MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS ISOLATES
Objective: Johneâ€™s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is highly prevalent in domestic ruminants. In India, the exact prevalence of MAP genotypes still remains unknown limited, and the systematic disease control programs are also limited. This study was undertaken to study the molecular characterization of MAP isolates.
Methods: About 22 MAP isolates were from cattle, sheep, and goat under gone the molecular characterization by three different methods (1) IS1311 polymerase chain reaction (PCR) with restriction enzyme analysis (REA), (2) GyrA and GyrB PCR with sequencing, and (3) digital microfluidic chip (DMC)-PCR. The study demonstrated that a) IS1311 PCR with REA (based on point mutations) identified all 22 MAP isolates as â€œintermediate typeâ€ irrespective of a host of origin and also belong to Indian Bison type. Molecular typing based on the gyrA and gyrB genes partial amplification and sequencing revealed that the MAP isolates exhibited more lineages toward the reference Type III, Intermediate strain.
Results: The MAP isolate of sheep origin showed more lineages toward the sheep type than the isolates of cattle and goats. This variation may be due to host-pathogen interactions and adaptation to different hosts and environmental conditions in the nature.
Conclusions: The DMC-PCR, which is based on sequence difference at 5` end of IS900 of MAP, differentiated rapidly all the isolates as sheep type. The application of DMC-PCR to differentiate sheep and Intermediate types is limited as the Intermediate type (Type III) and sheep type (Type I) are very closely related to each other and all the MAP isolates were confirmed as Intermediate or Type III by three different methods which are commonly present in India, Spain, and Iceland.
2. Ott SL, Wells SJ, Wagner BA. Herd-level economic losses associated with Johneâ€™s disease on US dairy operations. Prev Vet Med
3. Nielsen SS, GrÃ¸nbaek C, Agger JF, Houe H. Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture
for diagnosis of paratuberculosis. Prev Vet Med 2002;53(3):191-204.
4. Green EP, Tizard ML, Moss MT, Thompson J, Winterbourne DJ, McFadden JJ, et al. Sequence and characteristics of IS900, an insertion
element identified in a human Crohnâ€™s disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res 1989;17(22):9063-73.
5. Motiwala AS, Li L, Kapur V, Sreevatsan S. Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis. Microbes Infect 2006;8(5):1406-18.
6. Reddy YG, Prakash R, Anandakumar S. Isolation, cloning and expression of recombinant staphylokinase gene against thrombosis. Int
J Pharm Pharm Sci 2014;6(4):266-70.
7. Vansnick EL, de Rijk OP, Vercammen F, Geysen D, Rigouts L, Portaels F. Newly developed primers for the detection of Mycobacterium avium subsp. paratuberculosis. Vet Microbiol 2004;100:197-204.
8. Nath SS. Serological immune diagnosis of tuberculosis using PCR and in-house developed clinical isolate based ELISA. Int J Pharm Pharm Sci 2013;5(3):584-92.
9. Poupart P, Coene M, Van Heuverswyn H, Cocito C. Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johneâ€™s disease. J Clin Microbiol 1993;31(6):1601-5.
10. Pavlik I, Horvathova A, Dvorska L, Bartl J, Svastova P, du Maine R, et al. Standardisation of restriction fragment length polymorphism
analysis for Mycobacterium avium subspecies paratuberculosis. J Microbiol Methods 1999;38(1-2):155-67.
11. de Juan L, Alvarez J, Aranaz A, RodrÃguez A, Romero B, Bezos J, et al. Molecular epidemiology of Types I/III strains of Mycobacterium
avium subspecies paratuberculosis isolated from goats and cattle. Vet Microbiol 2006;115(1-3):102-10.
12. Sohal JS, Singh SV, Subodh S, Sheoran N, Narayanasamy K, Singh PK, et al. Mycobacterium avium subspecies paratuberculosis diagnosis andgeno-typing: Genomic insights. Microbiol Res 2009;164(3):330-7.
13. Whittington RJ, Hope AF, Marshall DJ, Taragel CA, Marsh I. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia. J Clin Microbiol 2000;38(9):3240-8.
14. Castellanos E, Aranaz A, Romero B, de Juan L, Alvarez J, Bezos J, et al. Polymorphisms in gyrA and gyrB genes among Mycobacterium avium subsp. paratuberculosis type I, II and III isolates. J Clin Microbiol 2007;45(10):3439-42.
15. Collins DM, Zoete MD, Cavaignac SM. Mycobacterium avium subsp. paratuberculosis strains from cattle and sheep can be distinguished by a PCR test based on a Novel DNA sequence difference. J Clin Microbiol (12):4760-2.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.