Detection of Shiga toxin-producing Escherichia coli by PCR in cattle in Argentina

Veterinary Microbiology 87 (2002) 301–313

Detection of Shiga toxin-producing Escherichia coli by PCR in cattle in Argentina Evaluation of two procedures A. Gioffre´a, L. Meichtrib, E. Miliwebskyc, A. Baschkierc, G. Chillemic, M.I. Romanoa, S. Sosa Estanid, A. Cataldia,*, R. Rodrı´guezb, M. Rivasc a

Instituto de Biotecnologı´a, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnologı´a Agropecuaria, Castelar, Argentina b Instituto de Tecnologı´a de Alimentos, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnologı´a Agropecuaria, Castelar, Argentina c Servicio Fisiopatogenia, Instituto Nacional de Enfermedades Infecciosas, ANLIS, Dr. Carlos G. Malbra´n, Buenos Aires, Argentina d Centro Nacional de Diagno´stico e Investigacio´n de Endemoepidemias, Instituto Nacional de Enfermedades Infecciosas, ANLIS, Dr. Carlos G. Malbra´n, Buenos Aires, Argentina Received 25 June 2001; received in revised form 26 March 2002; accepted 26 March 2002

Abstract 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 heatlysis 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 6 h, 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. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Shiga toxin; Escherichia coli; PCR; Cattle-bacteria * Corresponding author. Tel.: þ54-11-4621-1447; fax: þ54-11-4481-2975. E-mail address: [email protected] (A. Cataldi).

0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 0 7 9 - 2

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1. Introduction In the last two decades, Shiga toxin-producing Escherichia coli (STEC) strains have been recognized as an important cause of potentially life-threatening human diseases such as hemolytic uremic syndrome (HUS). These strains constitute one of the most important causes of food-borne disease in developed countries. Since the first report by Riley et al. (1983), STEC has been associated with both outbreaks and sporadic cases of human disease, ranging from uncomplicated diarrhea to hemorrhagic colitis and HUS. E. coli O157:H7 is the prototype STEC. However, there are over 100 other serotypes that share a similar pathogenic potential for humans (Griffin and Tauxe, 1991). Because E. coli O157:H7 infections are often associated with the consumption of contaminated ground beef, raw milk, and other bovine products, cattle are suspected to be a primary reservoir (Belongia et al., 1991; Blanco et al., 1994; Keene et al., 1997; Bielaszewska et al., 1997). Serological studies demonstrated that the majority of cattle have been exposed to STEC (Pirro et al., 1995), but its epidemiological significance is not known. STEC O157:H7 infects both adult animals and calves (Besser et al., 1997). STEC strains have been found to produce a family of related cytotoxins known as Stxs. Stxs have been classified into two major classes, Stx1 and Stx2. Whereas the Stx1 family is very homogenous, several Stx2 variants have been identified. These Stx2 variants are: Stx2c and Stx2d produced by human STEC isolates, Stx2e typically found in STEC pathogenic for pigs, and Stx2f, described recently in STEC isolates from feral pigeons (Pie`rard et al., 1998; Schmidt et al., 2000). An STEC strain can produce Stx1, Stx2 (or its variants), or both. Several virulence factors contribute to the pathogenicity: the eae gene that codes for the intimin required for the attaching-and-effacing lesion. Another putative virulence factor is an RTX toxin designated as EHEC-hemolysin, coded by the EHEC hly operon. It is considered that in North America and Europe around 90% of children with HUS show evidence of STEC infection, with the O157:H7 serotype responsible for 70% of cases (Caprioli et al., 1994; Lior et al., 1994). In Argentina, where HUS is endemic (WHO and Reilly, 1998), approximately 300 new cases are reported annually by hospital nephrology units. At present, the estimated annual incidence rate for HUS in children under five years of age is 9.2 per 100,000. However, the source of STEC infection in Argentina is still unknown. PCR is considered an extremely sensitive diagnostic test, able to detect the genome of only one bacterium. stx gene detection is an useful strategy in evaluating STEC strains by PCR. Due to the genetic heterogeneity of stx genes, different strategies have been developed. Karch and Meyer (1989) used a primer pair whose sequences were partially homologous to Stx1 and Stx2 and probes to differentiate stx genes. Other authors have used primers to distinguish between different genes (Brian et al., 1992). Multiplex PCR is an alternative used for detection of both loci in the same reaction (Paton and Paton, 1998). The sources of template can be as diverse as DNA from a single colony, or fecal or food cultures. However, the application of PCR for the direct detection of STEC in fecal samples has been hampered by the presence of Taq polymerase inhibitors in feces that affect the sensitivity of the assay (Saulnier and Andremont, 1992; Brian et al., 1992; Monteiro et al., 1997). In addition, the number of STEC in feces can be very small. To solve this problem, enrichment of fecal culture, immunomagnetic separation, and purification of DNA with

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silica particles, have also been used (Brian et al., 1992; Bonardi et al., 1999). Several investigators detected STEC bacteria by testing individual colonies, previously cultured in solid media with or without previous enrichment (Heuvelink et al., 1998). These procedures allow the identification of other virulence factors when a positive strain is isolated (Gannon et al., 1993; China et al., 1996; Paton and Paton, 1998). In this study, we evaluated different procedures for detection of STEC strains in beef cattle in Argentine by stx gene amplification. The phenotypic and genotypic characterization of the STEC strains isolated was performed to evaluate the presence of the main virulence-associated genes, toxin production, and antibiotic resistance.

2. Materials and methods 2.1. Cattle sample processing One hundred and sixty steers from the main beef cattle producing area of Argentina (Pampas Region), were sampled from September 1997 to May 1998 at the slaughter line. Fecal samples from the large intestine (cecum) were collected and placed in a sterile plastic bag. Upon arrival at the laboratory, samples were immediately processed and then frozen at 20 8C with the addition of 20% glycerol to further processing. In order to evaluate the presence of STEC in fecal samples, different culture protocols were applied. 2.2. Protocols for STEC detection in feces Protocol A. For primary culture of fecal samples, 1 g of feces was inoculated into 10 ml of m-TSB (modified Trypticase Soy Broth, Difco Laboratories, Detroit, USA), and incubated for 6 and 18 h at 37 8C. Identical conditions were used for Protocol B. The primary fecal culture was allowed to stand for 20 min at room temperature to eliminate insoluble materials, then 1 ml of supernatant was centrifuged. The pellet obtained by centrifugation of the primary fecal culture was processed using the commercial Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI), according to the manufacturer’s instructions. The DNA was resuspended in 20 ml of distilled water and 2 ml were used for PCR amplification. Samples with a positive PCR result, that were kept frozen, were plated onto MacConkey agar (MAC) and sorbitol-MacConkey (Merck, Darmstadt, Germany) supplemented with cefixime (50 ng/ml) and potassium tellurite (25 mg/ml) (bioMe´ rieux, Marcy l’Etoile, France) (CT-SMAC). For STEC isolation, 10 colonies, five from MAC (lactose positive) and five from CT-SMAC (sorbitol negative), were selected and suspended in water. Then, heat lysis and a centrifugation step at 12,000g for 5 min were performed to analyze the virulence factors by PCR using the supernatant as template. Colony blot hybridization assays were applied to confirm PCR results as described below (Fig. 1). Protocol B. Primary fecal cultures in m-TSB, after incubation for 6 or 18 h, were plated onto MAC and CT-SMAC plates. Following overnight incubation, a loop was taken from the bacterial confluent growth zone, suspended in 0.5 ml of distilled water, boiled for 10 min, and centrifuged at 12,000g for 5 min. Five microliters of the supernatant were used

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Fig. 1. Different procedures used to detect STEC in fecal samples of naturally infected healthy steers.

for PCR. From each PCR-positive sample, 10 lactose-fermenting and/or sorbitol nonfermenting colonies were selected and pooled in 500 ml of water, and processed by PCR. If a positive result was obtained, individual colonies were tested by PCR to identify positive isolates. If the pool was negative, another sample was taken from the confluent growth zone, and dilutions were performed in order to obtain individual colonies on MAC to identify STEC by PCR analysis (Fig. 1). Protocol C. Fecal samples were suspended in saline solution, an aliquot was inoculated onto MAC and CT-SMAC, and incubated for 18 h. Typical colonies from each media were selected, plated on TSA and analyzed by PCR as described for Protocol A (Fig. 1). This protocol was used as a Gold Standard (GS), because it is based on individual colony testing and it was used by many other authors (Bokete et al., 1993; Paton et al., 1993; Sanz et al., 1998; Stephan and Untermann, 1999; Pradel et al., 2000).

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PCR sensitivity. The PCR detection sensitivity of Protocols A, B and C was performed using an E. coli 026:H11 strain (stx1/stx2), isolated from a calf that was previously characterized by colony blot hybridization and Vero cell cytotoxicity biological assays. A negative fecal sample was spiked with 50 ml aliquots of a serially diluted 18 h culture of this strain and processed by Protocol A. The minimal number detected was 103 colony forming units (cfu) per gram. When fecal samples with 103 cfu/g were tested by Protocols B and C, similar results were obtained. 2.3. Detection of stx genes by PCR Multiplex PCR was performed to amplify stx1 and stx2 genes. The primers described by Pollard et al. (1990) and Blanco et al. (1996) for Stx1 and Stx2, respectively, were used. The mixture for the amplification of stx1 and stx2 genes consisted of 5 ml of PCR buffer (10 mM Tris–HCl pH 9, 50 mM KCl, and 0.1% Triton X-100), 2.5 mM MgCl2, 0.2 mM of each dNTP, 1 mM of each primer and 1.25 U of Taq polymerase, in a final volume of 50 ml. The amplification conditions consisted of an initial denaturation step at 95 8C for 5 min, and 30 cycles of 95 8C for 1 min (denaturation), 55 8C for 1 min (hybridization), and 72 8C for 1 min (extension), and a final step at 72 8C for 10 min. PCR products were analyzed by electrophoresis on 2% agarose gel and visualized by ethidium bromide staining. 2.4. Identification and characterization of isolates Each colony selected from MAC and CT-SMAC was identified as E. coli by indole production, methyl red, Voges-Proskauer and Simmons’ citrate tests (Hitchins et al., 1992). E. coli virulence factors, including stx1, stx2, and eae genes, were determined by colony blot hybridization assays using specific UTP-digoxigenin-labelled gene probes under stringent washing conditions (Thomas et al., 1991). Gene probes were provided by Drs. J. Kaper and J. Nataro, Center for Vaccine Development, University of Maryland, School of Medicine, Baltimore, Maryland, USA. To determine Stx production, bacterial supernatant fluids and periplasmic cell extracts were used in cytotoxicity assays on Vero cells (Karmali et al., 1985), using Stx1 and Stx2-specific monoclonal antibodies (MAb 13C4 and MAb BC5BB12, respectively), provided by Dr. N.A. Strockbine, Center for Diseases Control and Prevention (CDC), Atlanta, Georgia, USA. For differentiation of the Stx2 variants, the genotyping method of Tyler et al. (1991) was applied. This method is based on restriction fragment length polymorphism (RFLP) analysis of a B-subunitencoding DNA fragment obtained by PCR. Enterohemolysis was determined on TSA agar plates with 5% washed defibrinated sheep blood (Beutin et al., 1989), and the genotype was confirmed by PCR using the primers and conditions described by Schmidt et al. (1995). For eae gene amplification, oligonucleotide primers that amplify a sequence common to STEC and EPEC strains were used (Gannon et al., 1993). Antibiotic susceptibility patterns were assayed by the Kirby Bauer Method (Barry and Thornsberry, 1991) for ampicillin, cefixime, cefotaxime, cefuroxime, cephalothin, chloramphenicol, streptomycin, gentamicin, nalidixic acid, norfloxacin and tetracycline (National Committee for Clinical Laboratory Standards, 2000).

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Serotyping was performed by Dr. Lothar Beutin at the E. coli Reference Laboratory, Department of Microbiology, Robert Koch Institute, Germany, employing specific O and H antisera. 2.5. Reference strains In each assay, the E. coli O157:H7 933J (stx1) and 933W (stx2) strains were used as positive controls. 2.6. Estimation of sensitivity and specificity To assess the sensitivity and specificity of the different protocols for stx gene detection used in the present study, the results of the 64 samples that were processed by all of them were analyzed. The Protocol C was considered as GS. The concordance index (Kappa) was calculated. The statistics analysis was developed using Epiinfo 2000 and Epidat v2.1.

3. Results 3.1. Sampling of healthy steers Protocol A. Five out of 160 (3.1%) stx-positive samples were identified using this protocol. However, only one STEC isolate was recovered from a sample (Table 1). The low sensitivity of this procedure was demonstrated when one stx-positive sample (# 15) was identified during the analysis by probe hybridization of the negative-PCR samples. Furthermore, an additional stx-positive sample (# 17) was identified when individual colonies isolated from animal samples belonged to the same positive-herd, were analyzed by PCR. These false negative results, suggested that Taq inhibitors were not eliminated by this protocol. Protocol B. Eight out of 64 (12.5%) animals were identified as stx-positive by this methodology. STEC strains were isolated from five of eight (62.5%) positive samples from MAC and CT-SMAC media (Table 1). Three samples were identified as stx-positive in both media, whereas four were positive only in MAC and another only in CT-SMAC (Table 2). In three PCR-positive samples, STEC isolates were not recovered in spite of analyzing over 100 colonies per sample. The low number of STEC in these samples could be demonstrated by the fact that in all cases over 30 colonies had to be analyzed to detect the stx-positive isolate by PCR (data not shown). Table 1 Detection of STEC in fecal samples of healthy steers using different PCR-protocols Protocol assayed

No. of samples

No. of positive samples (%)

No. of STEC isolates (%)

A B C

160 64 80

5 (3.1) 8 (12.5) 1 (1.2)

1 (20) 5 (62.5) 1 (100)

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Table 2 PCR-positive fecal samples of healthy steers identified by Protocol B in different culture conditions Sample no.

Enrichment

STEC isolation

MAC

85 91 96 107 108 179 180 184 n¼8 a

CT-SMAC

6h

18 h

6h

18 h

   nda  nd nd  0/5

 þ þ þ þ þ þ þ 7/8

þ þ  þ   þ  4/8

   nd  nd nd  0/5

þ þ  þ   þ þ 5/8

Not done.

Protocol C. Eighty samples were analyzed by this direct culture procedure. Only one sample, gave a positive-PCR result (Table 1). This sample was also positive using Protocol B. The other seven samples identified as positive by Protocol B were negative by Protocol C. 3.2. Sensitivity and specificity of different protocols To assess the sensitivity and specificity of the three different protocols for stx gene detection used in the present study, the results of the 64 samples that were processed by all of them were analyzed. The sensitivity and specificity of the Protocol A were 0 and 100%, respectively. No positive samples by Protocol B (n ¼ 8) could be detected by Protocol A. The sensitivity and specificity calculated to Protocol B was 100 and 89%, respectively (Table 3). Among 64 samples analyzed by Protocols A, B and C, eight samples were Table 3 Sensitivity, specificity and concordance of the protocols evaluated to detect Stx genes in bovine fecal samples Protocol assayed GS—Protocol C (n ¼ 64)

Sensitivity (%) Specificity (%) Concordance (%) Kappa Standard error p-Value

Protocol A

Protocol B

Protocols A and B

0 100.0 98.4 nda – –

100.0 88.9 89.0 0.20 0.07 0.003

– – 87.5 ndb – –

a Not done for statistical impossibility. Results of Protocol A  Protocol C: ‘‘n’’; negneg ¼ 63; pospos ¼ 0; posneg ¼ 0; negpos ¼ 1. b Not done for statistical impossibility. Results of Protocol A  Protocol B: ‘‘n’’; negneg ¼ 56; pospos ¼ 0; pos-neg ¼ 0; neg-pos ¼ 8.

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Table 4 Genotypic and phenotypic characteristics of STEC recovered from fecal samples of healthy steers Sample no.

Genotype

Phenotype

stx1

stx2

eae

EHEC-hlyA

Vero cell cytotoxicity

EHEC-Hly activity

Serotype

15 17 70 85 91 107 180 184

 þ þ     

þ   þ þ þ þ þ

 þ      

þ þ   þ þ þ þ

þ þ þ þ þ þ þ þ

þ þ   þ þ þ þ

O8:H19 O26:H11 O116:NM O2:H25 O8:H19 O11:H14 O8:H19 O8: H19

Total (%)

2 (25)

6 (75)

1 (12.5)

6 (75)

8 (100)

6 (75)

positive (seven by Protocol B, and one by Protocols B and C). Isolates were recovered in five samples (four positive samples by Protocol B, and one positive sample by Protocols B and C). The concordances among Protocols A and C, B and C, A and C were 84.4, 89.0 and 87.5%, respectively. The Kappa index could be calculated only among Protocols B and C (Kappa index ¼ 0:20; S:E: ¼ 0:07; p ¼ 0:003). The Kappa index of the Protocol A could not be calculated because no samples were positive according to this protocol. 3.3. Characteristics of STEC isolates Six of eight STEC isolates harbored the stx2 gene, while two strains carried the stx1 gene. Stx2 variants were not detected by PCR–RFLP. Cytotoxicity assays on Vero cells confirmed the stx results obtained by genotyping techniques. Enterohemolytic activity was detected in six of eight strains, and only one strain carried the eae gene. This last strain belonged to the O26:H11 serotype and was characterized as stx1/eae/EHEC-hlyA (Table 4). In addition, four STEC strains were of the O8:H19 serotype. The other STEC serotypes identified were O2:H25, O11:H14 and O116:NM. The strains detected were susceptible to all of the antibiotics assayed, with the exception of one isolate characterized as STEC O8:H19 stx2/eae-negative/EHEC-hlyA, that was both ampicillin and cephalothin-resistant.

4. Discussion The main purpose of this study was to evaluate different procedures for the detection and isolation of STEC strains in cattle feces. PCR is a fast and sensitive technique that can be used without previous isolation of the pathogenic agent (Bastian et al., 1998). Nevertheless, the presence of PCR inhibitors in feces reported (Saulnier and Andremont, 1992; Brian et al., 1992; Monteiro et al., 1997) is the main limitation to the successful application of the PCR in this type of sample. The protocols that use PCR directly on feces can be suitable when they are used to detect STEC in human patients with clinical symptoms, since the bacterial number in clinical

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samples might be higher than in asymptomatic patients. In a first instance, we used a protocol for amplifying stx directly from feces using non-commercial reagents (data not shown). However, this protocol was unable to identify positive samples. To our knowledge, these kind of protocols have not been previously used on cattle feces. Although preliminary experimental infection of samples, a fundamental step to standardize protocols, gave satisfactory results, it is difficult to reproduce the conditions of a naturally infected sample. In addition, usually only one, or a reduced number, of artificially contaminated samples are used for this purpose. Saulnier and Andremont (1992) observed a PCR-sensitivity variation of up to three log among human feces samples inoculated with the same bacterial concentration. In our study, all protocols could identify stx gene sequences by PCR when a negative bovine fecal sample was spiked with 103 cfu/g of an STEC 026:H11 strain. However, a different sensitivity was observed when naturally contaminated fecal samples were processed by these three procedures. Immunoblot of colony lifts was reported as a sensitive detection of STEC (Atalla et al., 2000). However PCR was also used in many studies. Plating on solid media eliminates the interference of PCR inhibitors. These studies with samples from asymptomatic and symptomatic humans and animals, involved the culture of fecal specimens on solid media such as MAC, SMAC, or sheep blood agar, and the further selection of a number of representative colonies to perform phenotypic and/or genotypic analyses (Bokete et al., 1993; Wieler et al., 1998; Stephan and Untermann, 1999). In our study, those protocols that involved a plating step before PCR, were the most efficient. It is important to consider that although it is known that cattle both carry and excrete STEC, little is known about the ecology of the bacteria in the animal (number, shedding load, etc.). The selection of a low number of representative colonies (as in Protocol C) may underestimate STEC incidence, since the culture media commonly used are not selective enough, and allow the growth of organisms other than STEC. In the studies mentioned above, the number of selected colonies varied from 5 to 10. Heuvelink et al. (1998) collected at least 18 colonies per sample. However, our results suggest that this number may not be enough. A considerable number of the STEC positive samples would have been identified as negative if we had only selected 10 colonies per plate. From the result obtained in the present study, it can be concluded that STEC was present in low number in the asymptomatic cattle studied, since an enrichment step of 6 or 18 h was needed to identify most of the stx-positive samples. In addition, using Protocol C, only one sample was detected as positive without previous enrichment. Most of the STECpositive samples (7/8) from Protocol B, were identified on MAC, while a lower number (4/8) was detected on CT-SMAC, a fact that might be attributed to the inhibitory effect of the CT mix present in the second medium on non-O157 E. coli (Zadic et al., 1993). Protocol B detected the highest number of positive samples, even in samples that were negative by Protocol C (GS). According to these results, it is highly probable that specificity value obtained may be underestimated due to the GS method selected. Some of the samples found positive by Protocol B and negative by Protocol C were true positive as demonstrated by the identification of STEC isolates in these samples. Besides, the identification of STEC isolates using Protocol B is faster and simpler than using Protocols that perform PCR on individual colonies. This recovery is higher than those reported by other authors using different methodologies. Tambunan and Bensik (Robins-Browne et al., 1998) tested feces from 335 animals using PCR to detect stx genes and colony blot

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hybridization to identify positive bacteria. They found stx genes in 34% of samples, and were able to isolate STEC in 18% of these. Robins-Browne et al. (1998) reported that 30% of 576 animals were PCR positive, and that STEC could be isolated from 31% of the positive samples. In our study, STEC colonies could be identified by Protocol B in samples with a low STEC number, as demonstrated by the high number of colonies that were necessary to be analyzed to obtain an STEC isolate. In those positive samples, where the STEC strains were not recovered, it might be assumed that the STEC number was low and that a higher number of colonies would need to be tested to isolate them. We observed that Stx2-producing isolates were more frequent than Stx1, as reported for human STEC in Argentina (Rivas et al., 1996). These findings must be confirmed analyzing a higher number of isolates. Other authors (Blanco et al., 1997), also observed the prevalence of the stx2 genotype in cattle. No strains carrying both toxin genes were detected in our study. It is relevant to note that E. coli O157 was not isolated, a fact which also coincides with previous screening studies of healthy animals by other authors in Argentina (Sanz et al., 1998; Parma et al., 2000), suggesting that these strains are not common in healthy animals, or that they exist in very low proportion in feces, indicating that more sensitive protocols are needed. The more prevalent human EHEC serotypes reported are O157:H7, O157:H, O26:H11 and O111:H (Blanco et al., 1996). In Argentina, other serotypes in addition to O157, are present (Lo´ pez et al., 2000). In the present study, an STEC O26:H11 isolate possessing the eae and EHEC-hlyA genes was detected. In this work, four of the strains isolated belonged to the O8:H19 serotype. The epidemiological significance of this serotype, not frequently reported, is unknown. Isolation of O8:H19 from meat was reported in the United Kingdom and recently in France (Willshaw et al., 1993a,b; Pradel et al., 2000). No cases of HUS or HC associated with this serotype have been reported. A previous STEC prevalence estimation of 4% has been reported for a subset of the samples analyzed here (Meichtri et al., 2000). However, considering that the most sensitive procedure was Protocol B, this value should be established at around 8%. The value found in the current study is lower than that obtained by Sanz et al. (1998), who reported the presence of STEC in 44% of cows sampled at the slaughterhouse. However the comparison of prevalences is difficult when different methodologies are used. In addition to the detection method used, the cattle setting in which the sample is taken, the animal feeding, and the management of cattle, can influence the isolation frequency of STEC. In summary, we tested different protocols for STEC identification in healthy cattle and found that enrichment in liquid media for periods greater than 6 h, followed by plating onto a selective medium, and performing PCR in the confluent growth zone, was the procedure with the best recovery results, sensitivity and specificity. Consequently, we will perform a wider screening of STEC in Argentine cattle using this protocol, to determine the prevalence and the potential pathogenic role of these strains in Argentina.

Acknowledgements This study was partially supported by grants from Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´ gicas (CONICET) (PIP no. 0020/98) and Fundacio´ n Alberto J.

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