The experimental infection study demonstrated that pigs challenged with B. hyodysenteriae developed elevated concentrations of IgG and IgM in their serum to both WC and Bhlp29.7 antigen preparations. Antibody levels had a dramatic increase 4-6 weeks after infection, and hence this might be an optimal interval after infection to seek serological evidence of infection using these antigens. Thereafter the IgG levels to Bhlp29.7 decreased more rapidly than did those to WC. One of the pigs showed a secondary increase in Bhlp29.7-reactive IgG between 18 weeks and slaughter at 22 weeks, suggesting that a proliferation of the spirochaete may have occurred in that pig at around 18 weeks of age. Unexpectedly, an increase in antibody was not detected in this pig using the WC ELISA. As antibody levels to both antigen preparations remained elevated from 10 to 22 weeks, this confirms that sampling pigs at slaughter age should detect elevated antibody levels to the spirochaete.
In the cohort study, at no time were any of the sampled pigs found to be positive for B. hyodysenteriae by using culture and PCR. This was despite farm C being known to have SD, and having two colonized pigs detected amongst the additional 24 pigs from that farm that were sampled at the abattoir. There was no overt disease being expressed at farm C during the time of the study, and presumably the extent of colonization in individual pigs also was low. B. pilosicoli, B. innocens and/or other Brachyspira spp. were identified at different times in some pigs on both farms. This observation demonstrated that the culture methods used for detection were appropriate, but also indicated that the pulsed medication at farm C did not completely prevent colonization with Brachyspira species. Without careful veterinary surveillance of this farm, it would be difficult to recognize it as being infected with B. hyodysenteriae. Despite the lack of culture and PCR evidence for colonization, antibody levels to B. hyodysenteriae increased with time on farm C, to peak at around 18 weeks of age. Based on the experimental infection study, this suggests that there was a degree of spirochaete proliferation amongst the cohort of pigs on farm C at around 12-14 weeks of age. This sort of information could be helpful for the consulting veterinarian, who might decide to medicate the pigs at this time to prevent excessive proliferation of the spirochaete that otherwise could depress growth rates and affect feed conversion.
Differences were observed between the IgG response patterns using WC and Bhlp29.7 as plate-coating antigens. B. hyodysenteriae
WC preparations have been thought to potentially generate false positive results arising from cross-reactivities associated with exposure to other Brachyspira
species, or other bacteria with cross-reactive proteins [5
]. In the pigs from the cohort on farm A, where SD did not occur, there was no significant temporal change in IgG levels to the B. hyodysenteriae
WC preparation, but there were significant increases to WC preparations from both B. pilosicoli
and B. innocens
. The seroconversions in responses to the protein content of B. pilosicoli
and B. innocens
further confirmed the presence of these organisms in both farms, in accordance with the results of culture and PCR. Most importantly, the data provided additional evidence supporting the specificity of the B. hyodysenteriae
WC IgG ELISA as a diagnostic test. On the other hand, it is possible that exposure to B. hyodysenteriae
may generate antibodies that cross-react with WC preparations of B. pilosicoli
and B. innocens
. This was because antibody levels to antigen preparations from the latter two species were higher in the pigs from farm C than in those from farm A, despite these spirochaetes being present on both farms; moreover, their pattern of increase, peaking at around 18 weeks on farm C, closely resembled the pattern seen with the B. hyodysenteriae
Although IgG antibody levels to the B. hyodysenteriae
WC preparation and to Bhlp29.7 both gave evidence of infection amongst the cohort on farm C, and could be used as tools to monitor infection, the WC preparation gave a better discrimination between the two cohorts at 18 weeks. An apparent problem with the Bhlp29.7 ELISA in this study was the significant temporal increase in antibody levels in the pigs from farm A, reducing the discrimination between results for the two sets of sera. This is paradoxical, given that the use of specific recombinant antigens like Bhlp29.7 should reduce potential cross-reactivity [5
]. However, the gene for Bhp29.7 has been identified in B. innocens
strain B256T 
, and the presence of such strains might generate false positive cross-reactivities and reduce the discriminatory power of this ELISA. Even more problematic is the recent report that the gene encoding Bhlp29.7 could not be amplified from 33 (45%) recent German isolates of B. hyodysenteriae
]. Although recently Lobova et al reported that Immunoblotting using Bhlp29.7 in conjunction with culturing method was a valuable tool for detecting swine herds latently infected with B. hyodysenteria
], the specificity of the Bhlp29.7 is still not clear. Hence further work is required to identify alternative antigenic surface exposed proteins specific to B. hyodysenteriae
that can be used in serological assays. Recombinant forms of such proteins are useful as plate-coating antigens as they are easier to prepare as a standard product than are WC preparations.
The change in IgM also demonstrated great magnitude in the experimental animals after exposure to B. hyodysenteriae. The performance of the IgM ELISA systems based on WC and Bhlp29.7 was then evaluated using the field samples in the cohort study. However, IgM levels to both antigens demonstrated a significant increase at 10 week on farm A, indicating that IgM is less specific than IgG in providing evidence of exposure to the spirochaete.
To further explore the suitability of ELISA systems using WC and Bhlp29.7 as herd tests, swine sera were collected from the herds with high health status and herds with a history of SD. A sample size of 50 animals within each herd was chosen to achieve an appropriate confidence of 95% of detecting an individual infected pig based on the previous study [9
]. Due to considerable variation in antibody response observed in the non-infected pigs, the value of three standard deviations above the mean was applied as diagnostic thresholds. Although the high stringent cut-off value (mean + 3 SD) might prioritize the test specificity and compromise the test sensitivity, the preliminary study in a clinical setting demonstrated that the assays based on both antigens could discriminate positive herds from negative herds. Certainly testing more herds is required to accurately estimate the assay performance with more reliable cut-off established from a large population of non-infected herds in the future.
Additionally meat juice has been reported to be a useful alternative to serum as a source of antibodies, and has been used in a number of sero-epidemiological studies and in surveillance for porcine infections [17
]. A comparative study on serum and meat juice samples from the same source of herds was undertaken using the ELISA systems described above. The assays based on meat juice achieved equivalent sensitivity and specificity as serum specimens (Table ). It was recognized that the current study could be strengthened by including more samples from negative and positive farms, but nevertheless it was clear from the results that meat juice from some pigs on the infected farms had elevated IgG and IgM levels to B. hyodysenteriae
. This finding supported our hypothesis that testing meat juice for specific antibodies could be used as a surveillance tool to help detect farms infected with B. hyodysenteriae
. The noteworthy, in both serum and meat juice tests, the IgG WC ELISA had 100% sensitivity and specificity as a herd test and gave a high detection rate. This finding further confirmed the suitability of using a IgG ELISA coated with B. hyodysenteriae
WC antigen as an aid in the diagnosis of infected herds.