Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery

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Abstract

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n = 1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.

Introduction

Swine dysentery (SD) is a mucohaemorrhagic colitis of mainly grower/finisher pigs resulting from colonization of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae (Hampson et al., 2006a). Diagnosis of SD primarily relies on the herd history and observation of typical clinical signs of diarrhoea containing fresh blood and/or mucus. A definitive diagnosis of SD traditionally has required the isolation and identification of the spirochaete, although the use of PCR-based assays on faeces or on isolated spirochaetes now allows more rapid and reliable diagnosis (Atyeo et al., 1998, Fellström et al., 2001, La et al., 2003). Nevertheless, culture and/or PCR-based testing is time consuming, relatively costly, and may not identify pigs that intermittently excrete low numbers of spirochaetes.

Colonization by B. hyodysenteriae elicits a strong systemic immune response against the spirochaete that can last up to 17 weeks (Fisher and Olander, 1981, Fernie et al., 1983, Joens et al., 1979), and hence measuring circulating antibody titres can provide indirect evidence of exposure to B. hyodysenteriae. A number of tests have been developed that detect antibodies to B. hyodysenteriae (reviewed by La and Hampson, 2001). Amongst these, enzyme-linked immunosorbent assays (ELISA) using B. hyodysenteriae lipooligosaccharide (LOS) or sonicated cells as plate-coating antigens have shown the most potential (Joens et al., 1982, Wright et al., 1989), but they have tended to be serogroup-specific (in the case of LOS antigens) and/or lacking in specificity or sensitivity. Consequently there is a need for the use of a more species-specific plate-coating antigen that is applicable to all B. hyodysenteriae serogroups. Previously, it has been suggested that a 29.7 kDa outer membrane lipoprotein of B. hyodysenteriae might be a useful ELISA antigen as it is surface exposed, immunogenic and present in all strains of B. hyodysenteriae (La and Hampson, 2001). This molecule was originally called BmpB (Lee et al., 2000), then BlpA (Cullen et al., 2003), but is now designated as Bhlp29.7 (Hampson et al., 2006b).

The aim of this study was to develop and evaluate an ELISA for the serological detection of SD using recombinant His6-Bhlp29.7 as the discriminatory antigen. The test was targeted at detection of SD at the herd level, and used sera collected from commercial pigs slaughtered in abattoirs since this is a convenient source of sera for screening herd health status.

Section snippets

Permissions

The work was conducted with the approval of the Murdoch University Animals Ethics Committee. All procedures were carried out under Australian National Health and Medical Research Council guidelines.

Preparation of ELISA antigen

Recombinant Bhlp29.7 was expressed as an N-terminal histidine-tagged fusion protein (His6-Bhlp29.7), as described previously (La et al., 2004, La et al., 2005). The purified lyophilised antigen was re-hydrated and diluted to a working concentration of 200 μg/ml using sterile phosphate buffered saline

Immunoassay development

Optimized results were obtained with a 200-fold dilution of positive serum and 3000 ng/ml of antigen (Table 1). The reactivity of the serum from the pig immunized with B. hyodysenteriae B78T gave an ELISA value of 94.1, which was approximately 2.6-fold greater than the serum raised against B. pilosicoli 1648 (ELISA value of 36.3).

Determination of the cut-off value for the ELISA

The mean and standard deviation of the normalized ELISA absorbance value of sera from pigs in group A (SD not observed) were 16.9 and 9.4, respectively. The mean + three

Discussion

An indirect ELISA was established and standardized for the detection of antibodies to Bhlp29.7, an outer membrane lipoprotein of B. hyodysenteriae. As the disease history of individual animals tested was not recorded, it was not possible to investigate the potential usefulness of the ELISA at identifying individual infected pigs.

To determine the disease status of individual herds, a cut-off value was established using the mean and distribution of the serum reactivity of groups of pigs from

Acknowledgement

The authors thank Novartis Animal Health for financial support for this study.

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