腹泻犊牛大肠杆菌的分离鉴定和药敏试验

犊牛腹泻是犊牛常见病之一.犊牛腹泻大肠杆菌有多种血清型,主要有导致初生犊牛腹泻的O8、O9、O20、O101大肠杆菌,感染犊牛发生败血症的O78、O15、O35、O115、O117、O137等血清型大肠杆菌[1].2005～2007年我们从南京地区部分奶牛场犊牛腹泻和犊牛败血症病例的犊牛,无菌采集直肠棉拭及病死犊牛的脾脏和关节液作病料,同时无菌采集部分临床健康的犊牛和成年奶牛直肠棉拭,划线于麦康凯平板,37℃培养24 h;     

Characterization of eae+ Escherichia coli isolated from healthy and diarrheic calves.

Strains of Escherichia coli from 101 healthy and 114 diarrheic calves were screened by PCR for the eae (intimin) gene and Shiga toxin genes (stx). Each eae+ and eae/stx+ strain was examined for antimicrobial susceptibility, enterohemolysin activity, and the somatic O antigen was determined. An immunoassay was used to detect Shiga toxin antigens for the eae/stx+ E. coli. Significantly more (p = 0.005) of the healthy calves carried eae+ and eae/stx+ E. coli in their feces when compared to strains from diarrheic calves. Moreover, Shiga toxin antigens were detected significantly more (p = 0.001) often among the eae/stx+ strains from healthy calves when compared to eae/stx+ strains from diarrheic calves. However, significantly more (p = 0.001) of the eae+ and eae/stx+ strains from diarrheic calves were resistant to at least one of the antimicrobials tested, and the strains from diarrheic calves had a significantly (p = 0.05) higher rate of antimicrobial resistance to at least two different antimicrobial classes. No significant difference (p> or =0.05) was detected among the eae+ and eae/stx+ strains from healthy and diarrheic calves for enterohemolysin production. Serogroups O-negative, O5, O26, and O111 were predominate among both healthy and diarrheic calves.     

Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves

We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.     

Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves

Tropical Animal Health and Production - Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain...     

Prevalence and characteristics of necrotoxigenic Escherichia coli (NTEC) strains isolated from diarrhoeic dairy calves.

Fecal samples from 246, 1-90-days old diarrhoeic dairy calves in 72 herds were screened for the presence of cytotoxic necrotizing factors (CNF)-producing Escherichia coli (NTEC). NTEC were detected by tissue culture assays and PCR in 39 (15.8%) of the diarrheic calves, and the majority of these animals (34 of 39, ca. 87.2%) were infected by NTEC producing CNF2. Calves were grouped according to their age (1-7 days, 8-14 days, 15-21 days, 22-30 days and 31-90 days) and analyses of prevalence were done by the Mantel-Haenzsel chi2-test for trend. A significant age-associated increase in the prevalence of NTEC producing CNF2 (p<0.0001) was found. Eighty-one (8.4%) of the 958 E. coli isolates from the 246 diarrheic calves were positive for CNF in the tissue culture assays. These strains were analyzed by PCR and this technique showed that three (3.7%) strains were CNF1-positive and 75 (92.6%) were CNF2-positive. Moreover, three of the strains positive in the tissue culture assays were negative by PCR. These strains were subsequently assayed in several biological tests (rabbit skin test, mouse intraperitoneal test and mouse footpad test) which showed that they were really NTEC, probably producing CNF2, but with some different properties to classical strains producing CNF2. NTEC strains producing CNF2 belonged to different serogroups (O2, O7, O9, O14, O15, O41, O43, O45, O55, O76, O86, O88, O109, O115, O123, O128, O153 and O159) than strains producing CNF1 (O11 and O32) or PCR-negative strains (O111). Moreover, a strong association between CNF2 and F17 fimbriae was found (78.6% of CNF2-positive strains were F17-positive, whereas only 22.9% of CNF2-negative strains were F17-positive).     

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.     

Antibiogram,virulotyping and genetic diversity of Escherichia coli and Salmonella serovars isolated from diarrheic calves and calf handlers

Antimicrobial resistance profile of E. coli and Salmonella serovars isolated from diarrheic calves and handlers in Egypt is unknown due to the absence of monitoring. Therefore, this study aimed to determine the virulence, genetic and antimicrobial resistance profiles of E. coli and Salmonella serovars associated with diarrhea in calves and handlers in intensive dairy farms in Egypt. A total of 36 bacterial strains (20 E. coli and 16 Salmonella) were isolated from fecal samples of 80 diarrheic Holstein dairy calves (10 E. coli and 13 Salmonella) and hand swabs of 35 handlers (10 E. coli and 3 Salmonella) in two intensive dairy farms in Sharkia Governate in Egypt. E. coli strains belonged to six different serogroups and O114:K90 was the most prevalent serogroup (30%). However, Salmonella strains were serotyped into four different serogroups and S. Kiel was the most prevalent serotype (50%). Thirteen (65%) E. coli isolates were harbouring either stx2, eaeA and/or astA virulence-associated genes. However, stn and spvC virulence genes were detected in 2 (12.5%) and 4 (25%) of Salmonella isolates, respectively. E. coli isolates showed marked resistance to ampicillin (75%), while Salmonella strains exhibited high resistance to amikacin (100%), gentamicin (93.75%) and tobramycin (87.5%). Results of the present study showed that E. coli and Salmonella serovars isolated from diarrheic calves and handlers in intensive dairy farms in Egypt exhibited resistance to multiple classes of antimicrobials, which may pose a public health hazard. Thus, the continuous monitoring of antimicrobial resistance is necessary for both humans and veterinary medicine to decrease the economic losses caused by antimicrobial-resistant strains in animals as well as the zoonotic risk.     

A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves

We investigated the clonal relationships among 41 enterotoxigenic (ETEC) or non-enterotoxigenic (NETEC) Escherichia coli strains producing the F17 a fimbriae isolated from diarrheic calves in France or Belgium in the early 1980s. Twenty-three of the 26 ETEC strains were highly clonally related, most of them with a O101:K32:H9-serotype. The NETEC strains were also divided in clonal subgroups, most of them with O101:H-serotype. The F17 a positive ETEC strains are no longer isolated from diarrheic calves in these countries. It is postulated that the use of a vaccine including O101, K32 and H9 antigens in addition to K99 (F5) explains the strongly reduced isolation of the O101:K32:H9, K99 (F5) E. coli clone.     

Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil.

One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.     

Studies with an unclassified virus isolated from diarrheic calves

A transmissible agent (Breda agent) was isolated from a calf with diarrhea and shown to be infectious by inoculation orally into gnotobiotic and conventionally reared calves. The “Breda” agent had the morphology of a virus and possessed a hemagglutinin. Antigenic studies showed the virus to be antigenically different from bovine coronavirus, parainfluenza 3 virus, bovine rotavirus, bovine parvovirus and bovine pestivirus (BVD). Attempts to culture the virus in cell or organ cultures or in embryonated eggs, were unsuccessful. The virus was either spherical or kidney shaped, with 7–9 nm peplomers on the surface. A few particles possessed coronavirus processes of 17–20 nm, but these were arranged irregularly and were thought to be tissue debris. Three out of eight experimental calves developed severe diarrhea and the lesions in the small and large intestines were similar to those reported for coronavirus. The virus replicated in the jejunal and ileal regions of the small intestine and in the spiral colon, as judged by immunofluorescence. The virus multiplied in all experimental calves and was excreted in the feces; excretion correlating with the onset of diarrhea or a change in the appearance of the feces. There was little or no malabsorption measured by the uptake of D-xylose and the fact that infection of both the crypt and villus epithelial cells was observed, suggests that the pathogenesis may be different from rotavirus and coronavirus. Fourteen of fortyseven calves in the outbreak were infected with the virus, virus was not identified in other farm outbreaks of the disease.     

Haemolytic Escherichia coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains

Twenty-four haemolytic Escherichia coli strains were isolated from dogs with diarrhea. The strains were serotyped and analysed by polymerase chain reaction (PCR) for genes encoding virulence factors associated with E. coli that cause diarrhea in animals. Adhesion antigen production was deduced from haemagglutination experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat extracts was also used as an indication for the production of adhesive structures. The majority of the strains was shown to produce this type of virulence factor. Adhesion and invasion tests of the strains and Caco-2 cells showed that all strains adhered and that two were invasive. The two invasive strains were positive in the intimin PCR and one of them also contained genes encoding CS31A. The PCR for heat stable toxin (ST) was positive in only four strains, as was the presence of F17 fimbrial genes. Surprisingly, 19 strains had intact P fimbrial operons, coding for an adhesin involved in urinary tract infection (UTI). The cytotoxic necrotising factor 1 (CNF1) gene, also mainly found in UTI was likewise detected in these 19 strains. Cytolethal distending toxin (Cdt) genes were found in five strains. The high number of strains positive for CNF1 and P fimbriae prompted us to test the strains in a multiplex PCR used to test E. coli isolated from UTI in various species for 30 virulence associated genes. The data showed that the majority of the diarrhea isolates have virulence factor profiles highly similar to UTI E. coli isolates from dogs. This raises the question whether these isolates are real intestinal pathogens or "innocent bystanders". However, since CNF1 producing necrotoxic E. coli (NTEC) strains isolated from humans, pigs and calves with diarrhea appear to be highly related to our strains, it might be that in dogs this type of isolate is capable of causing not only UTI, but also diarrhea. If this is the case and this type of isolate is "bifunctional", domestic animals likely constitute a reservoir of NTEC strains which can be also pathogenic for humans.     

腹泻犊牛大肠杆菌血清型抗菌药物耐药性耐药基因鉴定及脉冲场凝胶电泳分型

本试验对2011年由哈尔滨周边地区采集的263份腹泻犊牛的肛拭子进行大肠杆菌的分离鉴定,并通过小鼠致病性试验鉴定出26株致病性大肠杆菌,用Kirby-Bauer法开展对12种药物的药敏试验,PCR检测了我国流行广泛的9种耐药基因,脉冲场凝胶电泳(PFGE)进行基因分型.检测结果,该地区主要流行的致病性血清型为O26、O78和O15,所有致病性菌株都存在多重耐药现象,其中阿莫西林-棒酸、链霉素、四环素、复方新诺明的耐药率达到了100％,共检出7种耐药基因,检出率符合我国平均分布趋势,且检出率基本上与耐药表型相一致.PFGE结果显示,该地区存在致病性菌株的克隆传播.     

Escherichia coli strains isolated from diarrheic piglets in the Province of Quebec.

During 1972 to 1974, 112 Escherichia coli strains isolated from diarrheic piglets were recieved from different parts of the Province of Quebec, Canada. Fifty-six strains elicited a positive gut loop response in three week old piglets and were then considered as Moon's class 1 enteropathogens, while four of the 56 remaining strains reacted only in ten day old piglets and were classified as class 2 enteropathogens. Forty-eight strains produced both a heat-labile and a heat-stable enterotoxin, while 12 isolates which included the four class 2 enteropathogens produced only a heat-stable enterotoxin. Fifty-one enterotoxigenic strains could be serogrouped using OK antisera against E. coli strains commonly associated with colibacillosis in piglets. The most common serogroups encountered were O157: K "V17"; 88a,c, O149:K91; 88a.c. O157:K"V1c, O149:K91; 88a.c, O157:K"V17"; 88 a,c or a,b and O45:K"E5"; 88a,c. No significant difference was observed in the fermentation patterns, antibiotic susceptibility, colicin production, production of a filterable hemolysin and transferable tetracycline resistance between the enterotoxigenic and the nonenterotoxigenic strains.     

Isolation of piliated Escherichia coli from diarrheic foals

Escherichia coli was isolated from the feces and intestines of foals with and without diarrhea. Piliation of isolates was demonstrated by electron microscopy and agglutination in antisera having specificity for K88, K99, P987 and F41 pili. Piliation was also demonstrated by electron microscopy on organisms which did not react with any of the antisera.     

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.     

Anti-microbial susceptibility for east1 + Escherichia coli isolated from diarrheic pigs in Korea

The in vitro susceptibilities of 128 isolates of east1 + Escherichia coli from pre-weaned and post-weaned pigs with diarrhoea were tested with nine commonly used anti-microbial agents by an agar dilution minimal inhibitory concentration (MIC) procedure according to National Committee for Clinical Laboratory Standards guidelines. For the isolates from preweaned and post-weaned pigs, most of them were susceptible to low concentrations (MIC90) of tetracycline (4 and 2 microg/ml), ceftiofur (2 and 2 microg/ml), and colistin (4 and 2 microg/ml). Marked resistance was found in others.     

Isolation of necrotoxigenic Escherichia coli from a dog with hemorrhagic pneumonia

A 7-month-old sexually intact male Cocker Spaniel was admitted to the North Carolina State University Veterinary Teaching Hospital for evaluation of lethargy, panting, and excessive salivation that had become progressively severe during a 5-hour period. Despite intensive medical care, the dog died within the first 24 hours of hospitalization, and death was attributed to acute, severe, necrotizing pneumonia. Lung tissue collected at necropsy by use of swabs was cultured and yielded an isolate of Escherichia coli; because of the rapid progression of illness in an otherwise healthy dog, the isolate underwent virulence typing and was determined to be a necrotoxigenic E. coli. Necrotoxigenic E. coli produce a toxin called cytotoxic necrotizing factor and are known to be involved in extraintestinal infections, including urinary tract infection, in humans and animals. Virulence typing of E. coli isolates from dogs with peracute pneumonia is recommended to further characterize the epidemiologic characteristics and public health importance of necrotoxigenic E. coli.     

Serobiotypes and virulence genes of Escherichia coli strains isolated from diarrheic and healthy rabbits in Brazil

A total of 178 Escherichia coli isolates from diarrheic and healthy rabbits in the S?o Paulo State (Brazil) were serobiotyped and investigated by PCR for the presence of virulence genes. Among the 90 (50.6%) isolates which possessed the eae gene, 74 were from diarrheic animals and all but one encoded intimin beta. Sixty five (72.2%) of the eae+ isolates had insertion of the locus of enterocyte effacement locus in the pheU locus, 11 (12.2%) in the selC and 14 (15.6%) did not insert in either of these loci. All isolates were negative for genes of the E. coli enterotoxins, Stx1, Stx2, CNF1, CNF2 and EHEC hemolysin. The O132:H2 serotype was dominant, being present in 63 isolates (70%) of the 90 eae+ isolates, and 57 of the 63 isolates of this serotype belonged to biotype 30. PCR detected the gene for AF/R2 fimbriae in 75 (83.3%) of the 90 eae+ isolates. Adherence to HeLa cells was best detected following 6h incubation and a positive fluorescence actin staining (FAS) test was given by 52 isolates. These data show that isolates of E. coli associated with diarrhea in rabbits in Brazil possess the genotype and phenotype typically associated with rabbit enteropathogenic E. coli (EPEC). We conclude that EPEC that possess the eae gene are a common cause of diarrhea in Brazilian rabbit farms and that the pathogenic eae+ AF/R2+ isolates of O132:H2:B30 serobiotype are especially predominant.     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.