Serotypes and analysis of distribution of Shiga toxin producing Escherichia coli from cattle and sheep in the lower North Island, New Zealand

AIMS: To serotype a subset of Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep to determine whether any corresponding serotypes have been implicated in human diarrhoeal disease, both in New Zealand and worldwide, and to examine the distribution of STEC and enteropathogenic Escherichia coli (EPEC) amongst cattle (calves, heifers and dairy) and sheep (lambs, rams and ewes), to assess whether carriage of identified bacterial genotypes may be associated with a particular age of animal. METHODS: Recto-anal mucosal swabs (RAMS) were taken from 91 calves, 24 heifers and 72 dairy cattle, and 46 lambs, 50 ewes and 36 rams, from four sites in the Manawatu and Rangitikei regions of New Zealand. Strains of E. coli selected from primary isolation plates were subjected to a multiplex polymerase chain reaction (PCR), to determine the presence of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Overall, 186/319 (58.3%) animals sampled were positive for stx1, stx2, or eae isolates. More sheep (43.9%) were stx1-positive than cattle (2.7%; p = 0.036), and amongst sheep more lambs and ewes were stx1-positive than rams (p = 0.036). Amongst cattle, more calves and heifers were eae-positive than dairy cows (p = 0.030). Two or more different STEC were isolated from at least 28 (9%) animals (three cattle and 25 sheep), based on their stx/eae genotype. Enterohaemolysin genes were found in 39/51 (76%) isolates serotyped. Twenty-one different serotypes were detected, including O5:H-, O9:H51, O26:H11, O84:H-/H2 and O149:H8 from cattle, and O26:H11, O65:H-, O75:H8, O84:H-, O91:H-, O128:H2 and O174:H8 from sheep; O84:H-, O26:H11, O5:H-, O91:H- and O128:H2 serotypes have been associated with human disease. CONCLUSIONS: If nationally representative, this study confirms that cattle and sheep in New Zealand may be a major reservoir of STEC serotypes that have been recognised as causative agents of diarrhoeal disease in humans. Distribution of STEC and EPEC in cattle and sheep indicates that direct contact with, in particular, calves or their faeces, or exposure to environments cross contaminated with ruminant faeces, may represent an increased risk factor for human disease in New Zealand.     

Shiga toxin-producing Escherichia coli in the feces of Alberta feedlot cattle

Shiga toxin-producing Escherichia coli (STEC) are a public health concern. Bacterial culture techniques commonly used to detect E. coli O157:H7 will not detect other STEC serotypes. Feces from cattle and other animals are a source of O157:H7 and other pathogenic serotypes of STEC. The objective of this study was to estimate the pen-level prevalence of Shiga toxins and selected STEC serotypes in pre-slaughter feedlot cattle. Composite fecal samples were cultured and a polymerase chain reaction (PCR) was used to detect genes for Shiga toxins (stx1 and stx2) and genes for O157:H7, O111:H8, and O26:H11 serotypes. Evidence of Shiga toxins was found in 23 pens (92%), O157:H7 in 2 (8%), O111:H8 in 5 (20%), and O26:H11 in 20 (80%) of the 25 pens investigated. Although pen-level prevalence estimates for Shiga toxins and non-O157 serotypes seem high relative to O157:H7, further effort is required to determine the human health significance of non-O157 serotypes of STEC in feedlot cattle.     

Association between animal age and the prevalence of Shiga toxin-producing Escherichia coli in a cohort of beef cattle

Even with advancements in pre- and post-harvest food safety, Shiga toxin-producing Escherichia coli (STEC) still present challenges to human health. Since cattle are the primary reservoir for STEC, lowering the prevalence of this pathogen in farm animals may reduce STEC outbreaks in humans. However, because many of the factors that modulate the colonization and persistence of STEC in cattle remain unknown, reducing STEC in this host is challenging. In this study, we evaluated a cohort of beef cattle one to eleven years of age to determine the effect of animal age on the prevalence of STEC. During the first year of sample collection, heifers had significantly lower STEC prevalence than cows (37.5% vs. 70%). In the second year of sample collection, STEC prevalence peaked in cows that were two years of age and tended to decrease as animals became older. In addition, by studying a subset of the animals in both years, we observed an increase in STEC prevalence from 40.6% to 53.1% in heifers, whereas cows had a net decrease in STEC prevalence from 71.4% to 61.9%. The results from this study indicate that animal age is a significant factor that influences the prevalence of STEC in cattle. These findings have implications for the development of on-farm mitigation strategies by targeting animals with the highest risk of shedding; it could be possible to reduce pathogen transmission among cattle and prevent zoonotic or foodborne transmission to humans.     

Virulence markers in Shiga toxin-producing Escherichia coli isolated from cattle.

This study identified potential virulence markers in 93 eae-positive and 179 eae-negative Shiga toxin-producing Escherichia coli (STEC), isolated from a random sampling of healthy cattle in southwestern Ontario. PCR amplification was used to identify genes for enterohemorrhagic E. coli (EHEC)-hemolysin, the EAF plasmid, and bundle-forming pili (Bfp); adherence to HEp-2 cells and to bovine colonocytes, and the fluorescent actin staining (FAS) test were used to characterize interaction of the bacteria with epithelial cells. The EHEC-hemolysin sequences were detected in 98% of eae-positive isolates compared with 34% of eae-negative isolates. All isolates were negative for EAF and bfp sequences. There was 100% correlation between localized adherence (LA) to HEp-2 cells and the FAS test. Forty-eight (52%) of the eae-positive isolates were LA/FAS-positive, whereas none of the 179 eae-negative isolates was positive in either test. Among the eae-negative isolates, 20 (11%) showed diffuse adherence and 5 (2.8%) showed enteroaggregative adherence to HEp-2 cells. Seventy-three percent of the eae-positive isolates adhered to bovine colonocytes, whereas only 26% of 120 eae-negative isolates that were tested adhered. All 13 O157:H7 isolates were positive for eae and EHEC-hemolysin gene sequences, LA/FAS, and adherence to bovine colonocytes. It is concluded that possession of genes for eae and EHEC hemolysin is correlated with the serotype of STEC, that production of EHEC hemolysin was highly correlated with serotypes implicated in human disease, and that none of the potential markers that were examined can be used to predict the potential virulence of an isolate.     

Serotypes of verotoxin-producing (Shiga toxin-producing) Escherichia coli isolated from healthy sheep

Using PCR techniques Shiga toxin-producing strains of Escherichia coli were isolated from the faeces of 45 out of 101 healthy sheep. These strains were serotyped and found to include O5:H-, O91:H- and O163:H19, which had previously been reported as being associated with human disease including haemolytic uraemic syndrome.     

The occurrence of Shiga toxin-producing Escherichia coli in humans and animals

Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause haemorrhagic colitis and the diarrhoea-associated form of the haemolytic-uraemic syndrome in humans. The main cause of STEC infections in humans is the consumption of contaminated food. Both sporadic cases and outbreaks of STEC infections are mainly associated with the consumption of undercooked (minced) beef (mainly hamburgers) and unpasteurized milk. Therefore cattle are regarded as the primary reservoir of STEC. In this article the occurrence of STEC infections in humans and the occurrence of STEC in food and food-producing animals in the Netherlands are discussed, followed by a brief discussion of possible ways to prevent STEC infections in humans.     

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.     

Pathogenic Shiga toxin-producing Escherichia coli in the intestine of calves

The purpose of this study was to compare the pathological effects of Shiga toxin-producing Escherichia coli (STEC) that vary in their association with bovine and human disease. Shiga toxin-producing E. coli of serotypes associated with both dysentery in calves and hemolytic uremic syndrome (HUS) in humans (O5:H-, O26:H11, O111:H-, O113:H21) were compared with O157:H7 STEC, which are associated with HUS in humans but not with disease in calves. The STEC were administered orally to 80 day-old chicks and into ligated loops in the ileum and colon of four 2- to 6-day-old calves. Examination of the ceca of the chickens 10 d postchallenge showed no adherence or tissue abnormality for any isolate. The calves were euthanized 8 to 10 h postinoculation, and sections of the intestinal loops were examined by light microscopy, transmission and scanning electron microscopy, and immunohistochemistry. All strains showed consistent focal adherence associated with mild lesions in the colon. Attaching and effacing lesions were observed with the eae-positive strains. Ileal lesions were similar to the colonic ones but were sometimes severe, with marked polymorphonuclear leukocyte proliferation in the lamina propria. It is concluded that chickens were unsuitable for studying interaction of STEC with the intestine and that there was no difference in the interaction of the ligated calf intestine with STEC of serotypes associated with disease in calves compared with O157:H7 STEC.     

Long-term survival of Shiga toxin-producing Escherichia coli in cattle effluents and environment: an updated review

Shiga toxin-producing Escherichia coli (STEC) are one of the most important emergent foodborne pathogens. STEC are common as colonizers in the intestine of healthy cattle and are spread into the environment by fecal shedding or following the surface application of farm effluent on soil. The bacteria can be transmitted to humans through food, such as inadequately cooked ground beef or unpasteurized milk. During the last decade, a wide variety of environmentally related exposures have emerged as new routes of transmission. Major outbreaks due to the consumption of raw fruits and vegetables or accidental ingestion of soil or water contaminated by STEC have been increasingly reported. STEC survival in cattle effluents, soil, plants and water is discussed in the light of new knowledge regarding both biotic and abiotic factors which may affect their survival or enhance their dissemination in the environment. The ability to persist in cattle production environments contributes to the contamination and recontamination of cattle, as well as for human infection. Consequently, effective control strategies must be considered on cattle farms, in order to limit entry of STEC cells into the environment.     

Shiga toxin-producing Escherichia coli (STEC) isolated from healthy dairy cattle in southern Brazil

Over a period of 1 year, the production of verotoxin was investigated in 1127 Escherichia coli isolated from 243 dairy cattle from 60 small farms in southern Brazil. Vero cell assay was used to detect toxins in culture supernatants from E. coli isolated from bovine feces. Shiga toxin-producing E. coli (STEC) detection rates were 95% (57 of 60) for farms and 49% (119 of 243) for cattle. Prevalence of STEC-positive cattle in the farms ranged from 0 to 100%. Ninety-six percent (315 of 327) of the STEC isolates did not react in the panel of sera used for typing. Twelve isolates, all non-motile, belonged to serogroups previously associated with human diseases, and 67% (8 of 12) were of only two serotypes (O91:H- and sorbitol-fermenting O157:H-). These results indicate that dairy cattle from the region surveyed may be a source of STEC potentially pathogenic for humans.     

The identity and prevalence of coccidia species in sheep and cattle in New Zealand

The results of a national survey of faeces from clinically normal deer for Salmonella sp. and Yersinia pseudotuberculosis are reported. Five isolates of Y. pseudotuberculosis and none of Salmonella sp. were made from 3810 faeces representing 122 farms.     

Detection and characterization of Shiga toxin-producing Escherichia coli in feral pigeons

Escherichia coli strains producing a variant of Shiga toxin 2 (Stx2), designated Stx2f, have been recently described in the stools of feral pigeons. During 1997-1998, 649 pigeons were trapped and examined in three different squares of Rome. Stool samples were collected from each bird and enrichment cultures were examined for the presence of Stx by the vero cell assay. Stx-producing E. coli (STEC) were isolated from the positive cultures and characterized by serotyping and PCR analysis of stx and other virulence-related genes. Stx was detected in 10.8% of the stool enrichment cultures. The percentage of positive birds did not differ significantly for the three flocks considered and the season of sample collection. Conversely, STEC carriage was significantly more frequent in young than in adult birds (17.9 versus 8.2%). None of the birds examined showed signs of disease. STEC strains were isolated from 30 of 42 Stx-positive cultures examined. All the strains produced Stx2f, and most of them possessed genes encoding for intimin and the cytolethal distending toxin (CLDT). Six serogroups were identified, but most of the isolates belonged to O45, O18ab, and O75. Molecular typing indicated that most of the isolates within a flock were clonally-related. This work confirms that pigeons represent a natural reservoir of STEC strains characterized by the production of the toxin variant Stx2f, and by the frequent presence of eae and cldt genes. Further work is needed to clarify whether these STEC may represent a cause of avian disease or even a potential health hazard for humans.     

A survey of the prevalence of emerging macrocyclic lactone resistance and of benzimidazole resistance in sheep nematodes in the lower North Island of New Zealand

AIM: To investigate the occurrence of emerging macrocyclic lactone (ML) resistance and of resistance to benzimidazole anthelmintics on a number of sheep farms in the North Island of New Zealand. METHODS: On commercial sheep farms (n=30) in the Taihape district in the North Island of New Zealand, 30 animals were randomly allocated to one of two equal-sized groups and treated with either half of the recommended dose rate of ivermectin (half of 0.2 mg/kg), or with the full recommended dose rate of oxfendazole (4.5 mg/kg). The ivermectin treatment only was used on a further six properties. Faecal egg counts, accompanied by pooled larval cultures, were conducted on all samples at the time of treatment and 7-10 days later. RESULTS: Resistance, as indicated by a <95% faecal egg count reduction (FECR) in both instances, was found to oxfendazole on 13/30 (43%) farms and to a half dose of ivermectin on 12/36 (33%) properties. For oxfendazole, such resistance was found to involve all six nematode genera whereas for ivermectin it was almost entirely restricted to Ostertagia and Cooperia infections. CONCLUSIONS: These results indicate that emerging ML resistance may be more common on sheep farms in New Zealand than is generally realised. They also suggest that the half-dose ivermectin faecal egg count reduction test (FECRT) may offer some very practical benefits for parasite control by providing early warning of developing resistance to ML drenches and by signalling the possible imminent failure of these at their therapeutic dose rates. The sensitivity and reliability of this procedure may be further enhanced by the inclusion of larval cultures.     

A survey of the prevalence of emerging macrocyclic lactone resistance and of benzimidazole resistance in sheep nematodes in the lower North Island of New Zealand

AIM: To investigate the occurrence of emerging macrocyclic lactone (ML) resistance and of resistance to benzimidazole anthelmintics on a number of sheep farms in the North Island of New Zealand.

METHODS: On commercial sheep farms (n=30) in the Taihape district in the North Island of New Zealand, 30 animals were randomly allocated to one of two equal-sized groups and treated with either half of the recommended dose rate of ivermectin (half of 0.2 mg/kg), or with the full recommended dose rate of oxfendazole (4.5 mg/kg). The ivermectin treatment only was used on a further six properties. Faecal egg counts, accompanied by pooled larval cultures, were conducted on all samples at the time of treatment and 7–10 days later.

RESULTS: Resistance, as indicated by a <95% faecal egg count reduction (FECR) in both instances, was found to oxfendazole on 13/30 (43%) farms and to a half dose of ivermectin on 12/36 (33%) properties. For oxfendazole, such resistance was found to involve all six nematode genera whereas for ivermectin it was almost entirely restricted to Ostertagia and Cooperia infections.

CONCLUSIONS: These results indicate that emerging ML resistance may be more common on sheep farms in New Zealand than is generally realised. They also suggest that the half-dose ivermectin faecal egg count reduction test (FECRT) may offer some very practical benefits for parasite control by providing early warning of developing resistance to ML drenches and by signalling the possible imminent failure of these at their therapeutic dose rates. The sensitivity and reliability of this procedure may be further enhanced by the inclusion of larval cultures.     

Detection of Shiga toxin-producing Escherichia coli by PCR in cattle in Argentina. Evaluation of two procedures

Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.     

The occurrence of anthelmintic-resistant sheep nematodes in the southern North Island of New Zealand

An analysis of veterinary diagnostic cases submitted to the Batchelar Animal Health Laboratory for faecal egg count reduction testing in sheep between 1986 and 1992 showed that 63% of them originated from properties carrying anthelmintic-resistant worms. Most of these cases involved resistance to a single drench type only, with the benzimidazoles being implicated most frequently. Overall, the figures suggested a frequency of occurrence of resistance to benzimidazole anthelmintics of 74%, to levamisole-type anthelmintics of 23% and to benzimidazo-lelevamisole combinations of 30%. No cases of resistance to milbemycin/avermectin-type drenches were recorded.     

Frequency of Shiga toxin-producing Escherichia coli in cattle at a breeding farm and at a slaughterhouse in Japan

To investigate the frequency of Shiga toxin-producing Escherichia coli (STEC) infected calves at a breeding farm and cattle at a slaughterhouse in Tohoku area of Japan, the polymerase chain reaction (PCR) was used for detection of genes for Shiga toxin(s). The fecal samples from a total of 204 calves and 306 cattle were examined. The prevalence rates in calves less than 2 months of age, cattle 2-8 months of age, and adults greater than 1 year of age were 39.4, 78.9, and 40.8%, respectively. Detection frequency of STEC in the fecal specimens from calves aged 0-8 months was not different among the breeds of cattle (Holstein: H, Japanese black cattle: B, and F1: HxB). On the other hand, for calves over 12 months of age, the frequency of STEC in Japanese black cattle and F1 were significantly higher than in Holstein cattle. Serogroups of STEC usually identified in human cases of food poisoning (O157, O26, and O111) were not frequently found in the feces of the cattle.