Evaluation of IS900 PCR for Detection and IS1311 PCR via Restriction Enzyme Analysis for Strain Typing of Mycobacterium avium subspecies paratuberculosis in Ruminant Fecal Samples
DOI:
https://doi.org/10.48165/ijvsbt.22.2.07Keywords:
IS900 PCR, IS1311 PCR, Mycobacterium avium subspecies paratuberculosis, PCR-REA, Phylogenetic analysis.Abstract
Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic granulamatous infectious enteritis known as Johne’s disease in ruminants and is characterized by progressive diarrhea, weight loss, and reduced productivity. MAP shedding in feces makes fecal samples valuable for diagnostic screening. Based on clinical symptoms, 220 fecal samples from cattle and buffaloes with a history of chronic diarrhea were collected. Each sample was stained with Ziehl- Neelsen staining technique to detect acid fast positive bacteria. The DNA was collected from highly acid fast bacterial samples and was subjected to isolation of Mycobacterium avium subsp. paratuberculosis (MAP). Out of 71 acid fast bacterial samples; 22 were highly acid fast positive. Only 2 samples were found to be positive in isolation for MAP. DNA from these isolates were then extracted and subjected to IS900 PCR hence, confirming the presence of MAP in the samples. The positive samples were also subjected to IS1311 PCR and were analyzed by Restriction enzyme analysis (REA). The PCR-REA confirmed the presence of ‘Cattle type’ of strain of the two isolates. Sequence analysis further demonstrated a high degree of similarity between the two isolates and strong phylogenetic identity with previously reported global MAP sequences, with the highest similarity (99.3%) to a Chinese MAP isolate (MW546854_China).Downloads
References
Chiodini, R. J., Van Kruiningen, H. J., & Merkal, R. S. (1984). Ruminant paratuberculosis (Johne’s disease): Current status and future prospects. Cornell Veterinarian, 74(3), 218–262.
Collins, D. M., Gabric, D. M., & De Lisle, G. W. (1989). Identification of a repetitive DNA sequence specific to Mycobacterium avium subsp. paratuberculosis. FEMS Microbiology Letters, 51(1), 175–178.
Collins, D. M., Gabric, D. M., & De Lisle, G. W. (1990). Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. Journal of Clinical Microbiology, 28(7), 1591–1596.
Garrido, J. M., Cortabarria, N., Oguiza, J. A., Aduriz, G., & Juste, R. A. (2000). Use of PCR on fecal samples for diagnosis of sheep paratuberculosis. Veterinary Microbiology, 77(3–4), 379–386.
Green, E. P., Tizard, M. L. V., Moss, M. T., Thompson, J., Winterbourne, D. J., McFadden, J. J., & Hermon-Taylor, J. (1989). Sequence and characteristics of IS900, an insertion element identified in human Crohn’s disease isolates of Mycobacterium paratuberculosis. Nucleic Acids Research, 17(22), 9063–9073.
Marsh, I., Whittington, R., & Cousins, D. (1999). PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on IS1311 polymorphisms. Molecular and Cellular Probes, 13(2), 115–126.
McAdam, R. A., Guilhot, C., & Gicquel, B. (1994). Transposition in mycobacteria. In B. R. Bloom (Ed.), Tuberculosis: Pathogenesis, protection and control (pp. 199–216). ASM Press.
Pavlík, I., Horvathova, A., Dvorska, L., Bartl, J., Svastova, P., Dupris, C., & Bull, T. J. (1999). Standardization of restriction fragment length polymorphism analysis for Mycobacterium avium subsp. paratuberculosis. Journal of Microbiological Methods, 38(1–2), 155–167.
Peek, S. F., McGuirk, S. M., Sweeney, R. W., & Cummings, K. J. (2018). Infectious diseases of the gastrointestinal tract. In Rebhun’s Diseases of Dairy Cattle (3rd ed., pp. 249–356). Elsevier.
Sevilla, I. X., Singh, S. V., Garrido, J. M., Aduriz, G., Rodriguez, S., Vittorini, M., Joseba, M., & Juste, R. A. (2005). Molecular typing of Mycobacterium avium subsp. paratuberculosis strains from different hosts and regions. Revue Scientifique et Technique, 24(3), 1061–1066.
Singh, S. V., Singh, A. V., Singh, R., Sharma, S., Singh, N., Misra, P. N., & Tyagi, A. K. (2008). Seroprevalence of bovine Johne’s disease in buffaloes and cattle of North India using an indigenous ELISA kit based on native Mycobacterium avium subsp. paratuberculosis ‘Bison type’ genotype. Comparative Immunology, Microbiology and Infectious Diseases, 31(5), 419–433.
Vary, P. H., Andersen, P. R., Green, E., Hermon-Taylor, J., Morris, Z., & McFadden, J. J. (1990). Use of highly specific DNA probes and PCR to detect Mycobacterium paratuberculosis in Johne’s disease. Journal of Clinical Microbiology, 28(5), 933–937.
Whittington, R., Marsh, I., Choy, E., & Cousins, D. (1998). Polymorphism in IS1311 insertion sequence for differentiation within Mycobacterium avium complex species. Molecular and Cellular Probes, 12(6), 349–358.
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