Van der Heijden, E.M.D.L. (Elisabeth)Chileshe, J.Vernooij, J.C.M.Gortazar, G.Juste, R.A.Sevilla, I.Crafford, Jan ErnstRutten, Victor P.M.G.Michel, Anita Luise2018-07-192018-07-192017-11-20van 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.01884481932-6203 (online)10.1371/journal.pone.0188448http://hdl.handle.net/2263/65792S1 Fig. Conventional PCR for the detection ofMycobacterium 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.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.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 ΔPPDBÐΔPPDA 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.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). Interferon- γ responsiveness 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.en© 2017 van der Heijden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License.HIV/AIDSInfectionVaccinationBovine tuberculosis (bTB)Human immunodeficiency virus (HIV)Acquired immune deficiency syndrome (AIDS)Immune responseCalvesMycobacterium bovisArticle