title: Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.
abstract: RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment.
authors.affiliation: CSIRO Livestock Industries, Australian Animal Health Laboratory, Private Bag 24 Geelong, Victoria 3220, Australia.
authors.forename: Kathy
authors.initials: K
authors.lastname: Granger
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Moore
authors.forename: John K
authors.initials: JK
authors.lastname: Davies
authors.forename: Jill A
authors.initials: JA
authors.lastname: Vaughan
authors.forename: Paula L
authors.initials: PL
authors.lastname: Stiles
authors.forename: David J
authors.initials: DJ
authors.lastname: Stewart
authors.forename: Mark L V
authors.initials: ML
authors.lastname: Tizard
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D001483:Base Sequence; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D020459:Genes, rRNA; D015511:Goat Diseases / Q000382:microbiology*; D006041:Goats / Q000382:microbiology; D007082:Ileum / Q000382:microbiology; D007413:Intestinal Mucosa / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology; D008969:Molecular Sequence Data; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D018384:Oxidative Stress / Q000502:physiology; D010283:Paratuberculosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S