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.