Abstract. Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. To ensure efficient and reproducible PCR results from a difficult sample matrix such as feces, there are many obstacles that a DNA extraction method must overcome, including the presence of inhibitors and the thick waxy cell wall of MAP. In the current study, 6 commercial DNA extraction kits were evaluated using fecal samples from naturally infected cattle shedding various amounts of MAP. Upon extraction, DNA purity and yield were measured, and real-time PCR was performed for detection of the insertion sequence (IS)900 and ISMAP02 targets. The kits evaluated showed significant differences in the purity and yield of DNA obtained. The best results were observed with kits E and A, having identified 94% (16/17) and 76% (13/17) of the positive samples by IS900 PCR, respectively. Both of these kits utilized bead beating in a lysis solution for cell disruption, followed by spin column technology (kit E) or magnetic bead-based technology (kit A) for nucleic acid isolation and purification. Two kits (A and F) demonstrated improved performance when used in conjunction with the respective manufacturer's PCR test. The present study demonstrates the importance of choosing the correct methodology for the most accurate diagnosis of paratuberculosis through fecal PCR.
Salmonella enterica serovar Typhimurium continues to be a major cause of foodborne illness worldwide and pork can serve as a source of infection. Co-infection of S. enterica with Lawsonia intracellularis, a common intestinal pathogen of swine, has been found as risk factor for increased S. enterica shedding. The objective of this study was to investigate if vaccination against L. intracellularis could lead to decreased S. Typhimurium shedding. To test this hypothesis, pigs were challenged with either S. Typhimurium or S. Typhimurium and L. intracellularis, with and without L. intracellularis vaccination (n = 9 per group). A non-challenged group served as a negative control. Vaccination decreased the shedding of S. Typhimurium in co-infected animals by 2.12 log10 organisms per gram of feces at 7 days post infection. Analysis of the microbiome showed that vaccination led to changes in the abundance of Clostridium species, including Clostridium butyricum, in addition to other compositional changes that may explain the protection mediated against S. Typhimurium. These results indicate that vaccination against L. intracellularis in co-infected herds may provide a new tool to increase food safety by helping to prevent S. enterica without the need for antibiotics.
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