks. Fifteen produced coli-septicemia (pathogenic) and six were innocuous (nonpathogenic). Both pathogenic and nonpathogenic E. coli were tested for their ability to selectively absorb Congo red (CR) dye incorporated into agar medium. Eight of 15 pathogenic E. coli (somatic antigen types O1, O78, O11, O88, and OX9) absorbed the dye and produced red colonies (CR+) between 48 to 72 hours of incubation. All serotypes of E. coli with homologous somatic antigen O78 were CR+, while those of O2 antigen were CR- (white colonies). Five of six nonpathogenic E. coli also were CR+. In contrast to pathogenic E. coli, however, nonpathogenic isolates absorbed CR early, between 18 to 24 hours of incubation. Although CR dye binding did not correlate well with pathogenicity, it may be an identifiable property of some serotypes of E. coli.     

53株禽致病性大肠杆菌的耐药表型及耐药基因的检测

为了解禽致病性大肠杆菌耐药表型及耐药基因的情况,选取江苏、安徽等地分离的53株禽致病性大肠杆菌,采用药敏纸片法对9种抗菌药物进行药敏试验,并对四环素类tet(A)、tet(B)、tet(C)、tet(W)、tet(M)、tet(O)、tet(K)、tet(L)耐药基因,喹诺酮类GryA、ParC耐药基因,磺胺类sulⅠ、sulⅡ、sulⅢ耐药基因,β-内酰胺类SHV、CTX-M、ompCA耐药基因,氨基糖苷类aac(3)-Ⅰ、aac(3)-Ⅱ、aac(6')-Ⅰb、aac(6')-Ⅱ、ant(3″)-Ⅰ、armA、rmtA、rmtB、rmtC、rmtD、npmA耐药基因进行PCR检测。结果显示:53株禽致病性大肠杆菌对磺胺异噁唑、环丙沙星、氨苄西林、多西环素的耐药率较高,分别为88.68%(47/53)、71.71%(38/53)、86.79%(46/53)、75.47%(40/53)。其中50株禽致病性大肠杆菌表现为多重耐药,耐4、5、6种药物的现象最为普遍,且不同地区菌株存在差异。tet(A)是四环素耐药基因中最为流行的一种耐药基因(52.83%,28/53),喹诺酮类耐药基因主要由gryA(94.33%,50/53)、parC(94.33%,50/53)基因编码,耐磺胺类药物sulⅠ、sulⅡ、sulⅢ基因均有检出,分别为96.23%(52/53)、98.11%(48/53)、86.79%(46/53),耐β-内酰胺类药物中仅检出ompC A基因(30.19%,16/53),在检测的11种耐氨基糖苷类耐药基因中,最为流行为aac(3)-Ⅱ、aac(6')-Ⅰb、ant(3″)-Ⅰ基因,分别为92.59%(49/53)、98.11%(52/53)、100%(53/53)。耐药基因与相关耐药菌株检出率基本呈正相关。试验结果表明:53株禽致病性大肠杆菌耐药性高,耐药谱广,耐药基因流行现象十分普遍。本试验结果能为禽致病性大肠杆菌的耐药现状与临床用药提供理论指导。     

Molecular markers for detection of pathogenic Escherichia coli strains belonging to serogroups O 138 and O 139

Escherichia coli strains belonging to O-serogroup 138 and 139 are important as disease agents in pigs causing post-weaning diarrhea and edema disease. Several types of shiga toxin-producing O 138 and O 139 strains were isolated from diarrheic humans and from cattle and food of bovine origin. Serotyping is the current method for detection of O 138 and O 139 strains but its applicability can be limited due to the presence of capsules and capsular-like bacterial surface antigens and in the case of rough LPS. To overcome these difficulties for diagnosis, we have developed a specific PCR method suitable for detection of different types of O 138 and O 139 strains. The O-antigen gene clusters of E. coli O 138 and O 139 type strains were sequenced, and the genes were identified on the basis of homology. By screening against 186 E. coli and Shigella type strains, two genes specific to each of E. coli O 138 and O 139 were identified, respectively, and were tested on 15 clinical and environmental isolates of those two serogroups in a double-blind test. The sensitivity of the PCR assays was determined, and the detection limits were 2 pg per mul of chromosomal DNA and 2 CFU per 10 g of water or pork samples. PCR-based detection of O-antigen specific genes of E. coli O 138 and O 139 was shown to be accurate, highly sensitive and rapid, and is suggested as a new diagnostic tool for investigations of infections and outbreaks with these strains in animals and humans and for control of food.