BACKGROUND
caprine tuberculosis , caused either by mycobacterium bovis or m. caprae, and paratuberculosis , caused by m. avium subsp.
paratuberculosis , are endemic diseases in goat herds of the iberian peninsula  <cit> .
both infections may have an impact in terms of economic loss.
moreover, m. caprae and map can be transmitted between domestic hosts and wildlife species  <cit> .
in addition, m. caprae is a zoonotic agent  <cit>  and map has been associated with crohn’s disease  <cit> .
control of ptb in small ruminants can be facilitated by vaccination because the bacterial burden is greatly reduced, containing the spread of the disease and preventing clinical expression  <cit> .
furthermore, vaccination against ptb with map-killed formulations has been demonstrated to be an economically efficient strategy to achieve control of the disease in small ruminants  <cit>  and cattle  <cit> .
on the other hand, it has been shown that exposure to m. avium can interfere with the diagnosis of m. tuberculosis complex organisms such as m. bovis or m. caprae <cit> .
similarly, there are some reports showing that natural infection with map compromises tb diagnosis in cattle  <cit>  and goats  <cit> .
therefore, ptb could also affect the specificity of diagnostic tests used in tb control programs.
the spanish eradication program of bovine tb expressly forbids the use of map-based vaccines in cattle because of this potential interference with tb diagnostics.
in contrast, vaccination against ptb in goats is permitted with the exception of a minority of herds that are subjected to tb control in some regions.
to date, the potential effect of map-based vaccines on the diagnosis of tb has only been investigated post hoc under field conditions  <cit> .
it has been reported that tb diagnostic tests currently used in cattle, such as the skin test or the interferon-gamma  assay, can be used for diagnosis of tb in goats  <cit> .
moreover, in recent years, new ifn-γ assays based on antigens secreted by active growing bacilli, such as the peptide cocktail esat-6/cfp- <dig>  or rv3615c  <cit> , have been developed as alternatives to bovine and avian tuberculins.
these antigens are not present in either m. bovis bcg or map and can be viewed as novel diva  regents able to distinguish tb-infected from tb- or ptb-vaccinated animals.
the aim of the present work was to assess how vaccination against map affects standard and novel diagnostic tests in goats.
thus, we evaluated the interference of a commercial map-killed vaccine on tb diagnosis in goats vaccinated and subsequently challenged with m. caprae.
the objectives of the present work were:  to investigate the possible interference of vaccination before and after m. caprae infection on tb diagnostic tests ;  to assess the usefulness of diva-peptide candidates e/c and rv3615c; and  to evaluate immunological and post-mortem indicators of the effects of ptb vaccination on m. caprae infection.
RESULTS
assessment of diagnostic tests
the effect of map vaccination and subsequent infection with m. caprae on tb diagnostic assays based on cell-mediated immunity was assessed during this study.
four vaccinated goats were subjected to the tuberculin cervical comparative intradermal test  at week  <dig>  and all were negative for tb and correctly classified as avian reactors.
however, the four goats were classified as positive for tb if only the bovine tuberculin  result  was considered , which indicated that the specificity of the sit was severely compromised by ptb vaccination.
the skin test was repeated in all goats at week  <dig> .
in the vaccinated group,  <dig> of  <dig> goats were bovine reactors  using the cit , but all goats were classified as tb reactors by applying the sit interpretation.
all unvaccinated goats were positive for tb with both tests .
differences between groups in the mean specific thickness increase for each tuberculin were statistically significant only for Δppd-a , with a mean increase of  <dig>  mm  in the vaccinated group versus a mean increase of  <dig>  mm  in the control group  .
differences were also found in the mean value of Δppd-b minus Δppd-a.
this value was significantly lower in the vaccinated group  than in the unvaccinated group   .
test results are scored as  negative or  positive.
a Δav, increase in skin-fold thickness   <dig> h after ppd-a application.
b Δbov, increase in skin-fold thickness   <dig> h after ppd-b application.
c cit, cervical comparative intradermal test.
d sit, single intradermal test.
e avian reactor.
f *p <  <dig> ; **p <  <dig>  .
the tuberculin-based ifn-γ test was performed every  <dig> weeks throughout the experiment.
the kinetics of ifn-γ responses of the vaccinated group showed a ppd-a-biased response, which switched to a ppd-b-biased mean response  <dig> weeks after m. caprae infection  .
in the unvaccinated control group, a ppd-b-biased response was obtained  <dig> weeks after m. caprae infection, whereas no response was observed before this time point in any of the goats .
when the results were analyzed individually , map-vaccinated goats began showing responses to ppd-a  at week  <dig> after vaccination , and all of them subsequently became avian reactors between weeks  <dig> and  <dig>  however,  <dig> of  <dig> goats became a “bovine reactor”  at week  <dig> of the experiment , and this figure rose to  <dig> of  <dig> at week  <dig>  in the blood samples taken just before infection with m. caprae.
interestingly, in the next blood sampling at  <dig> wpi , again  <dig> of  <dig> goats showed a stronger reaction to ppd-a than to ppd-b, and the  <dig> other vaccinated goats were negative to both tuberculins.
by contrast, at week  <dig> ,  <dig> of  <dig>  of the vaccinated goats were bovine reactors.
at week  <dig>  all goats were positive to ppd-b , and these results were maintained during the rest of the trial with the exception of one goat that tested bovine-negative at week  <dig> and another that was an avian reactor at week  <dig> .
in the unvaccinated group, all goats were bovine reactors at week  <dig> and remained so until the end of the experiment, with the exception of two goats that were negative at week  <dig> but returned to positivity at weeks  <dig> and  <dig> .
test results are classified as: av, positive response to ppd-a; bov, positive response to ppd-b; or n, negative.
next, we assessed the interference of map vaccination with the ifn-γ assay by employing the defined antigen reagents e/c and rv3615c.
responses were assessed at the same five time points described in the materials and methods section.
at weeks  <dig>  and  <dig>  all goats were defined as tb-negative  according to the test results.
at weeks  <dig>   <dig>  and  <dig>  90%, 100%, and 70% of vaccinated goats were positive with the e/c cocktail and 100%, 90%, and 80% were positive with rv3615c, respectively .
however, these proportions increased to 100%, 100%, and 90% at weeks  <dig>   <dig>  and  <dig>  respectively, when the e/c and rv3615c results were considered together.
very similar results were obtained in the unvaccinated group, in which 100%, 80%, and 90% of goats were positive to e/c at weeks  <dig>   <dig>  and  <dig>  respectively; 100%, 80%, and 80% of goats were positive to rv3615c; and 100%, 80%, and 90% of goats were positive again when combining the results for both antigens at the same time points .
test results are classified as  negative or  positive.
seropositivity to map was assessed using the paratub.serum-s elisa kit  applying the cut-off defined above.
all goats  were negative at week  <dig>  unvaccinated goats also remained negative during the experiment .
in contrast,  <dig> of  <dig>  and  <dig> of  <dig>  vaccinated goats were positive or doubtful at weeks  <dig>  and  <dig> , respectively, with different degrees of intensity .
the mean intensity of antibody response  increased moderately, but was statistically significant , from 20%  at week  <dig> to 55%  at week  <dig> .
test results are classified as  negative,  positive, or  doubtful.
a two weeks after cit.
b s/p, seroreactivity rates.
pathology and bacteriology
at necropsy on week  <dig> , visible pathology typical of tb was observed in the lungs and pulmonary lymph nodes  of all animals irrespective of their vaccination status.
of the seven lung lobes evaluated, the vaccinated group presented an average of three lobes with gross lesions , slightly lower than the mean of four lobes  found in the unvaccinated group, but this difference was not statistically significant .
the mean log10-transformed volume of gross lesions in lungs was similar in the unvaccinated  and vaccinated  groups , but values were spread over a wider range in the vaccinated group.
by contrast, statistically significant differences between vaccinated and control goats were found after assessing the gross pathology in ln: the unvaccinated animals presented with a mean log10-transformed volume of visibly affected tissue of  <dig>  log <dig> mm <dig>  compared with  <dig>  log <dig> mm <dig>  obtained in the vaccinated group .
the individual pathological parameters are represented in figure 2a and b.
in addition, none of the vaccinated goats showed extrapulmonary gross lesions, whereas dissemination was observed in  <dig> of  <dig> unvaccinated goats that presented with gross lesions beyond the thoracic area .
these lesions were histopathologically confirmed to be tuberculous.
in three animals, focal lesions were also found in the ln draining the map vaccine inoculation point .
these lesions were attributed to the vaccine inoculation because of their localization and histological characteristics: lesions predominantly necrotizing rather than granulomatous, without langerhans cells, but surrounded by dispersed polymorphonuclear cells and abundant fibrosis.
finally, the mycobacterial load in ln was calculated as log <dig> cfu/g.
the bacterial burden in the pulmonary and retropharyngeal ln of all animals  ranged from  <dig> to  <dig> log <dig> cfu/g .
the total bacterial load in the unvaccinated group was  <dig>  log <dig> cfu/g , slightly higher than that in the vaccinated group .
however, this difference was not statistically significant .
cross-sectional analysis
to evaluate the vaccine effect in terms of heterologous protection, post-mortem parameters were also assessed in combination.
a mild positive correlation between bacteriological and pathological findings was found by correlating individual logarithmic values of bacterial load and total volume of visible lesions .
however, a higher dispersion of individual values was observed in the vaccinated group compared with the unvaccinated group , and a stronger positive correlation was found when only vaccinated goats were assessed , whereas results obtained from unvaccinated goats did not show significant correlations .
to further analyze the reductions in post-mortem parameters associated with vaccination, a cross-sectional study was performed on the basis of the criteria described in a previous tb vaccine efficacy trial  <cit> .
the vaccinated animals were divided into two subgroups according to the severity of the pathology and the bacterial burden.
this division was established by combining the data of the total volume of gross lesions and the bacterial load in ln.
the mean between the volume of lesions  and the bacterial load  was thereby calculated for each animal, and the cut-off point to classify the animals into categories of distinct vaccination outcomes was defined as the minimum value obtained among the unvaccinated control animals .
subsequently, a “vaccine responders”  subgroup of five goats and another “vaccine non-responders”  subgroup of five goats were defined .
once the subgroup division was applied as described in the previous paragraph, the assumed pathological and bacteriological differences between the new subgroups were checked.
the total volume of visible lesions and the bacterial load were significantly lower in vr compared with vnr and unvaccinated control  animals .
the release of ifn-γ specific to e/c, rv3615c, and ppd-b at week  <dig>  was also compared between the vr, vnr, and uc groups .
a significantly lower ifn-γ to e/c response was found in vr animals than in vnr and uc animals .
the relationship between e/c-specific ifn-γ response and disease severity was confirmed by finding a positive correlation of ifn-γ-specific secretion to e/c at week  <dig> with the total volume of gross lesions  and the bacterial load .
discussion and
CONCLUSIONS
the present experimental trial in goats was designed with the aim to determine the effects of map vaccination on current tb diagnostic tests and assess the effects of ptb vaccination on tb infection.
all experimental goats became infected irrespective of vaccination status.
however, a reduction in pulmonary pathology was observed in some vaccinated individuals compared with the unvaccinated group.
another remarkable finding was that all vaccinated goats showed only tb lesions at the site of infection  in contrast to the increased dissemination frequency in non-vaccinated animals.
this finding is analogous to the results of subcutaneous vaccination against ptb with the map-killed vaccine in cattle subsequently challenged with map.
the vaccine in those studies induced systemic immunity, preventing bacteremia and, consequently, dissemination of mycobacteria from the primary infection site.
however, they did not prevent the establishment of the initial infection  <cit> .
similar results were obtained in another experimental infection with m. caprae in which three of six  infected goats showed gross tb lesions in mesenteric ln  <cit> .
this outcome is also consistent with findings observed in field cases of tb in goats  <cit> .
in this sense, the present results indicate a certain degree of containment of dissemination of the infection from the primary complex, even when a significant reduction in the bacterial load in pulmonary drainage ln has not been observed.
nevertheless, this containment may not be sufficient to effectively prevent excretion of mycobacteria and horizontal transmission within a herd.
the role of the immunological status of vaccinated animals, especially the t-cell response, can be critical in terms of control or spread of the infection from the primary respiratory focus.
similarly, hope et al.
found few lesions at necropsy and smaller bacterial loads in ln in calves inoculated with m. avium and subsequently challenged with m. bovis <cit> .
the authors suggested that t-cell responses resulting from m. avium infection enhanced a protective secondary response after challenge with m. bovis.
after conducting cross-sectional analysis, we found positive correlations of the ifn-γ response to e/c with pathology severity, and more weakly with bacterial load.
a similar result was obtained with the single esat-6-specific ifn-γ response compared with pathology scores in goats  <cit>  and calves  <cit> .
furthermore, a positive correlation between the e/c-specific ifn-γ response and bacterial load was also previously described in goats  <cit>  and cattle  <cit> .
moreover, after analyzing post-mortem data, clear differences in the pathological and bacteriological results were found within the vaccinated group.
the ifn-γ responses of vaccinated goats with low and high post-mortem scores  were compared.
the significantly lower e/c-specific ifn-γ responses obtained in vr animals demonstrated the capacity of this immunological biomarker to predict the severity of the disease and, by default, to predict the vaccine outcome.
in terms of assessment of tb diagnostic tests, we have adapted the diagnostic tests routinely used in cattle in bovine tb eradication programs.
we have also introduced new diva reagents to perform the ifn-γ assay.
sensitivities obtained in all tests were higher than those obtained in previous studies in naturally infected goats  <cit> .
notwithstanding this, the enhanced sensitivity described in these works when combining skin tests and ifn-γ tests was also confirmed in the present experimental trial.
the sensitivities of skin tests performed in vaccinated animals , were higher than those obtained in another study carried out with goat herds naturally co-infected with tb and ptb   <cit> .
in this work, the authors reported many animals with false-negative cit results that showed higher skin-fold thickness to ppd-a than to ppd-b.
it is important to note that the sensitivity of cit in the present study was achieved using strict interpretation of the test.
with the standard interpretation , the sensitivity would be reduced to 60%.
by contrast, in our trial, only a slightly higher sensitivity was found when applying the sit in comparison with the cit.
however, the results showed serious compromise of the specificity of the sit in vaccinated animals prior to challenge , which completely disappeared when using the cit.
these results are in accordance with those found in another study performed in a ptb-vaccinated dairy goat herd free of tb  <cit> .
similarly, compromise of the specificity of the sit in ptb-vaccinated deer has also been reported  <cit> .
compared with the sit, the standard ifn-γ assay seemed to be more robust in terms of specificity.
false-positive results were concentrated at the interval between weeks  <dig> and  <dig>  and then disappeared at week  <dig> when there was not yet a response to m. caprae infection.
after  <dig> wpv, the ifn-γ responses to ppd-a decreased faster than did those to ppd-b .
as a consequence, 60% of the animals were false-positive “bovine reactors” at week  <dig>  more long-term trials must be performed to study the kinetics of the ifn-γ response to map vaccination.
encouragingly, the sensitivity obtained for the standard ifn-γ test after m. caprae challenge was very high considering that elevated whole-blood ifn-γ responses to ppd-a were previously observed in map-vaccinated cattle  <cit> .
in this sense, from weeks  <dig> to  <dig> , we only detected a masking due to ppd-a in two vaccinated animals.
however, the high ifn-γ responses observed in our study  may decrease with the progression of natural tb infection under field conditions.
in the last decade, much effort has been focused on the development of novel antigens for bovine tb diagnosis that are more sensitive and specific than the avian and bovine tuberculins.
this research has already resulted in the identification of several antigens, such as esat- <dig>  cfp- <dig>  and rv3615c, that reduce cross-reactive immune responses to different mycobacterial infections or vaccinations in cattle  <cit>  and goats  <cit> .
in the present study, we showed the capacity of both e/c and rv3615c to distinguish ptb-vaccinated and tb-infected goats.
furthermore, no differences in sensitivity were observed between the experimental groups.
importantly, when combining the positive results of e/c and rv3615c ifn-γ assays, the sensitivity was identical to that obtained by the tuberculin-based ifn-γ assay, which is currently being employed as an ancillary test to the skin test in some eradication campaigns.
a similar pattern was previously described in cattle experimentally infected with m. bovis, in which the sensitivity increased from  <dig> % to 91% when considering the results of rv3615c and e/c ifn-γ assay together  <cit> .
by contrast, serological responses to the map vaccine were moderate; only one and four vaccinated goats were seropositive to ptb at weeks  <dig> and  <dig>  respectively.
these data are not in agreement with the 50% positivity previously obtained in ptb-infected goats  <cit> .
interestingly, a boost effect on the map elisa due to cit might be found at week  <dig>  when comparing the results with the test performed at week  <dig>  the boost effect on the igg response due to the skin test was previously described in m. bovis-infected cattle  <cit>  and m. caprae-infected goats  <cit> .
vaccination against map represents an important advance in controlling ptb and improving the economic balance of affected farms.
therefore, the pros and cons of its application must be exhaustively evaluated.
an attractive speculation is that the partial protection to tb infection observed in some ptb-vaccinated animals could indirectly facilitate the control of tb, although long-term field studies are required to confirm this perspective.
moreover, we have demonstrated that to some degree, ptb vaccination interfered  with the sensitivity of tuberculin-based tb diagnostic tests.
however, beyond the first  <dig> weeks post-infection, only 5% false-negative results were obtained in the ifn-γ assay.
in addition, these animals reacted positively in more than 80% of the remaining post-challenge tests.
thus, considering the collective basis on which tb tests are usually applied, it is unlikely that an infected herd could be undiagnosed upon extrapolation to a larger population.
on the other hand, our results confirm that the interference with the specificity can be fully overcome by using defined diva reagents.
thus, developing and subsequently introducing these reagents into routine diagnostics could represent an improvement in both strategies: control of ptb by vaccination and control of tb by rapid detection of infected animals.

 caprine tuberculosis, caused either by mycobacterium bovis or m. caprae, and paratuberculosis, caused by m. avium subsp. paratuberculosis, are endemic diseases in goat herds of the iberian peninsula. both infections may have an impact in terms of economic loss. moreover, m. caprae and map can be transmitted between domestic hosts and wildlife species. in addition, m. caprae is a zoonotic agent and map has been associated with crohn’s disease. control of ptb in small ruminants can be facilitated by vaccination because the bacterial burden is greatly reduced, containing the spread of the disease and preventing clinical expression. furthermore, vaccination against ptb with map-killed formulations has been demonstrated to be an economically efficient strategy to achieve control of the disease in small ruminants and cattle. on the other hand, it has been shown that exposure to m. avium can interfere with the diagnosis of m. tuberculosis complex organisms such as m. bovis or m. caprae. similarly, there are some reports showing that natural infection with map compromises tb diagnosis in cattle and goats. therefore, ptb could also affect the specificity of diagnostic tests used in tb control programs. the spanish eradication program of bovine tb expressly forbids the use of map-based vaccines in cattle because of this potential interference with tb diagnostics. in contrast, vaccination against ptb in goats is permitted with the exception of a minority of herds that are subjected to tb control in some regions. to date, the potential effect of map-based vaccines on the diagnosis of tb has only been investigated post hoc under field conditions. it has been reported that tb diagnostic tests currently used in cattle, such as the skin test or the interferon-gamma assay, can be used for diagnosis of tb in goats. moreover, in recent years, new ifn-γ assays based on antigens secreted by active growing bacilli, such as the peptide cocktail esat-6/cfp- or rv3615c, have been developed as alternatives to bovine and avian tuberculins. these antigens are not present in either m. bovis bcg or map and can be viewed as novel diva regents able to distinguish tb-infected from tb- or ptb-vaccinated animals. the aim of the present work was to assess how vaccination against map affects standard and novel diagnostic tests in goats. thus, we evaluated the interference of a commercial map-killed vaccine on tb diagnosis in goats vaccinated and subsequently challenged with m. caprae. the objectives of the present work were: to investigate the possible interference of vaccination before and after m. caprae infection on tb diagnostic tests; to assess the usefulness of diva-peptide candidates e/c and rv3615c; and to evaluate immunological and post-mortem indicators of the effects of ptb vaccination on m. caprae infection.  assessment of diagnostic tests the effect of map vaccination and subsequent infection with m. caprae on tb diagnostic assays based on cell-mediated immunity was assessed during this study. four vaccinated goats were subjected to the tuberculin cervical comparative intradermal test at week and all were negative for tb and correctly classified as avian reactors. however, the four goats were classified as positive for tb if only the bovine tuberculin result was considered, which indicated that the specificity of the sit was severely compromised by ptb vaccination. the skin test was repeated in all goats at week. in the vaccinated group, of goats were bovine reactors using the cit, but all goats were classified as tb reactors by applying the sit interpretation. all unvaccinated goats were positive for tb with both tests. differences between groups in the mean specific thickness increase for each tuberculin were statistically significant only for Δppd-a, with a mean increase of mm in the vaccinated group versus a mean increase of mm in the control group. differences were also found in the mean value of Δppd-b minus Δppd-a. this value was significantly lower in the vaccinated group than in the unvaccinated group. test results are scored as negative or positive. a Δav, increase in skin-fold thickness h after ppd-a application. b Δbov, increase in skin-fold thickness h after ppd-b application. c cit, cervical comparative intradermal test. d sit, single intradermal test. e avian reactor. f *p <; **p <. the tuberculin-based ifn-γ test was performed every weeks throughout the experiment. the kinetics of ifn-γ responses of the vaccinated group showed a ppd-a-biased response, which switched to a ppd-b-biased mean response weeks after m. caprae infection. in the unvaccinated control group, a ppd-b-biased response was obtained weeks after m. caprae infection, whereas no response was observed before this time point in any of the goats. when the results were analyzed individually, map-vaccinated goats began showing responses to ppd-a at week after vaccination, and all of them subsequently became avian reactors between weeks and however, of goats became a “bovine reactor” at week of the experiment, and this figure rose to of at week in the blood samples taken just before infection with m. caprae. interestingly, in the next blood sampling at wpi, again of goats showed a stronger reaction to ppd-a than to ppd-b, and the other vaccinated goats were negative to both tuberculins. by contrast, at week, of of the vaccinated goats were bovine reactors. at week all goats were positive to ppd-b, and these results were maintained during the rest of the trial with the exception of one goat that tested bovine-negative at week and another that was an avian reactor at week. in the unvaccinated group, all goats were bovine reactors at week and remained so until the end of the experiment, with the exception of two goats that were negative at week but returned to positivity at weeks and. test results are classified as: av, positive response to ppd-a; bov, positive response to ppd-b; or n, negative. next, we assessed the interference of map vaccination with the ifn-γ assay by employing the defined antigen reagents e/c and rv3615c. responses were assessed at the same five time points described in the materials and methods section. at weeks and all goats were defined as tb-negative according to the test results. at weeks and 90%, 100%, and 70% of vaccinated goats were positive with the e/c cocktail and 100%, 90%, and 80% were positive with rv3615c, respectively. however, these proportions increased to 100%, 100%, and 90% at weeks and respectively, when the e/c and rv3615c results were considered together. very similar results were obtained in the unvaccinated group, in which 100%, 80%, and 90% of goats were positive to e/c at weeks and respectively; 100%, 80%, and 80% of goats were positive to rv3615c; and 100%, 80%, and 90% of goats were positive again when combining the results for both antigens at the same time points. test results are classified as negative or positive. seropositivity to map was assessed using the paratub.serum-s elisa kit applying the cut-off defined above. all goats were negative at week unvaccinated goats also remained negative during the experiment. in contrast, of and of vaccinated goats were positive or doubtful at weeks and, respectively, with different degrees of intensity. the mean intensity of antibody response increased moderately, but was statistically significant, from 20% at week to 55% at week. test results are classified as negative, positive, or doubtful. a two weeks after cit. b s/p, seroreactivity rates. pathology and bacteriology at necropsy on week, visible pathology typical of tb was observed in the lungs and pulmonary lymph nodes of all animals irrespective of their vaccination status. of the seven lung lobes evaluated, the vaccinated group presented an average of three lobes with gross lesions, slightly lower than the mean of four lobes found in the unvaccinated group, but this difference was not statistically significant. the mean log10-transformed volume of gross lesions in lungs was similar in the unvaccinated and vaccinated groups, but values were spread over a wider range in the vaccinated group. by contrast, statistically significant differences between vaccinated and control goats were found after assessing the gross pathology in ln: the unvaccinated animals presented with a mean log10-transformed volume of visibly affected tissue of log mm compared with log mm obtained in the vaccinated group. the individual pathological parameters are represented in figure 2a and b. in addition, none of the vaccinated goats showed extrapulmonary gross lesions, whereas dissemination was observed in of unvaccinated goats that presented with gross lesions beyond the thoracic area. these lesions were histopathologically confirmed to be tuberculous. in three animals, focal lesions were also found in the ln draining the map vaccine inoculation point. these lesions were attributed to the vaccine inoculation because of their localization and histological characteristics: lesions predominantly necrotizing rather than granulomatous, without langerhans cells, but surrounded by dispersed polymorphonuclear cells and abundant fibrosis. finally, the mycobacterial load in ln was calculated as log cfu/g. the bacterial burden in the pulmonary and retropharyngeal ln of all animals ranged from to log cfu/g. the total bacterial load in the unvaccinated group was log cfu/g, slightly higher than that in the vaccinated group. however, this difference was not statistically significant. cross-sectional analysis to evaluate the vaccine effect in terms of heterologous protection, post-mortem parameters were also assessed in combination. a mild positive correlation between bacteriological and pathological findings was found by correlating individual logarithmic values of bacterial load and total volume of visible lesions. however, a higher dispersion of individual values was observed in the vaccinated group compared with the unvaccinated group, and a stronger positive correlation was found when only vaccinated goats were assessed, whereas results obtained from unvaccinated goats did not show significant correlations. to further analyze the reductions in post-mortem parameters associated with vaccination, a cross-sectional study was performed on the basis of the criteria described in a previous tb vaccine efficacy trial. the vaccinated animals were divided into two subgroups according to the severity of the pathology and the bacterial burden. this division was established by combining the data of the total volume of gross lesions and the bacterial load in ln. the mean between the volume of lesions and the bacterial load was thereby calculated for each animal, and the cut-off point to classify the animals into categories of distinct vaccination outcomes was defined as the minimum value obtained among the unvaccinated control animals. subsequently, a “vaccine responders” subgroup of five goats and another “vaccine non-responders” subgroup of five goats were defined. once the subgroup division was applied as described in the previous paragraph, the assumed pathological and bacteriological differences between the new subgroups were checked. the total volume of visible lesions and the bacterial load were significantly lower in vr compared with vnr and unvaccinated control animals. the release of ifn-γ specific to e/c, rv3615c, and ppd-b at week was also compared between the vr, vnr, and uc groups. a significantly lower ifn-γ to e/c response was found in vr animals than in vnr and uc animals. the relationship between e/c-specific ifn-γ response and disease severity was confirmed by finding a positive correlation of ifn-γ-specific secretion to e/c at week with the total volume of gross lesions and the bacterial load. discussion and  the present experimental trial in goats was designed with the aim to determine the effects of map vaccination on current tb diagnostic tests and assess the effects of ptb vaccination on tb infection. all experimental goats became infected irrespective of vaccination status. however, a reduction in pulmonary pathology was observed in some vaccinated individuals compared with the unvaccinated group. another remarkable finding was that all vaccinated goats showed only tb lesions at the site of infection in contrast to the increased dissemination frequency in non-vaccinated animals. this finding is analogous to the results of subcutaneous vaccination against ptb with the map-killed vaccine in cattle subsequently challenged with map. the vaccine in those studies induced systemic immunity, preventing bacteremia and, consequently, dissemination of mycobacteria from the primary infection site. however, they did not prevent the establishment of the initial infection. similar results were obtained in another experimental infection with m. caprae in which three of six infected goats showed gross tb lesions in mesenteric ln. this outcome is also consistent with findings observed in field cases of tb in goats. in this sense, the present results indicate a certain degree of containment of dissemination of the infection from the primary complex, even when a significant reduction in the bacterial load in pulmonary drainage ln has not been observed. nevertheless, this containment may not be sufficient to effectively prevent excretion of mycobacteria and horizontal transmission within a herd. the role of the immunological status of vaccinated animals, especially the t-cell response, can be critical in terms of control or spread of the infection from the primary respiratory focus. similarly, hope et al. found few lesions at necropsy and smaller bacterial loads in ln in calves inoculated with m. avium and subsequently challenged with m. bovis. the authors suggested that t-cell responses resulting from m. avium infection enhanced a protective secondary response after challenge with m. bovis. after conducting cross-sectional analysis, we found positive correlations of the ifn-γ response to e/c with pathology severity, and more weakly with bacterial load. a similar result was obtained with the single esat-6-specific ifn-γ response compared with pathology scores in goats and calves. furthermore, a positive correlation between the e/c-specific ifn-γ response and bacterial load was also previously described in goats and cattle. moreover, after analyzing post-mortem data, clear differences in the pathological and bacteriological results were found within the vaccinated group. the ifn-γ responses of vaccinated goats with low and high post-mortem scores were compared. the significantly lower e/c-specific ifn-γ responses obtained in vr animals demonstrated the capacity of this immunological biomarker to predict the severity of the disease and, by default, to predict the vaccine outcome. in terms of assessment of tb diagnostic tests, we have adapted the diagnostic tests routinely used in cattle in bovine tb eradication programs. we have also introduced new diva reagents to perform the ifn-γ assay. sensitivities obtained in all tests were higher than those obtained in previous studies in naturally infected goats. notwithstanding this, the enhanced sensitivity described in these works when combining skin tests and ifn-γ tests was also confirmed in the present experimental trial. the sensitivities of skin tests performed in vaccinated animals, were higher than those obtained in another study carried out with goat herds naturally co-infected with tb and ptb. in this work, the authors reported many animals with false-negative cit results that showed higher skin-fold thickness to ppd-a than to ppd-b. it is important to note that the sensitivity of cit in the present study was achieved using strict interpretation of the test. with the standard interpretation, the sensitivity would be reduced to 60%. by contrast, in our trial, only a slightly higher sensitivity was found when applying the sit in comparison with the cit. however, the results showed serious compromise of the specificity of the sit in vaccinated animals prior to challenge, which completely disappeared when using the cit. these results are in accordance with those found in another study performed in a ptb-vaccinated dairy goat herd free of tb. similarly, compromise of the specificity of the sit in ptb-vaccinated deer has also been reported. compared with the sit, the standard ifn-γ assay seemed to be more robust in terms of specificity. false-positive results were concentrated at the interval between weeks and and then disappeared at week when there was not yet a response to m. caprae infection. after wpv, the ifn-γ responses to ppd-a decreased faster than did those to ppd-b. as a consequence, 60% of the animals were false-positive “bovine reactors” at week more long-term trials must be performed to study the kinetics of the ifn-γ response to map vaccination. encouragingly, the sensitivity obtained for the standard ifn-γ test after m. caprae challenge was very high considering that elevated whole-blood ifn-γ responses to ppd-a were previously observed in map-vaccinated cattle. in this sense, from weeks to, we only detected a masking due to ppd-a in two vaccinated animals. however, the high ifn-γ responses observed in our study may decrease with the progression of natural tb infection under field conditions. in the last decade, much effort has been focused on the development of novel antigens for bovine tb diagnosis that are more sensitive and specific than the avian and bovine tuberculins. this research has already resulted in the identification of several antigens, such as esat- cfp- and rv3615c, that reduce cross-reactive immune responses to different mycobacterial infections or vaccinations in cattle and goats. in the present study, we showed the capacity of both e/c and rv3615c to distinguish ptb-vaccinated and tb-infected goats. furthermore, no differences in sensitivity were observed between the experimental groups. importantly, when combining the positive results of e/c and rv3615c ifn-γ assays, the sensitivity was identical to that obtained by the tuberculin-based ifn-γ assay, which is currently being employed as an ancillary test to the skin test in some eradication campaigns. a similar pattern was previously described in cattle experimentally infected with m. bovis, in which the sensitivity increased from % to 91% when considering the results of rv3615c and e/c ifn-γ assay together. by contrast, serological responses to the map vaccine were moderate; only one and four vaccinated goats were seropositive to ptb at weeks and respectively. these data are not in agreement with the 50% positivity previously obtained in ptb-infected goats. interestingly, a boost effect on the map elisa due to cit might be found at week when comparing the results with the test performed at week the boost effect on the igg response due to the skin test was previously described in m. bovis-infected cattle and m. caprae-infected goats. vaccination against map represents an important advance in controlling ptb and improving the economic balance of affected farms. therefore, the pros and cons of its application must be exhaustively evaluated. an attractive speculation is that the partial protection to tb infection observed in some ptb-vaccinated animals could indirectly facilitate the control of tb, although long-term field studies are required to confirm this perspective. moreover, we have demonstrated that to some degree, ptb vaccination interfered with the sensitivity of tuberculin-based tb diagnostic tests. however, beyond the first weeks post-infection, only 5% false-negative results were obtained in the ifn-γ assay. in addition, these animals reacted positively in more than 80% of the remaining post-challenge tests. thus, considering the collective basis on which tb tests are usually applied, it is unlikely that an infected herd could be undiagnosed upon extrapolation to a larger population. on the other hand, our results confirm that the interference with the specificity can be fully overcome by using defined diva reagents. thus, developing and subsequently introducing these reagents into routine diagnostics could represent an improvement in both strategies: control of ptb by vaccination and control of tb by rapid detection of infected animals. 
ninety percent of the animals reacted positively to the tuberculin cervical comparative intradermal test performed at weeks post-infection. although 60% of the animals were bovine reactors at week avian ppd-biased responses returned at week after challenge with m. caprae, the ifn-γ responses radically changed to show predominant bovine ppd-biased responses from week onward. evaluation of interferon-γ secretion induced by avian and bovine tuberculins showed a predominant avian ppd-biased response in the vaccinated group from week post-vaccination onward. our results suggest a degree of interference of ptb vaccination with current tb diagnostics that can be fully mitigated when using new diva reagents. furthermore, post-mortem analysis showed reductions in tuberculous lesions and bacterial burden in some vaccinated animals, particularly expressed in terms of the degree of extrapulmonary dissemination of tb infection. however, vaccination against paratuberculosis in goats is often allowed even when its effect on tb diagnosis has not been fully evaluated.
 most countries carrying out campaigns of bovine tuberculosis eradication impose a ban on the use of mycobacterial vaccines in cattle. however, vaccination against paratuberculosis in goats is often allowed even when its effect on tb diagnosis has not been fully evaluated. to address this issue, goat kids previously vaccinated against ptb were experimentally infected with tb.  evaluation of interferon-γ secretion induced by avian and bovine tuberculins showed a predominant avian ppd-biased response in the vaccinated group from week post-vaccination onward. although 60% of the animals were bovine reactors at week avian ppd-biased responses returned at week after challenge with m. caprae, the ifn-γ responses radically changed to show predominant bovine ppd-biased responses from week onward. in addition, cross-reactions with bovine ppd that had been observed in the vaccinated group at week were reduced when using the m. tuberculosis complex-specific antigens esat-6/cfp- and rv3615c as new diva reagents, which further maintained sensitivity post-challenge. ninety percent of the animals reacted positively to the tuberculin cervical comparative intradermal test performed at weeks post-infection. furthermore, post-mortem analysis showed reductions in tuberculous lesions and bacterial burden in some vaccinated animals, particularly expressed in terms of the degree of extrapulmonary dissemination of tb infection.  our results suggest a degree of interference of ptb vaccination with current tb diagnostics that can be fully mitigated when using new diva reagents. a partial protective effect associated with vaccination was also observed in some vaccinated animals. tuberculosisparatuberculosisgoatvaccinediagnosticinterferon gamma 