Occurrence of enterotoxigenic Escherichia coli in calves with acute neonatal diarrhoea

Enterotoxigenic Escherichia coli (ETEC) were isolated from 16.4% of diarrheic calves of up to 30 days old. Of 1-2-day-old calves nearly one half (45.0%) harboured ETEC, while this was the case with only 4.9% of 8-day-old calves. Among 206 9-30-day-old calves just three were found to harbour ETEC.     

Intestinal changes associated with rotavirus and enterotoxigenic Escherichia coli infection in calves

Newborn calves inoculated with rotavirus, enterotoxigenic Escherichia coli (ETEC) serotype 020:K' x 106':K99:HNM, either alone or in combination, became depressed, anorectic, diarrhoeic and dehydrated. ETEC did not adhere to the intestine although there was extensive proliferation in the lumen. Only slight mucosal changes were induced by ETEC and the activity of membrane bound lactase remained normal. More severe mucosal damage and a decrease in lactase activity were found in newborn calves inoculated with either rotavirus or rotavirus and ETEC in combination. The most severe clinical illness was found in calves inoculated with both rotavirus and ETEC. Calves inoculated at 1 week of age with either rotavirus or ETEC remained clinically normal. Rotavirus infection produced slight mucosal changes and a reduction of lactase activity. In contrast, colostrum-fed or suckling calves up to 2 weeks old inoculated with both rotavirus and ETEC became clinically affected, showed severe mucosal damage and decreased lactase activity. There was no bacterial adhesion to the intestinal mucosa as observed by immunofluorescent labelling and light microscopy.     

Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves with diarrhea

In vitro resistance to 8 antimicrobials among enterotoxigenic Escherichia coli from piglets and calves over a 13-year period was evaluated. Least resistance occurred against ceftiofur for all, followed by apramycin and gentamicin for porcine and florfenicol for bovine isolates. No significant differences were found between the first 8 and last 5 years.     

The prevalence of enterotoxigenic Escherichia coli in the feces of calves with diarrhea.

A study was undertaken to determine the prevalence of enterotoxigenicity among Escherichia coli isolated from calves with diarrhea and from a control group of normal calves. The test organisms consisted of 200 E. coli recovered from scouring calves less than two weeks of age and 100 E. coli from normal calves. The enterotoxigenicity of the cultures was evaluated by three methods, namely, injection of ligated segments of piglet intestine, injection of ligated segments of calf intestine and oral inoculation of suckling mice. Live cutures of all the test organisms were used for the ligated intestine studies whereas sterile broth culture supernatants were used in the suckling mouse tests. Of the isolates from scouring calves, 36% were enterotoxigenic in the piglet intestine and 28% in the calf intestine. Amongst the isolates from normal calves, none was enterotoxigenic in the piglet intestine and one was enterotoxigenic in the calf test system. The ligated piglet intestine was considered unsuitable for determining the enterotoxigenicity of bovine E. coli, whereas the ligated calf intestine test was satisfactory and correlated completely with the suckling mouse test. The enterotoxigenic E. coli of bovine origin produced an enterotoxin that resembled the heat stable enterotoxin of typical porcine enteropathogenic E. coli.     

Rotavirus and enterotoxigenic Escherichia coli infections of calves on a closed Finnish dairy farm

Rotavirus and enterotoxigenic Escherichia coli infections of calves were surveyed during 2 successive years on a closed Finnish dairy farm consisting of 90–105 milking cows. From a clinical standpoint, diarrhoea was of moderate to high severity during the first year, compared to the milder disease in the second year of the study. Diarrhoea or abnormal faeces were found only in calves less than 8 weeks old, with the peak occurring during the first 2 weeks of life.In the first year, rotavirus was detected throughout the calving season in diarrhoeic or abnormal faeces of calves aged 1 day to 7 weeks. In the second calving season, rotavirus was detected only during the 4 autumn months and in calves aged 11 days to 8 weeks. Rotavirus was detected in only 1 sample of normal faeces in both years. Electron microscopy revealed no enteropathogenic viruses other than rotaviruses. Enterotoxigenic K99 E. coli was found in about half of diarrhoeic or abnormal faeces in both years and throughout the calving seasons. K99 E. coli was also found in 5–10 % of normal faeces.In the second year of the study, 45 of 101 pregnant dams were vaccinated with 2 doses of E. coli antigen. The vaccination trial did not prevent or reduce altered faeces in calves whose dams had been vaccinated compared with calves whose dams had not been vaccinated in the same year. Comparing the 2 years, the earlier uptake of colostrum together with better cleaning and disinfection of the calf house, contributed to the later and rarer occurrence of rotavirus infection in the second year of the study. The earlier uptake of colostrum together with better cleaning and disinfection of the salf house, in the second year, could not prevent enterotoxigenic E. coli infections in calves but partly prevented and modified the disease.     

Biochemical characteristics of enterotoxigenic and nonenterotoxigenic Escherichia coli isolated from calves with diarrhea.

Eighteen isolates of enterotoxigenic Escherichia coli (ETEC) and 15 isolates of nonenterotoxigenic E coli (NETEC) obtained from calves with diarrheal disease were characterized biochemically. Of 64 biochemical tests employed, none allowed making differentiation of ETEC from NETEC. Eleven tests were used to separate ETEC isolates into 1 of 5 biotypes, although the ability to ferment dulcitol, salicin, sucrose, and sorbose gave sufficient information to identify the 5 biotypes of ETEC. The biotype data were confirmed upon testing 159 additional isolates of ETEC of bovine origin. All isolates of ETEC studied belong to serogroups O9:K35, O101:K30, O8:K85, O20:K? O8:K25, and O101:K28. The ETEC in different serogroups were also different biotypically, with the exception that isolates in serogroups O101:K28 and O101:K30 were of the same biotype. The K99 antigen was detected in 172 of the 177 isolates of ETEC and in 1 of 15 isolates of NETEC. Marked biochemical differences were not found between K99 + and K99- isolates of E coli.     

Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 (n=175) all isolates with ciprofloxacin MIC ≥ 0.125 mg/L (n=25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution.     

牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立

通过多重PCR扩增产肠毒素大肠杆菌（enterotoxigentic E.coli，ETEC）的毒力因子F41菌毛、K99菌毛和STa肠毒素的编码基因来检测和鉴定ETEC。试验中对影响PCR扩增的dNTP、Mg^2＋、引物浓度以及退火温度等因素进行优化，在优化条件的基础上，确定多重PCR的特异性和灵敏性，以此建立同时检测ETEC多个毒力因子的多重PCR方法。用该方法对分离于犊牛腹泻和犊牛肠毒血症的7株大肠杆菌进行检测，结果2株为F41、K99和STa阳性，4株为F41、STa阳性，1株为K99STa阳性。这与玻片凝集试验检测菌毛的结果一致。试验表明，该方法特异性强、敏感性高、简便、快速，适用于临床鉴定和检测牛ETEC菌株。     

Enzyme histochemistry of the small intestinal mucosa in experimental infections of calves with rotavirus and enterotoxigenic Escherichia coli

The effect of rotavirus and enterotoxigenic Escherichia coli, administered in different sequences, on alkaline and acid phosphatase, leucinaminopeptidase, beta-galactosidase, and succinicdehydrogenase of the intestinal mucosa of cesarian-derived, colostrum-deprived calves was investigated. Decrease in enzyme activity was most prominent in dual infections; it also occurred in parts of the small intestine in monoinfected animals. Increases in enzyme activity involved totally either one or all tissue compartments (crypt, basal villus area, villus tips). Increased activity was present in enteric mucosae that were either not affected or were only slightly affected by rotavirus or enterotoxigenic E. coli. We interpret the increase in enzyme activity as an adaptation of the enteric mucosa to maintain the absorptive function.     

Characterization of enterotoxigenic bovine Escherichia coli.

Among 300 isolates of bovine Escherichia coli, 56 which had been found enterotoxigenic in calf gut loops were characterized on the basis of O and K antigens, colonial morphology and resistance to seven antimicrobial drugs. The 56 isolates enterotoxigenic in the calf were compared with the nonenterotoxigenic ones. Of the 56 enterotoxigenic E. coli the majority possessed the A type of K antigen and had OK groups, O9:K(PS274) or O101:K(RVC118). Fourteen of these isolates had the K99 antigen. None of 27 isolates found enterotoxigenic in the piglet but not in the calf possessed the K99 antigen or belonged to OK groups O9:K(PS274) or O101:K(RVC118). Comparison of the patterns of resistance to seven antimicrobial drugs showed that all enterotoxigenic and nonenterotoxigenic isolates were susceptible to nitrofurantoin and sulphachlorphyridiazine and that there was no significant difference in the patterns between the two groups. The majority of enterotoxigenic isolates were mucoid, whereas most of the nonenterotoxigenic isolates were nonmucoid.     

Vaccination of pregnant cows with K99 antigen of enterotoxigenic Escherichia coli and protection by colostrum in newborn calves

The immune response to the K99 was tested in 45 pregnant cows, subcutaneously vaccinated, for protecting the newborn calves. Serological tests were performed in the blood sera of all animals and in the milk and colostrum sera; hemogram, inhibition of the adhesion to the brush border and histological tests were performed. The calves from vaccinated cows survived the experimental infection after the suction of colostrum in spite of the fact that the calves from control dams died with diarrhea.     

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.     

Antimicrobial drug resistance in porcine enterotoxigenic Escherichia coli of 0-group 149 and non-enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains (104) and 96 non-ETEC, all isolated from herds with piglet diarrhea in 1981 and 1982, were investigated with regard to antimicrobial drug resistance and colicinogeny. Eighty strains (77%) of the ETEC were drug resistant as compared to 66 strains (69%) of the non-ETEC. There were no significant differences between ETEC and non-ETEC in the frequencies of the drug resistance determinants investigated, except for tetracycline, to which 51 (49%) of the former and 30 (31%) of the latter strains were resistant (0.05 greater than P greater than 0.01). Of all strains 116 (58%) were resistant to streptomycin, 93 (47%) to sulphadimidin, 81 (41%) to tetracycline, 20 (10%) to trimethoprim, 14 (7%) to ampicillin, 11 (6%) to neomycin, 6 (3%) to chloramphenicol and none to nitrofurantoin. The frequencies of the different drug resistance determinants correlated well with the total amount of active substance of each drug used in farm animals in Sweden in 1981. Of the ETEC, 97 (93%) were colicinogenic whereas only 13 (14%) of the non-ETEC were colicinogenic.     

产肠毒素大肠杆菌感染的分子致病机制

产肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）是发展中国家人群腹泻的主要原因,一直是西方国家旅行者腹泻的最常见病因,也是引起动物（尤其是幼龄动物）腹泻的主要病原菌。根据ETEC产生热敏和（或）热稳定肠毒素特性可以将其分类成不同致病型。针对这类重要病原体的疫苗研发目前仍然面临着艰难的挑战。本文综述了病原体-宿主相互作用的分子机制,从基因组学和蛋白质组学两方面介绍致病菌的多种毒力因子以及作为靶标研发疫苗的潜力,分析了病原与不同宿主易感性的差异,为针对ETEC新型疫苗的研发和有效预防ETEC感染提供理论依据。     

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.     

Prevalence of eae and shiga toxin genes among Escherichia coli strains isolated from healthy calves

Strains of Escherichia coli (n = 390) isolated from 132 healthy, 4-8-week old calves, were tested by polymerase chain reaction (PCR) for the eae (intimin) gene and shiga toxin genes (stx1 and stx2). All strains were also analysed for F5, F17 and F41 fimbriae and for the heat-labile (LT) and heat-stable (STI and STII) genetic markers. Overall, the eae gene was detected in 84 (21.5%) of the strains tested. Only 21 (5.4%) isolates were positive for stx1 (18 strains) or stx2 (three strains); nine of the stx1-positive isolates also possessed the eae gene. A high percentage (29.2%) of the isolates tested expressed F17 but no enterotoxin genes were detected. None of the eae- or stx-positive strains belonged to the O157 serogroup.     

Prevalence of faecal excretion of verocytotoxigenic Escherichia coli O157 in cattle in England and Wales

During the decade to 1999, the incidence of human infections with the zoonotic pathogen verocytotoxin-producing Escherichia coli O157 (VTEC O157) increased in England and Wales. This paper describes the results of a survey of 75 farms to determine the prevalence of faecal excretion of VTEC O157 by cattle, its primary reservoir host, in England and Wales. Faecal samples were collected from 4663 cattle between June and December 1999. The prevalence of excretion by individual cattle was 4.2 per cent (95 per cent confidence interval [CI] 2.0 to 6.4) and 10.3 per cent (95 per cent CI 5.8 to 14.8) among animals in infected herds. The within-herd prevalence on positive farms ranged from 1.1 to 51.4 per cent. At least one positive animal was identified on 29 (38.7 per cent; 95 per cent CI 28.1 to 50.4) of the farms, including dairy, suckler and fattening herds. The prevalence of excretion was least in the calves under two months of age, peaked in the calves aged between two and six months and declined thereafter. The phage types identified most widely were 4, 34 and 2, which were each found on six of the 29 positive farms.