Discussion
In this work, we have compared the relative ability of Zebu and HF cattle to control BCG in vivo in an intranodal BCG challenge model (Villarreal-Ramos et al., 2014). We have also evaluated the immune responses of Zebu and HF cattle following vaccination and then intranodal challenge with BCG.
In terms of bacterial recovery, the data indicated that there was a strong trend, which almost reached statistical significance, for lower numbers of mycobacteria recovered from BCG-vaccinated HF cattle than from non-vaccinated HF cattle. The data indicated that the BCG-challenge model could, to a large extent, differentiate between HF vaccinated and not vaccinated groups. It is possible that environmental conditions, such as prior exposure to environmental mycobacteria (see discussion of IFNγ data below), may have conferred a degree of protection to all animals prior to BCG vaccination which may have reduced the expected difference in bacillary recovery between the vaccinated and unvaccinated HF calves and thus, the power of the BCG-challenge model to differentiate between vaccinated and non-vaccinated HF cattle. It is pertinent to state that under these conditions, increasing the number of experimental animals may have provided the necessary numbers with which to obtain statistically significant data. Given that this is the first time that the model has been carried out under these conditions, this is important data to bear in mind for future trials.
The data also indicated that there was no statistically significant difference in the number of mycobacteria recovered from vaccinated Zebu cattle compared to non-vaccinated Zebu cattle. No difference was observed between the number of mycobacteria recovered from non-vaccinated HF and non-vaccinated Zebu cattle or between vaccinated HF and vaccinated Zebu cattle. The current data would appear to suggest that HF cattle can be more effectively protected by BCG vaccination than Zebu cattle.
Although responses to PPD-B and PPD-A were low in all groups at week 0, peripheral blood responses to mycobacterial antigens after vaccination with BCG exhibited a bias towards PPD-A, rather than towards PPD-B in BCG vaccinated HF and Zebu cattle; these results would indicate that cattle had been and/or were concomitantly being exposed to environmental mycobacteria. Similar to our results, studies have shown that exposure of cattle to environmental mycobacteria prior to vaccination with BCG or infection with M. bovis has the effect of biasing the ensuing immune response towards PPD-A (Hope et al., 2005; Howard, Kwong, Villarreal-Ramos, Sopp, & Hope, 2002) (Coad, Clifford, Vordermeier, & Whelan, 2013; Jones, Whelan, Clifford, Coad, & Vordermeier, 2012),; it is possible that BCG vaccination boosted or synergistically increased immune responses to antigens shared with environmental mycobacteria.
In non-vaccinated animals, particularly non-vaccinated HF calves, responses to PPD-A increased at weeks 4 and 8 compared to week 0, which could be taken as confirmation that animals were responding to an on-going infection with environmental mycobacteria; however, whilst column medians were different, no statistically significant difference was detected between responses observed at week 0 with those observed at weeks 4 or 8 once corrections for multiple comparison were performed; nevertheless, the data indicated that these animals may also have been exposed to environmental mycobacteria and had therefore mounted an immune response to PPD-A. In the case of non-vaccinated Zebu cattle, as it would be expected with control animals, no significant responses to PPD-A or PPD-B were detected at weeks 4 and 8 compared to those observed at week 0.
Whilst it was not possible to observe differences in the responses to PPD-B induced by BCG vaccination or challenge, it is clear that the two breeds of cattle responded differently to PPD-A following inoculation with BCG. HF cattle showed higher IFNγ responses to PPD-A than Zebu cattle (supplementary figure 1); it has been shown that the magnitude of the response to mycobacteria is positively correlated to antigen load, which could be an indicator of mycobacterial load, in this case M. avium . Thus differences in the immune response to PPD-A could be an indication that Zebu cattle handle mycobacteria in different ways to the way HF cattle handle mycobacteria. A study aimed at determining the prevalence of M. avium infection in Uganda cattle, reported thatM. avium could be found in both breeds; however, despite examining almost twice as many Zebu cattle as HF cattle, the number of HF cattle determined as positive for M. avium was greater than the number of Zebu cattle (Okuni, Reinacher, Loukopoulos, & Ojok, 2013). These data suggest that indeed, HF cattle may be more susceptible to M. avium than Zebu cattle.
In conclusion, the observed immune responses appear to indicate that prior to, or after BCG vaccination, cattle had been exposed to environmental mycobacteria. Prior exposure to environmental mycobacteria may have conferred a degree of protection against subsequent mycobacterial exposure, which may have reduced the power of differentiating between vaccinated and non-vaccinated animals by the BCG-challenge model. Nevertheless, the data indicate that under conditions under which M. avium exposure is suspected, the BCG-challenge model could be a useful tool provided the number of animals per group being tested is increased. We have also established that the responses to mycobacteria induced by BCG vaccination are different, at least in terms of secretion of IFNγ by mycobacteria-stimulated peripheral blood cell between Zebu and HF cattle. To our knowledge, this is the first study that shows, in a statistically significant manner, that Zebu and HF cattle mount different responses to mycobacterial antigens.