High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil

Veterinary Microbiology 70 (1999) 111±121

High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de Janeiro State, Brazil Aloysio M.F. Cerqueiraa,*,1, Beatriz E.C. Gutha, RogeÂrio M. Joaquimb, JoaÄo R.C. Andradec a

Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, SaÄo Paulo, Brazil b Departamento de Microbiologia e Parasitologia, UFF, NiteroÂi, Rio de Janeiro, Brazil c Disciplina de Microbiologia e Imunologia, UERJ, Rio de Janeiro, Brazil Received 9 March 1999; accepted 29 July 1999

Abstract In order to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains, 197 fecal samples of healthy cattle from 10 dairy farms, four beef farms and one slaughterhouse at Rio de Janeiro State, Brazil, were examined for Shiga toxin (Stx) gene sequences by polymerase chain reaction (PCR). For presumptive isolation of O157:H7 E. coli, the Cefixime-potassium telluritesorbitol MacConkey Agar (CT-SMAC) was used. A high occurrence (71%) of Stx was detected, and was more frequently found among dairy cattle (82% vs. 53% in beef cattle), in which no differences were observed regarding the age of the animals. Dot blot hybridization with stx1 and stx2 probes revealed that the predominant STEC type was one that had the genes for both stx1and stx2 in dairy cattle and one that had only the stx1 gene for beef cattle. Three (1.5%) O157 : H7 E. coli strains were isolated from one beef and two dairy animals by the use of CT-SMAC. To our knowledge, this is the first report of O157:H7 isolation in Brazil. A PCR-based STEC detection protocol led to the isolation of STEC in 12 of 16 randomly selected PCR-positive stool samples. A total of 15 STEC strains belonging to 11 serotypes were isolated, and most of them (60%) had both stx1 and stx2 gene sequences. Cytotoxicity assays with HeLa and Vero cells revealed that all strains except two of serotype O157:H7 expressed Stx. The data point to the high prevalence of STEC in our environment and suggest the need for good control strategies for the prevention of contamination of animal products. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Shiga toxin Escherichia coli; Epidemiology; Polymerase chain reaction; Cattle; Stx *

Corresponding author. Tel.: 55-0216205266; fax: 55-0216205266. E-mail address: [email protected] (A.M.F. Cerqueira) 1 Departamento de Microbiologia e Parasitologia, Universidade Federal Fluminense, Rua Hernani Mello, 101, Centro, NiteroÂi, CEP 24210-130 Rio de Janeiro, Brazil. 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 1 2 3 - 6

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1. Introduction Shiga toxin-producing Escherichia coli (STEC) strains are recognized as important etiological agents of diarrheal disease and hemorrhagic colitis (HC) in humans (Nataro and Kaper, 1998). In some patients, bloody diarrhea and HC usually precede the occurrence of hemolytic-uremic syndrome (HUS), a life-threatening sequel characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia, and its variant form referred to as thrombotic thrombocytopenic purpura (TTP) (Paton and Paton, 1998). Unlike other enterovirulent E. coli categories, the natural hosts of STEC are wildlife and domestic animals, mainly cattle (Griffin and Tauxe, 1991; Beutin et al., 1993). Most animals carrying STEC in their gastrointestinal tracts are asymptomatic, but some STEC strains may be associated with diarrhea in calves (Dean-Nystron et al., 1998; Nataro and Kaper, 1998; Wieller et al., 1998). Fecal shedding is influenced by a range of factors such as diet, stress and season (Griffin and Tauxe, 1991; Diez-Gonzalez et al., 1998). STEC strains comprise a recently recognized food-borne pathogenic group transmitted specially by meat and dairy products contaminated by bovine intestinal contents and improperly cooked or processed (Meng and Doyle, 1998). Shiga toxins (Stx) are produced by E. coli belonging to a broad range of serogroups, although large outbreaks and cases of HC and HUS are mostly associated with STEC strains belonging to serogroup O157 (Strockbine et al., 1998). However, there is a growing body of evidence pointing to the role of non-O157 serogroups, such as O26, O91, O103, O104 and O111 in human disease (Johnson et al., 1996; Paton and Paton, 1998). STEC are distributed worldwide but, virtually all human outbreaks and most sporadic cases were reported from developed nations of the northern hemisphere, with high prevalence in USA (Griffin, 1998), Canada (Spika et al., 1998), United Kingdom (Smith et al., 1998), some Continental European countries (Caprioli and Tozzi, 1998), and Japan (Michino et al., 1998). STEC have also been described associated with human disease in some countries of the southern hemisphere, such as South Africa, Australia, Chile and Argentina (Lopez et al., 1998; Robbins-Browne et al., 1998; Nataro and Kaper, 1998). A high prevalence of STEC in bovine hosts is usually found in those countries where high rates of STEC-associated human illnesses are reported. However, in some countries, such as Spain, high rates of STEC occurrence in food and animal sources coexist with a low incidence of human illness. (Blanco et al., 1996).The available data from Brazil pointed to a very low occurrence of STEC in human disease (Giraldi et al., 1990; Guth et al., 1994; Rosa et al., 1998). However, in a recent study, a high frequency of non-O157 STEC was found in ground beef and meat products at Rio de Janeiro City, Brazil (Cerqueira et al., 1997 ). Given the high sensitivity and ability to detect STEC strains, PCR amplification of Stx gene sequences present in fecal samples has been accepted as the most sensitive and specific means for STEC screening (Paton and Paton, 1998). Inhibitory effects on PCR reaction by components present in feces are avoided by the use of primary fecal cultures. In this work, we used PCR analysis of primary fecal cultures and

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conventional isolation techniques to study STEC occurrence in feces from healthy dairy and beef cattle from rural counties which serve as milk and meat suppliers at Rio de Janeiro City. 2. Material and methods 2.1. Stool samples Rectal swabs were collected during the period of April 1996 to September 1997, from 197 healthy animals taken from 10 dairy farms (n = 121), four beef farms (n = 60) and two samplings from one slaughterhouse (n = 16). All those facilities are localized in six rural counties serving as milk and meat suppliers at Rio de Janeiro City. Animals from dairy herds belonged to three age groups (calves up to 4 months; heifers from 5 months to 2 years and cows >2 years of age), while all other animals were adults (>2 years.). Rectal swabs were collected and immediately transported in Cary±Blair medium (Difco Laboratories, Detroit, MI) in an insulated container. In the laboratory, swabs were kept at 48C and processed within seven days of collection. 2.2. Culture for E. coli O157 All stool samples were streaked onto Cefixime-potassium tellurite-sorbitol MacConkey (CT-SMAC) agar (Zadik et al., 1993) and the plates were incubated at 378C for 18 h. Non-sorbitol fermenting colonies were biochemically tested (Ewing, 1986) and those confirmed as E. coli were submitted to agglutination tests with O157 and H7 antisera (Probac do Brasil, SaÄo Paulo, Brazil). E. coli O157 : H7 strain E40705 (Central Public Health Laboratory [PHLS], London) was used as a positive control for culture procedures. 2.3. PCR primers and processing of samples for PCR analysis T h e p r i m e r s M 1 ( 5 0 ATAC AG AG ( G A ) G ( G A ) AT T C C G T 3 0 ) a n d M 2 (5 TGATG(AG)CAATTCAGTAT30 ) used were previously described by Paton et al. (1993). This single pair of primers is based on consensus sequences and allows amplification of a gene sequence encoding subunit A in stx1 ( 215-bp fragment; nucleotides 586 to 800) and in stx2 (212-bp fragment; nucleotides 583-794). Primers were synthesized by Gibco-BRL (Life Technologies, Gaithersburg, MD). Samples were processed for PCR analysis as described by Schultsz et al. (1994). Briefly, rectal swabs were streaked onto cystine lactose electrolyte deficient (CLED) agar (Difco) to obtain confluent growth. Polymicrobial growth was collected in 4 ml of 0.1 M phosphatebuffered saline (PBS; pH 7.4) and 100 ml was then diluted 10-fold in sterile distilled water. Target DNA to PCR was obtained by heating the diluted bacterial suspension for 10 min in a boiling water bath. At the same time, 1 ml samples of the thick bacterial suspension were mixed with 1 ml of 20% glycerol-tryptic soy broth (2 concentrated) and stored at ÿ708C. 0

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2.4. PCR protocol A `hot start' protocol including the following components (Gibco BRL) was used: 1.5 mM PCR buffer-Mg; 500 ng of each primer; 2.5 mM dNTPs; H2O; 5 ml of target DNA and 2.5 U of TaqDNA polymerase in a final volume of 50 ml. The amplification was processed in a DNA thermal Minicycler (MJ, Watertown, MA) and the cycling conditions consisted of 35 cycles with 94, 60 and 728C as the temperatures of denaturation, annealing and elongation, respectively. A reagent blank (without DNA template), Stxnegative E. coli DH5a (K12) and Stx-positive STEC strain E40705 (stx1) were used as controls in every PCR procedure. Stool samples showing positive results by PCR were retested at least once to evaluate the reproducibility of PCR testing. The amplicons were electrophoresed on 1.8% agarose gels at 130V/45 min and were stained with ethidium bromide. A 0.1 kb DNA ladder (Gibco BRL) was run with each gel. 2.5. Confirmation of PCR amplification products Amplicons from PCR assays were confirmed as stx products by dot-blot hybridization (Sambrook et al., 1989). Briefly, an aliquot of 3 ml of PCR amplification products was diluted in SSC 6X, denaturated in a water bath and spotted onto two pieces of nylon membranes (Gene Screen Plus, Du Pont, NEN, Boston, MA). The DNA was denaturated, neutralized and then fixed by exposure of membranes to UV light. stx1 and stx2 -labeled DNA probes (Newland and Neill, 1988) were used to distinguish between the Stx genes. Amplification products of STEC strains E40705 (stx1) and E30138 (stx2) (PHLS) and E.coli DH5a were used as positive and negative controls, respectively. 2.6. Recovery of STEC from PCR-positive stool samples From each farm or sampling point (n = 16), at least one PCR-positive stool sample was randomly chosen for STEC isolation. A PCR-based strategy was used to detect and isolate STEC from PCR-positive stool samples. Bacterial suspensions stored at ÿ708C were thawed and serially diluted 10-fold (10ÿ5±10ÿ7) in PBS and were streaked onto MacConkey agar (Difco). After overnight incubation at 378C, lactose-positive colonies were picked and spotted onto trypic soy agar (TSA; Difco) and the plates were incubated for 18 h at 378C. Portions of 20 colonies were pooled in microcentrifuge tubes containing PBS, diluted in water, heated and submitted to PCR. Individual colonies from at least one PCR-positive pool were further tested by PCR. A single PCR-positive colony was chosen from each sample as the representative STEC strain. STEC strains detected were biochemically confirmed as E. coli and retested for stx1 and stx2 genes by colony blot hybridization (Maas, 1983). 2.7. Cytotoxicity assay Filter-sterilized culture supernatants of the STEC strains grown overnight in Penassay broth (Antibiotic Medium No. 3, Difco) were tested for the presence of Stx in Vero (ATCC CCL 81) and HeLa (ATCC CCL 2) cells according to Gentry and Dalrymple

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(1980). Stx-gene bearing strains that gave negative results in the cytotoxicity assays were further tested after polymyxin treatment, as described by Evans et al. (1976). 2.8. E. coli serotyping The serotypes of the strains were determined according to standard methods (Ewing, 1986) using antisera O1 to O172 and H1 to H51, kindly supplied by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). 3. Results PCR analysis of stool samples isolated from healthy cattle revealed that 139 of 197 (71%) samples harbored stx sequences. A higher frequency was found among dairy cattle (99 of 121 [82%]) when compared with beef cattle (40 of 76 [53%]) (Fig. 1). High rates of stx carriage were found among dairy animals of all age groups (Fig. 2). Dot blot hybridization assays showed that 81 of 139 (58%) PCR positive stool samples carried stx1/stx2 simultaneously, and stx1- or stx2-only genotypes were found in 37 (27%) and 21 samples (15%), respectively. The relative distribution of toxigenic genotypes differed among dairy and beef cattle, stx1/stx2 type prevailed in dairy cattle (69%) while stx1 was more frequently found among beef animals (45%) (Fig. 3). Direct streaking of all 197 stool samples onto CT-SMAC yielded the isolation of 3 O157 STEC strains from two dairy calves and one beef cattle, and all these animals were from distinct farms. These three stool samples were also positive in the PCR assay and were not submitted to the PCR-based isolation protocol because STEC strains were already detected.

Fig. 1. Occurrence of stx sequences in bacterial growth from stool samples of healthy cattle at Rio de Janeiro State, Brazil, detected by PCR.

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Fig. 2. Relative frequencies of stx sequences in bacterial growth from stool samples of dairy cattle according to age group.

Fig. 3. Stx toxigenic types found in the PCR positive stool samples of healthy cattle.

Isolation of STEC from PCR-positive stool samples was limited to one sample from each farm or sampling point (n = 16), and this approach allowed the isolation of at least one STEC strain from 12 distinct stool samples. The serotypes and the toxigenic profiles of the 15 STEC strains isolated are shown in Table 1. Eleven distinct serotypes were found, and most of the STEC strains were stx1/

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Table 1 Shiga toxin-producing Escherichia coli (STEC) strains isolated from dairy and beef cattle at Rio de Janeiro State, Brazil Strain

Source

Toxin genotypea

Serotypeb

B1/1 B16/2 B18/1 B30/5 B40/23 B47/32 B55/1 B63/17 B73/36 B124/11 GC9/38 GC53/24 GC62/40 GC78/28 GC148

dairy calf dairy calf dairy calf dairy cow dairy calf dairy calf dairy heifer dairy calf dairy cow dairy calf beef cattle beef cattle beef cattle beef cattle beef cattle

stx2c stx1/stx2 stx2c stx1/stx2 stx1 stx2 stx1/stx2 stx2 stx2 stx1/stx2 stx1/stx2 stx1/stx2 stx1/stx2 stx1/stx2 stx1/stx2

O157:H7 O82:H8 O157:H7 NT:H21 NT:NM NT:H18 R:H19 O22:H16 NT:H28 O22:H16 R:H8 R:H19 R:H18 NT:H42 O157:H7

a

Colony hybridization assays with stx1 and stx2 DNA probes. NT, non-typable with O1 to O172 antisera; NM, non-motile; R, rough. c Cytotoxic effects were not detected on Vero and HeLa cells. b

stx2(60%), but five strains were stx2 (33%) and one was stx1(7%) as determined by colony hybridization assays. Cytotoxicity tests on Vero and HeLa cells showed the production of Stx in 13 of the 15 STEC strains. No cytotoxic effect was observed in filtrates from the 2 O157:H7 strains isolated from dairy cattle, even after polymyxin extraction. 4. Discussion In the present study, a high occurrence (71%) of stx sequences was found in healthy cattle from Rio de Janeiro in the first systematic survey of STEC strains in cattle carried out so far in Brazil. The prevalence of STEC in cattle has been described in several studies from different countries. In Canada, two surveys conducted in dairy cattle, in 1988 and 1992, showed STEC prevalence rates of 17% and 45%, respectively (Johnson et al., 1996). Wells et al. (1991) in Wisconsin, USA, detected STEC from 19% of calves and 8% of adult cows at dairy farms. In Germany, 21% of healthy cattle harbored STEC strains (Montenegro et al., 1990; Beutin et al., 1993), and in Thailand, Suthienkul et al. (1990), found STEC in 26 and 82% of cattle stool samples collected at farms and slaughterhouses, respectively. In Spain, the overall prevalence rates of STEC colonization were recently estimated to be 38% in calves and 27% in cows (Blanco et al., 1998), and in Argentina, Sanz et al. (1998) reported prevalence as high as 44% from cows. Although the frequency of stx detected in the present study fits among the higher rates already described, the data on the prevalence

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of STEC in cattle are difficult to compare due to the varying study designs and the isolation methods used. Stx gene sequences occurred at high rates among calves, cows and heifers, despite the low number of samples from animals of the latter age group. These findings were similar to the ones described by Blanco et al., 1998 and Sanz et al., 1998, but differed from others in which higher frequencies of STEC, mainly the O157:H7 serotype, were detected in calves (Griffin and Tauxe, 1991; Nataro and Kaper, 1998). Differences on the stx frequency between beef and dairy cattle were also observed in this study, confirming previous reports (Griffin and Tauxe, 1991), and probably reflecting the distinct management of those animals. The stx2 and stx1/stx2 genotypes prevailed among stool samples from dairy cattle, as reported also by Blanco et al. (1997) and Sanz et al. (1998), while stx1only was more common among beef cattle. However, it could not be ruled out that the origin and dispersion of animals may have influenced such results since samples of beef cattle were collected from animals of distinct rural counties, while all dairy animals came from unrelated farms, but which were located at the same county (data not shown). Three E. coli strains belonging to the serotype O157:H7 were isolated. To our knowledge, this is the first report of O157:H7 isolation in Brazil. Several authors have emphasized that the immunomagnetic separation (IMS) technique improves the isolation of O157 strains from food and stool samples (Strockbine et al., 1998; Patton and Patton, 1998). In the present study, no enrichment step like IMS was done, and one may therefore suggest that the prevalence of O157:H7 strains in bovine stools could be higher than the 1.5% presently detected. Two of the 3 O157:H7 strains did not express Stx, a characteristic also described in a study recently conducted in Italy (Bonardi and Maggi, 1998). The PCR-based detection protocol led to the isolation of STEC in 12 of the 16 bacterial suspensions derived from PCR-positive stool samples. These results suggest that STEC comprise at least 5% of the E. coli isolates analyzed per sample, representing a high rate of colonization in most of the animals studied. Several serotypes were identified among the 15 STEC strains isolated, most of which have been previously detected in human and food sources (Johnson et al., 1996; Cerqueira, et al., 1997; Strockbine et al., 1998). Some of these serotypes (O22:H16, O82:H8, NT:H21 and NT:H28) have also been associated with human disease besides serotype O157:H7. Moreover, several strains were rough or untypable, a common characteristic previously observed among STEC strains isolated from food and animal sources (Gonzalez and Blanco, 1989; Cerqueira et al., 1997). Recently, a high frequency of non-O157 STEC strains in ground beef and meat products was described at Rio de Janeiro (Cerqueira et al., 1997). The present study showed that STEC was also widely distributed in healthy beef and dairy cattle in this region, and for the first time O157:H7 strains were isolated and identified in Brazil. Despite the fact that human STEC associated diseases are infrequently described in our country so far, all the data presently obtained point to the great occurrence of STEC in our environment and emphasize the importance of good control strategies for the prevention of contamination of animal products.

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Acknowledgements This study was supported by grants from Fundac,aÄo de Amparo aÁ Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Financiadora de Estudos e Projetos/ MinisteÂrio da CieÃncia e Tecnologia/Programa de Apoio a NuÂcleos de ExceleÃncia (FINEP/MCT/Pronex). Cerqueira, AMF and Joaquim, RM acknowledges FAPERJ and Coordenac,aÄo de Aperfeic,oamento de Pessoal de NõÂvel Superior/ MinisteÂrio da Educac,aÄo e Desportos (CAPES) respectively, for research fellowships. We thank Expedito B. Coelho and Tania A.T. Gomes for sampling and colony hybridization facilities, respectively.

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