Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii

Weissenböck, Herbert; Maderner, A.; Herzog, Anna Maria; Lussy, Helga; Nowotny, Norbert

doi:10.1016/j.vetmic.2005.09.002

Cited by 36 publications

(37 citation statements)

References 25 publications

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“…During such treatment, the DNA strands break into fragments, and studies have shown that amplicons of more than 300 base pairs (bp) lead to a significantly decreased 1 sensitivity of the PCR assay. 7,19 Thus, the chosen amplicon, which had a length of approximately 122 bp that targeted a part of the 18S ribosomal (r)RNA gene, shows a sufficient number of nucleotide differences to allow identification of cryptosporidian species through sequencing. Due to the high demand of specific identification of cryptosporidian species especially in reptilian samples, the assay was then modified for live animal diagnostics.…”

Section: Introductionmentioning

confidence: 99%

Detection ofCryptosporidiumspecies in feces or gastric contents from snakes and lizards as determined by polymerase chain reaction analysis and partial sequencing of the 18S ribosomal RNA gene

et al. 2011

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Abstract. Cryptosporidiosis is a well-known gastrointestinal disease of snakes and lizards. In the current study, 672 samples (feces and/or gastric contents or regurgitated food items) of various snakes and lizards were examined for the presence of cryptosporidia by polymerase chain reaction (PCR) assay targeting a part of the 18S ribosomal RNA gene. A consecutive sequencing reaction was used to identify the cryptosporidian species present in PCR-positive samples. Cryptosporidium varanii (saurophilum) was detected in 17 out of 106 (16%) samples from corn snakes (Pantherophis guttatus) and in 32 out of 462 (7%) samples from leopard geckos (Eublepharis macularius). Cryptosporidium serpentis was found in 8 out of 462 (2%) leopard gecko samples, but in no other reptile. The Cryptosporidium sp. "lizard genotype" was present in 1 leopard gecko sample, and 1 sample from a corn snake showed a single nucleotide mismatch to this genotype. Pseudoparasitic cryptosporidian species were identified in 5 out of 174 (3%) ophidian samples, but not in lizards. Other sequences did not show complete similarity to previously published Cryptosporidium sequences. The results stress the importance for diagnostic methods to be specific for Cryptosporidium species especially in snakes and show a relatively high prevalence of C. varanii in leopard geckos and corn snakes.

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Section: Introductionmentioning

confidence: 99%

Detection ofCryptosporidiumspecies in feces or gastric contents from snakes and lizards as determined by polymerase chain reaction analysis and partial sequencing of the 18S ribosomal RNA gene

et al. 2011

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“…Growth on the plates was tested by PCR for B. hyodysenteriae and Brachyspira pilosicoli [3], and for Brachyspira innocens and general Brachyspira spp. using the method of Weissenbock et al [24]. …”

Section: Methodsmentioning

confidence: 99%

The use of ELISAs for monitoring exposure of pig herds to Brachyspira hyodysenteriae

2012

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BackgroundSwine dysentery (SD), a mucohaemorrhagic diarrhoeal disease of pigs, results from infection of the large intestine with the spirochaete Brachyspira hyodysenteriae. ELISA systems using whole spirochaete cells (WC) and the B. hyodysenteriae outer membrane lipoprotein Bhlp29.7 previously have been established as potential diagnostic tools for SD. However, their true value in identifying infected herds remains unclear. The present study aimed to compare the performance of whole-cell and Bhlp29.7 based ELISAs in detecting specific immunoglobulin class IgG and IgM to B. hyodysenteriae in growing pigs, and additionally evaluated whether meat juice could serve as a source of specific antibodies.ResultsLevels of circulating IgG and IgM reacting with WC spirochaete preparations and recombinant Bhlp29.7 peaked 4-6 weeks post-infection in the experimentally challenged pigs, and remained elevated in the present study. In a cohort of pigs on an infected farm levels of antibody directed against both antigens showed a progressive increase with time. However, other than for the level of IgG against WC antigen, a significant increase in antibody levels also was observed in a cohort of pigs on a non-infected farm. In addition, assays using meat juice had 100% specificity and equivalent sensitivity to those based on serum, and likewise the best performance was achieved using the WC IgG ELISA.ConclusionsIgG ELISAs using either WC or Bhlp29.7 as plate-coating antigens were shown to be useful for monitoring the dynamics of B. hyodysenteriae infection in grower pigs. Of the two antigens, the WC preparation tended to give better discrimination between pigs from infected and non-infected farms. Testing of meat juice was shown to have potential for identifying infected herds.

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“…by speciation through molecular methods such as PCR targeting the NADH oxidase (nox) gene 2,16 ; however, as these assays are often applied to primary cultures, which can take 6 days or more to complete, there is often a significant delay between receipt of clinical samples and the reporting of Brachyspira culture results.…”

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Fluorescent in situ hybridization for detection of “Brachyspira hampsonii” in porcine colonic tissues

et al. 2013

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Swine dysentery is classically associated with infection by the strongly beta-hemolytic Brachyspira hyodysenteriae; however, the proposed novel species “ Brachyspira hampsonii” has also been isolated from clinical cases of dysentery in the United States and Canada. Microbial culture is highly sensitive for detecting Brachyspira in clinical samples but requires several days for completion and is often followed by molecular testing for speciation. Alternatively, in situ hybridization using molecular probes applied to sections of formalin-fixed tissue can provide rapid, culture-independent identification of agents observed histologically. Accordingly, a fluorescent in situ hybridization assay was developed for confirmation of a clinical diagnosis of swine dysentery associated with infection by “ B. hampsonii.” An oligonucleotide probe (Hamp1210) targeting a specific 23S ribosomal RNA sequence of “ B. hampsonii” was developed following sequence analysis and comparison of numerous Brachyspira spp. clinical isolates with reference sequences available in GenBank. The application of Hamp1210 and a previously published probe for B. hyodysenteriae (Hyo1210) to diseased colonic tissues successfully detected the target species in both experimentally infected pigs and naturally infected pigs from field cases, and the Hamp1210 probe consistently detected both clade I and clade II isolates of “ B. hampsonii”; however, a strong positive signal was also observed in a single case where the Hamp1210 probe was applied to tissues infected with Brachyspira intermedia. In situ hybridization incorporating the Hamp1210 probe can reduce the delay from sample submission to pathogen identification in cases of swine dysentery associated with “ B. hampsonii” infection where formalin-fixed tissues are available.

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Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii

Cited by 36 publications

References 25 publications

Detection ofCryptosporidiumspecies in feces or gastric contents from snakes and lizards as determined by polymerase chain reaction analysis and partial sequencing of the 18S ribosomal RNA gene

Detection ofCryptosporidiumspecies in feces or gastric contents from snakes and lizards as determined by polymerase chain reaction analysis and partial sequencing of the 18S ribosomal RNA gene

The use of ELISAs for monitoring exposure of pig herds to Brachyspira hyodysenteriae

Fluorescent in situ hybridization for detection of “Brachyspira hampsonii” in porcine colonic tissues

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