Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia

• Published in: Journal of veterinary diagnostic investigation 2005-03, Vol.17 (2), p.103-109
• Main Authors: Townsend, K.M, Giang, V.N, Stephens, C, Scott, P.T, Trott, D.J
• Format: Article
• Language: English
• Subjects: Animals; Australia; bacterial infections; Brachyspira hyodysenteriae; Brachyspira pilosicoli; disease diagnosis; DNA Primers - chemistry; DNA, Bacterial - chemistry; DNA, Bacterial - isolation & purification; DNA, Ribosomal - chemistry; Intestinal Diseases - diagnosis; Intestinal Diseases - microbiology; Intestinal Diseases - veterinary; intestinal microorganisms; molecular sequence data; NADPH Oxidases - chemistry; NADPH Oxidases - genetics; nucleotide sequences; optimization; pathogen identification; Phylogeny; Polymerase Chain Reaction - veterinary; Polymorphism, Restriction Fragment Length; Poultry; poultry diseases; Poultry Diseases - diagnosis; Poultry Diseases - microbiology; rapid methods; restriction fragment length polymorphism; ribosomal RNA; RNA, Ribosomal, 16S - chemistry; RNA, Ribosomal, 16S - genetics; Species Specificity; Spirochaetales - classification; Spirochaetales - enzymology; Spirochaetales - genetics; Spirochaetales - isolation & purification; Spirochaetales Infections - diagnosis; Spirochaetales Infections - microbiology; Spirochaetales Infections - veterinary; Swine; Swine Diseases - diagnosis; Swine Diseases - microbiology; swine dysentery
• ISSN: 1040-6387; 1943-4936
• DOI: 10.1177/104063870501700202

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.