Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs

• Published in: Veterinary microbiology Vol. 118; no. 1; pp. 91 - 100
• Main Authors: Calderaro, A., Bommezzadri, S., Gorrini, C., Piccolo, G., Peruzzi, S., Dettori, G., Chezzi, C.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 26.11.2006; Elsevier Science
• Subjects: Animals; Bacterial diseases; Bacterial diseases of the digestive system and abdomen; bacterial infections; Base Sequence; Biological and medical sciences; Brachyspira; Diagnosis; diagnostic techniques; digestive system diseases; disease diagnosis; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; feces; Feces - microbiology; Fundamental and applied biological sciences. Psychology; genes; genetic markers; Human bacterial diseases; Infectious diseases; Intestinal Diseases - diagnosis; Intestinal Diseases - microbiology; Intestinal Diseases - veterinary; Intestinal spirochaetosis; Medical sciences; microbial genetics; Microbiology; molecular sequence data; NADPH Oxidases - genetics; nucleotide sequences; pathogen identification; pathogenicity; Phylogeny; polymerase chain reaction; Polymerase Chain Reaction - veterinary; Polymorphism, Restriction Fragment Length; restriction fragment length polymorphism; RFLP-PCR; 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; strain differences; strains; Swine; swine diseases; Swine Diseases - diagnosis; Swine Diseases - microbiology; Swine dysentery; Brachyspira; Swine dysentery; RFLP-PCR; Diagnosis; Intestinal spirochaetosis; Microbiology; Digestive system; Identification; Infection; Polymerase chain reaction; Vertebrata; Mammalia; Swine; Spirochaetosis; Bacteriosis; Digestive diseases; Intestinal disease; Dysentery; Artiodactyla; Veterinary; Ungulata
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2006.06.022

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10 2–10 3 cells/g of faeces for 16S rRNA gene PCR and 10 4 cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.