Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates

RESEARCH ARTICLE Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates E. M. D. L. van der Heijden 1,2 *, J. Chileshe 2 , J. C. M. Vernooij 3 , C. Gortazar 4 , R. A. Juste 5,6 , I. Sevilla 5 , J. E. Crafford 2 , V. P. M. G. Rutten 1,2 , A. L. Michel 2,7 1Department of Infectious Diseases & Immunology, Division of Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, 2Bovine Tuberculosis and Brucellosis Research Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa, 3Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, 4SaBio Instituto de Investigacio ´n en Recursos Cinege ´ticos IREC (CSIC-UCLMJCCM), Ciudad Real, Spain, 5NEIKER-Instituto Vasco de Investigacio ´n y Desarrollo Agrario, Animal Health Department, Bizkaia Science and Technology Park, Derio (Bizkaia), Spain, 6SERIDA, Villaviciosa, Asturias, Spain, 7National Zoological Gardens of South Africa, Pretoria, South Africa *e.m.d.l.vanderheijd[email protected] Abstract Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlifelivestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4–6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M.bovis BCG (Danish 1331), formalin-inactivated M.bovis BCG, heat-killed M.bovis or PBS/Montanide™(control). Interferonγresponsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M.bovis BCG. The animals in the heat-killed M.bovis group demonstrated strong and sustained cellmediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heatkilled M.bovis group. In conclusion, the parenteral heat-killed M.bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M.bovis and assessment of vaccine efficacy in field conditions. PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 1 / 20 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: van der Heijden EMDL, Chileshe J, Vernooij JCM, Gortazar C, Juste RA, Sevilla I, et al. (2017) Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates. PLoS ONE 12(11): e0188448. https://doi.org/10.1371/ journal.pone.0188448 Editor: Ann Rawkins, Public Health England, UNITED KINGDOM Received: July 11, 2017 Accepted: November 7, 2017 Published: November 20, 2017 Copyright: ©2017 van der Heijden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by NWOWOTRO Science for Global Development (grant W01.65.321.00) to VR & AL (https://www.nwo.nl/ en/about-nwo/organisation/nwo-domains/wotro), and NWO-WOTRO funding was used towards all experimental costs, including purchase of animals, consumables, test kits etc.; The Institutional Introduction Control of bovine tuberculosis (BTB), caused by Mycobacterium bovis (M.bovis), is urgently needed on a global scale. The detrimental effects on the cattle industry worldwide as well as on wildlife conservation [1] are noteworthy, with global losses of approximately $3 billion annually [2], and BTB is furthermore of great public health concern. Although “test-and-slaughter” is the conventional control strategy that allowed eradication of the infection in many developed countries, it has proven less effective or unaffordable in other countries facing a multitude of constraints [3,4], and vaccination of cattle and other species is being considered as a possible alternative approach to BTB control. To date, the only available vaccine is that produced with the Bacille Calmette-Gue ´rin (BCG) strain, which originated from virulent M.bovis through attenuation and was first used in humans in 1921 [5,6]. Live BCG is currently registered for use in humans and badgers only [7]. Its use in cattle is prohibited in the EU [8] due to induction of immune responsiveness that interferes with the standard diagnostic methods for BTB utilizing tuberculins and in addition because of the widely variable degree of protection it provides. Hence research efforts have focused on the development of diagnostic reagents that can differentiate between infected and vaccinated animals (DIVA) and on potentiation of the protective effect of BCG [8]. Different strategies using live BCG as either the priming or boosting vaccine in combination with a viral vector [9], recombinant DNA or sub-unit vaccine formulations [10] incorporating various Mycobacterium tuberculosis complex (MTBC) specific antigens, have been explored, with conflicting results. Skinner et al. [11] found that the use of a DNA primeBCG boost regimen using plasmids encoding Hsp65, Hsp70 and Apa was able to significantly improve protection against BTB compared to BCG alone. Likewise, a study by Vordermeier et al. [9], showed that the efficacy of BCG seemed to improve after boosting with a formulation of recombinant attenuated adenovirus expressing antigen 85A. However, vaccination with MPB70 or MPB83 DNA plasmids was not found to be protective in cattle [12]. The rise of the HIV/AIDS pandemic prompted a renewed interest in killed vaccine candidates for protection against tuberculosis [13], as immunocompromised individuals are at risk of developing disseminated disease (BCG-osis) after vaccination with live BCG [10]. This is of special importance in southern Africa, where HIV prevalence is amongst the highest in the world [14]. In these regions, a complex wildlife-livestock-human interface furthermore increases the risk of zoonotic transmission of infectious agents [15]. The use of an inactivated rather than a live vaccine against BTB in cattle, would eliminate the risk of potential propagation of the vaccine strain in food producing animals, a concern raised by the Department of Agriculture, Forestry and Fisheries (DAFF) in South Africa (DAFF, personal communication, November 2014). Vaccination with formalin-inactivated BCG in a Novasome™adjuvant conferred protection against tuberculosis in guinea pigs [13] and subsequent evaluation in cattle by Whelan et al. [16] demonstrated strong IFN-γand IgG responses. Promising results of parenteral and oral vaccination with a heat-killed Mycobacterium bovis vaccine have since been obtained in several species [17–19]. The aim of the present study was to compare the immune response profiles in response to the parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Cell-mediated and humoral immune response profiles resulting from vaccination, skin testing and BCG challenge were monitored over time and compared to a control group. Materials & methods Animals This study was carried out in strict accordance with the guidelines of the Animal Use and Care Committee of the University of Pretoria and the protocol was approved (Certificate number V066-15) prior to commencement of the study. Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 2 / 20 Research Theme of the University of Pretoria on Animal Zoonotic Disease and the Third Framework Agreement Programme (FA3-III) through the Institute of Tropical Medicine, Antwerp, Belgium to VR & AL (https://www.itg.be), and IRT funding was used towards all experimental costs, including purchase of animals, consumables, test kits etc.; MINECO and FEDER Plan Nacional (grant AGL2014-56305) to CG (http://www.idi.mineco. gob.es/portal/site/MICINN/menuitem.26172fcf 4eb029fa6ec7da6901432ea0/?vgnextoid=4d 494d861a299210VgnVCM1000001d04140a RCRD), and MINECO & FEDER funding was used towards coordination among IREC members; and the Erasmus Mundus Action 2 EUROSA scholarship to EvdH (https://www.uantwerpen.be/ en/projects/eurosa/), and EUROSA funding was used towards the stipendium of EvdH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Twenty-four mixed breed Bos taurus calves (4–6 months of age; 12 males and 12 females) from a local beef herd with a known BTB free history were used in this study. Exclusion criteria included prior infection with M.bovis as determined by the bovine IFN-γrelease assay (BOVIGAM 1 ) and serological testing (IDEXX TB ELISA). All animals were subjected to a full clinical examination, received prophylactic treatment to prevent parasitic and bacterial infections and were allowed to acclimatize to their new environment at the class II biological containment holding facilities of the University of Pretoria Biomedical Research Center (UPBRC) for a period of 6 weeks. The facilities comprised of individual stalls (physical contact between animals in adjacent stalls was not possible) that held the animals of the same vaccine group in pairs, in a spacious, closed and well ventilated cattle holding facility. In view of maturation of the animals in course of the experiment the animals were grouped in pairs based on their sex and each pair was randomly allocated to a pen. Each pen was assigned to one of the treatment groups beforehand. Animals of the four different groups were kept separated. Experimental timeline The experimental timeline for the study is depicted in Fig 1. Vaccination Calves were assigned to one of four groups (n = 6 per group). Groups 1–3 received different vaccines and group 4 served as an unvaccinated control group. In groups 2–4, the adjuvant Montanide™ISA 50 V 2 (SEPPIC, France) was used; a water-in-oil adjuvant, compatible with inactivated antigens, recommended for use in cattle. While Montanide™stimulates both humoral and cell-mediated immunity, it induces a milder local reaction as compared to other adjuvants (unpublished data). Fig 1. Experimental timeline for the study. Baseline values were established from T-4 until T0 with a sampling interval of 2–3 weeks. After vaccination (T0) until the end of the experiment (T12), the sampling interval was 1 week. Animals were skin tested at T9, challenged with BCG at T9 + 3 days and euthanized at T12. T(x) = time point (week number). https://doi.org/10.1371/journal.pone.0188448.g001 Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 3 / 20 Live M.bovisBacille Calmette-Gue ´rin (Group 1). Lyophilized live M.bovis BCG Danish 1331 (Statens Serum Institute, Denmark) was reconstituted with the diluent provided by the manufacturer and made up to a concentration of approximately 2 x 10 6 CFU per ml. One milliliter was administered subcutaneously in the left mid cervical area to all animals in group 1 at T0. Formalin-inactivated M.bovisBacille Calmette-Gue ´rin (Group 2). The formalin-inactivated M.bovis BCG vaccine was prepared as previously described [13], with a 50% reduction in the formalin treatment time. Briefly, reconstituted live M.bovis BCG Danish 1331 were suspended in 1.5% formalin (v/v) in phosphate buffered saline (PBS) (Sigma-Aldrich, South Africa). The suspension was stirred continuously at 4˚C for 48h and subsequently centrifuged at 14,000 x gfor 15 minutes at 4˚C. The cells were then washed twice, resuspended in sterile PBS to a concentration of 4 x 10 7 cells/ml and stored at 4˚C until use (no longer than three days). Successful inactivation of the BCG was demonstrated by inoculation of aliquots of the vaccine on Lo¨wenstein-Jensen (LJ) slants enriched with pyruvate, followed by incubation for 10 weeks. No growth was observed. The vaccine consisted of formalin-inactivated BCG and Montanide™ISA 50 V 2 (SEPPIC, France) adjuvant carefully emulsified and made up to a concentration of 2 x 10 7 cells/ml. One milliliter was administered subcutaneously in the left mid cervical area to all animals in group 2 at T0 and again at T3 (booster). Heat-killed M.bovis(Group 3). The heat-killed M.bovis vaccine was prepared as previously described [17]. The vaccine consisted of heat-inactivated M.bovis (Neiker strain) and Montanide™ISA 50 V 2 (SEPPIC, France) adjuvant carefully emulsified and made up to a concentration of 1 x 10 7 CFU/ml. One milliliter was administered subcutaneously in the left mid cervical area to all animals in group 3 at T0 and again at T3 (booster). Control group inoculum (Group 4). Phosphate buffered saline (Sigma-Aldrich, South Africa) and 50% Montanide™ISA 50 V 2 (SEPPIC, France) adjuvant (v/v) were carefully emulsified to serve as an inoculum for the control group. One milliliter was administered subcutaneously in the left mid cervical area to all animals in group 4 at T0. Sample collection Whole blood was collected from the jugular vein using a vacutainer system at several time points prior to vaccination for the BOVIGAM 1 assay and IDEXX TB ELISA and outcomes were averaged in order to establish baseline values. Thereafter whole blood samples were collected every week for a total of twelve weeks (Fig 1). Bovine IFN-γrelease assay Heparinized whole blood samples from all calves were individually processed within 2–3 hrs after collection. Antigen stimulations for the BOVIGAM 1 assay were carried out in 48-well cell culture plates (Cellstar 1 Greiner Bio One, Germany) as previously described [20]. At each time point, undiluted heparinized blood was aliquoted into 1 ml per well and stimulated with pokeweed mitogen (PWM; 5 μg/ml) as a positive sample control, PPD-B (purified protein derivative of M.bovis; 600 IU/ml), PPD-A (purified protein derivative of M.avium; 1000 IU/ ml) and PPD-F (purified protein derivative of M.fortuitum; 28.5 μg/ml, ARC-Onderstepoort Veterinary Institute) [19]. One aliquot of whole blood was left unstimulated to serve as a negative control. At three time points (T0, T3 and T9), additional aliquots of whole blood were stimulated with the recombinant mycobacterial proteins ESAT-6 (5 μg/ml) and CFP-10 (5 μg/ ml) (LIONEX GmbH, Germany). The samples were incubated at 37˚C for 20 hrs, after which supernatants were harvested. Interferon-γdetection was carried out according to the manufacturer’s protocol (Thermo Fisher Scientific, South Africa). Criteria for sample validity were an Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 4 / 20 optical density value (OD) 0.45 for PWM and OD 0.35 for the negative control (OD neg ). Responses elicited by TB antigens were corrected by subtracting the OD-value of the negative control (OD bov minus OD neg , OD av minus OD neg , OD fort minus OD neg , OD ESAT6 minus OD neg and OD CFP10 minus OD neg ). Serology Blood samples were collected without anticoagulants and left to clot overnight at ambient temperature. Sera were harvested the next day and subsequently tested for the presence of M.bovis specific antibodies with the IDEXX TB ELISA, using a 1:50 dilution of the samples and controls, according to the manufacturer’s protocol (IDEXX, USA). Criteria for the test validity were an OD 0.3 for the positive control and OD 0.2 for the negative control. Sample/positive control (S/P) ratios were calculated according to the manufacturer’s protocol. Skin test At T9, the skin test was performed according to OIE [21] recommendations, with the addition of two protein cocktails alongside the standard tuberculins. Briefly, hair was clipped at 4 sites on the left mid-cervical region that were injected intradermally with 0.1 ml of PPD-B (30,000 IU/ml), PPD-A (25,000 IU/ml), protein cocktail 1 (PC1; containing 10 μg/ml of ESAT-6, CFP10 and Rv3615c each) and protein cocktail 2 (PC2; containing 10 μg/ml of ESAT-6, CFP-10, Rv3615c and Rv3020c each) [22], respectively. After 72 hrs the injection sites were inspected and palpated for signs of a delayed-type hypersensitivity reaction and the skin fold thickness measured to calculate the difference (Δmm) between pre-injection (0hrs) and post-injection (72hrs) measurements. Differences in increase of skin fold thickness of 4 mm between the bovine and avian injection sites and/or the presence of typical clinical signs (necrosis, edema, heat, pain) in combination with a lower increase were considered positive skin test reactions; a difference of 2–4 mm between bovine and avian injection sites was considered a suspect result; while a difference of 0 to 2 mm between bovine and avian injection sites was considered a negative skin test. The reactions to the protein cocktails were interpreted according to Jones et al. [22]; an increase in skin fold thickness of 1 mm was considered positive. Intranodular BCG challenge As an alternative challenge approach, the animals were inoculated in the right prescapular lymph node with live M.bovis BCG Danish 1331 at T9 + 3 days (after reading of the skin test). This approach has previously been shown to be a viable alternative to challenge with pathogenic M. bovis without the need for biosafety level 3 facilities [23] to serve as a preliminary evaluation of protection. Briefly, the lyophilized live M.bovis BCG Danish 1331 vaccine strain was reconstituted in the diluent provided by the manufacturer (Statens Serum Institute, Denmark), inoculated on LJ slants containing pyruvate and incubated for 4 weeks at 37˚C. A challenge inoculum was prepared through suspension of the fresh mycobacterial culture in PBS to a concentration of 2 x 10 8 CFU/ml and 1 ml was injected. At T12 the animals were euthanized by means of a captive bolt and the left (control) and right (inoculated) prescapular lymph nodes were harvested. Mycobacterial culture The weights of the prescapular lymph nodes, collected at T12, were measured to assess inflammation and cellular congestion. Each sample was weighed and inspected for the presence of lesions. In the absence of lesions, a representative sample of ±2 g from the center of each lymph node (the site of BCG inoculation) was collected aseptically for culture. The samples Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 5 / 20 were homogenized in a final volume of 7 ml sterile distilled water and decontaminated with an equal volume of 2% hydrogen chloride (HCl) during 10 minutes. The samples were then centrifuged at 2550 x gfor 10 minutes and the supernatant discarded. Subsequently the samples were resuspended in 7 ml of sterile distilled water and centrifuged at 2550 x gfor 10 minutes, in order to remove remaining HCl. The supernatant was discarded and the pellet resuspended in 3 ml of distilled water and inoculated onto LJ slants containing pyruvate and incubated at 37˚C for 10 weeks. Bacterial counts were determined as CFU per gram of lymph node. Confirmation of mycobacterial growth as being M.bovis BCG was done using the polymerase chain reaction (PCR) targeting the regions of difference (RD) RD1, RD4 and RD9 as previously described [24]. Data analysis Statistical analyses of the data gathered in this study were conducted in R version 3.3.0 [25], as described below. Bovine IFN-γrelease assay. A linear mixed effects model [26] was used to analyze the results of the BOVIGAM 1 assay in the different groups as compared to the control group. For analysis of PPD-B, PPD-B/PPD-A, PPD-B/PPD-F, ESAT-6 and CFP-10, data were log transformed, after adding 0.5, 0.5, 0.75, 0.05 and 0.05 to each value of the outcome variable (to achieve positive values), respectively, to meet the model assumptions of normality and homoscedasticity. Explanatory variables were time, vaccination group and the interaction between both, which proved to be the final model. A variance function was added to the models of PPD-B, ESAT-6 and CFP-10 to allow for different standard deviations in the vaccination groups. Correlated observations within animals were accounted for by using a random intercept and slope for the animal ID. The Akaike Information Criterion (AIC) was used to select the best model. Serology. A linear mixed effects model [26] was used to analyze the results of the IDEXX TB ELISA in the different groups as compared to the control group. For analysis of the S/P ratio data were log transformed, after adding 0.1 to each value of the outcome variable (to achieve positive values), to meet the model assumptions of normality and homoscedasticity. The other criteria and parameters of the model were the same as for the model of PPD-B. Skin test. In order to account for the heteroscedasticity of the variances, a double generalized linear model [27] was used to analyze the skin reactions to the tuberculins (ΔPPD-B minus ΔPPD-A in mm). A simple general linear model was used to analyze the skin reactions to the protein cocktails (ΔPC1 and ΔPC2 in mm). The explanatory variable was vaccination group. Intranodular BCG challenge. Prescapular lymph node weights in the treatment groups were compared to the control group using a linear mixed effects model [26]. The outcome variable was log transformed to meet the model assumptions of normality and homoscedasticity. Explanatory variables were lymph node side, vaccination group, the interaction between both and gender which proved to be the final model. Correlated observations within animals were accounted for by using a random intercept and slope for the animal ID. A negative binomial generalized linear model was used to compare the bacterial counts from the right prescapular lymph nodes of the treatment groups to the control group. The explanatory variable was vaccination group. Results Bovine IFN-γrelease assay Cell-mediated immunity (CMI) as a consequence of vaccination with the different vaccines was monitored by means of IFN-γresponses using the BOVIGAM 1 assay for nine weeks Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 6 / 20 (T1-T9) in all animals and compared to the control group. Interferon-γresponses following skin test and BCG challenge (at T9 and T9 + 3 days, respectively) were monitored for two weeks (T10-T11). One animal from the formalin-inactivated BCG group was excluded from this analysis due to unresponsiveness of white blood cells to stimulation with PWM from T1 to T9. The PPD-B specific IFN-γresponses (corrected for the OD neg ), expressed as OD values (OD bov ), in the four treatment groups over time are presented in Fig 2A. The response to PPD-B was analyzed using a linear mixed effects model [26] and compared to the control group (Table A in S2 Dataset). The mean (n = 6) responses to PPD-B prior to vaccination (T0) were below an OD-value of 0.5 for all groups. Mean OD-values for PPD-B in the control group ranged from 0.311 to 0.820 after vaccination (T1-T9). A similar trend was observed in the formalin-inactivated BCG group, with slightly lower mean OD bov values ranging from 0.275 to 0.594 and no significant differences to the control group. The live BCG group showed slightly elevated mean OD bov values as compared to the control group in response to vaccination (T1-T9), ranging from 0.802 to 1.353, and differences were significant at T4 only (Table A Fig 2. Interferon-γresponses in the BOVIGAM ® assay. Mean OD-values in response to (A) PPD-B, (B) PPD-B minus PPD-A, (C) PPD-B minus PPD-F, (D) ESAT-6 and (E) CFP-10. ●= Live M.bovis BCG; = Formalin-inactivated BCG; ▲= Heat-killed M.bovis;4= Control group. Error bars indicate the standard error of the mean (±SEM) for each time point. Arrows at T0, T3 and T9 indicate priming vaccination, booster vaccination and SICTT and BCG challenge, respectively. https://doi.org/10.1371/journal.pone.0188448.g002 Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 7 / 20 in S2 Dataset). The heat-killed M.bovis group showed the highest OD bov values after vaccination, ranging from 1.996 to 3.374, peaking at T4, and significant differences compared to the control group were observed from early on in the experiment and were sustained up to and including T9 (Table A in S2 Dataset). After skin test and BCG challenge, the mean OD bov values were elevated as compared to T9 in all groups except the heat-killed M.bovis group. In the control group, mean OD bov values ranged from 1.833 to 1.953. No significant differences to the control group were found in the live BCG and heat-killed M.bovis groups, with mean OD bov values ranging from 2.296 to 2.578 and 2.349 to 2.903, respectively. In the formalininactivated group OD bov values ranged from 0.793 to 1.014 and this was significantly lower as compared to the control group at T11 (Table A in S2 Dataset). Reactivity to two predominant environmental mycobacteria (M.avium and M.fortuitum) was taken into account in the analysis of the IFN-γresponses and presented as OD bov -OD av and OD bov -OD fort (both corrected for the OD neg ) in Fig 2B and 2C, respectively. The ratios of PPD-B/PPD-A and PPD-B/PPD-F were analyzed using a linear mixed effects model [26] and compared to the control group (Table A in S2 Dataset). In the PPD-B/PPD-A model, both the live and the formalin-inactivated BCG vaccination groups showed no significant differences when compared to the control group. In the heat-killed M.bovis group, however, responses significantly higher as compared to the control group were observed at several time points (T4-T9) after vaccination (Table A in S2 Dataset). In the PPD-B/PPD-F model, the immune responses in the live and formalin-inactivated BCG groups were largely comparable to those in the control group, but differences significantly higher as compared to the control group were seen in the live BCG group at T6 (Table A in S2 Dataset). Again, the heat-killed M.bovis group showed significantly higher responsiveness as compared to the control group at multiple time points (T1 and T3-T9) after vaccination as well as after skin test and challenge (T10) (Table A in S2 Dataset). In addition to the classical PPDs as stimulating antigens in CMI testing, ESAT-6 and CFP10, two antigens widely used in TB research and diagnosis, as they are assumed to be specific for the MTBC, were included in the BOVIGAM 1 assay at T0, T3 and T9. Fig 2D and 2E present the IFN-γresponses specific to these antigens in the four vaccination groups during the course of the experiment. The responses to ESAT-6 and CFP-10 were analyzed using a linear mixed effects model [26] and compared to the control group (Table B in S2 Dataset). One animal in the heat-killed M.bovis group showed an extremely high value at week 9 and this entry was excluded from the model. Mean OD-values for ESAT-6 (OD ESAT6 ) in the control group ranged from -0.026 to 0.064. Similar trends were observed in the other vaccination groups with OD ESAT6 values ranging from -0.034 to 0.011 (live BCG), from -0.049 to 0.022 (formalininactivated BCG) and from -0.019 to 0.094 (heat-killed M.bovis). There were no significant differences in the responses to ESAT-6 in any of the vaccination groups as compared to the control group (Table B in S2 Dataset). Mean OD-values for CFP-10 (OD CFP10 ) in the control group ranged from -0.021 to 0.016. Similar trends were observed in the vaccination groups receiving the live and formalin-inactivated BCG vaccines with OD CFP10 values ranging from -0.023 to 0.036 and from -0.047 to 0.034, respectively. The mean response to CFP-10 in the formalin-inactivated BCG group was significantly lower as compared to the control group at T9 (Table B in S2 Dataset). Mean OD CFP10 values in the heat-killed M.bovis group showed a slight increase as compared to the control group ranging from -0.008 to 0.462 at T3 and from 0.001 to 0.557 at T9, and these were significantly different (Table B in S2 Dataset). Serology Humoral immune (HI) responsiveness to the different vaccine candidates was monitored for nine weeks (T1-T9) in all animals and compared to the control group. Responses following Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 8 / 20 skin test and BCG challenge (at T9 and T9 + 3 days, respectively) were monitored for further three weeks (T10-T12). Fig 3 presents the mean S/P ratios for the vaccination groups over time. The S/P ratios were analysed using a linear mixed effects model [26] and compared to the control group (Table C in S2 Dataset). Prior to vaccination, the mean S/P ratios in all groups were approximately -0.04. Mean S/P ratios in the control group ranged from -0.022 to 0.007 after vaccination (T1-T9). Humoral responses after vaccination in the groups receiving the live and formalin-inactivated BCG preparations mirrored those in the control group (Fig 3) and thus no significant differences to the control group were found (Table C in S2 Dataset). In the heat-killed M.bovis group, a humoral response was detected from as early as T2, which increased and was sustained during the course of the experiment (Fig 3), with values ranging from 0.003 to 9.213 after vaccination (T1-T9). The S/P ratios in this group were found to be significantly higher compared to those in the control group at several time points (T2-T9) (Table C in S2 Dataset). After skin testing and BCG challenge (at T9 and T9 + 3 days, respectively), mean S/P ratios in the control group showed a very slight increase, ranging from 0.086 to 0.245. In contrast, there appeared to be no response to skin test and BCG challenge in the Fig 3. Antibody responses in the IDEXX TB ELISA. Mean sample/positive (S/P) ratios. ●= Live M. bovis BCG; = Formalin-inactivated BCG; ▲= Heatkilled M. bovis; 4= Control group. Error bars indicate the SEM for each time point. Arrows at T0, T3 and T9 indicate priming vaccination, booster vaccination and SICTT and BCG challenge, respectively. https://doi.org/10.1371/journal.pone.0188448.g003 Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 9 / 20 pathological changes as a function of vaccine efficacy of the heat-killed M.bovis vaccine candidate. The use of multiple statistical models increases the chance of observing a significant result. However, we believe that the significant results described in this study are not only statistically but also biologically relevant. The wide confidence intervals that were found in several analyses carried out in this study, can be explained by the fact that a relatively low number of animals was used, which was the main limitation of the current study. Nevertheless, strong evidence for a high immunogenicity of the heat-killed M.bovis vaccine was demonstrated which justifies further vaccine trials to evaluate its usefulness in future BTB control strategies. Lastly, our results indicate that there was exposure to environmental mycobacteria during the course of the experiment, as demonstrated by the responses to PPD-A and PPD-F in the BOVIGAM 1 assay, as well as PPD-A reactivity in the skin test. The true M.bovis specific effects of vaccination might be masked by this responsivity. However, it is important to note that non-tuberculous mycobacteria occur ubiquitously in the environment on a global scale. Therefore, it is promising that the heat-killed M.bovis vaccine performed well under these circumstances, as they should closely resemble field conditions. Conclusion The results obtained in the present study clearly indicate that subcutaneous vaccination with the heat-killed Mycobacterium bovis vaccine elicits strong and sustained cell-mediated and humoral immune responses in cattle, indicating excellent immunogenicity of the vaccine. Although exact correlates of protection are not known and this remains an important point of discussion between research groups, it may be assumed that an IFN-γresponse such as demonstrated in this study, forms part of the protective immune profile against BTB. The role of humoral immunity in providing protection against BTB is even less well established, but more evidence is becoming available suggesting its value. Therefore, the finding of both strong CMI and HI responses to the heat-killed M.bovis vaccine, in combination with a lesser degree of M. bovis BCG replication after challenge, highlights the potential of this vaccine candidate. In further studies, challenge with virulent M.bovis is recommended as well as assessment of the vaccine in field studies to further evaluate vaccine efficacy under natural conditions. Supporting information S1 Fig. Conventional PCR for the detection of Mycobacterium bovis.PCR targeting RD1, RD4 and RD9 as previously described. PCR products of +- 268bp (RD4 absent), +- 196bp (RD1 absent) and +- 108bp (RD9 absent) indicate M.bovis BCG. Animals 18, 21 and 31 belong to group 1 (live M.bovis BCG), animals 2, 6, 7, 16 and 29 belong to group 2 (formalin-inactivated M.bovis BCG), animals 8, 9, 10, 11 and 26 belong to group 3 (heat-killed M.bovis) and animals 12 and 15 belong to group 4 (control). R = right prescapular lymph node. (TIF) S1 Dataset. Tables containing the raw data of the immunological assays. (A) BOVIGAM assay. OD-values for all stimulations and controls. (B) IDEXX TB ELISA. OD-values for the samples and controls as well as S/P-ratio. (C) Skin test. Skin fold thickness measurements at 0hrs, 72hrs and the difference (Δmm) in mm. Avian = PPD-A; Bovine = PPD-B; PC1 = protein cocktail 1; PC2 = protein cocktail 2. (D) Culture. Weights (g) and bacterial counts (CFU/g of PLN) of left and right PLNs. (XLSX) Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 16 / 20 S2 Dataset. Tables describing the statistical models and their outcomes. (A) Linear mixed effects models describing PPD-B and the ratios of PPD-B/PPD-A and PPD-B/PPD-F. Outcome = a + b1 time + b2 group + b3 (time group). Data were log transformed in order to meet the model assumptions of normality and homoscedasticity. Back-transformed estimates and 95% confidence intervals are given. Significant results are in bold. (B) Linear mixed effects models describing ESAT-6 and CFP-10. Outcome = a + b1 time + b2 group + b3  (time group). Data were log transformed in order to meet the model assumptions of normality and homoscedasticity. Back-transformed estimates and 95% confidence are given. Significant results are in bold. (C) Linear mixed effects model describing the S/P ratio. Outcome = a + b1 time + b2 group + b3 (time group). Data were log transformed in order to meet the model assumptions of normality and homoscedasticity. Back-transformed estimates and 95% confidence intervals are given. Significant results are in bold. (D) Double generalized linear model describing ΔPPDB—ΔPPDA in the skin test. Outcome = a + b1 group. Estimates and 95% confidence intervals are given. Significant results are in bold. (E) A simple general linear model describing ΔPC1 and ΔPC2. Outcome = a + b1 group. Estimates and 95% confidence intervals are given. Significant results are in bold. (F) Linear mixed effects model describing the PLN weights. Outcome = a + b1 LN side + b2 group + b3 (LN side group) + b4 gender. Data (PLN weights) were log transformed in order to meet the model assumptions of normality and homoscedasticity. Estimates and 95% confidence intervals are given. Significant results are in bold. (G) Negative binomial generalized linear model describing the bacterial counts. Outcome = a + b1 group. Back-transformed estimates and 95% confidence intervals are given. Significant results are in bold. (XLSX) Acknowledgments We acknowledge Martin Vordermeier and Gareth Jones at the Animal and Plant Health Agency (UK) for providing the recombinant protein cocktails used in the skin test and for technical support, respectively. We furthermore acknowledge personnel of the UPBRC, who were instrumental in the care of the animals and support with animal handling during sampling occasions. Author Contributions Conceptualization: C. Gortazar, R. A. Juste, J. E. Crafford, V. P. M. G. Rutten, A. L. Michel. Data curation: E. M. D. L. van der Heijden, J. Chileshe, J. C. M. Vernooij, V. P. M. G. Rutten. Formal analysis: E. M. D. L. van der Heijden, J. C. M. Vernooij. Funding acquisition: V. P. M. G. Rutten, A. L. Michel. Investigation: E. M. D. L. van der Heijden, J. Chileshe, J. E. Crafford, A. L. Michel. Methodology: E. M. D. L. van der Heijden, J. C. M. Vernooij, C. Gortazar, R. A. Juste, I. Sevilla, V. P. M. G. Rutten, A. L. Michel. Project administration: J. E. Crafford, V. P. M. G. Rutten, A. L. Michel. Resources: E. M. D. L. van der Heijden, J. Chileshe, J. C. M. Vernooij, C. Gortazar, R. A. Juste, I. Sevilla, J. E. Crafford, V. P. M. G. Rutten, A. L. Michel. Software: E. M. D. L. van der Heijden, J. C. M. Vernooij. Supervision: V. P. M. G. Rutten, A. L. Michel. Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 17 / 20 Validation: E. M. D. L. van der Heijden, J. C. M. Vernooij. Visualization: E. M. D. L. van der Heijden. Writing – original draft: E. M. D. L. van der Heijden. Writing – review & editing: E. M. D. L. van der Heijden, J. Chileshe, J. C. M. Vernooij, C. Gortazar, R. A. Juste, J. E. Crafford, V. P. M. G. Rutten, A. L. Michel. References 1. de Lisle GW, Mackintosh CG, Bengis RG. Mycobacterium bovis in free-living and captive wildlife, including farmed deer. Rev Sci Tech. 2001; 20(1):86–111. PMID: 11288522 2. Garnier T, Eiglmeier K, Camus JC, Medina N, Mansoor H, Pryor M, et al. The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A. 2003; 100(13):7877–82. https://doi.org/ 10.1073/pnas.1130426100 PMID: 12788972 3. Michel AL. Improving specific disease outcomes through a One Health approach—tuberculosis. Rev Sci Tech. 2014; 33(2):583–92. PMID: 25707185 4. Fitzgerald SD, Kaneene JB. Wildlife reservoirs of bovine tuberculosis worldwide: hosts, pathology, surveillance, and control. Vet Pathol. 2013; 50(3):488–99. https://doi.org/10.1177/0300985812467472 PMID: 23169912 5. Buddle BM, Parlane NA, Wedlock DN, Heiser A. Overview of vaccination trials for control of tuberculosis in cattle, wildlife and humans. Transbound Emerg Dis. 2013; 60 Suppl 1:136–46. https://doi.org/10. 1111/tbed.12092 PMID: 24171859 6. Buddle BM, Pollock JM, Skinner MA, Wedlock DN. Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens. Int J Parasitol. 2003; 33(5–6):555–66. PMID: 12782055. 7. Chambers MA, Carter SP, Wilson GJ, Jones G, Brown E, Hewinson RG, et al. Vaccination against tuberculosis in badgers and cattle: an overview of the challenges, developments and current research priorities in Great Britain. Vet Rec. 2014; 175(4):90–6. https://doi.org/10.1136/vr.102581 PMID: 25059963 8. Vordermeier HM, Perez de Val B, Buddle BM, Villarreal-Ramos B, Jones GJ, Hewinson RG, et al. Vaccination of domestic animals against tuberculosis: review of progress and contributions to the field of the TBSTEP project. Res Vet Sci. 2014; 97 Suppl:S53–60. https://doi.org/10.1016/j.rvsc.2014.04.015 PMID: 24908189 9. Vordermeier HM, Villarreal-Ramos B, Cockle PJ, McAulay M, Rhodes SG, Thacker T, et al. Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis. Infect Immun. 2009; 77(8):3364–73. https://doi.org/10.1128/IAI.00287-09 PMID: 19487476 10. Ottenhoff TH, Kaufmann SH. Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathog. 2012; 8(5):e1002607. https://doi.org/10.1371/journal.ppat.1002607 PMID: 22589713 11. Skinner MA, Ramsay AJ, Buchan GS, Keen DL, Ranasinghe C, Slobbe L, et al. A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis. Immunology. 2003; 108(4):548–55. https://doi.org/10.1046/j.1365-2567.2003.01589.x [pii]. PMID: 12667217 12. Wedlock DN, Skinner MA, Parlane NA, Vordermeier HM, Hewinson RG, de Lisle GW, et al. Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 DNA prime-protein boost does not protect cattle against bovine tuberculosis. Tuberculosis (Edinb). 2003; 83(6):339–49. https://doi.org/10.1016/ s1472-9792(03)00055-6 13. Chambers MA, Wright DC, Brisker J, Williams A, Hatch G, Gavier-Wide ´n D, et al. A single dose of killed Mycobacterium bovis BCG in a novel class of adjuvant (Novasome™) protects guinea pigs from lethal tuberculosis. Vaccine. 2004; 22(8):1063–71. https://doi.org/10.1016/j.vaccine.2003.05.002 PMID: 15161084 14. UNAIDS. Global AIDS update [Report]. 2016. Available from: http://www.unaids.org/en/resources/ documents/2016/Global-AIDS-update-2016. 15. Michel AL, Muller B, van Helden PD. Mycobacterium bovis at the animal-human interface: a problem, or not? Vet Microbiol. 2010; 140(3–4):371–81. https://doi.org/10.1016/j.vetmic.2009.08.029 PMID: 19773134. Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 18 / 20 16. Whelan AO, Wright DC, Chambers MA, Singh M, Hewinson RG, Vordermeier HM. Evidence for enhanced central memory priming by live Mycobacterium bovis BCG vaccine in comparison with killed BCG formulations. Vaccine. 2008; 26(2):166–73. https://doi.org/10.1016/j.vaccine.2007.11.005 [pii]. PMID: 18055073 17. Garrido JM, Sevilla IA, Beltran-Beck B, Minguijon E, Ballesteros C, Galindo RC, et al. Protection against tuberculosis in Eurasian wild boar vaccinated with heat-inactivated Mycobacterium bovis. PloS One. 2011; 6(9):e24905. https://doi.org/10.1371/journal.pone.0024905 PMID: 21935486 18. Lopez V, Gonzalez-Barrio D, Lima-Barbero JF, Ortiz JA, Dominguez L, Juste R, et al. Oral administration of heat-inactivated Mycobacterium bovis reduces the response of farmed red deer to avian and bovine tuberculin. Vet Immunol Immunopathol. 2016; 172:21–5. https://doi.org/10.1016/j.vetimm.2016. 03.003 PMID: 27032499. 19. Juste RA, Alonso-Hearn M, Garrido JM, Abendano N, Sevilla IA, Gortazar C, et al. Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria. PLoS One. 2016; 11(11):e0165607. https://doi.org/10.1371/journal.pone.0165607 PMID: 27820836; PubMed Central PMCID: PMCPMC5098821. 20. Michel AL, Cooper D, Jooste J, de Klerk LM, Jolles A. Approaches towards optimising the gamma interferon assay for diagnosing Mycobacterium bovis infection in African buffalo (Syncerus caffer). Prev Vet Med. 2011; 98(2–3):142–51. https://doi.org/10.1016/j.prevetmed.2010.10.016 PMID: 21122932 21. World Organisation for Animal H. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris: World Organisation for Animal Health (OiE), 2014 Contract No.: Report. 22. Jones GJ, Whelan A, Clifford D, Coad M, Vordermeier HM. Improved skin test for differential diagnosis of bovine tuberculosis by the addition of Rv3020c-derived peptides. Clin Vaccine Immunol. 2012; 19 (4):620–2. https://doi.org/10.1128/CVI.00024-12 PMID: 22301696 23. Villarreal-Ramos B, Berg S, Chamberlain L, McShane H, Hewinson RG, Clifford D, et al. Development of a BCG challenge model for the testing of vaccine candidates against tuberculosis in cattle. Vaccine. 2014. https://doi.org/S0264-410X(14)01122-0 [pii]. 24. Warren RM, Gey van Pittius NC, Barnard M, Hesseling A, Engelke E, de Kock M, et al. Differentiation of Mycobacterium tuberculosis complex by PCR amplification of genomic regions of difference. Int J Tuberc Lung Dis. 2006; 10(7):818–22. PMID: 16850559 25. R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; 2016. 26. Pinheiro J, Bates D, DebRoy S, Sarkar D, Team RC. nlme: Linear and Nonlinear Mixed Effects Models. R package version 3. 1–128 ed2016. 27. Dunn PK, Smyth GK. dglm: Double Generalized Linear Models. R package version 1.8.3. ed2016. 28. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol. 1996; 178(5):1274–82. PMID: 8631702 29. Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B. A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology. 1998; 144 (Pt 11):3195–203. https://doi.org/10.1099/00221287-144-11-3195 PMID: 9846755. 30. Beltran-Beck B, de la Fuente J, Garrido JM, Aranaz A, Sevilla I, Villar M, et al. Oral vaccination with heat inactivated Mycobacterium bovis activates the complement system to protect against tuberculosis. PLoS One. 2014; 9(5):e98048. https://doi.org/10.1371/journal.pone.0098048 PMID: 24842853; PubMed Central PMCID: PMCPMC4026474. 31. Haile M, Schroder U, Hamasur B, Pawlowski A, Jaxmar T, Kallenius G, et al. Immunization with heatkilled Mycobacterium bovis bacille Calmette-Guerin (BCG) in Eurocine L3 adjuvant protects against tuberculosis. Vaccine. 2004; 22(11–12):1498–508. https://doi.org/10.1016/j.vaccine.2003.10.016 PMID: 15063575 32. Santema W, Rutten V, Segers R, Poot J, Hensen S, Heesterbeek H, et al. Postexposure subunit vaccination against chronic enteric mycobacterial infection in a natural host. Infect Immun. 2013; 81 (6):1990–5. https://doi.org/10.1128/IAI.01121-12 PMID: 23509147; PubMed Central PMCID: PMCPMC3676026. 33. Hope JC, Thom ML, Villarreal-Ramos B, Vordermeier HM, Hewinson RG, Howard CJ. Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis. Clin Exp Immunol. 2005; 139(1):48–56. https://doi.org/10.1111/j.1365-2249.2005. 02668.x PMID: 15606613; PubMed Central PMCID: PMCPMC1809274. 34. Shimokata K, Kawachi H, Kishimoto H, Maeda F, Ito Y. Local cellular immunity in tuberculous pleurisy. Am Rev Respir Dis. 1982; 126(5):822–4. https://doi.org/10.1164/arrd.1982.126.5.822 PMID: 6293353. Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 19 / 20 35. Barnes PF, Mistry SD, Cooper CL, Pirmez C, Rea TH, Modlin RL. Compartmentalization of a CD4+ T lymphocyte subpopulation in tuberculous pleuritis. J Immunol. 1989; 142(4):1114–9. PMID: 2464638. 36. Barnes PF, Fong SJ, Brennan PJ, Twomey PE, Mazumder A, Modlin RL. Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis. J Immunol. 1990; 145(1):149–54. PMID: 2113553. 37. Vordermeier M, Hewinson RG. Development of cattle TB vaccines in the UK. Vet Immunol Immunopathol. 2006; 112(1–2):38–48. doi: S0165-2427(06)00087-0 [pii]. https://doi.org/10.1016/j.vetimm. 2006.03.010 PMID: 16682085 38. Millington KA, Fortune SM, Low J, Garces A, Hingley-Wilson SM, Wickremasinghe M, et al. Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection. Proc Natl Acad Sci U S A. 2011; 108(14):5730–5. https://doi.org/10. 1073/pnas.1015153108 [pii]. PMID: 21427227 39. Pym AS, Brodin P, Brosch R, Huerre M, Cole ST. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol Microbiol. 2002; 46 (3):709–17. PMID: 12410828. 40. Jones GJ, Hewinson RG, Vordermeier HM. Screening of predicted secreted antigens from Mycobacterium bovis identifies potential novel differential diagnostic reagents. Clin Vaccine Immunol. 2010; 17 (9):1344–8. https://doi.org/10.1128/CVI.00261-10 PMID: 20668139; PubMed Central PMCID: PMCPMC2944449. 41. de la Rua-Domenech R, Goodchild AT, Vordermeier HM, Hewinson RG, Christiansen KH, Clifton-Hadley RS. Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques. Res Vet Sci. 2006; 81(2):190–210. https://doi. org/10.1016/j.rvsc.2005.11.005 PMID: 16513150 42. Hewinson RG, Vordermeier HM, Buddle BM. Use of the bovine model of tuberculosis for the development of improved vaccines and diagnostics. Tuberculosis (Edinb). 2003; 83(1–3):119–30. https://doi. org/10.1016/s1472-9792(02)00062-8 43. Buddle BM, Wedlock DN, Denis M. Progress in the development of tuberculosis vaccines for cattle and wildlife. Vet Microbiol. 2006; 112(2–4):191–200. https://doi.org/10.1016/j.vetmic.2005.11.027 PMID: 16326043. 44. Buddle BM, Wards BJ, Aldwell FE, Collins DM, de Lisle GW. Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis. Vaccine. 2002; 20(7–8):1126– 33. doi: S0264410X01004364 [pii]. PMID: 11803073 45. Fine PE. Variation in protection by BCG: implications of and for heterologous immunity. Lancet. 1995; 346(8986):1339–45. doi: S0140-6736(95)92348-9 [pii]. PMID: 7475776 46. Gcebe N. The occurrence and molecular characterization of non-tuberculous mycobacteria in cattle, African buffalo (Syncerus caffer) and their environments in South Africa and genomic characterization and proteomic comparison with Mycobacterium bovis: University of Pretoria; 2015. Inactivated Mycobacterium bovis vaccine candidates in cattle PLOS ONE | https://doi.org/10.1371/journal.pone.0188448 November 20, 2017 20 / 20