Prevalence of Escherichia coli O157:H7 on dairy farms in Shiraz, Iran by immunomagnetic separation and multiplex PCR

Abstract

To identify the reservoirs of shiga toxin-producing Escherichia coli O157, sensitive detection andisolation methods are necessary. The sensitivity of traditional culture methods can be improved significantlyby the inclusion of an immunoconcentration step, resulting in less false-negative results. In this study,enrichment procedure and immunomagnetic separation (IMS) were compared for use in conjunction with amultiplex polymerase chain reaction (M-PCR) method for detection of genes stx1, stx2, eaeA and hlyA. Atotal number of 975 faecal samples were collected from 26 dairy farms in Shiraz area, Shiraz, southern Iran.The samples were cultured at 37°C for 18–24 hrs in modified tryptic soy broth (m-TSB). Each of fiveenriched samples were pooled and examined in two ways—direct PCR and IMS. The detection limit of theM-PCR protocol for seeded E. coli O157:H7:ATCC:43895 in m-TSB without stool was 1.23 × 102 CFU/ml,whereas it was 1.23 × 106 CFU/ml with enriched faecal sample. In direct PCR of enriched samples, nopositive sample was detected. However, in IMS of enriched samples one specimen was positive. Theprevalence of E. coli O157:H7 in faeces of cows in examined farms was 0.51% and the herd prevalence was3.86%. Isolation of this serotype from faecal samples indicates that cattle are reservoirs of this pathogen andpotentially a source of human infection. This finding is of considerable public health importance