Development and Validation of Real-Time PCR assays for diagnosis of Equine Infectious Diarrhoea
Introduction
Diarrhoea is a major problem in young and adult horses. Apart from non-infectious causes for diarrhoea such as foal heat diarrhoea, dietary imbalance, lactose intolerance and parasitic infections several bacteria and viruses are associated with diarrhoea.
Objective
To monitor and detect these infections we developed four duplex PCRs for detection of six viral and bacterial pathogens: equine coronavirus (ECoV), equine rotavirus (ERV), Lawsonia intracellularis, toxinogenic Clostridium difficile species (TcdB gene), toxinogenic Clostridium perfringens type A and type C species (CPA, CPB and CPB2 genes) and Salmonella enterica species. The foal and yearling diarrhoea PCR panel included all PCRs, whereas in the diarrhoea PCR panel for adult horses ERV and Lawsonia intracellularis were excluded.
Materials and methods
DNA/RNA isolation was carried out with the MagMax-96 Total Pathogen Isolation kit. The extraction platform used was the KingFisher 96 Magnetic Particle processor. For each PCR the AgPath-ID One-Step RT-PCR Kit was used. Primer and probe sets were selected from the literature (references available upon request) or designed in-house and specificity and inclusivity were analysed in silico. In every duplex PCR one probe was labelled with FAM and one probe was labelled with Cy5. For each pathogen a well-defined strain with known titre in CFU/ mL or TCID50/mL was used, and decimal dilution series were made in threefold in PBS and in pooled faecal Eswab (Copan) suspensions and were tested in three different test runs. The undiluted samples were also used to check for cross-reactivity in a panel of 20 different gastrointestinal viruses and bacteria.
Results
Detailed results of the technical validation are shown in table 1. Detection limits for the viruses and bacteria ranged from 0.01 – 1 TCID50/PCR reaction and from 1-10 CFU/PCR reaction, respectively. All PCRs showed a linear range of at least 4 log10 values, and efficiency of all PCRs was between 90-110%, allowing reliable quantification. Repeatability and reproducibility were well within limits with variation coefficients of < 5% and < 7%, respectively. Although Ct-values of undiluted strains were low in the homologous PCRs, no cross-reactivity was observed with other pathogens represented in the inclusivity/exclusivity panel. Preliminary results of the diarrhoea PCR panels for adult horses and foals/ yearlings after introduction into routine diagnoses are shown in table 2. We did not detect Clostridium perfringens type C (CPB gene) in any of the samples, we detected ECoV only
in adult horses and Clostridium difficile almost exclusively in foals/yearlings and we detected Clostridium perfringens type A and Salmonella enterica in both categories.
Conclusion
These duplex PCRs as building stones for diarrhoea PCR panels for foals and adult horses form rapid, sensitive and cost-effective assays to diagnose and quantify simultaneously the main pathogens involved in equine infectious diarrhoea. The interpretation of the clinical relevance of some of these findings, also in relation to the quantitative results, needs further analysis and discussion. For example, the presence of toxinogenic genes in Clostridium species does not necessarily imply the production of the relevant toxins and the clinical relevance of an ERV diagnosis strongly depends on the virus concentration.