| dc.description.abstract | Introduction.
C. perfringes type A, E. coli and Rotavirus type A (RVA) are the most important pathogens causing diarrhea in suckling piglets. These three pathogens may be present in feces or enteric content simultaneously. Therefore it is hard to determine the causative agent of piglet diarrhea in some cases based on bacterial culture or PCR. The general objective of this project is to determine a cut-off value for the combined “diarrhea package PCR test” of the Animal Health Service with respect to diagnosing diarrheic cases. The objective is broken down into several research questions,
1) At which value for alpha toxin, beta 2 toxin, or RVA is the specificity of the qPCR higher than 90%.
2) Is there interference between RVA, C. perfringens type A and E. coli in qPCR.
3) Which toxin, alpha or beta 2, is preferred to infer conclusions over C. perfringens type A.
Material & methods.
An observational retrospective cross sectional study was conducted.
Data were collected at the Animal Health Service (GD Deventer) from submitted samples. The data consist of quantitative PCR (qPCR) results for RVA and alpha/beta 2 toxin genes for C. perfringens and a qualitative PCR for E. coli fimbriae genes. In addition, veterinarians and pathologists decided based on clinical data which pathogen was the probable cause of diarrhea. A comparison was made between the veterinarian’s interpretation and the qPCR results.
Results
1) ROC curves showed a specificity of 90% at values of 104.50 Cfu/ml for alpha toxin, 104.02 Cfu/ml for beta 2 toxin and 105.66 copies/ml for RVA.
2) A significant lower mean for beta 2 toxin was detected in samples that tested positive for a combination of RVA and E. coli when compared to samples that tested only positive for C. perfringens.
3) C. perfringens type A detected by qPCR only (C. perfringens-qPCR) results showed a larger deviation between alpha toxin and beta 2 toxin when compared to C. perfringens what was detected by qPCR and veterinarians (C. perfringens-AD). This was supported by the correlation coefficients of 0.572 for C. perfringens-qPCR and 0.896 for C. perfringens-AD. Besides, beta 2 toxin had a higher area under the curve (AUC) compared to alpha toxin.
Discussion.
1) High viral loads (>105.66 copies/ml) of RVA or high copy numbers for alpha (>104.50 Cfu/ml) and beta 2 toxin (>104.02 Cfu/ml) give a good indication for the identification of the causative agents of diarrhea. Furthermore, qPCR test for RVA has a higher analytic sensitivity compared to the commonly used fast-antigen test. Conversely, qPCR tests detecting alpha and beta 2 toxin should be used with a certain caution, since C. perfringens type A is part of the normal microbiome of the piglet intestine.
2) The presented data indicates interference between pathogens in qPCR test in the group where all pathogens are simultaneously present. However, it is unclear whether the interference is test, pathogen or piglet related.
3) This study shows that beta 2 toxin may be a preferred gene for identifying C. perfringens type A in cases of piglet diarrhea if one toxin has a low copy number value. | |
