title: IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep.
abstract: Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.
authors.affiliation: Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine, Komenskeho-73, Kosice, Slovakia. mangeshbhide@hotmail.com
authors.forename: Mangesh
authors.initials: M
authors.lastname: Bhide
authors.forename: Eaknath
authors.initials: E
authors.lastname: Chakurkar
authors.forename: Ludmila
authors.initials: L
authors.lastname: Tkacikova
authors.forename: Sukhadeo
authors.initials: S
authors.lastname: Barbuddhe
authors.forename: Michal
authors.initials: M
authors.lastname: Novak
authors.forename: Ivan
authors.initials: I
authors.lastname: Mikula
mesh_terms: D000818:Animals; D001483:Base Sequence; D001793:Blood Preservation / Q000379:methods / Q000662:veterinary; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000737:chemistry; D005838:Genotype; D008969:Molecular Sequence Data; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D018807:Polymorphism, Single-Stranded Conformational; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000382:microbiology; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial