Clinical and sero-molecular characterization of Escherichia coli with an emphasis on hybrid strain in healthy and diarrheic neonatal calves in Egypt

Abstract

The present study was carried out to characterize pathogenic E. coli in apparently healthy and diarrheic neonatal calves with special reference to the hybrid E. coli strains and evaluate their clinical and hematobiochemical consequences. One hundred and seventy calves (age 1-30 days) were divided into two groups: apparently healthy (n = 70) and diarrheic (n=100). Animals were subjected to thorough clinical, hematobiochemical and bacteriological examinations. Clinically, diarrheic calves showed various degree of diarrhea with the presence of cardinal signs of dehydration in moderate and severe cases. There was a significant increase (p<0.05) in the hemogram parameters with uremia and hyperkalemia in calves with severe diarrhea. The O-H serotyping of cultural and biochemically positive isolates identified 31 isolates belonging to 12 serotypes including O44:H18, O55:H7, O146:H21, O113:H4, O121:H7, O26:H11, O91:H21, O111:H2, O8, O127: H6, O86 and O128:H2. Molecular characterization of E. coli isolates on three toxin genes: heat-stable enterotoxin (sta), shiga toxin type 1 and 2 (stx1 and stx2) revealed two well-known pathotypes (EPEC O44:H18, O55:H7, O146:H21, O113:H4, O121:H7 and EHEC O26:H11 O91:H21 O111:H2) with high frequency of enterohemorrhagic E. coli (EHEC). Molecular analysis also showed a number of E. coli isolates that carry sta and stx1 or sta and stx2 gene and belonged to O8, and O127:H6, O86 and O128:H2. These isolates were identified as hybrid E. coli strains (ETEC-STEC) and found in both apparently healthy and diarrheic calves. In conclusion, the present study identified high frequency of pathogenic E. coli in both apparently healthy and diarrheic calves. Serological and molecular analysis of E. coli isolates showed that high frequency of EHEC and presence of a new phenotype, STEC-ETEC hybrid, revealing their importance in the etiopathogenesis of diarrhea in calves and reinforcing the role of these animals as a reservoir of potentially pathogenic E. coli for humans.

Keywords: Calf; Diarrhea; Escherichia coli; Hybrid.

Fig. 1

Cultural identification of E. coli. Inoculated MacConkey agar shows characteristic pink colonies (a), inoculated EMB agar shows characteristic green metallic sheen colonies (b) and Gram stain shows Gram negative coccobacilli (c).

Fig. 2

Agarose gel electrophoresis of multiplex PCR of stx1 (614bp) and stx2 (779) genes for characterization of Enteropathogenic E. coli. 100 bp ladder as molecular size DNA marker (M), positive control (Sakai) for stx1 and stx2 genes (C+), negative control (E. coli K12DH5α) (C-), positive E. coli isolates (O8, O44, O55, O127 & O146) for stx1 gene (lanes 1, 3, 4, 11 & 13, respectively), (O26, O91 & O111) for stx1 and stx2 genes (lanes 2, 6 & 7, respectively), (O86, O113, O121 & O128) for stx2 gene (5, 8, 9 & 12, respectively), negative E. coli isolate (O124) for both stx1 and stx2 genes (Lane 10).

Fig. 3

Agarose gel electrophoresis of uniplex PCR of sta (244bp) gene for characterization of enterotoxogenic E. coli. 100 bp ladder as molecular size DNA marker (M), control positive E. coli for sta gene (C+), control negative (C-), positive E. coli isolates (O8, O86, O127 and O128) for sta gene (lanes 1, 5, 11 and 12, respectively).