Streamlined computational pipeline for genetic background characterization of genetically engineered mice based on next generation sequencing data.

BACKGROUND: Genetically engineered mice (GEM) are essential tools for understanding gene function and disease modeling. Historically, gene targeting was first done in embryonic stem cells (ESCs) derived from the 129 family of inbred strains, leading to a mixed background or congenic mice when crossed with C57BL/6 mice. Depending on the number of backcrosses and breeding strategies, genomic segments from 129-derived ESCs can be introgressed into the C57BL/6 genome, establishing a unique genetic makeup that needs characterization in order to obtain valid conclusions from experiments using GEM lines. Currently, SNP genotyping is used to detect the extent of 129-derived ESC genome introgression into C57BL/6 recipients; however, it fails to detect novel/rare variants. RESULTS: Here, we present a computational pipeline implemented in the Galaxy platform and in BASH/R script to determine genetic introgression of GEM using next generation sequencing data (NGS), such as whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. The pipeline includes strategies to uncover variants linked to a targeted locus, genome-wide variant visualization, and the identification of potential modifier genes. Although these methods apply to congenic mice, they can also be used to describe variants fixed by genetic drift. As a proof of principle, we analyzed publicly available RNA-Seq data from five congenic knockout (KO) lines and our own RNA-Seq data from the Sall2 KO line. Additionally, we performed target validation using several genetics approaches. CONCLUSIONS: We revealed the impact of the 129-derived ESC genome introgression on gene expression, predicted potential modifier genes, and identified potential phenotypic interference in KO lines. Our results demonstrate that our new approach is an effective method to determine genetic introgression of GEM.

Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells.

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin.

AIMS: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. METHODS AND RESULTS: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1.3x10(-8) mol l(-1), failed in the detection of some of these isolates. CONCLUSION: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. SIGNIFICANCE AND IMPACT OF THE STUDY: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil.

AIMS: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. METHODS AND RESULTS: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. CONCLUSIONS: This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil.

Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in São Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H(-), O119:H(-) and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H(-), and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type theta/gamma2 was the most frequent among STEC, whereas the intimin beta1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin muB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H(-), O113:H21, O118:H16, and O174:H21 were isolated.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans.

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin.

The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

Inhibition of drug transport by genistein in multidrug-resistant cells expressing P-glycoprotein.

It has been claimed that the flavonoid genistein could be used to distinguish multidrug-resistant tumors expressing the multidrug resistance-associated protein (MRP) from those expressing P-glycoprotein (Pgp). Genistein would be block drug transport by MRP without affecting Pgp-mediated drug transport. However, we found that exposure to 200 microM genistein elicited an elevation in intracellular accumulation of rhodamine 123 (R123) and daunorubicin (DNR) in Pgp-expressing cell lines. Genistein inhibited R123 efflux in a rapidly reversible manner (ca. 2 min). The flavonoid also decreased photoaffinity labeling of Pgp by [3H]azidopine, a Pgp substrate. The present results show that genistein interacts with Pgp and inhibits Pgp-mediated drug transport. Hence, genistein cannot be used in simple assays to distinguish MRP- and Pgp-expressing cells.

Mechanism of inhibition of P-glycoprotein-mediated drug transport by protein kinase C blockers.

P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport hy mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.

Erythrocyte receptors for cholera and heat-labile enterotoxins of Escherichia coli.

Many serological reactions using red blood cells (RBC) such as radial immune haemolysis (RIH) and indirect haemagglutination (IH) tests have often been used for the detection of cholera toxin (CT) and heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains. In these tests, the enterotoxins bind to sheep, bovine and guinea-pig RBC without any ligand. We studied several factors which might interfere with such binding, as well as the nature of the receptors involved. Treatment of erythrocytes with different enzymes revealed that proteolytic enzymes had no effect on the adsorption of enterotoxins to RBC. Conversely, treatment with neuraminidase increased the adsorption. Experiments carried out with delipidized RBC revealed that none of the enterotoxins under study bound to the cells thus treated. Pre-incubation of ganglioside fractions with the enterotoxins blocked RIH and IH reactions and the biological effect of them on Vero cells. Assaying RBC ganglioside fractions by thin-layer chromatography revealed the presence of GM1. Our results suggest that the receptors for GT and LT enterotoxins in sheep, bovine and guinea pig RBC are gangliosides: mainly GM1.

eae-negative attaching and effacing Escherichia coli from piglets with diarrhea.

One hundred and ninety strains of Escherichia coli that were isolated from pigs with diarrhea in the state of São Paulo, Brazil, and that were negative for enterotoxins and cytotoxins were investigated. Strains which adhered to HeLa cells were examined for fluorescence actin staining (FAS), the ability to induce attaching and effacing (A/E) lesions on HEp-2 cells detectable by transmission electron microscopy and the presence of eae gene sequences detected by PCR. Intimin production was detected by western blot and serogrouping was performed. Forty-seven isolates adhered to HeLa cells in several patterns, but none adhered in a localized adherence pattern. However, seven of the 47 adherent strains were positive for the FAS reaction, although the reactions were usually weak or atypical. One FAS-negative and three FAS-positive strains, which were examined for their ability to induce A/E lesions, were all positive. Subsequently, testing of these strains for the eae gene showed that they all lacked this gene. These findings, along with earlier reports of eae-negative A/E E. coli, suggest that higher quantities of E. coli in this category might be detected if more reliance were placed on phenotypic tests rather than on gene detection tests alone.

Bluetongue virus: production and study of viral antigen for serological diagnosis.

A soluble antigen, produced from the culture supernatant of VERO cells infected with bluetongue virus serotype 4 (BTV-S4) and concentrated by sequential ultrafiltration with membranes with cut-off values 10(3) and 25 x 10(3) NMWP, showed complete identity to standard antigens when compared by agar gel immunodiffusion (AGID) and SDS-PAGE profiles, revealing that the main protein component responsible for the AGID reaction has a molecular weight of about 60 kDa corresponding probably to the NS1 protein.

Characteristics associated with pathogenicity of avian septicaemic Escherichia coli strains.

Seventeen strains of E. coli, isolated from chickens with colisepticaemia, were studied with respect to their pathogenic characteristics including: serum resistance, toxin production, pathogenicity for one-day-old chicks, colicin production, adherence to and invasiveness of HeLa cells, plasmid DNA profile and SDS-PAGE electrophoresis of membrane proteins, as well as electron microscope studies and hemagglutination tests for fimbriae. We concluded that the adherence to and the invasiveness of HeLa cells were not related to the pathogenicity of these strains for chickens. Plasmid profiles were not related to the bactericidal activity of the serum. Toxin production was correlated to the highest levels of pathogenicity. Some of the strains had mannose-resistant fimbriae. SDS-PAGE of membrane proteins of all the strains which were either not pathogenic or which had a very high LD50 lacked two major protein subunits of 40.7 kDa and 28.8 kDa found only in pathogenic strains.

Rotavirus excretion in naturally infected pigs with and without diarrhoea.

Seven hundred and fifty faecal samples from piglets ranging from 1 to 60 days old were studied for the presence of group A rotavirus by polyacrylamide gel electrophoresis (PAGE) and by enzyme immunoassay (EIA). From 451 diarrhoeic pigs, 117 (25.94%) were positive for rotavirus and only 45 (15.05%) of 299 pigs without diarrhoea excreted the virus (P < 0.005). When these animals were separated into four age groups with regard to the presence or absence of diarrhoea, it was observed that the excretion of rotavirus was associated with diarrhoea in piglets, both before and after weaning.

Properties of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep in the State of São Paulo, Brazil.

Fecal samples from 48 sheep from two farms in São Paulo, SP, Brazil, were examined to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC). Forty-two STEC strains were isolated from 25 (52.1%) of 48 sheep feces, and were examined for the presence of genes encoding STEC-related virulence factors. Twenty-one (50.0%) of the 42 STEC isolates were positive for stx(1) and stx(2), 16 isolates (38.1%) were stx(1), and five (11.9%) were stx(2). Expression of Shiga toxins was demonstrated by the Vero cell toxicity test for all the strains carrying stx. Fourteen of the STEC strains (33.3%) carried the enterohemolysin gene (ehly) and presented the enterohemolytic phenotype, and five (11.9%) were positive for the plasmid encoded katP gene. The eae gene was not present in any of the isolates. STEC strains presenting stx(1), stx(2) and ehly were most commonly (23.8%) recovered from these sheep. The predominant STEC serotype found was ONT:H8, and others included O5:H-, O16:H-, O75:H-, O75:H8, O87:H16, O91:H-, O146:H21, O172:H-, OR:H-, ONT:H- and ONT:H16. This is the first report on ovine STEC in South America, and identifies a number of ovine non-O157 STEC that belong to serotypes implicated in human disease.

Frequency of Shiga toxin-producing Escherichia coli (STEC) isolates among diarrheic and non-diarrheic calves in Brazil.

The occurrence of Shiga toxin (Stx) gene sequences was examined in 344 fecal samples from diarrheic (n=139) and non-diarrheic (n=205) calves from 12 beef farms in São Paulo State, Brazil to study the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains. Forty-four (12.7%) animals were found to be positive for stx. The frequency of carriage of stx was higher in diarrheic calves (28/139, 20%) than in non-diarrheic animals (16/205, 7.8%) (P<0.001). Among the 24 STEC strains recovered from the animals, 12 isolates carried stx1, four stx2, and 8 carried both stx1 and stx2 genes. The eae and the enterohaemolysin (Ehly) gene sequences occurred at high frequencies in these STEC strains (41.6 and 50.0%, respectively). A total of 16 serotypes were identified. The serotypes O111:NM (four isolates), O111:H8 (two) and O118:H16 (one), currently described as enterohaemorrhagic E. coli (EHEC), were isolated from cattle in Brazil for the first time. These findings reinforce the importance of cattle as a reservoir of EHEC strains in Brazil.

Genes coding for Shiga-like toxins in bovine verotoxin-producing Escherichia coli (VTEC) strains belonging to different O:K:H serotypes.

Forty-six verotoxin-producing Escherichia coli (VTEC) strains isolated from diarrhoeic and healthy calves in Spain were examined for DNA sequences homologous to genes for verotoxins (VT1 and VT2) and enterotoxins (LT-I, LT-II, STaH, STaP and STb). Hybridisation showed that 26 (57%) of VTEC strains carried VT1 genes, 13 (28%) possessed VT2 genes, and 7 (15%) carried both VT1 and VT2 genes. No VTEC strains hybridised with DNA probes for enterotoxins. A correlation was found between the serotype and type of VT produced. Thus, all strains of serotypes O26:K-:H11 (13 strains), O103:K-:H2 (3 strains) and O128:K?:H- (4 strains) hybridised with the VT1 probe only, whereas all strains of serotypes O4:K-:H4 (3 strains) and O113:K-:H21 (4 strains) were positive with the VT2 probe only. By contrast, O81:K?:H28 (2 strains) and O157:K-:H- (2 strains) strains hybridised with both VT1 and VT2 probes. One strain of serotype O157:K-:H7 was VT2 positive.

Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans.

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.

Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil.

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC).

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of São Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.