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

• Published in: Journal of applied microbiology 2022-10, Vol.133 (4), p.2516-2527
• Main Authors: Pansri, Potjamas, Svensmark, Birgitta, Liu, Gang, Thamsborg, Stig Milan, Kudirkiene, Egle, Nielsen, Henrik Vedel, Goecke, Nicole Bakkegård, Olsen, John Elmerdahl
• Format: Article
• Language: English
• Subjects: Animals; Antimicrobial agents; Bacteria; Bacteria - genetics; Calf diarrhoea; calf scours; Calves; Cattle; detection; Detection limits; Diarrhea; Diarrhea - diagnosis; Diarrhea - microbiology; Diarrhea - veterinary; Feces; Feces - microbiology; Laboratories; Methods; Multiplex Polymerase Chain Reaction - methods; multiplex qPCR; Multiplexing; Nucleic Acids; Original; Parasitic diseases; Pathogens; Polymerase chain reaction; quantification; Sensitivity and Specificity
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.15722

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.