Development of a Multiplex PCR Assay for Selected Escherichia coli Virulence Genes, Clostridium perfringens cpa and Cryptosporidium 18S rRNA in Faecal Samples from Diarrheic Dairy Calves

Escherichia coli, Clostridium perfringens and Cryptosporidium are common diarrhea-related pathogens in dairy calves, posing considerable economic losses to animal husbandry and threatening public health. A previous study in our lab found the frequent occurrence of Escherichia coli virulence genes (eaeA, stx1 and stx2), Clostridium perfringens cpa and Cryptosporidium in diarrheic dairy calves in Ningxia Hui Autonomous Region, China. The present study aimed to develop a multiplex PCR for simultaneous detection of these virulence genes and Cryptosporidium in diarrheic dairy calves. The multiplex PCR demonstrated sensitivities of 2060 copies, 18200 copies, 1300 copies, 1990 copies and 974 copies for stx1, stx2, eaeA, cpa and 18S rRNA, respectively. Moreover, the method showed no cross-reactivity with Giardia duodenalis, Enterocytozoon bieneusi, Eimeria, Haemonchus contortus, Oesophagostomum, Moniezia, Salmonella, Proteus mirabilis and Staphylococcus aureus. Further application of the multiplex PCR in 20 clinical faecal samples from diarrheic dairy calves found that the positive rates of the multiplex PCR assay were 55% (11/20), 50% (10/20), 60% (12/20), 45% (9/20) and 25% (5/20) for stx1, stx2, eaeA, cpa and 18S rRNA, respectively, which were not significantly lower than those of the conventional PCR targeting stx1 (60%, 12/20) and eaeA (65%, 13/20), but higher than those of the reported PCR targeting stx2 (45%, 9/20) and cpa (40%, 8/20), and were consistent with those of the reported nested PCR targeting 18S rRNA (25%, 5/20). Taken together, the present study preliminarily developed a multiplex PCR assay for the rapid detection of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in dairy calves, which could provide basic data and technical support for the diagnosis and prevention of calf diarrhea. However, more samples from divergent clinical settings are needed to validate the assay in the diagnosis of selected E. coli virulence genes, C. perfringens cpa and Cryptosporidium 18S rRNA in future studies.