Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA

Barcellos, David Emílio Santos Neves de; Uzeda, Milton de; Ikuta, Nilo; Lunge, Vagner Ricardo; Fonseca, Aline Segeren; Kader, Ivonyr Irene Tróglio Abdel; Duhamel, G

doi:10.1016/s0378-1135(00)00212-1

Cited by 13 publications

(11 citation statements)

References 27 publications

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“…This pathogen and B. pilosicoli, B. innocens/B. intermedia were also detected and differentiated utilising generic Brachyspira primers and subsequent restriction fragment length polymorphism (RFLP) analysis of TaqI digested amplicons (Barcellos et al, 2000). PCR product of the Brachyspira sp.…”

Section: Sample Processingmentioning

confidence: 99%

Prevalence of selected enteropathogenic bacteria in Hungarian finishing pigs

Biksi

Lőrincz

Beáta

et al. 2007

Acta Veterinaria Hungarica

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The aim of this study was to obtain prevalence estimates about the most important enteropathogenic bacteria: Lawsonia intracellularis, Brachyspira hyodysenteriae, Brachyspira pilosicoli, Salmonella enterica and Clostridium perfringens A and C in Hungarian farrow-to-finish pig herds. A total of 31 herds were selected, from where six pooled faecal samples, each containing three individual rectal faecal samples were collected from fattening pigs of 5-6 months of age. All 186 samples were examined by polymerase chain reaction (PCR) for the presence of the pathogens mentioned above. Lawsonia intracellularis was found in 29 herds (93.55%) and in 108 samples (58.06%); B. hyodysenteriae in 14 herds (45.16%) and in 23 samples (12.37%); B. pilosicoli in 19 herds (61.29%) and in 53 samples (28.49%); S. enterica in 17 herds (54.83%) and in 40 samples (21.50%). We detected the presence of C. perfringens A in 19 herds (61.29%) and in 46 samples (24.73%), while C. perfringens C was found in 8 herds (25.81%) and in 11 samples (5.91%). All examined herds were infected with one or more of these agents. Herds with diarrhoea in the mid-to late finishing phase had almost 10 times higher prevalence of B. hyodysenteriae than herds without such a history.

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Section: Sample Processingmentioning

confidence: 99%

Prevalence of selected enteropathogenic bacteria in Hungarian finishing pigs

Biksi

Lőrincz

Beáta

et al. 2007

Acta Veterinaria Hungarica

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“…Historically, hemolysis patterns on blood agar, during primary isolation, have been used for general identification to distinguish pathogenic from non-pathogenic intestinal spirochetes [6]. However, there are some strains, which produce weak and/or intermediate hemolysis and can not be assigned to a specific group [3]. PCR-RFLP has been shown to produce accurate, rapid and reproducible results for the identification of porcine intestinal spirochetes [3,16,19].…”

Section: Discussionmentioning

confidence: 99%

“…However, there are some strains, which produce weak and/or intermediate hemolysis and can not be assigned to a specific group [3]. PCR-RFLP has been shown to produce accurate, rapid and reproducible results for the identification of porcine intestinal spirochetes [3,16,19]. For nox -based PCR-RFLP experiment [16], four sets of primers were used; this implies that the target sequences for primer binding are not highly conserved, though the restriction pattern was highly distinct.…”

Section: Discussionmentioning

confidence: 99%

“…Many attempts have been made to characterize the porcine intestinal spirochete isolates by serological diagnosis [9], restriction endonuclease analysis (REA) [11,18], PCR [1,7,15], sequence analysis of genes [4,14], multilocus enzyme electrophoresis (MLEE) [10,12,13] and RAPD [5]. Recently, PCR-RFLP methods have been developed and used for 23S rRNA genes [3], fla A1 [8] and NADH oxidase ( nox ) [2,16,19]. In this study, the 23S rRNA gene PCR-RFLP was used for the characterization of Korean isolates.…”

Section: Introductionmentioning

confidence: 99%

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The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates

Kim

Lee

2006

J Vet Sci

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Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.

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“…But there are few reports on diagnostic techniques for the detection of antibodies to B. pilosicoli or B. alvinipulli in infected animals. Although polymerase chain reaction (PCR) with 16S rDNA [12] or 23S rRNA [2] is a useful tool for the identification of B. pilosicoli, it cannot detect less than 10 3 spirochete cells in the fecal samples [8]. On the other hand, PCR tests and immunological tests have never been developed for the diagnosis of B. alvinipulli [15].…”

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A Possibility of Application of the 105-Kilodaltons Protein of <i>Brachyspira alvinipulli</i> Cross-Reacting with Antisera to Five Species in the Genus <i>Brachyspira</i> to Diagnosis

Abe

Adachi

2004

The Journal of Veterinary Medical Science

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ABSTRACT. The antigenic properties of Brachyspira (B.) Brachyspira (B.) alvinipulli is an anaerobic and weakly beta-hemolytic spirochete [14], and a causal agent of the deterioration of chicks and reduction in egg production [17]. The spirochete colonized the mucosal surface of ceca in one-day-old chicks and 14-month-old hens, and the ceca affected with the spirochetes were dilated and contained pale-yellow watery contents [17]. Lymphocytic typhlitis and proctitis in the infected chicks were observed as histological changes [17].Many studies on the antigens of B. hyodysenteriae have been published [7,9,11,13,19]. Amongst the reports, a 45-kilodaltons (kDa) polypeptide located in the outer envelope of B. hyodysenteriae P18A is more interesting, because it has the ability to produce antibody in gnotobiotic pig serum [13]. A 45-kDa protein in the B. hyodysenteriae B204 was related to endotoxin [19]. A 44-kDa periplasmic flagellar sheath protein of B. hyodysenteriae was confirmed as a species-specific antigen consisting of glycoprotein, and the protein could produce antibody in adult New Zealand White rabbits [9]. Ochiai et al. [11] reported that 22-and 17-kDa proteins present in eight strains of B. hyodysenteriae reacted strongly with convalescent pig sera and were intimately related to the antigens responsible for the strong stimulation in pigs [11]. Amongst the protein antigens, the periplasmic flagella have been studied intensively. The hyperimmune pig serum to periplasmic flagella derived from B. hyodysenteriae P18A cross-reacted with strains B78, S75/1, B169, P18A, KF9, VS1, MC52/80 and P35/2 of B. hyodysenteriae. Convalescent serum of a pig affected with dysentery also reacted strongly with the periplasmic flagella of B. hyodysenteriae P18A. These results mentioned above demonstrated that the periplasmic flagella of B. hyodysenteriae were major antigens in the spirochete [7], but there are no reports on the immunochemical characteristics of proteins of B. alvinipulli. In this study we analyzed the proteins immunochemically and compared the antigens with those of the other Brachyspira species. MATERIALS AND METHODSStrains used: B. alvinipulli ATCC 51933 and strain C2, B. hyodysenteriae ATCC 27164 and ATCC 31212, B. pilosicoli ATCC 51139, B. innocens ATCC 29796 and B. aalborgi NCTC 11492 were used in this study. All strains were grown at 37°C for 72 hr on trypticase soy agar (BBL, U.S.A.) containing 5% (volume/volume) sheep blood under anaerobic conditions with the GasPak System (BBL, U.S.A.). The cells were harvested and washed twice with physiological saline by centrifugation at 15,000 rpm for 10 min at 4°C, and the pellets were stored at -80°C before use.Hyperimmune serum: Hyperimmune sera were prepared as previously described [1]. The cells in 0.01 M phosphate buffer (pH7.2) were adjusted to approximately 1 × 10 9 cells ml -1 , and were inoculated intravenously into rabbits at intervals of four days. When the antibody titer to the inoculum, reached the maximum titer in agglutination and immunodiffusion t...

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Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA

Cited by 13 publications

References 27 publications

Prevalence of selected enteropathogenic bacteria in Hungarian finishing pigs

Prevalence of selected enteropathogenic bacteria in Hungarian finishing pigs

The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates

A Possibility of Application of the 105-Kilodaltons Protein of <i>Brachyspira alvinipulli</i> Cross-Reacting with Antisera to Five Species in the Genus <i>Brachyspira</i> to Diagnosis

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