Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs.
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.
Calderaro
A
A
Department of Pathology and Laboratory Medicine, Section of Microbiology, Faculty of Medicine and Surgery, University of Parma, Viale A. Gramsci, 14-43100 Parma, Italy. adriana.calderaro@unipr.it
Bommezzadri
S
S
Gorrini
C
C
Piccolo
G
G
Peruzzi
S
S
Dettori
G
G
Chezzi
C
C
D000818:Animals; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology; D007410:Intestinal Diseases / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D019255:NADPH Oxidases / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D013144:Spirochaetales / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D013145:Spirochaetales Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019255:NADPH Oxidases