Antigen detection in enteropathogenic Escherichia coli using secretory immunoglobulin A antibodies isolated from human breast milk

Enteropathogenic Escherichia coli (EPEC) produces a characteristic attaching and effacing (A/E) lesion in the small intestines of infected children. The immune response to EPEC infection remains poorly characterized. The molecular targets that elicit protective immunity against EPEC disease are unknown. In this study protein antigens from EPEC were identified using secretory immunoglobulin A (sIgA) antibodies isolated from milk from Mexican women by Western blot analysis. Purified sIgA antibodies, which inhibit the adherence of EPEC to cells, reacted to many EPEC proteins, the most prominent of which were intimin (a 94-kDa outer membrane protein) and two unknown proteins with apparent molecular masses of 80 and 70 kDa. A culture supernatant protein of 110 kDa also reacted strongly with the sIgA antibodies. The molecular size of this protein and its reactivity with specific anti-EspC antiserum suggest that it is EPEC-secreted protein C (EspC). These EPEC surface protein antigens were consistently recognized by all the different sIgA samples obtained from 15 women. Screening of clinical isolates of various O serogroups from cases of severe infantile diarrhea revealed that all EPEC strains able to produce the A/E lesion showed expression of intimin and the 80- and 70-kDa proteins. Such proteins reacted strongly with the purified sIgA pool. Moreover, nonvirulent E. coli strains were unable to generate a sIgA response. The immunogenic capacities of the 80- and 70-kDa proteins as virulence antigens have not been previously reported. The strong sIgA response to intimin and the 80- and 70-kDa proteins obtained in this study indicates that such antigens stimulate intestinal immune responses and may elicit protective immunity against EPEC disease.     

Purification and measurement of secretory IgA in mouse milk

An important factor limiting better understanding of the protective role of sIgA at mucosal surfaces is the limited availability of the purified immunoglobulin. Among other things, purified sIgA is needed for use as a standard in measurements of the concentration of this immunoglobulin in mucosal secretions, particularly in mice, where several models of mucosal infections are available. We describe here a simple method by which one can obtain a mean of 3.5 ml of milk per mouse without a breast pump. Immunoblotting studies after native PAGE demonstrated that the milk contained mainly 420 kDa dimeric sIgA and higher polymeric forms of sIgA; only a trace of monomeric IgA was present. Similar immunoblotting studies after SDS-PAGE revealed that a portion of the sIgA was dissociated by this treatment. The 420 kDa sIgA was purified by salt fractionation, gel filtration, and affinity chromatography, and the purity of the final product was demonstrated by immunoblot analysis of biotinylated polypeptides after reduction of biotinylated protein. The concentration of 420 kDa sIgA in whey was measured by densitometry of immunoblot bands, using the purified 420 kDa sIgA as a standard, and found to be 1.0 ± 0.3 mg/ml.     

Presence in bovine enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli of genes encoding for putative adhesins of human EHEC strains

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections are characterised by the formation of attaching and effacing lesions on intestinal epithelial cells. The first step of EPEC and EHEC pathogenesis involves the initial adherence of the bacterium to the intestinal epithelium. A collection of bovine EPEC and EHEC strains belonging to different serogroups was tested by colony blot hybridization with gene probes for putative adhesins (BFPA, LPFA, IHA, LIFA) of human EPEC and EHEC, and also for fimbrial and afimbrial adhesins (AFA8, F17, Cs31A) of bovine necrotoxigenic E. coli (NTEC). In the bovine EPEC and EHEC strains tested, sequences homologous to lifA, ihA, and lpfA genes were detected, sometimes in association with particular serogroups. Bovine 026 EPEC also possessed a sequence homologous to a gene of the c/p operon, coding for the CS31A adhesin, associated with bovine NTEC. Overall results showed that different genes encoding for putative adhesins of human EHEC strains are present in bovine EPEC and EHEC strains, but not one of them is present in all strains.     

Study of enteropathogenic Escherichia coli(EPEC) diarrhoea in children

Enteropathogenic E. coli (EPEC) are one of the predominant aetiological agents of diarrhoea in children. A study was undertaken to determine the incidence of EPEC diarrhoea in children and to assess the clinical spectrum. Faecal samples from 300 children below 12 years of age, hospitalised with acute diarrhoea were processed for isolation of bacterial pathogens. EPEC were isolated from 36(12%) of the diarrhoeal cases. O86 was the predominant serogroup isolated. EPEC diarrhoea was common in children below two years of age (86.1%). Vomiting, fever and dehydration were the common presenting features. Faecalleucocytes were observed in 17(47.2%)stool samples. EPEC were found to be resistant to several antibiotics. Since EPEC are known to belong to restricted number of 'O' sergroups, serogrouping with 'O' antisera to predominant EPEC serogroups in a particular area remains the most convenient method for early detection of EPEC diarrhoea in children.     

Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers.     

Characterization of monkey enteropathogenic Escherichia coli (EPEC) and human typical and atypical EPEC serotype isolates from neotropical nonhuman primates

Enteropathogenic Escherichia coli (EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries. While diarrhea is also a major problem among primates kept in captivity, the role of E. coli is unclear. This study was designed to characterize diarrheagenic E. coli recovered from the feces of 56 New World nonhuman primates, primarily marmosets (Callithrix spp.). Seventeen of the 56 primates had signs of diarrhea and/or enteritis. E. coli recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic E. coli and for patterns of adherence to HeLa cells. In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions. PCR for the eae gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis. Colonies of eae(+) E. coli were serotyped and examined by PCR for genes encoding EPEC virulence markers. The eae(+) E. coli isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC. The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation. The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans.     

Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactotransferrin and secretory immunoglobulin A isolated from human milk.

Immunoglobulins from bovine and human colostrum and milk and lactotransferrin (LTF) from human milk were investigated for bacteriostatic activity against Escherichia coli growing in a tissue culture medium. When tested separately, LTF or secretory immunoglobulin A (sIgA) from pooled human milk showed only slight bacteriostatic activity against human commensal or enteropathogenic strains of E. coli. Together, they had a considerable bacteriostatic effect, but only against strains of enteropathogenic serotype. This activity of the sIgA from pooled human milk was consistent for all enteropathogenic serotypes tested, but sIgA isolated from individual milk samples was inactive against some serotypes, and this specificity was associated with antibody to the O antigens. The activity of the sIgA was stable to heat at 56 degrees for 2 h but was lost progressively on heating at 65 degrees for 10 min or longer. Bovine colostral IgGl was without bacteriostatic effect alone. Together with LTF, it was active against a strain pathogenic to calves but not against human enteropathogenic strains. Tests on rabbit antisera raised against commensal enteropathogenic strains of E. coli showed that for the enteropathogens the bacteriostatic activity (in association with LTF) was high and was specific for the serotype of the eliciting strain, but bacteriostatic activity was low or absent in the antisera to commensal strains in spite of the presence of high titres of agglutinating antibodies to these strains.     

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called 'attaching and effacing' lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.     

Tracheal and bronchial polymeric immunoglobulin secretory immune system (PISIS) development in a porcine model

Polymeric immunoglobulins (pIgs) mucosal secretion is mediated by the pIg secretory immune system (PISIS), which is composed of J-chain (JC) and antibody (IgM/IgA) producing cells (JC-AbPC), pIg receptor (pIgR) epithelial cell expression and the efficient release of secretory Igs (SIgs) to the mucosal lumen. A poor development or disturbances in this system may cause higher infection susceptibility, as observed in young and elderly people. In spite of this system's importance, few detailed studies regarding its development have been described in the lower respiratory tract of humans. Because the porcine model has been reported as an option for translational medicine to humans, we studied the tracheal and bronchial PISIS development in healthy, non-vaccinated, SPF, miniature Vietnamese pigs from birth to adulthood using immunohistochemistry and ELISAs. Our results demonstrated that pIgR was present at birth, and its expression increased with age. In contrast, JC-AbPC were low in neonatal pigs; however, colostrum was a source of IgM, SIgA, total IgA and IgG in respiratory secretions (trachea and bronchoalveolar lavages, nasal secretion and saliva) in piglets. JC-AbPC steadily increased in post-weaned, young and adult pigs, correlating with considerable increases in secretory and total Igs in the trachea and bronchi. These data suggest a compensatory role of maternal Igs at the respiratory mucosa in the absence of a structured PISIS before weaning. Furthermore, monomeric Igs (IgG and IgA) may also play an important role in respiratory protection and deserves a more thorough study.     

Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential – an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.     

Calcium-calmodulin dependence of actin accretion and lethality in cultured HEp-2 cells infected with enteropathogenic Escherichia coli.

Infection of cultured HEp-2 cells with enteropathogenic Escherichia coli causes substantial actin accretion at points of bacterial contact and cell death. Loss of viability was delayed by chelating intracellular free calcium. Actin accretion was partially inhibited by preventing elevation of free cytosolic calcium and prevented by treatment with a calmodulin inhibitor.     

Molecular detection and antibacterial susceptibility of enteropathogenic Escherichia coli (EPEC) and shigatoxigenic Escherichia coli (STEC) strains isolated from healthy and diarrhoeic dogs

Animal contacts have been regarded as an emerging rout of Shiga toxin-producing Escherichia coli (STEC) infection in humans. Diarrhoeic and asymptomatic dogs have been recognised as a reservoir of atypical enteropathogenic Escherichia coli (EPEC), and STEC in some investigations. In this study E. coli isolates from 100 faecal samples of healthy (n = 50) and diarrhoeic (n = 50) dogs were screened by polymerase chain reaction (PCR) for the presence of determining virulence genes of STEC and EPEC pathotypes including stx and eaeA. The confirmed virulence-positive strains were subjected to antimicrobial susceptibility testing against 12 antibacterial using disc diffusion method. Resistance profiles were also determined for the STEC and EPEC strains. Ten isolates from 10 dogs (10%) were shown to possess at least one of the tested virulence genes. Six of these isolates (6%) harboured only the eaeA gene and were considered as EPEC. Four isolates (4%) were stx+ and regarded as STEC, of which two were stx+/eae+. The resistance was specially observed against penicillin, ampicillin, sulfomethoxazole, streptomycin and oxytetracyclin. Altogether, nine resistance profiles were observed among 10 isolates. In conclusion, dogs can act as a reservoir for EPEC and STEC strains, and close contacts of children with companion animals can be a potential risk factor in development of diarrhoea and haemolytic uremic syndrome. In rural areas shepherd dogs can also be a transient carrier of STEC strains that they may acquire from ruminants. To our knowledge this is the first study which reports the faecal shedding of STEC and EPEC from dogs in Iran.     

Plasmid coding for drug resistance and invasion of epithelial cells in enteropathogenic Escherichia coli 0111:H−

EnteropathogenicEscherichia coli(EPEC) can adhere to, invade and multiply in human epithelial cells. To define the elements required for bacterial invasion, we isolated from an 0111:H EPEC a 6.6 kb plasmid that is capable of conferring to an avirulent, non-adherentE. coliK12 strain (DK1) the capacity to invade epithelial cells. With this system a dissociation was possible between bacterial invasion and adherence to epithelial cells. Bacteria containing this plasmid synthesise a protein of 32 kDa (pI 4.93) which seemed to be required for cell invasion. The results provide a new basis for strategies to prevent EPEC infections.     

Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation

Neutralising inhibitors to respiratory syncytial (RS) virus have been demonstrated in the whey of most samples of human milk tested. Although high titres were secreted in colostra of some mothers (1/10–1/2,560; median 1/40) inhibitor levels in milk collected after the first week of lactation were uniformly low (median 1/10). High neutralising titres correlated with high colostral levels of specific antiviral IgA but, unlike neutralising activity, IgA antiviral antibody persisted in the milk of only four of 18 mothers. Similarly, antiviral IgG and IgM antibodies were not generally detected after the first postpartum week. Differences in antibody secretion among mothers did not correlate with differences in total protein or total immunoglobulin secretion, and appeared to reflect maternal immune status. In one mother a marked rise in specific antiviral IgA and IgG secretions during the second and third months of lactation suggested a response to virus infection. The relevance of maternal immunity and colostral and milk antiviral antibody to protection of breast-fed babies from RS-virus bronchiolitis is discussed.     

A novel serotype of enteropathogenic Escherichia coli (EPEC) as a major pathogen in an outbreak of infantile diarrhoea

An outbreak of infantile diarrhoea was investigated in 32 children, all <2 years old, in the tropical north of Australia. Rotavirus (63%) and enteropathogenic Escherichia coli (EPEC) (59%) were the most common pathogens identified. Of the 19 EPEC isolates, 14 (74%) were of serotype O126:H12, hitherto unreported as an EPEC serotype. Other pathogens isolated included Salmonella spp. (16%), Campylobacter spp. (3%), Giardia (3%) and Shigella spp. (3%). EPEC-related gastro-enteritis is an uncommon but recognised cause of diarrhoeal outbreaks in Australia and clinicians need to be aware of the possibility of this serotype being implicated. This report highlights the disadvantages of relying on serotyping alone for the recognition of EPEC.     

Unidentified serogroups of enteropathogenic Escherichia coli (EPEC) associated with diarrhoea in infants in Londrina, Parana, Brazil

Digoxigenin-labelled DNA probes were used to characterise enteropathogenic Escherichia coli (EPEC) isolated in Londrina (Brazil) from faeces samples of 102 children with diarrhoea, and the results were compared with those obtained by serogrouping and adherence to HEp-2 cells. The probes employed detect the gene coding EPEC adherence factor (EAF) and the virulence genes for bundle-forming pilus (bfp) and entero-attaching-effacing (eae) factor. Twenty-one isolates hybridised with at least one probe, and 11 of them were classified as typical EPEC because they hybridised with all three probes, showed a pattern of localised adherence (LA) and carried no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype 0119:H6 and one to O111:H6; O antigens could not be determined in four strains with antisera against 01-0173. All typical EPEC strains carried a 70-MDa plasmid plus two other large plasmids. These data showed that typical EPEC virulence traits may be found in strains not belonging to classical serogroups/serotypes and that molecular identification is required for studying the epidemiology of diarrhoea in children.     

Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq

Purpose: Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of diarrhoea. The study was carried out to investigate the occurrence of EPEC as a cause of infectious diarrhoea in children younger than 2 years of age and characterize their virulence genes. Materials and Methods: During the study period, a total of 656 faecal specimens from children with diarrhoea and 54 from healthy children were analyzed. E. coli isolates were serotypically identified with EPEC polyvalent and monovalent antisera. The isolated EPEC were examined for the presence of the attaching and effacing (eaeA), bundle-forming pilus (bfpA), Shiga like toxins (stx₁ and stx₂), enterohaemorrhagic E. coli enterohaemolysin (EHEC hlyA) and EPEC adherence factor (EAF) genes by the PCR assay. Results: The study has shown that 22 (3.4%) had diarrhoea due to EPEC, while no EPEC isolates were detected in asymptomatic children. The highest number of the EPEC isolated belonging to polyvalent 2. The primers encoding virulence genes were subjected to all the EPEC isolates. Only 9.1%, 27.3%, and 9.1% isolates gave positive re sults with intimin (eaeA), bfbA and (EAF) genes, respectively. None of the isolates were positive for stx₁, stx₂, and hlyA genes. Typical EPEC (eaeA⁺, bfpA⁺) was diagnosed in two isolates, while, atypical EPEC was manifested in four isolates. Conclusions: According to the results, the frequency of EPEC isolates in Najaf was lower than what has been suspected and the investigation including the use of molecular technique and serotyping, are necessary to allow precise identification and epidemiological study of these pathogens.     

Bacterial aetiology of diarrhoea in young children: high prevalence of enteropathogenic Escherichia coli (EPEC) not belonging to the classical EPEC serogroups

Diarrhoeal disease continues to be one of the most common causes of admittance in Children hospital emergency. The aim of the present study was to investigate the relative contribution of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) as a cause of infectious bacterial diarrhoea in children from the region of Toulouse. We analysed 280 samples of stools from 280 children (<2 years) with diarrhoea admitted in the "Hopital des Enfants" from January to August 2005. Classic pathogens (Salmonella, Campylobacter, Yersinia, Shigella, Aeromonas and Vibrio) were detected by standard culture methods. Enterotoxigenic Clostridium difficile were identified after culture by immuno-enzyme assay (IEA). Virulence genes of EPEC and EHEC were detected by using PCR. Shiga-toxin production of EHEC strains was confirmed with an IEA test. Potential enteric pathogens were identified in 55 patients. EPEC was the most frequently identified agent (30 patients), followed by Campylobacter (9 cases: 7 C. jejuni and 2 C. coli) and C. difficile (8 patients), then EHEC (5 patients) and Salmonella (3 patients). No Shigella, Yersinia, Aeromonas or other pathogenic bacteria were detected during this period in that class of children. EPEC not belonging to the classical EPEC serogroups were highly prevalent (24 versus 6). EHEC possessed different genotypes and serogroups: O26 (2 strains), O157 (2 strains) and one un-typable strain. This study demonstrates the importance of EPEC (55 % of positive cases) and of EHEC (more frequent than Salmonella) in the aetiology of diarrhoeal diseases of young children. We confirm the usefulness of the PCR methodology: it allows the detection of virulent E. coli and thus increases by two fold the diagnosis of bacterial diarrhoea.     

Secretory immunoglobulin A (sIgA) deficiency in serum of patients with GALTectomy (appendectomy and tonsillectomy)

INTRODUCTION: In adult human beings, 80-85% of the immune cells are located in the digestive tract mucosa; hence the importance of the Gut Associated Lymphoid Tissue (GALT) in host defence. We studied the influence of the surgical removal of two important parts of the gut associated with lymphoid tissue (tonsillectomy and appendectomy) on immune parameters. METHODS: One hundred and sixty patients were enrolled in this study. They were divided into four groups of forty patients each and matched for gender and age: group 1, appendectomized and tonsillectomized; group 2, only appendectomized; group 3, only tonsillectomized; and group 4, control group, neither tonsillectomized nor appendectomized. We analysed in blood: hemogram, protein electrophoresis, lymphocytic populations (T4 cells, T8 cells, NK cells), IgG, IgM, IgA immunoglobulin, and their fractions IgA1, IgA2, and secretory IgA. RESULTS: Levels of secretory IgA in serum of patients in group 1 were significantly lower than in the other three groups (1.89 mg/dl, group 1; 2.32 mg/dl, group 2; 2.19 mg/dl, group 3 and 4.97 mg/dl, group 4; p<0.0001). Also, the values found in the two groups that had undergone only one of the operations were clearly lower than in control patients (p<0.0001). In this study, the reduction was sustained for a period of between 3 months and 3 years in appendectomized patients, and more than 20 years in tonsillectomized patients. IN SUMMARY: GALTectomy (appendectomy and tonsillectomy) significantly decreases secretory IgA levels in serum. The decrease is more intense when both operations have been done.     

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called ‘attaching and effacing’ lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eaeγ-tirα-espAα-espBα (65%), eaeβ-tirβ-espAβ-espBβ (29%), eaeα-tirα-espAα-espBα (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.