Evaluation of a novel multiplex qPCR method for rapid detection and quantification of pathogens associated with calf diarrhoea

Abstract

Aims: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea.

Methods and results: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%-103% and detection limits of 100-1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83.

Conclusion: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples.

Significance and impact of study: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

Keywords: Calf diarrhoea; calf scours; detection; multiplex qPCR; quantification.

FIGURE 1

Relationship between log₁₀CFU per 0.1 gram faeces and Cq values for Clostridium perfringens, C. perfringens type C, Escherichia coli F5 and Salmonella enterica serovar Dublin. Bacteria were spiked into pathogen free faecal samples in the numbers indicated and Cq valued in Enterit4Calves was determined. The correlation coefficients (R ²) of plots was 0.998, 0.9938, 0.9965 and 0.9989. Error bars show SD of Cq values and the dotted lines indicate the 95% confidence interval of the fitted line.