Enteropathogenic Escherichia coli O80:H2 in Young Calves with Diarrhea, Belgium

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Damien Thiry, Marc Saulmont, Shino Takaki, Klara De Rauw, Jean-Noël Duprez, Atsushi Iguchi, Denis Piérard, and Jacques G. Mainil

Author affiliations: University of Liège, Liège, Belgium (D. Thiry, S. Takaki, J.-N. Duprez, J.G. Mainil); Association Régionale de Santé et d’Identification Animales, Ciney, Belgium (M. Saulmont); Universitair Ziekenhuis Brussel, Brussels, Belgium (K. De Rauw, D. Piérard); University of Miyazaki, Miyazaki, Japan (S. Takaki, A. Iguchi)

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Abstract

Serogroup O80 was detected in 40% of 104 enteropathogenic Escherichia coli isolates from calves with diarrhea from 42 farms in Belgium during 2008‒2015. These isolates harbored the eae-ξ and fliCH2 genes, similar to the O80 attaching-effacing Shigatoxigenic E. coli isolates found in humans in France. This strain might be emerging.

Enteropathogenic and attaching-effacing Shigatoxigenic Escherichia coli (EPEC and AE-STEC) cause bloody diarrhea in humans and young calves. For clarity, we use the term AE-STEC instead of enterohemorrhagic E. coli, similar to a previous publication (1), to refer to STEC isolates from animals that produce attaching-effacing lesions. EPEC and AE-STEC that infect humans are diverse and comprise scores of serotypes (2); in contrast, most calf AE-STEC strains comprise a few serotypes, mostly O5:H-, O26:H11, O111:H-, and O118:H16 (3). The O26:H11 serotype is also the most common among calf EPEC. However, most serotypes that infect calves have not been identified (3). Therefore, during November 2008‒June 2015, we conducted a study on 104 EPEC and 153 AE-STEC isolates collected from the feces or the intestinal contents of calves suffering diarrhea (1 isolate/calf) at the Association Régionale de Santé et d’Identification Animales in Ciney, Belgium. Isolates were screened by PCR for genes of the 10 most pathogenic and common calf and human O serogroups: O5, O26, O103, O104, O111, O118, O121, O145, O157, and O165. Confirming published results (3), 80% (122/153) of AE-STEC isolates and only 21% (22/104) of EPEC isolates tested positive for 1 of these (J.G. Mainil, unpub. data) (4). We sought to further characterize this collection of calf EPEC with unidentified O serogroups.

We submitted 9 calf EPECs with unidentified serogroups to the O-typing multiplex PCR platform (5); 6 of 9 EPEC isolates contained the O80 serogroup‒encoding gene, and 3 belonged to 3 other O serogroups. We subsequently performed an O80 serogroup‒specific PCR (5) of all 31 AE-STEC and 82 EPEC isolates with unidentified serogroups, along with one O80-positive E. coli strain and negative controls; 42 EPEC isolates and the O80-positive E. coli strain but no AE-STEC isolates or negative controls tested positive.

We further tested the calf EPEC isolates and 3 human Shiga toxin 2‒encoding gene (stx2)‒positive AE-STEC O80 isolates from the STEC National Reference Center (Brussels, Belgium) by PCR for fliCH2 and eae-ξ genes found in human AE-STEC O80 strains. For amplifying eae-ξ, we used previously published PCR conditions (6), and for amplifying fliCH2, we used primers H2_F (5′-TGATCCGACATCTCCTGATG-3′) and H2_R (5′-CCGTCATCACCAATCAACGC-3′) and the following thermocycler conditions: initial denaturation at 94°C for 1 min; 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and elongation for 1 min at 72°C; and final elongation at 72°C for 2 min. All 42 calf EPEC and 3 human AE-STEC isolates tested positive by both PCRs.

Among the 104 calf EPEC isolates, O80:H2 was frequently found (40% were PCR positive) and, thus, could be considered emerging. Indeed, the EPEC O80 isolates were isolated from calves from 42 farms. The yearly EPEC O80:H2 isolation rate varied from 12% in 2009 to 40%–50% during 2010‒2013 to as high as 73% for the first 6 months of 2015 (Table). In comparison, the rate for EPEC O26 serotype was 5%‒25%. Although prevalence data before 2009 are lacking, EPEC O80 isolates have been found infrequently in animals: 8 in dead poultry (7,8), 1 in a piglet with diarrhea (9), 1 in a healthy cow (9), and 5 in lambs with diarrhea (10). However, even fewer AE-STEC O80 isolates have been found: 2 in healthy cattle (6); 1 in a calf with diarrhea in January 1987 (J.G. Mainil, unpub. data); and 1 in raw cow’s milk cheese (9). According to the literature, the O80:H2 serotype might be emerging in France, where human cases of AE-STEC O80:H2 have been reported (9).

Molecular virulotyping results indicate that our calf EPEC O80 isolates appeared to be more closely related to human AE-STEC (because they all harbored eae-ξ and fliCH2) than to ovine and poultry EPEC O80 (which usually harbor eae-β and fliCH26) (J.G. Mainil, unpub. data) (8,10). Further studies are needed to characterize these calf EPEC O80:H2 isolates, and the isolation rate of EPEC O80:H2 in calves with diarrhea must be tracked. Additional PCR virulotyping should be performed with our isolates to identify, if present, other EPEC-related virulence genes and extraintestinal E. coli‒related virulence genes. Some genes could be located on plasmids, like those that were found in AE-STEC O80:H2 patients with bacteremia and internal organ infections (9), although the infected calves from which our isolates were taken did not show evidence of septicemia before or after necropsy. The relationship among calf EPEC O80:H2 isolates (which are all independent isolates, not constituting a single strain until proven otherwise) and between calf and human isolates needs to be further characterized with pulsed-field gel electrophoresis and whole-genome sequencing. The prevalence of O80:H2 EPEC and AE-STEC in healthy cattle at slaughterhouses and farms in Belgium should be examined. Finally, we need to determine whether these calf EPEC O80:H2 isolates are true EPEC, AE-STEC derivatives that have lost stx genes, or AE-STEC precursors that could acquire stx genes in the future. This work will aid in the detection, prevention, and control of this potentially emerging pathogen.

Dr. Thiry is an assistant professor at the Bacteriology Laboratory at the Faculty of Veterinary Medicine of the University of Liège. His areas of research are the identification of enteropathogenic and Shigatoxigenic Escherichia coli isolated from young diarrheic calves and bacteriophages as an alternative treatment for bacterial infections (Escherichia coli and Staphylococcus aureus) in cattle.

Acknowledgments

The authors thank Maika Furukawa for her efficient technical support and Melha Mellata for helpful discussions.

The University of Liège provided financial support (Fonds Spéciaux de la Recherche, 2016‒2018). S.T. is a trainee veterinary student from the University of Miyazaki, Japan, under the Japan Public-Private Partnership Student Study Abroad Program of the Japan Student Services Organization.

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