Molecular screening of virulence genes in extraintestinal pathogenic Escherichia coli isolated from human blood culture in Brazil.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.

Prevalence of the paa gene (porcine attaching and effacing associated) in porcine enteropathogenic Escherichia coli (PEPEC) associated with postweaning diarrhea in south Brazil.

Paa (porcine attaching and effacing associated) may be an important virulence factor E. coli of piglets with diarrhea. This study showed for the first time in Brazil the prevalence of the paa gene (22%) in E. coli strains isolated from piglets and these isolates also harboured genes for other adhesins and toxins LT II, STa and STb.

EcoR phylogenetic analysis and virulence genotyping of avian pathogenic Escherichia coli strains and Escherichia coli isolates from commercial chicken carcasses in southern Brazil.

Escherichia coli strains designated as avian pathogenic E. coli (APEC) are responsible for avian colibacillosis, an acute and largely systemic disease that promotes significant economic losses in poultry industry worldwide because of mortality increase, medication costs, and condemnation of carcasses. APEC is a subgroup of extraintestinal pathogenic E. coli pathotype, which includes uropathogenic E. coli, neonatal meningitis E. coli, and septicemic E. coli. We isolated E. coli from commercial chicken carcasses in a Brazilian community and compared by polymerase chain reaction-defined phylogenetic group (A, B1, B2, or D) with APEC strains isolated from sick chickens from different poultry farms. A substantial number of strains assigned to phylogenetic E. coli reference collection group B2, which is known to harbor potent extraintestinal human and animal E. coli pathogens, were identified as APEC (26.0%) in both commercial chicken carcasses and retail poultry meat (retail poultry E. coli [RPEC]) (21.25%). The majority of RPEC were classified as group A (35%), whereas the majority of APEC were groups B1 (30.8) and A (27.6%). APEC and RPEC presented the genes pentaplex, iutA, hly, iron, ompT, and iss, but with different virulence profiles. The similarity between APEC and RPEC indicates RPEC as potentially pathogenic strains and supports a possible zoonotic risk for humans.

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains.

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh(s)) and a 33 kDa ß-domain (Tsh(ß)). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh(s) (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh(ß) (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37°C or 42°C. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26°C, 37°C, or 42°C, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37°C in mucin agar, and it was not detected when the bacteria were grown at 26°C in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.

[Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its application in a competitive enzyme-linked immunosorbent test]. / Clonagem, expressão, caracterização molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicação em um teste de ELISA por competição.

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The sequencing of the msp5 gene of PR1 isolate showed 98% of identity with the Florida and Saint Maries isolates, 97% with Brazil (Pernambuco) and Havana isolates; and 91% with A. centrale. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 380 bovine sera were used, whereas 245 truly positives and 135 truly negatives sera tested by the cELISA-USA. In the standardization of the cELISA-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3% specificity, 100 and 98%, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1% specificity, 98.9 e 96.3% sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically detect cattle naturally infected with A. marginale and would be recommended for epidemiological studies, eradications program, and regulation of international cattle movement, while IFI, which presented lower specificity should not be used in situations that demand more specific diagnosis.

Clonagem, expressão, caracterização molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicação em um teste de ELISA por competição / Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its application in a competitive enzyme-linked immunosorbent test

No presente trabalho, um teste imunoenzimático por competição (cELISA) foi padronizado com a proteína recombinante de superfície rMSP5, clonada a partir do gene msp5 do isolado PR1 de A. marginale. O sequenciamento mostrou que o gene que codifica a rMSP5/PR1 tem 98 por cento de identidade com os isolados Flórida e Santa Maria, 97 por cento com isolados de Pernambuco, Brasil e Havana, Cuba e 91 por cento com A. centrale. O teste de cELISA-PR1 foi comparado com os testes de IFI e cELISA-USA. Para a padronização e validação do cELISA-PR1, foram utilizados 380 soros bovinos comprovadamente positivos ou negativos pelo cELISA-USA. Desse total, 245 soros positivos eram de animais de área endêmica e 135 soros eram negativos, de área livre de anaplasmose. Na padronização, foram utilizados 283 soros de bovinos, dos quais 135 eram negativos e 148 positivos. Os testes de cELISA-PR1 e IFI apresentaram especificidade de 100 e 99,3 por cento, sensibilidade de 100 e 98 por cento, com coeficiente kappa de 0,993 e 0,978, respectivamente. Na validação do teste, foram utilizados 245 soros de bovinos de áreas endêmicas para anaplasmose, testados pelo cELISA-PR1 e IFI e apresentaram especificidade de 96,7 e 69,1 por cento, sensibilidade de 98,9 e 96,3 por cento e coeficiente kappa de 0,956 e 0,699, respectivamente. Esses resultados permitem afirmar que o teste de cELISA-PR1 apresentou performance equivalente ao cELISA-USA, podendo também ser utilizado em estudos epidemiológicos, programas de controle e movimentação internacional de animais, enquanto a IFI, com os resultados menos precisos apresentados, não deve ser utilizada em situações que requerem maior rigor no diagnóstico.

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The sequencing of the msp5 gene of PR1 isolate showed 98 percent of identity with the Florida and Saint Maries isolates, 97 percent with Brazil (Pernambuco) and Havana isolates; and 91 percent with A. centrale. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 380 bovine sera were used, whereas 245 truly positives and 135 truly negatives sera tested by the cELISA-USA. In the standardization of the cELISA-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3 percent specificity, 100 and 98 percent, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1 percent specificity, 98.9 e 96.3 percent sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically detect cattle naturally infected with A. marginale and would be recommended for epidemiological studies, eradications program, and regulation of international cattle movement, while IFI, which presented lower specificity should not be used in situations that demand more specific diagnosis.

Frequency of virulence genes in Escherichia coli strains isolated from piglets with diarrhea in the North Parana State, Brazil / Frequência de genes de virulência em Escherichia coli isolada de suínos com diarréia no norte do Estado do Paraná, Brasil

Identification of Escherichia coli causing porcine postweaning diarrhea requires knowledge regarding the prevalent pathotypes within a given region. A total of 100 Escherichia coli isolates from piglets with diarrhea in Londrina city, Parana State, South Brazil, were screened for the presence of genes for F4, F5, F6, F18, F41 fimbrial antigens by specific probes and for enterotoxins (STa, STb, LT and STx2e) by polymerase chain reaction (PCR). The results showed that 60% of the isolates were positive for one or more of the fimbrial antigens and 92% were positive at least for one of the virulence factors examined. Virulence factor genesdetected were F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa (40%), STb (47%) andSTx2e (3%). Twenty four patterns of virulence factor according to the different virulence genes form werefound and the most frequent virulence gene pattern was F4, F18, F41, STa, STb and LT. Most of the isolates that carried genes for adhesins also harboured genes for toxins.

A identificação de amostras de Escherichia coli responsáveis por diarréia pós-desmame em suínos requerconhecimento dos patotipos prevalentes dentro de uma dada região. Cem amostras de Escherichia coli isoladas de leitões com diarréia no Estado do Paraná, Brasil, foram testadas para apresença dos genes que codificam antígenos fimbriais F4, F5, F6, F18, F41 e para a produção de enterotoxinas (STa, STb, LT and STx2e), através de sondas e da técnica da PCR (polymerasechain reaction). Os resultados mostraram que 60% dos isolados foram positivos para um ou mais antígenos fimbriais e 92% foram positivos para pelo menos um dos fatores de virulência examinados. Os genes de virulência detectados foram F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa(40%), STb (47%) e STx2e (3%). Vinte e quatro padrões de virulência, de acordo com as diferentes combinações dos genes de virulência, foram encontrados e o mais prevalente foi F4,F18, F41, STa, STb e LT. A maioria das amostras que carreiam genes para adesinas também transportam genes para produção de toxinas.

Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil.

The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1beta gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1alpha and msp1beta genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70kDa to 105kDa and 100kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1beta gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.

Frequency of virulence genes in Escherichia coli strains isolated from piglets with diarrhea in the North Parana State, Brazil.

Identification of Escherichia coli causing porcine postweaning diarrhea requires knowledge regarding the prevalent pathotypes within a given region. A total of 100 Escherichia coli isolates from piglets with diarrhea in Londrina city, Parana State, South Brazil, were screened for the presence of genes for F4, F5, F6, F18, F41 fimbrial antigens by specific probes and for enterotoxins (STa, STb, LT and STx2e) by polymerase chain reaction (PCR). The results showed that 60% of the isolates were positive for one or more of the fimbrial antigens and 92% were positive at least for one of the virulence factors examined. Virulence factor genes detected were F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa (40%), STb (47%) and STx2e (3%). Twenty four patterns of virulence factor according to the different virulence genes form were found and the most frequent virulence gene pattern was F4, F18, F41, STa, STb and LT. Most of the isolates that carried genes for adhesins also harboured genes for toxins.

Induced immune response of DNA vaccine encoding an association MSP1a, MSP1b, and MSP5 antigens of Anaplasma marginale.

The outer membrane proteins of Anaplasma marginale have been the focus of research to obtain an improved vaccine against bovine anaplasmosis. We evaluated the capacity of the recombinant plasmids pcDNA-msp1alpha, pcDNA-msp1beta, and pcDNA-mp5 to express MSP1a, MSP1b, and MSP5 proteins, and to determine the immunogenicity of BALB/c mice immunized with these plasmids individually or in association. Expression of proteins was confirmed in Vero cells by IFA. The combination of recombinant plasmids showed high antibodies response, produced better induction of Th1 response than individual plasmids, and induced significant proliferation of splenocytes. The mice sera immunized with A. marginale showed seroconversion and reacted with all native MSPs, but demonstrated predominance of the humoral IgG1 isotype and did not induce significant proliferation of splenocytes. The use of association of recombinant plasmid can be an effective strategy for the immunoprophylaxis of anaplasmosis.

Immune response of BALB/c mouse immunized with recombinant MSPs proteins of Anaplasma marginale binding to immunostimulant complex (ISCOM).

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.

Detection of Tsh protein mucinolytic activity by SDS-PAGE.

The temperature-sensitive hemagglutinin (Tsh, 140 kDa) produced by Escherichia coli is cleaved into a fragment (106 kDa) containing mucinase activity, and an agglutinin fragment (33 kDa). By incorporating mucins into SDS-PAGE gels stained by Schiff's periodic acid, we could simultaneously detect about 0.5 microg of mucinase activity and the fragment molecular mass.

Clonagem, sequenciamento, expressão e caracterização do gene tsh de uma cepa de Escherichia coli patogênica de aves / Cloning, sequencing, expression, and characterization of the tsh gene from an avian pathogenic Escherichia coli strain

A hemaglutinina temperatura sensível (Tsh) pertence à família das serino-proteases autotransporte de Enterobacteriacea (SPATE), as quais são capazes de clivar diferentes substratos. Nós isolamos e caracterizamos o gene de Escherichia coli patogênica aviária (APEC) amostra APEC 13, sorotipo O2:H9, clonado em pET 101. A região de 4.2 kb do DNA clonado codificou uma proteína de aproximadamente 140 kDa (r-Tsh). O plasmídio recombinante pET 101-tsh conferiu um fenótipo de hemaglutinação positivo para a linhagem BL21 (tsh) para eritrócitos de galinha. A proteína r-Tsh foi purificada em coluna de níquel e utilizada na produção de anticorpos anti-Tsh. Um fragmento de 1.6 kb foi amplificado e subclonado em pCR4, e a seqüência parcial mostrou alta homologia com outras seqüências analisadas. O anti-Tsh reagiu com as proteínas r-Tsh e Tsh nativa da amostra APEC13, como demonstrado pela técnica de Western blot, mostrando que a r-Tsh tem epitopos conservados e que sua antigenicidade foi preservada. O anti-Tsh também inibiu a atividade hemaglutinante das amostras APEC 13 e BL12/pET 101-tsh

The temperature-sensitive hemagglutinin (Tsh) belongs to a family of high-molecular-weight serineprotease autotransporters of Enterobacteriaceae (SPATEs), which can cleave different substrates. Weisolated and characterised the tsh gene from an avian pathogenic Escherichia coli (APEC) strain, APEC13serotype O2:H9, which was cloned in pET101. The 4.2 kb region of cloned DNA coded one protein ofapproximately 140 kDa (r-Tsh). The recombinant plasmid pET101-tsh conferred to E. coli BL21 strain(tsh) the hemagglutination-positive phenotype against chicken erythrocytes. The r-Tsh was purified byNi-NTA column and used to produce antibody anti-Tsh. A 1.6 kb fragment of the tsh sequence was alsoamplified and cloned in pCR4, and a partial sequence showed high homology with other sequenceanalysed. The anti-Tsh reacted with the protein r-Tsh and native Tsh of APEC13, as demonstrated byWestern blot, showing that r-Tsh has conserved epitopes and that its antigenicity was preserved. Theanti-Tsh also inhibited the hemagglutinating activity of strains APEC13 and BL21/pET101-tsh

Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis: correction.

Several virulence genes of avian Escherichia coli were detected in 200 colibacillosis isolates from our region by PCR. However, the genes sfaDE and facA were not detected in that study. In this work we correct those data, showing by colony hybridization that sfaDE and facA are present in 40% and 30% of those isolates, respectively.

Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil.

Ehrlichia canis has a worldwide distribution, but clinical manifestations may vary geographically. We selected 129 dogs to determine prevalence of ehrlichiosis in dogs with anemia, thrombocytopenia, or ticks presented to a Veterinary Teaching Hospital in South Brazil. Of the 129 dogs, 68 carried the brown dog tick (Rhipicephalus sanguineus), 61 had thrombocytopenia (platelet count <150,000/microl), and 19 had anemia (PCV < 22%). Twenty dogs fulfilled more than one inclusion criteria. Ehrlichiosis was diagnosed by positive amplification of ehrlichial DNA by PCR using primers ECC and ECB that amplify a sequence of the 16S rRNA gene. Presence of E. canis was confirmed by cleavage of the amplified DNA using endonucleases HaeIII and AvaI. Fourteen of 68 (21%) dogs with ticks had ehrlichiosis, whereas 12 of 61 (20%) dogs presented with thrombocytopenia and 4 of 19 (21%) anemic dogs had ehrlichiosis. Similar results were obtained in dogs with thrombocytopenia and anemia (one of eight positive) and in dogs with thrombocytopenia and ticks (two of seven positive). All four dogs with anemia and ticks, and the dog that fulfilled all inclusion criteria yield no amplification of ehrlichial DNA by PCR. Based on our results, one in each five dogs infested by the brown dog tick, with anemia or thrombocytopenia had ehrlichosis. Contrary to widespread believe, ehrlichiosis was not the main cause for thrombocytopenia in our region.

Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis.

In this study, we compared Escherichia coli isolates from chickens with avian cellulitis with those from feces of healthy chickens. Cellulitis-derived strains presented phenotypic and genotypic characteristics of greater virulence than did the fecal isolates. Phylogenetic analysis by repetitive extragenic palindromic-PCR showed that, in agreement with their virulence characteristics, the cellulitis isolates form two clonal groups distinct from the fecal isolates.

Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis.

Avian pathogenic Escherichia coli, the causative agent of colibacillosis, harbors several putative virulence genes. In this study we examined by polymerase chain reaction (PCR) the presence of 16 of those genes in 200 colibacillosis isolates from our region. The seven virulence genes iutA, iss, cvaC, tsh, papC, papG and felA were detected significantly more often amongst colibacillosis isolates than in fecal isolates from healthy birds, thereby confirming their worldwide occurrence and possible pathogenic role in colibacillosis. However, several of those genes were not detected in many colibacillosis isolates, and none of them were detected in 27.5% of those isolates, which suggests that variants of those genes and yet undetected virulence factors should be searched for.

Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli.

In this study, we determined the occurrence of the tsh gene among 305 Escherichia coli isolates from chickens by means of the polymerase chain reaction and agglutination of chicken erythrocytes; 200 of those isolates were obtained from chickens with colisepticemia, 52 isolates were from lesions of cellulitis, and 53 were from feces of normal chickens. The tsh gene was found in 79 (39.5%) isolates from colisepticemia, in 10 (19%) cellulitis-derived E. coli isolates, and in two (3.8%) fecal isolates. Among the tsh+ strains, 68 (86%) isolates from colisepticemia and nine (90%) from cellulitis agglutinated chicken erythrocytes in the presence of mannose, after growing the strains on colonization factor antigen agar plates at 26 C, which confirms a correlation between mannose-resistant hemagglutination and expression of hemagglutinin Tsh. These results show, for the first time, the presence of the gene tsh in cellulitis-derived E. coli isolates; the high frequency of this gene among avian pathogenic E. coli isolates in Brazil indicates that its putative role as a virulence factor should be studied more thoroughly.

Molecular identification of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea in Londrina, Parana, Brazil

In this work, the prevalence of enteropathogenic Escherichia coli (EPEC) in children in Londrina-PR, Brazil, was evaluated by means of digoxigenin-labelled DNA probes which identify the plasmid responsible for EPEC adherence factor (EAF), and virulence genes for EPEC as bundle-forming pilus (bfp) and E. coli attaching-effacing factor (eae). In addition, the isolated strains were serotyped and tested for adherence to HEp-2 cells. From 102 children with diarrhoea, 19 strains hybridized with at least one probe, and eleven of them were identified as typical EPEC because they hybridized with the three probes used, showed a localized adherence (LA) pattern, and presented no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype O119:H6 43(per cent); in four strains O antigens could not be determined using antisera against O1 to O173, they were all ONT:H7 29(per cent); one strain belonged to O111:H6. Three strains were classified as atypical EPEC: O26H-, O111:H9 and O119:HNT. Strains O26H- and O111:H9 hybridized with the eae probe only and showed localized adherence like (LAL) pattern; strain O119:HNT hybridized with the bfp and eae probes, and showed a localized adherence/diffuse adherence (LA/DA) pattern after 6 h. A DA pattern was observed in two strains isolated from children with diarrhoea (ONT:H11 and O142:H34), which hybridized with the eae probe. From 46 controls, five strains hybridized with one or two probes, but none hybridized with all probes or presented the LA pattern. Three strains with the DA pattern hybridized with the eae probe. No EPEC strain belonging to classical EPEC serotypes was isolated from faeces of control children.