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

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Abstract

Brachyspira infections are significant causes of enterocolitis in pigs. In order to differentiate pathogenic species (Brachyspira (Br.) hyodysenteriae, Brachyspira pilosicoli) from less pathogenic or non-pathogenic species (Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii) in paraffin-embedded tissue samples a polymerase chain reaction (PCR) protocol allowing identification of Brachyspira at species level in archival material was developed. This approach was complemented by sequencing of the PCR amplification products. All seven cases presented with clinical and morphological Brachyspira-associated enterocolitis. Br. hyodysenteriae was not identified in any of the cases, while Br. pilosicoli was identified in a single case in conjunction with Br. murdochii. One case each was found positive for Br. innocens and Br. intermedia. Interestingly, the majority of cases presented as single or double infections with Br. murdochii. In some of the pigs other pathogens, like porcine circovirus-2 or Lawsonia intracellularis were present. These observations point at the possibility that under certain conditions even Brachyspira species of low pathogenicity can multiplicate extensively and lead to Brachyspira-associated enterocolitis.

Introduction

Intestinal infections with Brachyspira species are responsible for severe economic losses in the pig industry worldwide. Brachyspira (Br.) hyodysenteriae is considered the most pathogenic species and causes swine dysentery (Moxley and Duhamel, 1999, ter Huurne and Gaastra, 1995, Wilcock and Olander, 1979). Brachyspira pilosicoli has been linked to a less severe condition known as porcine intestinal spirochetosis (Duhamel, 2001, Muniappa et al., 1997, Oxberry and Hampson, 2003, Thomson et al., 1998, Trott et al., 1996). The pathogenicity of the three other defined species of Brachyspira in pigs, Brachyspira intermedia, Brachyspira murdochii, and Brachyspira innocens is controversially discussed. While Kinyon and Harris (1979) and Lee et al. (1993) considered these species as non-pathogenic, other studies by Fellström and Gunnarsson (1995) and Neef et al. (1994) provided some evidence that even these species may induce enterocolitis.

Diagnosis and species differentiation of Brachyspira has to be done by culture and subsequent biochemical tests, restriction fragment length polymorphisms of the bacterial genome, or polymerase chain reaction (PCR) using species-specific primers (Atyeo et al., 1999, Barcellos et al., 2000, Fellström and Gunnarsson, 1995, Hommez et al., 1998, Leser et al., 1997, Rohde et al., 2002). These techniques are time consuming and rather expensive for routine bacteriology. Alternatively, PCR amplification directly from fecal samples without prior cultivation has been described, with the advantage of being quick and comparatively cheap, but the disadvantage that DNA extraction from feces is rather tedious due to the need for removing PCR inhibitors (La et al., 2003, Moalic et al., 2001). Thus, in routine settings, exact determination of involved Brachyspira species is frequently not done, and the detection of any Brachyspira is sometimes equated with the presence of Br. hyodysenteriae.

Usually the diagnosis is made on fresh fecal material or mucosal scrapings; for retrospective studies, however, the only material available is paraffin-embedded tissue. Thus, we are currently exploring different methods for differentiating Brachyspira in such material. On the one hand we evaluate in situ hybridization (ISH) with digoxigenin-labeled RNA probes (Herzog et al., 2005), on the other hand PCR amplification of short DNA fragments using species-specific primer sets.

During these experiments Br. hyodysenteriae could not be identified in paraffin-embedded colon tissue of pigs with clinically severe diarrhea and histological evidence of Brachyspira-associated colitis. In the present study, the identity of the involved Brachyspira species is revealed by PCR with genus-specific primers and subsequent sequencing of the obtained amplification products as well as by PCRs employing species-specific primer sets allowing the identification of Brachyspira at species level.

Section snippets

Samples

From the necropsy files of the Institute of Pathology and Forensic Veterinary Medicine seven cases previously diagnosed as swine dysentery or Brachyspira infection were selected. Paraffin-embedded tissue blocks from large intestine were subjected to hematoxylin and eosin staining, silver impregnation according to Warthin-Starry for morphological screening, and ISH with a genus-specific riboprobe for the detection of Brachyspira species nucleic acid (Herzog et al., 2005). The porcine

Necropsy findings

The seven pigs investigated in the present study originated from six different farms. Age ranged from 2.5 to 3 months; three animals were castrated males, two were females and for two the sex was not recorded.

Case histories revealed herd problems with diarrhea or enteritis, often associated with wasting.

At necropsy, all animals were in poor nutritional state. All showed gray colored watery to mucoid contents of the large intestine; in some cases the colonic subserosa und submucosa was

Discussion

Although all cases included in the present study exhibited marked macroscopic and histologic lesions consistent with dysentery or spirochetal diarrhea (Duhamel, 2001, Dünser et al., 1997, Moxley and Duhamel, 1999), in none of the samples was Br. hyodysenteriae identified and only one sample contained Br. pilosicoli. In the majority of the samples Br. murdochii was identified, and in one sample each Br. intermedia and Br. innocens were present. These findings clearly indicate that the occurrence

Acknowledgements

We thank Gudrun Kurz for excellent technical assistance and Klaus Bittermann for professional digital artwork. This study was partially funded by the Hochschuljubiläumsstiftung of the City of Vienna. We are grateful to Tim K. Jensen and Ronny Lindberg, who provided paraffin-embedded tissue samples of Br. hyodysenteriae infected pigs as positive controls.

The nucleotide sequences determined in this study have been deposited in GenBank database under the accession numbers DQ149198 –DQ149220.

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