PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.
Animals ; Bacteriological Techniques/economics/*veterinary ; Cattle ; Cattle Diseases/diagnosis/*microbiology ; DNA, Bacterial/chemistry/genetics ; Female ; Mycobacterium avium subsp. paratuberculosis/*genetics ; Paratuberculosis/diagnosis/*microbiology ; Real-Time Polymerase Chain Reaction/veterinary ; Reproducibility of Results

The presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis (Johne's disease) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease. Mycobacterium avium subsp. paratuberculosis infection was histochemically confirmed using Ziehl-Neelsen staining and molecularly diagnosed through rpoB DNA sequencing. Galectin-3 was detected in the majority of inflammatory cells, possibly macrophages, in the granulomatous lesions within affected tissues, including the ileum. These findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis, probably through cell adhesion and anti-apoptosis mechanisms.
Animals ; Cattle ; Cattle Diseases/*pathology ; Chronic Disease ; Galectin 3/*metabolism ; Immunohistochemistry ; Intestine, Small/microbiology/*pathology ; Mycobacterium avium subsp. paratuberculosis/growth & development/isolation & purification ; Paratuberculosis/*pathology ; RNA Polymerase II/genetics