Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves.

Abstract

Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain reaction (PCR) assays with different specific primers. The used PCR systems were designed to detect sequences representing the group-specific virulence genes encoding fimbriae f5 (K99), Shiga toxins (stx1 and stx2), heat-stable enterotoxins (st), heat-labile enterotoxins (lt), intimin (eae), hemolysin (hylA), and EAEC heat-stable enterotoxin (astA). In the present work, a total of 150 E. coli field isolates were recovered from 150 fecal swabs collected from 100 diarrheic and 50 apparently healthy in-contact cattle and buffalo calves under 3 months old. Out of these 150 isolated E. coli, 106 isolates from 77 diarrheic and 29 in-contact calves harbored one or more of the investigated virulence genes. The pathotyping of the isolates could classify them into shigatoxigenic E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) with a 30.7, 2.7, 12.7, and 7.3% distribution, respectively. Meanwhile, the detection rates of f5, stx1, stx2, st, lt, eae, hylA, and astA genes were 17.3, 27.3, 6.7, 10, 37.3, 17.7, 9.3, and 20.7%, respectively. These virulence genes were found either single or in different combinations, such as stx/eae, stx/st/f5, eae/st/f5, or st/lt/f5. Four attaching-effacing shigatoxigenic E. coli isolates (AE-STEC) harboring stx/eae were retrieved from diarrheic calves. Although none of the stx-or eae-positive isolates was verified as O157:H7, STEC isolates detected in apparently healthy calves have potential pathogenicity to humans highlighting their zoonotic importance as reservoirs. Atypical combinations of ETEC/STEC and ETEC/EPEC were also detected in percentages of 14.7 and 2.7%, respectively. Most of these atypical combinations were found more in buffalo calves than in cattle calves. While STEC and EPEC isolates were detected more in cattle calves than in buffalo calves, ETEC isolates were the same in the two species. The pathogenic E. coli infection in calves was recorded to be higher in the first weeks of life with the largest numbers of virulence factor-positive isolates detected at the age of 4 weeks. Histopathological examination of five intestinal samples collected from four dead buffalo calves revealed typical attaching and effacing (AE) lesion which was correlated with the presence of intimin encoding virulence gene (eae). Other lesions characterized by hemorrhagic enteritis, shortening and fusion of intestinal villi and desquamation of the lining epithelium of intestinal mucosa had also been detected.