Virulence factors of Escherichia coli isolated from bovine clinical mastitis.

Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors. The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E. coli, clinical pictures of coliform mastitis, environmental conditions and herd management. The genes of nine virulence factors were detected by polymerase chain reaction. Presence of K1 and K5 capsules was assessed by use of specific bacteriophages. Serum resistance was tested by a turbidimetric assay. Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E. Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17. Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants. Genes for F17a, CS31A, Afa7D and Afa7E were not detected. Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates. Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113). K1 and K5 capsules were not detected.     

Comparison of virulence gene profiles of Escherichia coli isolates from sows with coliform mastitis and healthy sows

Coliform mastitis (CM) is not only a serious economical and animal welfare touching problem in dairy cattle, but also in sows after farrowing. Due to this disease, the essential adequate supply with colostrum for the growth and the health of the piglets is not ensured. Besides other influencing factors, Escherichia (E.) coli is of great importance as a causative agent of this multifactorial disease. In this study, E. coli isolates from milk samples of healthy and CM-affected sows were examined for the presence of virulence genes associated with extraintestinal E. coli strains, enterotoxigenic E. coli and other pathogenic E. coli. The isolated E. coli harbored mainly virulence genes of extraintestinal E. coli strains (especially fimC, ompA, traT, hra, kpsMTII, iroN). The virulence gene spectrum for both samples from CM-affected and healthy sows did not differ significantly. Particular virulence gene profiles of E. coli isolates from diseased sows were not detected. This study provides novel insights into the role of E. coli in association with mastitis in sows since it is the first time E. coli isolates from CM-affected sows' milk were analysed for virulence genes. Because there were no differences in the prevalence of E. coli and their virulence-associated genes between healthy and diseased sows, other causative factors seem to have greater influence on the pathogenesis of porcine CM.     

新疆石河子地区奶牛乳房炎大肠杆菌的分离鉴定及耐药性分析简

本试验旨在调查新疆石河子地区奶牛乳房炎源大肠杆菌的某些生物学特性及其耐药状况,以提高药物疗效,减少牛奶中药物的残留。试验从新疆石河子地区患乳房炎奶牛的乳样中分离纯化并鉴定出21株大肠杆菌,并对大肠杆菌分离株进行抗菌药物敏感性分析。结果表明,21株大肠杆菌对21种抗菌药物中的6种药物耐药率超过50%,其中最多的耐药15种,最少的耐药5种,耐药6及6种以上的菌株共占到76.19%。提示,该地区奶牛乳房炎源大肠杆菌对多种药物已产生了不同程度的耐药性,且存在严重的多重耐药情况。     

A case-control study of verocytotoxigenic Escherichia coli infection in cats with diarrhea.

The objectives of this study were to determine the prevalence of enteric verocytotoxigenic E. coli (VTEC) infection in a population of cats in Ontario, and to determine whether an association exists between the presence of VTEC and feline diarrhea. Fecal samples from 179 cats, representing 113 cats with diarrhea and 66 cats with normal feces, were cultured for E. coli. The fecal cultures were screened for verocytotoxin activity with a Vero cell assay. Confirmation of the presence of verocytotoxin (VT) genes was done with polymerase chain reaction (PCR) amplification; the frequency of occurrence of the genes for generic VT, VT1, and VT2 was determined. VTEC-positive samples were defined as those that demonstrated cytotoxicity on the Vero cell assay and yielded E. coli possessing one or more of the VT genes. All VTEC-positive isolates were serotyped. The overall prevalence of enteric VTEC infection in the cats was 12.3% (22/179). Statistical analysis of the case-control data showed no significant association between VTEC infection and diarrheal illness. The majority of the cats with VT-positive E. coli were positive for the presence of the generic VT, rather than for VT1 or VT2; it is therefore possible that a novel verocytotoxin gene may exist in E. coli isolated from cats. Eight VTEC strains were identified by serotyping; 4 of these serotypes have previously been isolated from humans, and 2 from cattle, suggesting that cats may be capable of acting as reservoirs for human and bovine VTEC serotypes.     

Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences

Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.     

Prevalence of verotoxin-producing Escherichia coli (VTEC) in bovine coli mastitis and their antibiotic resistance patterns.

Between December 1996 and October 1997, milk samples from a total of 145 cows with coli mastitis were screened for the presence of verotoxin-producing E. coli (VTEC). VTEC were found in four (2.8%) out of the 145 samples. The four isolated strains proved to be verotoxin (VT) 1-, VT2- or VT1- and VT2-positive. However, no strain contained all three virulence factors tested. Further strain characterization was carried out by serotyping as well as by resistance pattern analysis.     

Bacterial adherence in mastitis caused by Escherichia coli.

The possible role of bacterial adherence in the pathogenesis of experimental mastitis in the mouse was examined with four strains of Escherichia coli. Two of these strains had a known adhesion antigen (K88) and two did not. The K88 antigen did not play a significant role in the virulence or infectivity of E. coli either in the murine or bovine mammary gland. Two E. coli strains, W1 (K88+) and J2 (K88-) were virulent in the mouse but did not adhere to epithelial cells. Both these strains produced clinical mastitis in the cow. A third strain, D282 (K88-), produced mild disease in the mouse but was avirulent in the cow. The fourth strain, 233/ID (K88+), was avirulent in both the mouse and the cow. Strains D282 and 233/1D were killed rapidly by bovine serum whilst J2 and W1 were more resistant. All strains were more sensitive than the control resistant strain E. coli P4, which is known to be highly virulent for the lactating udder.     

Surveillance of dairy production holdings supplying raw milk to the farmhouse cheese sector for Escherichia coli O157, O26 and O111

Clinically healthy domestic animals can harbour Escherichia coli O157 and other verocytotoxigenic E. coli (VTEC) strains in their faeces. Milk filters can be used to microbiologically monitor direct milk secretion and environmental contamination for these pathogens. The aim of this study was to establish baseline data on the prevalence and characteristics of VTEC organisms in lactating animals (bovine, ovine and caprine) supplying milk to the farmhouse cheese sector, with particular emphasis on serogroups O157, O111 and O26. Fifty-six bovine, 13 caprine and 5 ovine herds/flocks, the majority of which supplying milk for farmhouse cheese production, were surveyed from May 2004 to July 2005. Milk filters were analysed by immunomagnetic separation followed by PCR, on a serogroup-specific basis for E. coli O157, O26 and O111. Positive isolates were examined using a multiplex PCR protocol, for their potential to produce verocytotoxins (vt1/vt2), the haemolysin-encoding gene (hlyA) and the gene encoding attaching and effacement (eae). Five verocytotoxigenic and 22 non-virulent E. coli O157 isolates were detected. Seventeen E. coli O26 isolates were also detected, four of which were verocytotoxigenic, seven isolates contained the eae gene only and six isolates were devoid of any of the virulence factors. The VTEC O157 and O26 isolates contained the hlyA and eae genes along with the verocytotoxin genes. No E. coli O111 isolates were detected. Some of the herds were positive on more than one occasion and multiple E. coli serogroups were isolated from the same milk filter sample. Although all food products tested were VTEC negative, routine surveillance for such pathogens in raw milk/raw milk products is of public health importance. Herd-level surveillance along with subsequent risk management action may be a cost-effective component of risk reduction strategies for food production, drinking water supplies and the protection of public health.     

Virulence genes of Escherichia coli strains isolated from mastitic milk

Escherichia coli, a Gram-negative environmental pathogen associated with bovine mastitis was isolated from the milk of 34 symptomatic cows that had been diagnosed with clinical mastitis. Eighty isolates were obtained over a 17-month period and these isolates were screened by DNA amplification for the following E. coli virulence genes: cnf1, cnf2, eaeA, eagg, einv, ltx1, stx1, stx2 and vt2e. Thirty of the bacterial isolates, obtained from 23 different cows, had toxin genes identified in their DNA. The most common virulence gene detected was stx1, with a prevalence of 31%, followed by cnf2 (7.5%), vt2e (6.25%) and eaeA (4%). The possession of different virulence genes by the bacterial isolates had no discernable impact on the health status of the cows as there was no correlation between the potential for toxin production by the E. coli isolates and the systemic clinical condition of the respective infected cows.     

宠物源大肠杆菌的血清型和毒力基因及耐药性调查

为研究宠物源致病性大肠杆菌(Escherichia coli)的血清型、毒力基因和耐药性之间的相关性,本研究由健康和患病犬、猫直肠拭子样品中分离177株E.coli,并采用玻片凝集法鉴定其血清型,结果显示分离株中定型菌株135株,分别属于20个不同的血清型,其中O1、O2和O8为主要的流行血清型。PCR方法检测11种毒力相关基因,并采用琼脂稀释法测定分离菌株对14种抗菌药的敏感性,结果表明3个血清型的菌株拥有相似的耐药表型,但毒力基因谱不同。62%的分离菌株携带fimH基因,并且毒力基因组合iroN+hlyF、iroN+fimH和traT+sitA比较流行。55%的O1血清型携带fimH基因,并且多数耐受四环素、多西环素、头孢噻吩和庆大霉素;20.8%的O2血清型菌株携带traT和sitA基因;50%的O8血清型携带traT和fimH基因。3个血清型分离菌株多数对安普霉素和阿米卡星敏感。     

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.     

Identification of a bovine mastitis Escherichia coli subset

Eleven Escherichia coli isolates from clinical bovine mastitis cases (mastitic strains) and 11 from the cowshed environment (environmental strains) were compared, to determine if the former were a subset of the latter. The mastitic and environmental strains could not be distinguished according to O antigen and antibiotic sensitivity. All mastitic isolates showed significantly (P<0.0001) faster growth in milk and faster lactose fermentation than most (approximately 64%) environmental strains, but growth rates in nutrient broth did not differ. The rates of lactose fermentation and growth in milk were positively correlated. Adhesion and phagocytosis of mastitic strains by bovine PMN were significantly (P<0.0001) lower than those of environmental strains, and correlated negatively with growth in milk and lactose fermentation. The average percentages of killing by bovine leukocytes in the two sources were not statistically different. All mastitic strains were serum sensitive, whereas most ( approximately 72%) environmental ones were resistant. Finally, pulse-field gel electrophoresis revealed two main pulse type clusters, sharing a similarity coefficient of 79%. Cluster 1 comprised only environmental strains, whereas cluster 2 comprised mostly mastitic strains and only three environmental ones. Four mastitic strains shared a similarity coefficient of less than 74% with the other strains and were not included in the clusters. Our results suggest that clinical bovine mastitis E. coli isolates may form a subset of the general environmental E. coli population; they seem better able to multiply in the udder medium and to evade the host cellular innate immune response, and are genetically distinct from most environmental strains.     

Short Communication - Prevalence of Verotoxin-Producing Escherichia coli (VTEC) in Bovine Coli Mastitis and their Antibiotic Resistance Patterns

Between December 1996 and October 1997, milk samples from a total of 145 cows with coli mastitis were screened for the presence of verotoxin-producing E. coli (VTEC). VTEC were found in four (2.8 %) out of the 145 samples. The four isolated strains proved to be verotoxin (VT) 1-, VT2- or VT1- and VT2-positive. However, no strain contained all three virulence factors tested. Further strain characterization was carried out by serotyping as well as by resistance pattern analysis.     

Occurrence and characteristics of CS31A antigen-producing Escherichia coli in calves with diarrhoea and septicaemia in Argentina

CS31A is a K88-related non-fimbrial adhesin first described on Escherichia coli strains isolated from diarrhoeic and septicaemic calves. In this report, CS31A antigen was screened by immunological methods and confirmed by PCR among bovine E. coli isolates. In addition, CS31A-producing strains were characterized with respect to different fimbrial antigens, O-serogroup and other properties related to virulence. Faecal or tissue specimens of 100 diarrhoeic or septicaemic calves and 27 older cattle with different pathologies from 71 outbreaks or individual cases that occurred in Buenos Aires province, Argentina, were examined. CS31A + E. coli strains were isolated from 21 (21.0%) calves from 16 outbreaks or individual cases. No CS31A + E. coli was detected in samples from cattle more than 1 year old. Fimbriae F5, F41, F17a and F17b were not detected among the CS31A-producing strains. Three (14.3%) of the CS31A+ E. coli strains expressed the F17c fimbria. All of the 21 isolates exhibited at least one property of septicaemic strains (resistance to serum, production of aerobactin or colicins) but none of them demonstrated heat-stable enterotoxigenic activity. CS31A + E. coli isolates belonged to 10 serogroups, more commonly O8, O7, O17 and O21. The results obtained here confirm the worldwide distribution of CS31A antigen in bovine E. coli strains. However, CS31A + or CS31A + /F17c + E. coli were less frequently isolated than they were in North hemisphere countries.     

Prevalence of fimbial colonization factors F18ab and F18ac in Escherichia coli isolates from weaned piglets with edema and/or diarrhea in China

F18ab and F18ac are important fimbrial colonization factors of verotoxigenic Escherichia coli (VTEC) and/or enterotoxigenic E. coli (ETEC) in weaned piglets with edema disease and/or diarrhea. To further investigate their prevalence and correlation to pathogenic E. coli, a duplex PCR, using three primers derived from the nucleotide sequence of the F18 major fimbrial subunit gene (fedA), and a direct agglutination test, using a monoclonal antibody specific for the antigenic factor 'a' of F18, were performed. Among 60VTEC, 24VTEC/ETEC and 24 ETEC isolates tested from weaned piglets with edema disease and/or diarrhea in different pig farms in the Jiangsu Province of China, 52 isolates (48.15%) were positive in the direct agglutination test and 63 isolates (58.33%) were positive in the duplex PCR. Among 63 PCR-positive isolates, 53 isolates (49.07%) were F18ab-positive and 10 isolates (9.26%) were F18ac-positive. In addition, the F18ab gene was more frequently detected in VTEC (61.67%) or VTEC/ETEC (62.50%) than in ETEC (4.17% only), while the F18ac gene was more frequently detected in VTEC/ETEC (33.33%) than in ETEC (8.33%) or VTEC (0%). Furthermore, F18ab was more frequently associated with Shiga toxin 2e (Stx2e), whereas F18ac was more frequently associated with enterotoxin ST I. These results suggest that the duplex PCR performed in this experiment is a more reliable method for identification of F18+E. coli, and that F18 is a more important virulence factor of VTEC and VTEC/ETEC.     

张家口地区奶牛临床型乳腺炎病原菌的分离鉴定

采集具有临床型乳腺炎奶牛的奶样,经细菌的分离培养、纯培养和生化试验,以确定奶牛临床型乳腺炎的致病菌种类。结果发现患临床型乳腺炎奶牛的致病菌和占分离菌株的百分率为:腐生葡萄球菌19.7%、金黄色葡萄球菌16.5%、兽疫链球菌17.3%、停乳链球菌9.4%、乳房链球菌3.9%、无乳链球菌1.6%、产气肠杆菌17.3%、聚团肠杆菌4.7%、奇异变形杆菌3.0%、大肠杆菌3.0%、蜡样芽胞杆菌3.0%,样品中单纯感染与混合感染的百分率分别为66.0%和27.8%。     

traT and CNF2 genes of Escherichia coli isolated from milk of healthy cows and sheep

The objectives of the present study were to isolate Escherichia coli from milk of apparently healthy cows and sheep and to investigate the presence of traT and cytotoxic necrotising factor-2 (CNF2) virulence genes by multiplex polymerase chain reaction (PCR). Milk samples collected from a total of 1028 apparently healthy ruminants (737 cows and 291 sheep) in eastern Turkey were streaked onto 5% sheep-blood agar. E. coli was isolated and identified by biochemical tests in 5.9% (61/1028) of milk samples. Correct amplification with the molecular length of 232 bp was obtained from all E. coli isolates by the species-specific PCR. The isolation rates of the agent were calculated to be 7.6% (56/737) in cows and 1.7% (5/291) in sheep. The difference between these proportions was statistically significant (p < 0.001). Multiplex PCR showed that traT and CNF2 genes were present in 62.3% and 6.6% of all isolates, respectively. Both genes were present in 16.4% of the isolates, with only 14.7% having neither gene.     

Virulence factors of Escherichia cofi from cellulitis or colisepticemia lesions in chickens

This study was designed to compare virulence factors of cellulitis-derived Escherichia coli to colisepticemic E. coli in order to clarify whether E. coli associated with cellulitis comprise a unique subset of pathogenic E. coli. Isolates were tested for serotype, capsule, aerobactin production, colicin production, the presence of the iss gene, and serum resistance. Untypable isolates made up the greatest percentage of each group. Serotypes O2 and O78 were the most commonly identified among both groups of isolates. No statistical differences in the distribution of aerobactin or colicin production, capsule, or iss gene were observed between groups. Cluster analysis showed that 90% of the E. coli isolates had greater than 42% livability in serum-resistance tests. No separation of colisepticemic vs. cellulitis E. coli isolates was observed on the basis of SR. Colicin production by E. coli was highly correlated with serum resistance (P = 0.0029). These data suggest that cellulitis E. coli have virulence traits similar to those of colisepticemic E. coli.     

鸡大肠杆菌traT基因的扩增及TraT-DIG探针制备

以致病性Ｅ．ｃｏｌｉ的质粒为模板,用人工合成引物扩增出致病性相关的ｔｒａＴ基因核心部分;将菌株的致病性与ｔｒａＴ基因的扩增结果进行比较,强致病力菌株均扩增出ｔｒａＴ基因,无致病力菌株未扩增出ｔｒａＴ基因,约大多数中等致病力菌株亦扩增出ｔｒａＴ基因。扩增获得的ｔｒａＴ基因经纯化后标记地高辛（Ｄｉｇｏｘｉｎ,ＤＩＧ）研制出ｔｒａＴ－ＤＩＧ基因探针,该探针与鸡沙门氏菌、鸡白沙门氏菌、副伤寒沙门氏菌不发生     

Simultaneous occurrence of selected food-borne bacterial pathogens on bovine hides, carcasses and beef meat

The aim of this study was to determine the simultaneous occurence of Salmonella spp., L. monocytogenes, verotoxigenic E. coli (VTEC), and Campylobacter spp. in slaughtered cattle and in beef meat subjected for human consumption. A total of 406 bovine hides and 406 corresponding carcasses were used to collect the samples with a swab method after exsanguination and evisceration of animals, respectively. Furthermore, 362 beef meat samples were purchased in local retail shops over the same period of time as for the bovine samples. Food-borne bacterial pathogens were identified with standard ISO methods with some modification by the use of PCR for VTEC. The isolated bacteria were then molecularly speciated (Campylobacter), serotyped (L. monocytogenes) and characterized for the presence of several virulence marker genes (VTEC and Campylobacter). It was found that 49 hide (12.1%) and 3 (0.7%) carcass samples were contaminated with more than one bacterial pathogen tested. Most of the hides were positive for Campylobacter spp. and VTEC (27 samples) and Campylobacter spp. together with L. monocytogenes (12 samples). Eight bovine hides contained L. monocytogenes and VTEC while L. monocytogenes and Salmonella spp. were detected in one sample. Furthermore, 3 pathogens (Campylobacter spp., L. monocytogenes and VTEC) were simultaneously identified in one bovine hide tested. In case of bovine carcasses 2 samples contained Campylobacter spp. and VTEC whereas one carcass was positive for L. monocytogenes and VTEC. On the other hand, 10 out of 362 (2.8%) minced beef samples were contaminated with at least two pathogens tested. The majority of these samples were contaminated with L. monocytogenes and Salmonella spp. (6 samples). It was noticed that equal number of C. jejuni and C. coli were found, irrespective of the origin of the samples. Most of the strains possessed more than one pathogenic factor as identified by PCR. Molecular serotyping of L. monocytogenes revealed that the majority of the isolates (27 out of 31; 87.1%) belonged to 1/2a serogroup. It was found that most of the VTEC isolates possessed the Shiga toxin stx2 gene (12 strains) whereas only 2 strains were str1-positive. The eneterohemolysin and intimin markers were identified only in 7 and 2 isolates, respectively. PCR analysis revealed that 4 VTEC belonged to O91 serogroup, 2 strains were O145 and 1 isolate was identified as O113. None of the VTEC detected in the study was O157 serogroup.