Occurrence and characteristics of enterohemorrhagic Escherichia coli O157 in calves associated with diarrhoea

Little is known of the association of enterohemorrhagic Escherichia coli O157:H7/NM (EHEC O157) with disease in naturally infected calves, although cattle have been known as a major source for EHEC O157 outbreaks in humans. In this study, we investigated the occurrence of EHEC O157 in calves associated with/without diarrhoea to examine if EHEC O157 is involved in calf diarrhoea and to characterize the isolates. Four hundred and ninety eight diarrhoeic and non-diarrhoeic young calves from 115 different farms were examined. Of 244 diarrhoeic calves, 24 (9.8%) were positive for EHEC O157, and of 254 non-diarrhoeic calves, 7 (2.8%) were positive. EHEC O157 was recovered from 12/76 (15.79%) of diarrhoeic calves less than 2-week-old, and no EHEC O157 was detected in this age group of non-diarrhoeic calves. This implicates EHEC O157 as a possible cause of the disease in naturally infected neonatal calves. The occurrence of EHEC O157 was relatively lower in the older calves (aged older than 8 weeks) and no significant difference was found in the occurrence rates between these diarrhoeic and non-diarrhoeic calves. PCR analysis of virulence markers revealed that the isolates carried various virulence genes such as Ehly, eae, stx1 and stx2, which underlines the potential importance of these attributes for the infection, colonization and possible pathogenesis of calf diarrhoea.     

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.     

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.     

Occurrence and properties of FY(Att25)+ Escherichia coli associated with diarrhoea in calves

Escherichia coli producing the adhesive antigen FY(Att25) were isolated from 46 of 1341 (3.4 per cent) E coli isolated from calves on 20 of 164 (12.1 per cent) farms in Scotland and England. Twenty of the 46 calves had diarrhoea and in nine of these animals there were mixed infections with rotavirus, coronavirus, cryptosporidium and Salmonella typhimurium. The F41 fimbrial adhesin was found on one of the FY(Att25)+ E coli. This strain also produced heat stable enterotoxin. The remaining FY(Att25)+ isolates did not produce other adhesins, enterotoxins or verotoxins. The FY(Att25) antigen was not detected on 109 pathogenic E coli isolated from calves, chickens, lambs, pigs or man.     

Occurrence and characteristics of enterohemorrhagic Escherichia coli O26 and O111 in calves associated with diarrhea

The aims of this study were: (1) to examine whether or not enterohemorrhagic Escherichia coli O26 and O111 (EHEC O26 and O111) are involved in neonatal calf diarrhea; (2) to determine the specific age periods at which the calves are vulnerable to these organisms, and (3) to reveal the biochemical, genetic and cytotoxic characteristics of the isolates. The study investigated the occurrence of EHEC O26 and O111 in calves associated with or without diarrhea. A total of 442 diarrheic and non-diarrheic young calves from 115 different farms were examined. Of the 257 calves with diarrhea, 37 (14.4%) and 32 (12.5%) tested positive for EHEC O26 and EHEC O111, respectively. Of the 185 non-diarrheic calves, 14 (7.6%) and 11 (5.9%) tested positive for EHEC O26 and EHEC O111, respectively. EHEC O26 and O111 were recovered from 14/69 (20%) and 11/69 (16%) diarrheic calves <2-weeks-old, respectively, and no EHEC O26 and O111 were detected in the non-diarrheic claves of this age group, suggesting that EHEC O26 and O111 are possible causes of the disease in infected neonatal calves. However, there were similar rates of occurrence in the diarrheic and non-diarrheic calves in the older animals (particularly, aged >10 weeks). PCR analysis showed that the isolates carried various virulence genes such as Ehly, eae, stx1 and stx2, which highlight the potential importance of these attributes for the infection, colonization and the possible pathogenesis of calf diarrhea. Cytotoxicity analysis revealed that many of the EHEC isolates showed high cytotoxicity to Vero cells, re-emphasizing the potential for cattle being a direct source of EHEC infections in humans.     

Non-O157 Shiga toxin-producing Escherichia coli isolated from diarrhoeic calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.     

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.     

Necrotoxigenic Escherichia coli type-2 invade and cause diarrhoea during experimental infection in colostrum-restricted newborn calves

There exists experimental evidence that necrotoxigenic Escherichia coli (NTEC) strains producing the cytotoxic necrotising factor 1 cause intestinal and extra-intestinal disease in piglets. On the other hand, no experimental model has been developed with NTEC strains producing the cytotoxic necrotising factor 2. In all, 14 colostrum-restricted calves were orally challenged with two strains isolated from the faeces of a diarrheic calf (B20a) or from the heart blood of a septicaemic calf (1404). All calves had diarrhoea which lasted until euthanasia in eight of them. In those calves, diarrhoea was correlated with the faecal excretion of the challenge strains. At necropsy, vascular congestion of the intestinal mucosa, hypertrophy of the mesenteric lymph nodes (MLN) and some congestion of the lungs were observed. Bacteriology confirmed the colonisation of the intestine by the challenge strains which were also recovered from the heart blood, the lungs and/or the liver. Histological sections confirmed enterocolitis, lymphadenitis and limited bronchopneumonia. In the intestinal tissue sections, bacteria testing positive in an in situ DNA hybridisation assay with a CNF2 probe were observed. Those results were confirmed by immunohistochemistry with a polyclonal anti-O78 and a monoclonal anti-F17b antisera. Three of the five control calves receiving either saline or a CNF(-), F17a strain (25KH09) had no clinical signs or lesions. The other two presented a profuse liquid diarrhoea but those calves were positive for the presence of K99(+) E. coli. In this model, both NTEC2 strains were thus, able to colonise the intestine, to cause long-lasting diarrhoea and to invade the blood stream with localisation in various internal organs in colostrum-restricted conventional newborn calves.     

Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in galicia (north-western Spain)

A total of 289 Escherichia coli colonies isolated from 78 diarrhoeic calves were studied for production of heat-labile (LT) and heat-stable (STa) enterotoxins, verotoxin (VT), cytotoxic necrotizing factor (CNF) and K99 antigen, and they were serotyped. Production of STa was detected in a single strain possessing both K99 and F41 antigens; the serotype was 09:K (A) 35. LT-producing strains were not detected. From 16 (20.5%) calves, 51 VT-producing colonies of E. coli were isolated. Production of the necrotic factor was detected in 33 E. coli colonies isolated from 14 (17.9%) calves. Serotype was a useful marker for production of VT and CNF. Among the 51 VT-producing colonies, 24 were untypable and the remainder belonged to serotypes O2:K?, O103:K--, O104:K?, O128:K?, O153:K-- and O157:K--:H7. Four of the 33 CNF-producing colonies were untypable and the majority of the remaining colonies belonged to serotypes O15:K14, O78:(K80), O123:K-- and O139:K--. Both VT and CNF were lethal for mice, but only CNF showed necrotizing reaction in rabbit skin. Our results indicate that VT-producing and CNF-producing E. coli strains are frequently isolated from diarrhoeic calves and that according to the serotypes exhibited, some of them might be considered potential pathogens for humans. The role of VT-producing and CNF-producing strains in calf diarhoea remains to be established.     

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.     

Escherichia coli and diarrhoea in the rabbit.

An outbreak of severe diarrhoea and death in young rabbits was associated with many nonenterotoxigenic Escherichia coli in the caecum. The severe clinical, pathological and bacteriological features of the diesase, acute diarrhoea associated with typhlitis and many E. coli in the caecum, could be reproduced either by the intraintestinal inoculation of many bacteria recovered aerobically or anaerobically from the caecum of these rabbits or by the intestinal inoculation of large numbers of a serogroup of E. coli, 0153, recovered from the caecum. Further experiments showed that this serogroup of E. coli, as well as a nonenteropathogenic serotype recovered from human faeces, would cause typhlitis and diarrhoea if inoculated in large numbers into the jejunum; pathological changes also were seen in the liver and kidney. Similar changes also could be induced by intravenous inoculation of a freeze-thaw (endotoxic) extract prepared from these strains. Any factor that allows rapid multiplication of E. coli in the rabbit caecum may be followed by absorption of endotoxin and subsequent typhlitis and so metimes by severe diarrhoea; this effect is seen in some field cases of diarrhoea in the rabbit.     

Isolation and characterisation of Shiga toxin-producing Escherichia coli O157:H7 from calves in Argentina

Four hundred and twenty-two calves were examined for intestinal carriage of Shiga toxin-producing Escherichia coli O157:H7 using conventional plating. Two (0.5%) E. coli O157 were recovered. They were compared with 96 Argentine strains of different origin by pulsed-field gel electrophoresis, phage typing and PCR-RFLP of stx2 genes. One strain isolated from a calf, was closely related with 18 strains of clinical origin.     

Prevalence of enteropathic Escherichia coli in dogs with acute and chronic diarrhoea

Samples of faeces from 57 dogs with acute diarrhoea, 82 dogs with chronic diarrhoea, 34 clinically healthy household dogs and 88 kennelled control dogs were analysed by hybridisation, using DNA probes to detect enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E coli (ETEC), verocytotoxin-producing E coli (VTEC), enterohaemorrhagic E coli (EHEC), enteroinvasive E coli (EIEC) and enteroaggregative E coli (EAggEC). Samples of duodenal juice from 60 of the 82 dogs with chronic diarrhoea were also examined. Significantly more of the dogs with diarrhoea were excreting EPEC (acute 35.1 per cent, chronic 31.7 per cent) and VTEC (acute 24.6 per cent, chronic 28 per cent) than the kennelled dogs (EPEC 17.1 per cent, VTEC 0 per cent) or the household control dogs (EPEC 6 per cent, VTEC 5.9 per cent). Enteropathic E coli was also detected in the duodenal juice of 23 of 60 (38.3 per cent) of the dogs with chronic diarrhoea. The EPEC attaching and effacing A (eaeA) gene and the verocytotoxin 1 (VR1) gene coding for VTEC were often found together. There was good agreement between in vitro studies and hybridisation for the detection of eaeA and VT1. Isolates from the dogs with diarrhoea adhered significantly more to Hep-2 cells, and VT1-positive strains from the dogs with diarrhoea consistently killed more than 50 per cent of Vero cells.     

Efficacy of danofloxacin 18% injectable solution in the treatment of Escherichia coli diarrhoea in young calves in Europe

The efficacy of danofloxacin 18% against naturally occurring Escherichia coli diarrhoea was investigated in calves at seven European sites. Treatment commenced on day 0, with either a single subcutaneous injection of danofloxacin 18% (n=267) at 6 mg/kg repeated on day 2 if required, or reference product containing baquiloprim/sulphadimidine (n=37) or gentamicin (n=98) administered as recommended. E. coli was isolated from 90% to 100% of calves pre-treatment, and the prevalence of serotypes K99 and F41 was 8-46% and 46-92%, respectively. In both treatments, the majority of calves (93.2-93.9%) showed clinical improvement and completed the studies. There were significant reductions for both treatments, in severity of clinical signs on days 4 and 10 compared to day 0 (P<0.0001), and between days 4 and 10 (P<0.05), but no significant differences between treatments (P>0.05). Danofloxacin 18% was clinically safe, and as effective as the reference products in the treatment of E. coli diarrhoea in calves.     

Isolation of enterotoxigenic Escherichia coli from camels with diarrhoea.

E. coli serogroups 02, 08, 083, 0103 and 0120 were isolated from seven camels with diarrhoea of which 02, 08, and 083 were found to be enterotoxigenic on rabbit ligated ileal loop test. Out of 125 apparently healthy camels, 75 strains of E. coli were isolated. The majority of isolates were susceptible to gentamycin, nitrofurantoin, trimethoprim plus sulphonamide, neomycin, kanamycin and chloramphenicol.