DETECTION OF eaeA AND hlyA GENES IN ESCHERICHIA COLI-CAUSING DIARRHOEA IN YOUNG CALVES BY MULTIPLEX PCR

E.coli was isolated from the faeces of 28/31 (90.3%) diarrhoeic and 9/11 (81.8%) apparently healthy calves. E. coli O157 was the most prevalent serotype, detected from 5/31 diarrhoeic calves (16.1%). No O157 E. coli was revealed from the apparently healthy calves. Other serotypes were detected in diarrhoeic calves as O136, O111, O118, O103, O91, O121, O145, O87, O22 and O8 whereas O91, O113, O87, and O8 were revealed from the apparently healthy. There was also 3 untypable E. coli isolates. Screening virulence genes eaeA and hlyA in identified isolates or in faecal culture crude extract revealed clear PCR products, eaeA gene of 384bp and hlyA of 534bp. Isolates from diarrhoeic calves possessed 20/29 (69%) eaeA gene and 21/29 (72.4%) hlyA gene.  Detection of the same genes in the faecal samples from apparently healthy calves was manifested in significantly (p<0.01) lower pattern as eaeA was detected only in 2/9 isolates (22.2%) and hlyA in 3/9 (33.3%). Use of 2-4h faccal culture crude extract in direct PCR assay for the detection of eaeA and hlyA genes demonstrated higher incidence of both genes than their detection from isolate lysates, as in diarrhoeic specimens, eaeA was detected in 23/31 (74.2%) and hlyA in 25/31 (80.6%), whereas in samples collected from apparently healthy eaeA was detected in 3/11 (27.3%) and hlyA in 4/11 (36.4%). All E. coli isolates from diarrhoeic calves, as well as the specimens from which they were isolated, were 100% positive for either single detection of one gene (eaeA or hlyA) or for double possession of both genes, as 12/29 (41.4%) isolates harbored both genes at the same time, whereas 17/29 (58.6%) possessed only one of either genes eaeA or hlyA. Faecal samples from diarrhoeic calves showed signifiantly (p<0.01) higher detection of double possession of both genes eaeA and hlyA, 16/31 (51.6%), whereas single possession of one of either genes was 15/31 (48.4%). Isolates from apparently healthy calves, as well as faecal specimens from which they were isolated, in all cases, possessed only one gene either eaeA or hlyA (5/9, 55.6% isolates, and 7/11, 63.6% faecal samples). There was no double possession of both genes in any case of samples from apparently healthy calves.