Tuberculosis vaccination: Microbiological and immunological summary of a series of experimental challenge studies

Tuberculosis vaccination: Microbiological and immunological summary of a series of experimental challenge studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tuberculosis vaccination: Microbiological and immunological summary of a series of experimental challenge studies Ramon A Juste, Iker A Sevilla, Esmeralda Minguijon, Miguel Fuertes, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6032013/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract Background : Bovine tuberculosis (TB) caused by Mycobacterium bovis remains a persistent zoonotic and agricultural challenge, despite advances in eradication programs targeting cattle. The complexity of TB epidemiology, especially in extensive livestock systems and wildlife reservoirs, necessitates novel control strategies. Vaccination has re-emerged as a promising tool, with growing interest in inactivated vaccines for both efficacy and diagnostic compatibility. Objectives : This study evaluates the comparative efficacy of homologous and heterologous inactivated vaccines against the standard live Bacille Calmette–Guérin (BCG) vaccine in calves, focusing on M. bovis isolation, gross TB lesions, immune responses, and compatibility with Differentiating Infected from Vaccinated Animals (DIVA) strategies. Methods : Data from four controlled vaccination and challenge trials involving 41 calves were analyzed. Animals were vaccinated with live BCG or heat-inactivated M. bovis via the oral or parenteral routes, with immunologic assays, skin tests, and post-mortem analyses conducted to evaluate vaccine performance. Results : All vaccination strategies significantly reduced M. bovis bacterial loads in the lungs compared to non-vaccinated controls, achieving up to 99% reductions. However, bacterial loads in lymphoid tissues increased, underscoring tuberculosis as a primarily lymphatic disease. Diagnostic interference varied by vaccine type and administration route, with oral administration showing lower interference. Gross lesion scores were inconsistent across groups, suggesting limited utility as a measure of vaccine efficacy. Immune responses revealed enhanced detection of infection post-vaccination, particularly with inactivated vaccines, which showed promising compatibility with DIVA strategies. Lung bacterial load appeared to be decoupled from cellular immune responses and lymph node lesions and bacterial load which were negatively correlated among themselves. Conclusions : This study demonstrates that inactivated vaccines offer a safe and effective means of reducing TB transmission by confining bacterial presence to lymphoid tissues and minimizing diagnostic interference. Vaccination programs should shift from eradication goals to transmission control, prioritizing reductions in reproductive rate (R₀) over total bacterial clearance. These findings highlight the need for revised evaluation criteria and support the integration of inactivated vaccines into TB control strategies. tuberculosis cattle skin test interferon gamma infection transmission vaccine inactivated vaccine Interferon gamma M. bovis route Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Animal tuberculosis (TB) is caused by species within the Mycobacterium tuberculosis complex (MTC), primarily M. bovis and M. caprae , which affect a wide range of hosts, including cattle. Since TB has long been recognized as a zoonotic disease, eradication programs targeting cattle have been implemented in many countries. These programs have been largely successful, particularly in reducing TB prevalence in the primary zoonotic reservoir, dairy cattle. However, in recent decades, the epidemiology of TB has proven to be more complex than previously understood. Beyond dairy cattle, other livestock such as beef cattle, goats, pigs, and sheep managed in extensive systems—often in contact with wildlife—serve as significant hosts. These hosts, frequently outside the scope of traditional cattle-centered control programs, play a critical role in maintaining MTC in the environment [ 1 – 9 ]. Additionally, wildlife hosts contribute significantly to the MTC maintenance community [ 10 ] Compounding this challenge is growing evidence of the limited sensitivity of traditional diagnostic methods [ 11 – 14 ], highlighting the need for alternative TB control strategies. Among these alternatives, vaccination has emerged as a promising tool. However, cattle vaccination has been banned since the 1950s due to concerns about interference with immune-based diagnostic tests used in eradication programs and doubts about its efficacy [ 15 , 16 ]. Notably, research conducted in Ethiopia has demonstrated the potential of cattle vaccination against TB. Even a nominal vaccine efficacy of 50% was shown to reduce the basic reproduction number (R₀) to less than 1, driving infection towards extinction at a significantly lower economic and social cost compared to test-and-cull strategies [ 17 ]. Despite this, research efforts have predominantly focused on live BCG vaccines, while inactivated (killed) vaccines have been largely neglected [ 18 – 20 ]. The demonstrated success of inactivated vaccines in wildlife [ 21 – 26 ], their inherent safety, and insights gained from heat-inactivated paratuberculosis vaccines have rekindled interest in their use for TB control [ 27 – 30 ]. Furthermore, the recent concept of trained immunity (TRAIM) [ 31 ] provides theoretical support for the use of killed vaccines. Despite early observations suggesting TRAIM effects were exclusive to live vaccines [ 32 – 34 ], the use of adjuvants in killed vaccines has shown potential to enhance their infection-fighting ability. Additionally, oral administration of inactivated vaccines, which avoids eliciting specific immune responses [ 35 ], presents a promising opportunity for a Differentiating Infected from Vaccinated Animals (DIVA) strategy. This approach may protect against infection without interfering with official diagnostic tests. Traditionally, tuberculosis has been considered a respiratory disease due to its primary localization in the thorax. However, in cattle, the lungs—histologically and functionally distinct from lymph nodes—are less commonly affected. In Australia, for example, only 14.1% of gross lesions were located in the lungs, with 3.9% as single lesions [ 36 ]. In Spain, prior to the implementation of test-and-cull programs in the 1950s, 48.8% of 1,561 slaughtered cattle displayed gross lesions in the lungs [ 15 ]. By contrast, in the United States, no gross lesions were observed in a small sample of 15 cattle, although 6.7% were positive by isolation [ 37 ]. These findings, combined with evidence of immune response compartmentalization[ 38 , 39 ] supports the view of tuberculosis being a lymphatic disease with lungs as its portal[ 40 ], and suggest that giving the same weight to lymphatic infection as to lung infection may lead to an underestimation of epidemiological risks in this slow-progressing, low-transmissibility disease [ 41 ]. Furthermore, the lower vaccine efficacy observed in experimental challenge trials[ 25 , 42 , 43 ] compared to field conditions[ 9 , 17 , 21 , 24 ] may be partially attributed to differences in infection routes. Experimental models often bypass natural respiratory infection pathways, potentially biasing efficacy assessments. Based on these premises, we aimed to compare homologous and heterologous killed vaccines with the current standard live BCG vaccine. Our study evaluates these vaccines in terms of M. bovis isolation as proxy of transmission risk, gross TB lesions, specific immune responses, and DIVA performance. 2. Materials and Methods 2.1. Animals, Vaccination, and Sampling This meta-analysis revisits data from four experimental vaccination and challenge trials involving a total of 41 calves. Results from these trials have been partially published in two reports: Trial 2 [44] and Trial 4 [45] . 2.1.1. First Trial: Vaccine Diagnostic Interference and Protection with a Heterologous Vaccine Six Friesian calves, aged 2–3 months, were selected from farms in northern Spain with no known history of tuberculosis (TB) and tested negative for interferon-gamma release assay (IGRA). The calves were recruited at a feedlot and subcutaneously vaccinated with the commercial paratuberculosis vaccine Silirum™ (CZ Vaccines, Porriño, Spain) (CPVP). After 1.5 months, the calves were transported to the biosafety level 3 (BSL-3) facilities at Neiker-BRTA. Following a 32-day acclimatization period, the calves were challenged intratracheally with a recent M. bovis isolate. Blood samples were collected from the jugular vein at days -72, 0, 13, 26, 62, 74, 91, and 146 post-infection. Blood was drawn into lithium heparin-coated tubes for cellular immunity assays (Figure 1). On day 146, all calves were euthanized following standard approved procedures and subjected to a full necropsy. Gross lesions were scored based on the method described by Palmer et al., 2007[46]. Samples were collected from seven lymph nodes, the tonsils, lungs, liver, and spleen for M. bovis isolation using the Mycobacterial Growth Indicator Tube (MGIT) system (Becton Dickinson). 2.1.2. Second Trial: Vaccine Diagnostic Interference and Protection with Homologous and Heterologous Vaccines This experiment is described in detail in Serrano et al. (2017a, 2017b)[44, 45]. Briefly, ten male Friesian calves, similar in characteristics to those used in Experiment 1, were randomly assigned to one of two groups: five calves received the commercial Silirum™ paratuberculosis vaccine (CPVP), and five calves were administered only phosphate-buffered saline (PBS) (NoVac group). After 110 days, all calves were transported to the NEIKER-BRTA biosafety level 3 (BSL-3) facilities. Ten days after arrival, the animals were challenged intratracheally with M. bovis. Blood samples were collected on days -133, 0, 15, 29, 57, and 84 post-infection (DPI). 2.1.3. Third Trial: Vaccine Protection with Live and Inactivated Homologous Vaccines Fifteen calves were randomly assigned to one of three treatment groups: NoVac : Non-vaccinated calves received 2 mL of plain PBS. BCGO : Calves received an oral BCG vaccine (n=5). HIMBO : Calves received an oral heat-inactivated M. bovis (HIMB) vaccine (n=5). For the HIMB vaccine, a wild boar M. bovis isolate (NEIKER Strain #1403) previously used in experiments was propagated and inactivated as described by Balseiro et al. (2017) [42], Garrido et al. (2011)[25], and Jones et al. (2016)[35]. 2.1.4. Fourth Trial: Vaccine Protection with Live and Inactivated Homologous Vaccines by Different Routes To assess differences in protection and diagnostic interference related to antigen type, dose, and administration route, fifteen calves were randomly assigned to one of three treatment groups: BCGO : Calves received an oral BCG vaccine. HIMBO : Calves received 2 mL of an oral HIMB suspension containing 10^7 CFU of heat-inactivated M. bovis . HIMBP : Calves received 1 mL of an intramuscular HIMB suspension containing 10^3 CFU of heat-inactivated M. bovis . This vaccine was formulated as a water-in-oil emulsion using 0.5 mL of Montanide™ ISA 50V2 adjuvant (Seppic, Paris, France) and 0.5 mL of PBS containing the inactivated bacterial suspension heated at 80°C for 20 minutes. 2.2. Challenge Procedures In Experiments 1, 2, and 4, all calves were challenged on day 0 DPI via the endotracheal route with 2 mL of PBS containing approximately 5×10^6 CFU of an M. bovis field isolate. The isolate, NEIKER Strain #2575/08, was originally obtained from a naturally infected wild boar and had been used in previous studies [23, 45, 47, 48]. Calves were sedated intramuscularly with XILAGESIC® 2% (10 mg/50 kg; Laboratorios Calier, S.A., Barcelona, Spain) prior to inoculation. The bacterial suspension was delivered with a syringe by inserting a needle between two consecutive tracheal rings (positions 25–30). Endotracheal air was aspirated to confirm placement before injecting the inoculum. In Experiment 3, the bacterial suspension was administered orally without sedation. 2.3 Interferon -γ release assay (IGRA) Blood was collected from the caudal vein into BD Vacutainer™ tubes containing lithium heparin (Becton Dickinson and Company, Sparks, MD, USA) (Figure 1). Lithium heparinized blood was aliquoted into cell culture plate wells for antigenic stimulation, which was initiated within 4 hours of collection. Samples were incubated overnight at 37°C in a 5% CO₂ incubator with different antigens and a nil control (Nil). The antigens used included: Standard tuberculins : avian purified protein derivative ( avPPD ) and bovine purified protein derivative ( boPPD ) (CZ Veterinaria, Porriño, Spain). Protein complex : boPPD -derived protein P22 [49] Defined antigens : Protein cocktails APHA-1 (ESAT-6 CFP-10 and Rv3615c; 10 µg each protein) and APHA-2 (ESAT-6, CFP-10, Rv3615c and Rv3020c; 10 µg each protein) kindly provided by Drs G Jones and M Vordermeier[35, 50–53]. Both PPDs and P22 were used at a final concentration of 20 µg/mL, while synthetic peptides were used at 5 µg/peptide/mL. Following incubation, samples were centrifuged, and interferon-gamma (IFN-γ) levels in the blood supernatants were measured using either the Bovigam™ TB kit (Thermo Fisher Scientific, Inc.) for the first and second experiments or the IDScreen® Ruminant IFN-γ ELISA kit (IDvet, Grabels, France) for the third and fourth experiments, as per the manufacturers' instructions. Optical densities (OD) were measured at 450 nm using a Multiskan™ FC photometer (Thermo Scientific, Vantaa, Finland). For P22 and Defined antigens: Sample-to-positive (S/P) % values were calculated using the formula: ([(boPPD or p22 OD–avPPD OD)/(positive control X̄OD–negative control X̄OD)]×100). For E/C, Rv3615c, and Rv3020c peptides, S/P% was calculated similarly, as: ([(E/C, Rv3615c or Rv3020c OD–NIL OD)/(positive control X̄OD–negative control X̄OD)]×100). Values were interpreted according to Arrieta-Villegas et al. (2020). For the Bovigam Test: A positivity cut-off of 0.1 OD difference between the sample and Nil control was used. For the IDScreen® Kit: A standard cut-off value of S/P% ≥ 16 was used to classify samples as positive for all antigens, including specific peptides. IGRA assays for P22 and Rv3020c in the control group could not be conducted due to technical issues. 2.4. Skin test At the last sampling all calves in all the experiments were submitted to a cervical intradermal test with both the standard avian (2,500 IU avPPD) and bovine (2,500 IU boPPD) in 0.1 ml doses as well as with two more defined antigens: protein cocktails APHA-1 (ESAT-6 CFP-10 and Rv3615c; 10 µg each protein) and APHA-2 (ESAT-6, CFP-10, Rv3615c and Rv3020c; 10 µg each protein) kindly provided by Drs G Jones and M Vordermeier [53–57] . The skin thickness was measured before injection and 72 h after inoculation. The results of PPD skin thickness increase were interpreted according to the standards of official criteria (EU Council Directive 64/432/CEE and Spanish RD 2611/1996) for both Single Intradermal Test (SIT) and Comparative Intradermal Test (CIT). A calf was considered SIT positive when the skin thickness increase at the boPPD site was of more than 4 mm. For CIT, animals were deemed positive when the thickness increase of the boPPD injection site exceeded that of the avPPD site by more than 4 mm. With regard to APHA reagents, animals were considered APHA-1 and APHA-2 positive when the skin thickness increase was equal to or bigger than 2 mm. 2.5 Clinical signs and post-mortem lesions Animals were housed in the NEIKER biosafety facilities and were fed a standard hay and concentrate diet with free access to an automatic drinking bowl. They had a daily overlayered straw bed and were supervised for clinical signs. At the end of the experiment, right after skin test reading, animals were sedated (10 mg/50 kg XILAGESIC® 2%; Laboratorios Calier, S.A., Barcelona, Spain) and then euthanized (4-6 ml/50 kg T61; Intervet International GMBH, Unterschleissheim, Germany). Upon standard necropsy procedure, calves were thoroughly inspected for TB-compatible lesions and samples from the head (mandibular, parotid and retropharyngeal lymph nodes (LNs) and tonsils), the thorax (prescapular, tracheobronchial and mediastinal LNs), the lungs (right and left cranial and caudal lobes and medium and accessory lobes), the abdomen (hepatic, jejunal and ileocecal LNs as well as liver and spleen) and other body areas (prefemoral and popliteal LNs) were collected for pathological and microbiological analysis. Both isolation and gross lesions results were grouped according to tissue as the sum of the individual samples counts or scores for lung, lymphoid, or other. Further grouping of lung and other tissues was made and defined as non-lymphoid compartment. 2.5.1 Gross pathology Tissues were visually inspected, palpated and sliced in search of TB-compatible lesions. A previously described pathological scoring system was used for LNs, lungs and other organs. LNs were scored as follows: 0, no visible lesions; 1, small focal lesion (1–2 mm); 2, several small foci; 3, extensive lesions. Lungs and other organs were ranked as follows: 0, no visible lesions; 1, no external lesions but lesions detected upon slicing; 2, up to 5 lesions of less than 10 mm in diameter; 3, more than 5 lesions of less than 10 mm in diameter; 4, more than 1 distinct gross lesion bigger than 10 mm in diameter; 5, coalescing gross lesions. Histopathological evaluation was not used as it was considered that it added very little in quantitative terms to gross pathology for a comparative study like this one. 2.5.2 M. bovis isolation Lymph nodes (retropharyngeal, prescapular, tracheo-bronchial, mediastinal, hepatic and popliteal) and lung samples were systematically taken for isolation with or without gross lesions. Any other lymph node, liver, spleen and kidney showing gross lesions were sampled for isolation as well. Mycobacterial isolation was attempted simultaneously in Coletsos (Difco, Francisco Soria Melguizo SA, Madrid, Spain) and in BBL Mycobacteria growth indicator tubes (MGIT) (Becton Dickinson, Franklin Lakes, NJ, USA) as previously described [25] . Two grams of sample were homogenized in 10 ml of sterile distilled water. The homogenate was separated in two aliquots of 5 ml. One aliquot was processed for culture in supplemented MGIT (BACTEC MGIT growth supplement with PANTA) following manufacturer’s instructions. Five ml of 1.5% RonaCare Cetylpyridinium Chloride (Merck, Darmstadt, Germany) (w/v) was added to the remaining half of the homogenate, thoroughly mixed and incubated at room temperature for 12–18 h. After centrifugation (2,500 × g, 5 min) the pellets were cultured in Coletsos tubes. MGIT tubes were incubated for 42 days in a BACTEC MGIT 960 System and Coletsos tubes at 37°C for 4 months. Time to detection was transformed into CFU according to an exponential power equation according to Sevilla et al. 2021. Tubes with a time to detection longer than 15 days but shorter than 42 days (end of incubation) were assigned a value of 1 CFU. Tubes without growth within 42 days were scored as negative, unless there was growth in the solid media in which case, a value of 1 CFU was assigned to that sample. Results of isolation were grouped according to tissue type (lymph node, lung and other locations) and sum of CFU count per tissue type for each animal was calculated and retained as the basic experimental measure for statistical analysis. DNA was extracted from all positive cultures for PCR confirmation [58] and spoligotyping of M. tuberculosis positives [59] . 2.6 Statistics Immunologic variables were analysed as dependent variables with a general linear model with treatment group and time as independent variables. In order to simplify and prioritize the global view, individual missing data for experiment and control cells were filled with the corresponding previous control data. Differences between groups were assessed for each main effect and interaction versus the NoVac group with the corresponding treatment group post-hoc comparisons. Isolation counts and gross lesion scores were used as the primary dependent variables. Even though from an individual perspective, pathology is the most important outcome, from an epidemiological view it is bacterial load what really determines population health. Therefore, all statistical testing was focused on bacterial counts in both compartments with occasionally more detailed focus on the lung. The sum of all samples isolations was chosen because it was deemed to be the most representative and the one variable that allowed a best discrimination between the two variables of interest. Given the high variability of bacterial counts and lesion scores between experiments tissue and animals, sums were corrected according to a ratio of each experiment mean to the fourth experiment mean. Bacterial counts and scores were submitted to a generalized linear model for tissue and treatment based in the recommendation to use a negative binomial distribution model to assess zero inflated count results with equidispersion according to the quotient Chi square by degrees of freedom overdispersion indicator (ODI) [60, 61] . To fall within the mathematical ranges of this type of distribution, all counts larger than 100 were transformed by division by 2000. This model had a higher proportion of reduction of error (R^2) than the Gaussian and the Poisson. Specifically, for CFU counts the treatment and tissue Gaussian, Poisson and negative binomial had an R^2 value of 0.1614, 0.3965 and 0.4538, respectively. Their ODI was 105.6735, 13.3576 and 0.9601, respectively. Post-hoc statistical pairwise comparisons were submitted to an uncorrected Student’s t test of significance when applied to vaccination versus non-vaccinated control or to the Bonferroni correction when applied to other comparisons [62] . Correlation between in vivo tests (IGRA, Ab-ELISA, SIT) results and isolation count and lesion score were calculated using both the Pearson and Spearman correlation tests. Only correlations with coefficients of the same sign in both tests and a p<0.1 were retained for discussion. Standard statistical significance was considered at p values <0.05, but p values <0.10 are also reported as of potentially relevant but of lower significance due to group low experimental units. In order to more graphically describe the relationships between immunological, microbiological and pathological variables final control results were submitted to a Principal Components Analysis (PCA) reduced to each one of the IGRA antigens (7 variables), and the microbiological (3) and pathological (3) ones by compartment. All statistical analyses were carried out using the Generalized Linear Model, GAMLj, Factor and frequency modules of jamovi application [62, 63] (The jamovi project (2022). jamovi. (Version 2.3) [Computer Software]. Retrieved from https://www.jamovi.org. R Core Team (2021). R: A Language and environment for statistical computing. (Version 4.1) [Computer software]. Retrieved from https://cran.r-project.org. (R packages retrieved from MRAN snapshot 2022-01-01). Gallucci, M. (2019). Factor. Retrieved from https://cran.r-project.org/package=psych. GAMLj: General analyses for linear models. [jamovi module]. Retrieved from https://gamlj.github.io/.)(Gallucci, 2019; The jamovi project, 2022). 3. Results 3.1 IGRA Figure 2 displays the dynamics of immune response in the IFNγ release assay with different antigens including the Nil control. Panel A in Fig. 2 shows the levels of IFNγ in unstimulated blood. Overall, only time of sampling had a statistically significant effect on the basal blood IFNγ levels (p = 0.0009), while neither treatment (p = 0.1963) or interaction effects (p = 0.2851) were significant. The control group showed peaks at 15 DPI, 75 DPI and 90 DPI that were marginally significantly different from the initial mean (p = 0.0654, p = 0.0581 and p = 0.0357) and the other groups means (p = 0.1272, 0.0637, 0.1840, and 0.1236 for BCGO, CPV P, HIMB-O and HIMB-P at 15 DPI, p = 0.3482, 0.1816, 0.6238 and 0.1349 on 75 DPI, and p = 0.4616, p = 0.0724, p = 0.5153 and p = 0.09621 on day 90 respectively). The vaccinated groups showed also some increase in the IFNγ means between at the 75 and 90 DPI that were significantly different from pre-vaccination controls (p = 0.0113 BCGO, p = 0.0237 CPVP, 0.0066 HIMBO, p = 0.030 HIMBP). These raised levels of basal IFNγ in untreated blood would be suggestive of an active immune response following challenge in the animals at these time points. Regarding the diagnostic antigens, time of sampling had the main effect on the aviPPD IGRA (p < 0.0001) (Fig. 2 , panel B), followed by interaction (p = 0.0293) and treatment (p = 0.0401). No differences were found before challenge in any of the groups (BCG p = 0.2342, CPVP p = 0.0536, HIMBO p = 0.2433, HIMBP p = 0.6860, NoVac p = 0.0528) compared to each group’s first control, nor compared to NoVac (BCGO p = 0. 9378, CPVPE p = 0.3296, HIMBO p = 0.2043, HIMBP p = 0.9866). After vaccination and challenge, all groups significantly increased their avPPD IGRA mean readings, that never differed between them. Both vaccination and time of control were highly significant effects (p < 0.0001), as was their interaction (p = 0.0014) regarding the boPPD IGRA (Fig. 2 , panel C). No significant differences were observed in the two pre-challenge controls (BCG 0.3565, CPV p = 0.2741, HIMBO p = 0.6092, HIMBP p = 0.9770. NoVac p = 0.4409), nor relative to NoVac group (BCG p = 0.3860, HIMBO p = 0.5212, HIMBPE p = 0.9896). Afterwards, all groups had statistically significant increased means with respect to their initial readings and no differences between groups were observed until the end of the experiment except for the CPV groups that, starting on 30 DPI, sustained decreased readings relative to the NoVac group (p = 0.0118, p < 0.0001, p = 0.0104, p = 0.0538). The more specific antigens behaved similarly. For p22 (Fig. 2 , panel D), there were no significant differences in the pre-challenge controls between BCG and HIMB (no CPVP group for this antigen) and NoVac group (0.7585 > = p = 0.8153). At 15 DPI, the BCG group had larger values than the NoVac (p = 0.0305), while the HIMB did not differ (p = 0.2847). From then on, BCG 0.0038, HIMB 0.0288 30 DPI; 0.9005 BCG, 0.3908 HIMB 60 DPI; 0.0082 BCG 0.0180 HIMB 75 DPI; 0.4231 BCG, 0.4412 HIMB 105 DPI. For ESAT/CFP (Fig. 2 , panel E), no significant differences were observed in the first two controls (0.7216 > = p <=0.9827) between NoVac and the vaccinated groups. At 15 DPI, both BCG (p = 0002) and HIMB (p = 0.0140) groups had higher readings than the NoVac which was not different from CPVP (p = 0.9089). Afterwards, both BCG and HIMB (p < 0.0001) had higher readings than NoVac which had higher ones than CPVP (p = 0.0193) for 30 DPI. These differences were maintained < 0.0001 for both M. bovis vaccines and for the CPVP (p = 0.0002) by 60 DPI. By 75 DPI, the differences started to diminish p = 0.0248 and p = 0.0537) until at the last control there were no differences between groups (p = 0.3589 and p = 0.7459 for BCG and HIMB). Differences with the initial IGRA values were observed as soon as at 15 DPI (p = p <=0.9463) and the rest of the groups except at 60 DPI when HIMB showed a smaller value (p = 0.0417) while BCG was not significant (p = 0.1999). Concerning Rv3615c (Fig. 2 , panel G) no differences were observed during the first three controls (0.5095 =0.9803). By 30 DPI, the BCG group showed a larger IGRA response p = 0.0054) and CPVP smaller (p = 0.0390) than NoVac which did not differ from HIMB (p = 0.2969). By 60 DPI, CPVP had an even larger difference with NoVac (p = 0.0018), while the other groups had values closer to the NoVac (p = 0.2846 and p = 0.9332 for BCG and HIMB, respectively). At the last control, no differences were observed between groups (p = 0.7733 and p = 0.7939 for BCG and HIMB: there was 150 DPI control for the CPVP group). The NoVac group increased the IGRA response from 30 DPI (p = 0.0172) onwards (p < 0.0100). The differences were even larger in the BCG, HIMB and CPVP groups that also started showing statistically different mean values from the same 30 DPI (p < 0.0001) until the end when some convergence occurred (p < 0.0500). Panel I in Fig. 2 shows the bovine tuberculosis diagnostic positive results according to the supplier. Only one animal of the CPVPE group at the first control and another different one in the second (p = 0.5165 and p = 0.6338, respectively) were classified as positive by the IGRA in the first experiment. After challenge, positivity frequencies ranged between 20 and 50 in the 15 DPI control (p = 0.8087), while in the 30 DPI and 60 DPI controls the HIMBP reached 100% positivity but CPVPE decreased to 9.1% and 18.2% (p = 0.0039 and p = 0.0040, respectively). This would indicate that HIMBP experienced no diagnostic interference from the 30 DPI onwards, while the CPVPE seemed to inhibit the specific cell immune reactivity at least until 90 DPI to boPPD and even more to the more specific antigens. 3.2. Skin test Figure 3 displays the skin tests results at the last control. It shows that the HIMBO was the group with the least reactivity. However, differences between groups were not statistically significant. NoVac and BCGO groups showed the highest reactivity with in the comparative test (100%) and CVPE the lowest (36%). 3.3. Clinical signs and post mortem lesions No clinical signs were observed after vaccination or challenge. All but five animals presented gross lesions compatible with TB in at least one location (one each from BCGO, CPVPE and HIMBO and two from NoVac) and ten did not yield any M. bovis isolation (one from BCGO, six from CPVPE, one from HIMBO and two from NoVac). The two negatives both to gross lesions and to isolation belonged to the BCGO and to the CPVPE groups. Figure 4 shows the mean scores and SEM of pathological and microbiological variables for each group and tissue. Both main effects, treatment (p = 0.0140) and tissue (p < 0.0001) and their interaction were significantly different (p = 0.0007) for isolation. For lesion score, treatment (p = 0.3113) was not statistically significant, but tissue (p < 0.0001) and their interaction (0.0796) were (Fig. 4 ). This implies vaccination would induce different effects depending on the tissue. Indeed, taken together, all vaccinated groups but the HIMBO had higher bacterial counts than the non-vaccinated controls (data not shown). However, there was a much lower mean count in lung for all vaccinated groups, accounting to between 84% and 95% reductions in lung but to 1019%, 144%, 276% and 855% increases (BCGO, CPVPE, HIMBO and HIMBP, respectively) in the lymphoid tissue. Right the opposite occurred with the lesion score that showed lower values in the NoVac groups than in any other ones in the non-lymphoid compartment (Fig. 4 ). 3.4. Correlation analyses No clear pattern appeared after submitting to correlation analysis the individual immune test results with the post-mortem pathological and microbiological results (Table 1 ). Considering all groups together, only 9 correlations out of 224 fulfilled the selection criteria (both Pearson and Spearman of the same sign and with a p < 0.1). All were negatively related to lesions except one that correlated early ESCF response at 15 DPI with bacterial counts in lung. Four related specific and generic M. bovis antigen reactions at late samplings with lung lesions and some were lost when including other non-lymphoid locations. Treatment groups seemed to behave differently each one showing many correlations that were lost when treated together in the all-groups analysis. The non-vaccinated controls only showed a significant correlation between Rv3615 and bacterial isolation in the lymphoid tissues, while in the vaccinated groups IGRA post-challenge responses to different antigens tended to correlate with lesion scores. A notable exception were correlations of basal IFNγ, avPPD and boPPD at the first control of the BCGO group. Since no treatment had been applied at that time, this could be interpreted the other way round, that is, that non-specific reactivity at vaccination time might interact with bacterial multiplication at the end of the experiment. The CPVPE group showed few correlations and somewhat contradictory. Even though the correlation of the avPPD with bacterial load correlated negatively both in lung and in lymphoid tissues by 90 DPI, there was a positive correlation of basal IFNγ at 90 DPI with bacterial load in lung. This group did not show any correlation of the immune response with the lesion scores. The HIMBO vaccinated group showed a positive relation between one of the specific antigens and the lung bacterial load at 30 DPI. This correlation was even more stable regarding the basal IFNγ levels, since it appeared by 30 DPI and persisted until the end of the experiment. Table 1 Correlations between IGRA and IDR results and post-mortem isolations and lesions according to location and compartment Lung Other Lymphoid Non-Lymphoid CFU Lesion score CFU Lesion score CFU Lesion score CFU Lesion score A/T r p A/T r p A/T r P A/T r p A/T r p A/T r p A/T r p A/T r p All groups - - - p22/60 -0.8985 < .0001 ESCF/15 0.3532 0.0296 - - - - - - ESCF/75 -0.3696 0.0224 - - - p22/60 -0.8236 < .0001 - - - Rv3020/60 -0.5333 0.0035 - - - - - - - - - Rv3615/75 -0.3374 0.0383 - - - Rv3020/60 -0.5803 0.0012 - - - boPPD/90 -0.8076 < .0001 - - - - - - - - - - - - - - - - - - - - - boPPD/150 -0.6752 < .0001 - - - - - - - - - - - - - - - - - - NoVac - - - - - - - - - - - - Rv3615 -0.8004 0.0054 - - - - - - - - - BCGO Nil/00 0.7834 0.0125 - - - - - - Rv3615/30 -0.7466 0.0208 p22/150 -0.9681 < .0001 avPPD/60 -0.6815 0.0432 avPPD/00 0.8694 0.0011 Rv3615/30 -0.6846 0.0419 avPPD/00 0.9607 < .0001 - - - - - - - - - - - - - - - boPPD/00 0.9874 < .0001 - - - boPPD/00 0.8494 0.0038 - - - - - - - - - - - - - - - ESCF/00 0.8229 0.0035 - - - boPPD/60 0.689 0.0401 - - - - - - - - - - - - - - - - - CPV Nil/A75 0.6965 0.0173 - - - - - - - - - avPPD/90 -0.618 0.0427 - - - Nil/A75 0.6965 0.0173 avPPD/A75 0.6863 0.0197 avPPD/90 -0.7214 0.0122 - - - - - - - - - - - - avPPD/90 -0.7214 0.0122 - - - HIMBO RV3020/30 0.7529 0.0192 p22/60 -0.9404 0.0002 Nil/30 0.8671 0.0025 - - - Nil/00 0.6948 0.0378 - - - Rv3020/30 0.753 0.0192 p22/60 -0.9311 0.0003 - - - Rv3615/30 -0.7824 0.0127 Nil/60 0.8181 0.007 - - - - - - - - - - - - Rv3615/30 -0.7982 0.0099 - - - boPPD/30 -0.723 0.0277 Nil/75 0.7632 0.0167 - - - - - - - - - - - - Rv3020/60 -0.738 0.0232 - - - boPPD/75 -0.9892 < .0001 Nil/90 Missing Missing - - - - - - - - - - - - boPPD/75 -0.9892 < .0001 - - - boPPD/90 Missing Missing Nil/150 0.7616 0.0171 - - - - - - - - - - - - boPPD/90 Missing Missing - - - avPPD/150 -0.7389 0.0229 AP2Dif 0.7669 0.0159 - - - - - - - - - - - - boPPD/150 -0.9887 < .0001 - - - boPPD/150 -0.9883 < .0001 - - - - - - - - - - - - - - - avPPD/150 -0.7598 0.0175 - - - ESCF/150 -0.8624 0.0028 - - - - - - - - - - - - - - - ESCF/150 -0.8839 0.0016 HIMBP - - - Nil/30 0.9068 0.0337 - - - ESCF/00 0.9416 0.0168 boPPD/15 -0.9924 0.0008 avPPD/00 0.9234 0.0251 - boPPD/15 0.8737 0.0529 - - - Nil/75 0.9599 0.0096 - - - Rv3615/00 0.8452 0.0714 boPPD/75 -0.9118 0.031 - - - - - - boPPD/75 0.9479 0.0141 - - - Nil/90 Missing Missing - - - Rv3020/00 0.8279 0.0834 boPPD/90 Missing Missing - - - - - - boPPD/90 Missing Missing - - - Nil/150 -0.9118 0.031 - - - Rv3020/15 0.8147 0.0931 boPPD/150 Missing Missing - - - - - - boPPD/150 Missing Missing - - - Rv3615/30 0.9895 0.0013 - - - - - - - - - - - - - - - - - - A/T: Antigen and time 3.5. Principal Components Analysis The factor analysis clearly shows that the specific immune response as assessed by the IGRA test with different antigens is strongly and negatively associated with the gross lesion scores defining the first component of data variability that accounts to 38.6% of it. The lymphoid bacterial load also aligns to this axis in positive association with pathology in both lung and lymphoid tissue. Strikingly, bacterial burden in lung and other tissues lay nearly at the 0 correlation with component 1, and define nearly completely the second component (15.3% of variability) along with gross lesions in other tissues and the basal IFNγ levels. 4. Discussion The most significant finding of this study is the substantial reduction in lung bacterial load achieved through vaccination. This result provides a mechanistic basis for epidemiological evidence that vaccination can reduce the basic reproduction rate (R₀) below 1, leading to a progressive decrease in tuberculosis prevalence in vaccinated populations [ 17 ]. A similar observation has been reported for paratuberculosis, another relevant mycobacteriosis in ruminants [ 27 , 64 ]. This conclusion is partly attributable to a refined vaccine efficacy assessment strategy that differentiates between lymphoid and environment-interface compartments[ 40 ], further elucidating the complex pathogenesis of mycobacteriosis. The experimental assays summarized here were designed to assess the immune mechanisms involved in the pathogenesis of M. bovis infection. They also evaluated sensitizations caused by various TB vaccine candidates, their delivery routes, potential interference with standard bovine tuberculosis diagnostic tests, and the protection offered by inactivated vaccines. The study tested several types of antigens (live and heat-inactivated homologous, as well as heterologous inactivated antigens) and two delivery routes for the inactivated vaccines. All tested antigens and routes demonstrated potential to elicit protective immune responses compared to non-vaccinated controls when challenged with a field strain of M. bovis . However, the nature of the response varied depending on the antigen, route, and the parameters measured, occasionally leading to higher bacterial loads or lesion scores in vaccinated groups. This variability underscores the need for tailored approaches to vaccine efficacy evaluation based on intervention goals. Unstimulated IFN-γ levels in blood remained at baseline during the pre-challenge period but increased by day 15 post-infection (DPI) across all groups for generic PPD antigens. Conversely, responses to specific antigens remained unchanged until 30 DPI, only reaching comparable levels across all groups by 60 DPI, with exceptions in the NoVac and BCGO groups for ESAT-CFP and Rv3615 antigens, respectively. This indicates that some vaccines may not induce false positives without infection, while enhancing the detection capabilities of in vitro cellular immunity tests during early post-infection stages, particularly for PPD antigens. However, delayed responses to defined antigens may reduce the sensitivity of cell-immunity-based tests. BCG and HIMBP vaccination showed minimal interference with standard diagnostic antigens but significantly reduced detectability with specific antigens. This aligns with previous findings in non-vaccinated animals [50–52, 65, 66], highlighting their potential for Differentiating Infected from Vaccinated Animals (DIVA) strategies in BCG or parenterally vaccinated HIMB individuals. Skin tests were evaluated only for sensitivity in this study, as they were performed solely at the final post-infection control. The standard boPPD appeared to benefit from vaccination, as significant differences relative to the NoVac control group were observed in all groups except HIMBO. The only difference observed with the defined antigen APHA1 was an increased mean thickening in the HIMBPE group, while no differences were noted with APHA2. The comparative test appeared to perform satisfactorily, particularly with respect to the MAP vaccine. In summary, vaccine interference either enhanced the detectability of true infections or was irrelevant when compared to the official skin test responses in non-vaccinated animals. Differentiating lung and lymphoid responses revealed distinct patterns of vaccination response possibly related to pathological and immune responses decoupling with bacterial load outside the lymphoid tissue according to the PCA. This might be an important observation that needs further attention as it could substantially change tuberculosis pathogenetic mechanism models. Vaccination appeared to influence bacterial load and lesion development in the lung but have less impact on lymphoid tissues. This supports the tuberculosis model of a lymphatic disease with entry and exit points in the lungs[ 40 ]. While protecting the lungs may reduce transmission, bacteria may persist in lymph nodes in a latent form. This dual effect supports a shift from eradication goals toward coexistence and control of transmission, as long as R₀ remains below 1 [ 17 ] and economic impacts are manageable. Oral administration of HIMB was associated with lower IFN-γ responses compared to parenteral routes, potentially indicating an anti-inflammatory effect. However, specific immune responses varied based on antigen type and delivery route, suggesting the need for tailored strategies to optimize protective efficacy. Vaccination reduced lung bacterial loads but did not consistently impact gross lesion scores, challenging their reliability as a measure of vaccine efficacy. Instead, focusing on bacterial load reductions in transmission-critical tissues such as lung is may provide a more accurate evaluation for epidemiological purposes. The study confirms discrepancies between experimental and field outcomes observed in previous research [25, 67]. Field studies often report higher vaccine efficacy, likely due to lower infectious doses generated by lower transmission from vaccinated but infected animals and reduced animal-to-animal contact. This supports the need for revised evaluation criteria that better reflect real-world conditions. The results suggest vaccination induces epidemiological latency, confining bacteria to lymphoid tissues and reducing transmission. This aligns with recent findings by Fromsa et al. (2024)[ 17 ], which demonstrated additive effects of vaccination in preventing dissemination and enhancing resistance in naïve individuals. Wider adoption of vaccines, particularly those offering higher lung protection and having lower biological risks, could significantly reduce control program costs. Additionally, the non-specific immune enhancement provided by these vaccines may reduce antibiotic usage and the associated risk of resistance development. 5. Conclusions This study demonstrates that vaccination significantly reduces lung bacterial loads while exerting minimal effects on lymphoid tissues. Gross lesion evaluations alone are insufficient to measure vaccine efficacy, particularly for transmission risk. The findings support the hypothesis that vaccination induces compartmentalized immune responses, providing a mechanistic explanation for its efficacy in reducing transmission. Effective control of chronic, multi-host infections like tuberculosis may benefit from shifting from eradication to strategies that strengthen barriers to spread and enhance population-level resilience. Vaccination goals in animals should prioritize reducing transmission rather than total sterilization, especially in multi-host ecosystems, in the initial stages and in those that have shown to be refractory to eradication. These findings underscore the potential of inactivated vaccines as biosafe and cost-effective tools for improving bovine tuberculosis control. They also highlight the need for revised evaluation methods to accurately assess vaccine efficacy in both experimental and field settings. Declarations Ethical Considerations All experimental procedures involving animal care and housing complied with European, national, and regional laws and ethical guidelines. The experimental design was reviewed and approved by NEIKER’s Animal Care and Use Committee (OEBA-NEIKER-2015-010) and the Department of Agriculture of Diputación Foral de Bizkaia (MYCOINVAC-39/2015-BFA). Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This study was supported with funds from the Spanish Ministry of Economy and Competitiveness (Research Project AGL2014-56305-C3-3-R) and the Department of Economic Development and Competitiveness of the Basque Government. MS held a fellowship from the Department of Education of the Basque Government (PRE_2017_2_0043). Contributions RAJ, JMG, ISE designed the experiments, obtained the funding and dealt with animal procurement, vaccination, sampling and general supervision. EMI, MFU, JMG and ISE carried out the necropsies and sample collection at post-mortem. MGE, EMO, AET, RAR, PVA and NEL helped in the samplings and laboratory processing. CGO helped in the manuscript drafting. RAJ summarized and standardized the data, performed the statistical analyses and wrote the manuscript. All authors read and approved the successive drafts. Then the manuscript was submitted to ChatGPT 4o for improving language and further changes were manually made. All authors read and approved the final version. Acknowledgements The authors would like to thank Gareth Jones and Martin Vordermeier (Animal and Plant Health Agency, UK) for supply of the defined antigen peptide cocktails used in IGRA and protein cocktails used in skin tests. We are indebted to the NEIKER-BRTA high biosafety animal facilities for their careful handling of the experimental animals. Data availability Final data used for evolution of immune response (EvolImmuneTests.xlsx) and post-mortem findings (Post-Mortem.xlsx) analyses are included as two Excel spreadsheet files. 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Juste","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIie2PMQrCQBBFRwKbMu0G9A4JgdiE3GUIpLQRZAuLFSGWtnZeIeAB3LCQatEDpPQCESsbNbGzGtMJ7it+Ne/zB8Bi+UlUHwmAw4YoCvJBytvSXX6rTFcmurXiPPNchhcBCe4loYyVibkyzdxfMx0ayLFUhMLBxFAVDZbaLXwJGktqWKdEbfU44VG7m7uEJz2sUwJeSYWlw+qRBIWSHlYvuKkz3GmW+TLIIvoXrg+tWKa43dbhVYp0Qg4D/lEaUOc9HllqsVgsf88LEURFTb/8zOYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6037-5873","institution":"NEIKER-BRTA","correspondingAuthor":true,"prefix":"","firstName":"Ramon","middleName":"A","lastName":"Juste","suffix":""},{"id":425505927,"identity":"a4400a2a-fd70-4317-9dac-d698f61dbd37","order_by":1,"name":"Iker A Sevilla","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Iker","middleName":"A","lastName":"Sevilla","suffix":""},{"id":425505928,"identity":"4e92b76a-0755-409f-9318-cd7be9279dac","order_by":2,"name":"Esmeralda Minguijon","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Esmeralda","middleName":"","lastName":"Minguijon","suffix":""},{"id":425505929,"identity":"415dab9f-7cb2-473d-99c6-1e487e513524","order_by":3,"name":"Miguel Fuertes","email":"","orcid":"","institution":"University of Leon Faculty of Veterinary Medicine: Universidad de Leon Facultad de Veterinaria","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Fuertes","suffix":""},{"id":425505930,"identity":"a192aa69-a7f5-4fb8-a00d-cb8fb1b58c98","order_by":4,"name":"Natalia Elguezabal","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Elguezabal","suffix":""},{"id":425505931,"identity":"343cc687-81ae-4706-ac58-2978008afaf4","order_by":5,"name":"Marivi Geijo","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Marivi","middleName":"","lastName":"Geijo","suffix":""},{"id":425505932,"identity":"88e703e6-2ee4-45af-8040-8955e52c4636","order_by":6,"name":"Patricia Vazquez","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Vazquez","suffix":""},{"id":425505933,"identity":"03ce830a-a49c-43ec-a9af-e81fc06375eb","order_by":7,"name":"Miriam Serrano","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Miriam","middleName":"","lastName":"Serrano","suffix":""},{"id":425505934,"identity":"e0c38ca1-ad06-4643-9e9a-8d5000ca0bdf","order_by":8,"name":"Rakel F Arrazuria","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Rakel","middleName":"F","lastName":"Arrazuria","suffix":""},{"id":425505935,"identity":"ed2d232f-cd95-4575-9384-3c2402e39f75","order_by":9,"name":"Christian Gortazar","email":"","orcid":"","institution":"IREC SaBio: Instituto e Investigacion en Recursos Cinegeticos Grupo de Investigacion Sanidad y Biotecnologia","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Gortazar","suffix":""},{"id":425505936,"identity":"be8629fb-38e2-4e0e-bbd3-75c4d02f31c5","order_by":10,"name":"Amaia Etxezarreta","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Amaia","middleName":"","lastName":"Etxezarreta","suffix":""},{"id":425505937,"identity":"ccf3fbff-f664-49b3-8fbc-68e9d469e771","order_by":11,"name":"Elena Molina","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Molina","suffix":""},{"id":425505938,"identity":"22eedf0a-8839-4c56-91f3-546fcf2afa91","order_by":12,"name":"Joseba M Garrido","email":"","orcid":"","institution":"NEIKER Basque Institute of Agricultural Research and Development Derio Center: Neiker Instituto Vasco de Investigacion y Desarrollo Agrario Centro de Derio","correspondingAuthor":false,"prefix":"","firstName":"Joseba","middleName":"M","lastName":"Garrido","suffix":""}],"badges":[],"createdAt":"2025-02-14 16:01:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6032013/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6032013/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-025-01637-2","type":"published","date":"2025-10-24T16:16:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78234057,"identity":"bd1ae79b-b973-4c8c-9863-9c3706f8fd96","added_by":"auto","created_at":"2025-03-11 08:07:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88509,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental Design. Study groups and experimental timeline. \u003cstrong\u003eNoVac:\u003c/strong\u003e Infected, non-vaccinated animals; \u003cstrong\u003eCPVP:\u003c/strong\u003eParenteral commercial paratuberculosis vaccine; \u003cstrong\u003eBCGO:\u003c/strong\u003e Oral BCG vaccine; \u003cstrong\u003eHIMBO:\u003c/strong\u003eOral heat-inactivated \u003cem\u003eM. bovis\u003c/em\u003e (HIMB) vaccine; \u003cstrong\u003eHIMBP:\u003c/strong\u003e Parenteral (intramuscular) HIMB vaccine. \u003cstrong\u003e00AV:\u003c/strong\u003e Day 0 of the experiment, pre-vaccination; \u003cstrong\u003e00V75:\u003c/strong\u003e Standardized day 75 post-vaccination; \u003cstrong\u003e015DPI:\u003c/strong\u003eStandardized day 15 post-infection; \u003cstrong\u003e030DPI:\u003c/strong\u003e Standardized day 30 post-infection; \u003cstrong\u003e060DPI:\u003c/strong\u003e Standardized day 60 post-infection; \u003cstrong\u003e075DPI:\u003c/strong\u003eStandardized day 75 post-infection; \u003cstrong\u003e090DPI:\u003c/strong\u003e Standardized day 90 post-infection; \u003cstrong\u003e150DPI:\u003c/strong\u003e Standardized day 150 post-infection. Numbers within arrows indicate actual days post-vaccination (DPV) and post-infection (DPI) for each experiment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/309b5852f8ff5f8f422b7bb6.png"},{"id":78232874,"identity":"1d65099d-149b-4575-8339-30d953469965","added_by":"auto","created_at":"2025-03-11 07:51:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":983806,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of IFNγ release assay (IGRA) and specific antibody responses by group and time. A) IGRA with no specific antigen, only PBS (notice that the y-axis scale is larger than for the antigen responses); B) IGRA with avian PPD tuberculin; C) IGRA with bovine PPD tuberculin; D) IGRA with p22 antigen (NoVac group was not processed); E) IGRA with ESAT6/CFP10 antigens; F) IGRA with Rv3615c; G) IGRA with Rv3020c antigen (CPVPE group was not processed). H) Frequency of positives in the standard IGRA diagnostic test with bovine PPD; I) PPD- Frequency of positives in the standard IGRA diagnostic test with avian PPD. NoVac: Non vaccinated control. CPVP: Parenteral commercial paratuberculosis vaccination. BCGO: Oral BCG vaccination. HIMBO: Oral vaccination with heat-inactivated M. bovis. HIMBP: Parenteral vaccination with heat-inactivated M. bovis. Error bar have been omitted to avoid overlapping and overall cluttering. Instead, relevant differences are reported in the text.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/23922bdd39f94b4b3f83c9c1.png"},{"id":78233700,"identity":"308a1983-6e25-4158-a993-4819b01752a1","added_by":"auto","created_at":"2025-03-11 07:59:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":532969,"visible":true,"origin":"","legend":"\u003cp\u003eSkin test results with different antigens aIDR: avian PPD; bIDR: bovine PPD; APHA1: peptide cocktail 1 (ESAT-6 CFP-10 and Rv3615c); APHA2: peptide cocktail 2 (ESAT-6, CFP-10, Rv3615c and Rv3020c). Only groups BCG and HIMBP had 100% positive results in the bPPD comparative test. No group had 100% positive results with a comparative criterion and APHA1 antigen. The APHA2 however detected 100% of non-vaccinated but failed to detect infected in the vaccinated groups. * APH2 not carried out for this treatment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/b3c57708f62e3340d773b1fd.png"},{"id":78232872,"identity":"a67313db-5552-4d48-85e8-0c441d32b337","added_by":"auto","created_at":"2025-03-11 07:51:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120476,"visible":true,"origin":"","legend":"\u003cp\u003ePlot of \u003cem\u003eM. bovis\u003c/em\u003e CFU and gross lesion score group means (with SEM) and reduction relative to NoVac according to treatment and compartment. CFU and lesion score reductions percent reduction below the compartment label. Notice the strong interaction for isolation of the NoVac groups versus the vaccinated ones.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/c60ad8ef93f7037419390dc2.png"},{"id":78233696,"identity":"55466fec-8d54-4fe7-9074-2525428ae5f5","added_by":"auto","created_at":"2025-03-11 07:59:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66585,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal components analysis of the final control IFNγ results with each antigen, gross lesion score and bacterial load according to tissue. Notice the alignment of immune response in IGRA with different antigens with lesions and lymphoid tissue bacterial load defining component 1 (38.6% of variability) and their decoupling with bacterial load in lung and other tissues. Lung_MTC: M. bovis isolation from lung; Lymp_MTC: \u003cem\u003eM. bovis\u003c/em\u003eisolation from lymph nodes. Other_MTC: \u003cem\u003eM. bovis\u003c/em\u003e isolation from other tissues. Lung_PAL: Lung lesion score; Lymp_PAL: Lymph node lesion score. Other_PAL: Other tissues lesion score.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/05cbc7e8d9c4bca9072ea58f.png"},{"id":94490242,"identity":"44717d39-f333-46d4-a9cd-42218dab1940","added_by":"auto","created_at":"2025-10-27 17:08:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2675705,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/c6537497-9222-4445-8b0f-2d7ed0aaa5ad.pdf"},{"id":78233702,"identity":"0816c9d8-af9d-4930-8854-ac17327e80d7","added_by":"auto","created_at":"2025-03-11 07:59:18","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":131652,"visible":true,"origin":"","legend":"","description":"","filename":"EvolImmuneTests.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/aa1a1623623bcfbbcc26d54a.xlsx"},{"id":78234058,"identity":"e0380410-f8c0-4050-a371-f73cde7b60f2","added_by":"auto","created_at":"2025-03-11 08:07:18","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":22766,"visible":true,"origin":"","legend":"","description":"","filename":"PostMortem.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6032013/v1/efe86d07dba1506f868aa490.xlsx"}],"financialInterests":"","formattedTitle":"Tuberculosis vaccination: Microbiological and immunological summary of a series of experimental challenge studies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAnimal tuberculosis (TB) is caused by species within the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex (MTC), primarily \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eM. caprae\u003c/em\u003e, which affect a wide range of hosts, including cattle. Since TB has long been recognized as a zoonotic disease, eradication programs targeting cattle have been implemented in many countries. These programs have been largely successful, particularly in reducing TB prevalence in the primary zoonotic reservoir, dairy cattle.\u003c/p\u003e \u003cp\u003eHowever, in recent decades, the epidemiology of TB has proven to be more complex than previously understood. Beyond dairy cattle, other livestock such as beef cattle, goats, pigs, and sheep managed in extensive systems\u0026mdash;often in contact with wildlife\u0026mdash;serve as significant hosts. These hosts, frequently outside the scope of traditional cattle-centered control programs, play a critical role in maintaining MTC in the environment [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, wildlife hosts contribute significantly to the MTC maintenance community [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCompounding this challenge is growing evidence of the limited sensitivity of traditional diagnostic methods [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], highlighting the need for alternative TB control strategies. Among these alternatives, vaccination has emerged as a promising tool. However, cattle vaccination has been banned since the 1950s due to concerns about interference with immune-based diagnostic tests used in eradication programs and doubts about its efficacy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, research conducted in Ethiopia has demonstrated the potential of cattle vaccination against TB. Even a nominal vaccine efficacy of 50% was shown to reduce the basic reproduction number (R₀) to less than 1, driving infection towards extinction at a significantly lower economic and social cost compared to test-and-cull strategies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite this, research efforts have predominantly focused on live BCG vaccines, while inactivated (killed) vaccines have been largely neglected [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The demonstrated success of inactivated vaccines in wildlife [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], their inherent safety, and insights gained from heat-inactivated paratuberculosis vaccines have rekindled interest in their use for TB control [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, the recent concept of trained immunity (TRAIM) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] provides theoretical support for the use of killed vaccines. Despite early observations suggesting TRAIM effects were exclusive to live vaccines [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the use of adjuvants in killed vaccines has shown potential to enhance their infection-fighting ability. Additionally, oral administration of inactivated vaccines, which avoids eliciting specific immune responses [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], presents a promising opportunity for a \u003cem\u003eDifferentiating Infected from Vaccinated Animals\u003c/em\u003e (DIVA) strategy. This approach may protect against infection without interfering with official diagnostic tests.\u003c/p\u003e \u003cp\u003eTraditionally, tuberculosis has been considered a respiratory disease due to its primary localization in the thorax. However, in cattle, the lungs\u0026mdash;histologically and functionally distinct from lymph nodes\u0026mdash;are less commonly affected. In Australia, for example, only 14.1% of gross lesions were located in the lungs, with 3.9% as single lesions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In Spain, prior to the implementation of test-and-cull programs in the 1950s, 48.8% of 1,561 slaughtered cattle displayed gross lesions in the lungs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By contrast, in the United States, no gross lesions were observed in a small sample of 15 cattle, although 6.7% were positive by isolation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These findings, combined with evidence of immune response compartmentalization[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] supports the view of tuberculosis being a lymphatic disease with lungs as its portal[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and suggest that giving the same weight to lymphatic infection as to lung infection may lead to an underestimation of epidemiological risks in this slow-progressing, low-transmissibility disease [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Furthermore, the lower vaccine efficacy observed in experimental challenge trials[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] compared to field conditions[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] may be partially attributed to differences in infection routes. Experimental models often bypass natural respiratory infection pathways, potentially biasing efficacy assessments.\u003c/p\u003e \u003cp\u003eBased on these premises, we aimed to compare homologous and heterologous killed vaccines with the current standard live BCG vaccine. Our study evaluates these vaccines in terms of \u003cem\u003eM. bovis\u003c/em\u003e isolation as proxy of transmission risk, gross TB lesions, specific immune responses, and DIVA performance.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Animals, Vaccination, and Sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis meta-analysis revisits data from four experimental vaccination and challenge trials involving a total of 41 calves. Results from these trials have been partially published in two reports: Trial 2\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003e[44]\u003c/span\u003e and Trial 4\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003e[45]\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1. First Trial:\u0026nbsp;Vaccine Diagnostic Interference and Protection with a Heterologous Vaccine\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix Friesian calves, aged 2\u0026ndash;3 months, were selected from farms in northern Spain with no known history of tuberculosis (TB) and tested negative for interferon-gamma release assay (IGRA). The calves were recruited at a feedlot and subcutaneously vaccinated with the commercial paratuberculosis vaccine \u003cem\u003eSilirum\u0026trade;\u003c/em\u003e (CZ Vaccines, Porri\u0026ntilde;o, Spain) (CPVP).\u003c/p\u003e\n\u003cp\u003eAfter 1.5 months, the calves were transported to the biosafety level 3 (BSL-3) facilities at Neiker-BRTA. Following a 32-day acclimatization period, the calves were challenged intratracheally with a recent \u003cem\u003eM. bovis\u003c/em\u003e isolate. Blood samples were collected from the jugular vein at days -72, 0, 13, 26, 62, 74, 91, and 146 post-infection. Blood was drawn into lithium heparin-coated tubes for cellular immunity assays (Figure 1).\u003c/p\u003e\n\u003cp\u003eOn day 146, all calves were euthanized following standard approved procedures and subjected to a full necropsy. Gross lesions were scored based on the method described by Palmer et al., 2007[46]. Samples were collected from seven lymph nodes, the tonsils, lungs, liver, and spleen for \u003cem\u003eM. bovis\u003c/em\u003e isolation using the Mycobacterial Growth Indicator Tube (MGIT) system (Becton Dickinson).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2. Second Trial: Vaccine Diagnostic Interference and Protection with Homologous and Heterologous Vaccines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment is described in detail in Serrano et al. (2017a, 2017b)[44, 45]. Briefly, ten male Friesian calves, similar in characteristics to those used in Experiment 1, were randomly assigned to one of two groups: five calves received the commercial Silirum\u0026trade; paratuberculosis vaccine (CPVP), and five calves were administered only phosphate-buffered saline (PBS) (NoVac group).\u003c/p\u003e\n\u003cp\u003eAfter 110 days, all calves were transported to the NEIKER-BRTA biosafety level 3 (BSL-3) facilities. Ten days after arrival, the animals were challenged intratracheally with M. bovis. Blood samples were collected on days -133, 0, 15, 29, 57, and 84 post-infection (DPI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.3. Third Trial: Vaccine Protection with Live and Inactivated Homologous Vaccines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFifteen calves were randomly assigned to one of three treatment groups:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNoVac\u003c/strong\u003e: Non-vaccinated calves received 2 mL of plain PBS. \u003cstrong\u003eBCGO\u003c/strong\u003e: Calves received an oral BCG vaccine (n=5). \u003cstrong\u003eHIMBO\u003c/strong\u003e: Calves received an oral heat-inactivated M. bovis (HIMB) vaccine (n=5). For the HIMB vaccine, a wild boar \u003cem\u003eM. bovis\u003c/em\u003e isolate (NEIKER Strain #1403) previously used in experiments was propagated and inactivated as described by Balseiro et al. (2017) [42], Garrido et al. (2011)[25], and Jones et al. (2016)[35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.4. Fourth Trial: Vaccine Protection with Live and Inactivated Homologous Vaccines by Different Routes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess differences in protection and diagnostic interference related to antigen type, dose, and administration route, fifteen calves were randomly assigned to one of three treatment groups: \u003cstrong\u003eBCGO\u003c/strong\u003e: Calves received an oral BCG vaccine. \u003cstrong\u003eHIMBO\u003c/strong\u003e: Calves received 2 mL of an oral HIMB suspension containing 10^7 CFU of heat-inactivated \u003cem\u003eM. bovis\u003c/em\u003e. \u003cstrong\u003eHIMBP\u003c/strong\u003e: Calves received 1 mL of an intramuscular HIMB suspension containing 10^3 CFU of heat-inactivated \u003cem\u003eM. bovis\u003c/em\u003e. This vaccine was formulated as a water-in-oil emulsion using 0.5 mL of Montanide\u0026trade; ISA 50V2 adjuvant (Seppic, Paris, France) and 0.5 mL of PBS containing the inactivated bacterial suspension heated at 80\u0026deg;C for 20 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Challenge Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn Experiments 1, 2, and 4, all calves were challenged on day 0 DPI via the endotracheal route with 2 mL of PBS containing approximately 5\u0026times;10^6 CFU of an \u003cem\u003eM. bovis\u003c/em\u003e field isolate. The isolate, NEIKER Strain #2575/08, was originally obtained from a naturally infected wild boar and had been used in previous studies [23, 45, 47, 48]. Calves were sedated intramuscularly with XILAGESIC\u0026reg; 2% (10 mg/50 kg; Laboratorios Calier, S.A., Barcelona, Spain) prior to inoculation. The bacterial suspension was delivered with a syringe by inserting a needle between two consecutive tracheal rings (positions 25\u0026ndash;30). Endotracheal air was aspirated to confirm placement before injecting the inoculum. In Experiment 3, the bacterial suspension was administered orally without sedation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Interferon\u003c/strong\u003e\u003cstrong\u003e-\u0026gamma; release assay (IGRA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood was collected from the caudal vein into BD Vacutainer\u0026trade; tubes containing lithium heparin (Becton Dickinson and Company, Sparks, MD, USA) (Figure 1). Lithium heparinized blood was aliquoted into cell culture plate wells for antigenic stimulation, which was initiated within 4 hours of collection. Samples were incubated overnight at 37\u0026deg;C in a 5% CO₂ incubator with different antigens and a nil control (Nil).\u003c/p\u003e\n\u003cp\u003eThe antigens used included:\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eStandard tuberculins\u003c/strong\u003e: avian purified protein derivative (\u003cem\u003eavPPD\u003c/em\u003e) and bovine purified protein derivative (\u003cem\u003eboPPD\u003c/em\u003e) (CZ Veterinaria, Porri\u0026ntilde;o, Spain).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eProtein complex\u003c/strong\u003e: \u003cem\u003eboPPD\u003c/em\u003e-derived protein P22 [49]\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDefined antigens\u003c/strong\u003e: Protein cocktails APHA-1 (ESAT-6 CFP-10 and Rv3615c; 10 \u0026micro;g each protein) and APHA-2 (ESAT-6, CFP-10, Rv3615c and Rv3020c; 10 \u0026micro;g each protein) kindly provided by Drs G Jones and M Vordermeier[35, 50\u0026ndash;53].\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eBoth PPDs and P22 were used at a final concentration of 20 \u0026micro;g/mL, while synthetic peptides were used at 5 \u0026micro;g/peptide/mL.\u003c/p\u003e\n\u003cp\u003eFollowing incubation, samples were centrifuged, and interferon-gamma (IFN-\u0026gamma;) levels in the blood supernatants were measured using either the Bovigam\u0026trade; TB kit (Thermo Fisher Scientific, Inc.) for the first and second experiments or the IDScreen\u0026reg; Ruminant IFN-\u0026gamma; ELISA kit (IDvet, Grabels, France) for the third and fourth experiments, as per the manufacturers\u0026apos; instructions. Optical densities (OD) were measured at 450 nm using a Multiskan\u0026trade; FC photometer (Thermo Scientific, Vantaa, Finland).\u003c/p\u003e\n\u003cp\u003eFor P22 and Defined antigens: Sample-to-positive (S/P) % values were calculated using the formula: ([(boPPD or p22 OD\u0026ndash;avPPD OD)/(positive control X̄OD\u0026ndash;negative control X̄OD)]\u0026times;100). For E/C, Rv3615c, and Rv3020c peptides, S/P% was calculated similarly, as: ([(E/C, Rv3615c or Rv3020c OD\u0026ndash;NIL OD)/(positive control X̄OD\u0026ndash;negative control X̄OD)]\u0026times;100). Values were interpreted according to Arrieta-Villegas et al. (2020). For the Bovigam Test: A positivity cut-off of 0.1 OD difference between the sample and Nil control was used. For the IDScreen\u0026reg; Kit: A standard cut-off value of S/P% \u0026ge; 16 was used to classify samples as positive for all antigens, including specific peptides.\u003c/p\u003e\n\u003cp\u003eIGRA assays for P22 and Rv3020c in the control group could not be conducted due to technical issues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Skin test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the last sampling all calves in all the experiments were submitted to a cervical intradermal test with both the standard avian (2,500 IU avPPD) and bovine (2,500 IU boPPD) in 0.1 ml doses as well as with two more defined antigens: protein cocktails APHA-1 (ESAT-6 CFP-10 and Rv3615c; 10 \u0026micro;g each protein) and APHA-2 (ESAT-6, CFP-10, Rv3615c and Rv3020c; 10 \u0026micro;g each protein) kindly provided by Drs G Jones and M Vordermeier \u003cspan lang=\"EN-GB\"\u003e[53\u0026ndash;57]\u003c/span\u003e. The skin thickness was measured before injection and 72 h after inoculation. The results of PPD skin thickness increase were interpreted according to the standards of official criteria (EU Council Directive 64/432/CEE and Spanish RD 2611/1996) for both Single Intradermal Test (SIT) and Comparative Intradermal Test (CIT). A calf was considered SIT positive when the skin thickness increase at the boPPD site was of more than 4 mm. For CIT, animals were deemed positive when the thickness increase of the boPPD injection site exceeded that of the avPPD site by more than 4 mm. With regard to APHA reagents, animals were considered APHA-1 and APHA-2 positive when the skin thickness increase was equal to or bigger than 2 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Clinical signs and post-mortem lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were housed in the NEIKER biosafety facilities and were fed a standard hay and concentrate diet with free access to an automatic drinking bowl. They had a daily overlayered straw bed and were supervised for clinical signs.\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, right after skin test reading, animals were sedated (10 mg/50 kg XILAGESIC\u0026reg; 2%; Laboratorios Calier, S.A., Barcelona, Spain) and then euthanized (4-6 ml/50 kg T61; Intervet International GMBH, Unterschleissheim, Germany). Upon standard necropsy procedure, calves were thoroughly inspected for TB-compatible lesions and samples from the head (mandibular, parotid and retropharyngeal lymph nodes (LNs) and tonsils), the thorax (prescapular, tracheobronchial and mediastinal LNs), the lungs (right and left cranial and caudal lobes and medium and accessory lobes), the abdomen (hepatic, jejunal and ileocecal LNs as well as liver and spleen) and other body areas (prefemoral and popliteal LNs) were collected for pathological and microbiological analysis. Both isolation and gross lesions results were grouped according to tissue as the sum of the individual samples counts or scores for lung, lymphoid, or other. Further grouping of lung and other tissues was made and defined as non-lymphoid compartment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.1 Gross pathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were visually inspected, palpated and sliced in search of TB-compatible lesions. A previously described pathological scoring system was used for LNs, lungs and other organs. LNs were scored as follows: 0, no visible lesions; 1, small focal lesion (1\u0026ndash;2 mm); 2, several small foci; 3, extensive lesions. Lungs and other organs were ranked as follows: 0, no visible lesions; 1, no external lesions but lesions detected upon slicing; 2, up to 5 lesions of less than 10 mm in diameter; 3, more than 5 lesions of less than 10 mm in diameter; 4, more than 1 distinct gross lesion bigger than 10 mm in diameter; 5, coalescing gross lesions. Histopathological evaluation was not used as it was considered that it added very little in quantitative terms to gross pathology for a comparative study like this one.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5.2 M. bovis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Lymph nodes (retropharyngeal, prescapular, tracheo-bronchial, mediastinal, hepatic and popliteal) and lung samples were systematically taken for isolation with or without gross lesions. Any other lymph node, liver, spleen and kidney showing gross lesions were sampled for isolation as well. Mycobacterial isolation was attempted simultaneously in Coletsos (Difco, Francisco Soria Melguizo SA, Madrid, Spain) and in BBL Mycobacteria growth indicator tubes (MGIT) (Becton Dickinson, Franklin Lakes, NJ, USA) as previously described \u003cspan lang=\"EN-GB\"\u003e[25]\u003c/span\u003e. Two grams of sample were homogenized in 10 ml of sterile distilled water. The homogenate was separated in two aliquots of 5 ml. One aliquot was processed for culture in supplemented MGIT (BACTEC MGIT growth supplement with PANTA) following manufacturer\u0026rsquo;s instructions. Five ml of 1.5% RonaCare Cetylpyridinium Chloride (Merck, Darmstadt, Germany) (w/v) was added to the remaining half of the homogenate, thoroughly mixed and incubated at room temperature for 12\u0026ndash;18 h. After centrifugation (2,500 \u0026times; g, 5 min) the pellets were cultured in Coletsos tubes. MGIT tubes were incubated for 42 days in a BACTEC MGIT 960 System and Coletsos tubes at 37\u0026deg;C for 4 months. Time to detection was transformed into CFU according to an exponential power equation according to Sevilla et al. 2021. Tubes with a time to detection longer than 15 days but shorter than 42 days (end of incubation) were assigned a value of 1 CFU. Tubes without growth within 42 days were scored as negative, unless there was growth in the solid media in which case, a value of 1 CFU was assigned to that sample. Results of isolation were grouped according to tissue type (lymph node, lung and other locations) and sum of CFU count per tissue type for each animal was calculated and retained as the basic experimental measure for statistical analysis. DNA was extracted from all positive cultures for PCR confirmation \u003cspan lang=\"EN-GB\"\u003e[58]\u003c/span\u003e and spoligotyping of \u003cem\u003eM. tuberculosis\u003c/em\u003e positives \u003cspan lang=\"EN-GB\"\u003e[59]\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Statistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunologic variables were analysed as dependent variables with a general linear model with treatment group and time as independent variables. In order to simplify and prioritize the global view, individual missing data for experiment and control cells were filled with the corresponding previous control data. Differences between groups were assessed for each main effect and interaction versus the NoVac group with the corresponding treatment group post-hoc comparisons. Isolation counts and gross lesion scores were used as the primary dependent variables. Even though from an individual perspective, pathology is the most important outcome, from an epidemiological view it is bacterial load what really determines population health. Therefore, all statistical testing was focused on bacterial counts in both compartments with occasionally more detailed focus on the lung. The sum of all samples isolations was chosen because it was deemed to be the most representative and the one variable that allowed a best discrimination between the two variables of interest. Given the high variability of bacterial counts and lesion scores between experiments tissue and animals, sums were corrected according to a ratio of each experiment mean to the fourth experiment mean. Bacterial counts and scores were submitted to a generalized linear model for tissue and treatment based in the recommendation to use a negative binomial distribution model to assess zero inflated count results with equidispersion according to the quotient Chi square by degrees of freedom overdispersion indicator (ODI) \u003cspan lang=\"EN-GB\"\u003e[60, 61]\u003c/span\u003e. To fall within the mathematical ranges of this type of distribution, all counts larger than 100 were transformed by division by 2000. This model had a higher proportion of reduction of error (R^2) than the Gaussian and the Poisson. Specifically, for CFU counts the treatment and tissue Gaussian, Poisson and negative binomial had an R^2 value of 0.1614, 0.3965 and 0.4538, respectively. Their ODI was 105.6735, 13.3576 and 0.9601, respectively. Post-hoc statistical pairwise comparisons were submitted to an uncorrected Student\u0026rsquo;s t test of significance when applied to vaccination versus non-vaccinated control or to the Bonferroni correction when applied to other comparisons \u003cspan lang=\"EN-GB\"\u003e[62]\u003c/span\u003e. Correlation between in vivo tests (IGRA, Ab-ELISA, SIT) results and isolation count and lesion score were calculated using both the Pearson and Spearman correlation tests. Only correlations with coefficients of the same sign in both tests and a p\u0026lt;0.1 were retained for discussion. Standard statistical significance was considered at p values \u0026lt;0.05, but p values \u0026lt;0.10 are also reported as of potentially relevant but of lower significance due to group low experimental units. In order to more graphically describe the relationships between immunological, microbiological and pathological variables final control results were submitted to a Principal Components Analysis (PCA) reduced to each one of the IGRA antigens (7 variables), and the microbiological (3) and pathological (3) ones by compartment. All statistical analyses were carried out using the Generalized Linear Model, GAMLj, Factor and frequency modules of jamovi application \u003cspan lang=\"EN-GB\"\u003e[62, 63]\u003c/span\u003e(The jamovi project (2022). jamovi. (Version 2.3) [Computer Software]. Retrieved from https://www.jamovi.org. R Core Team (2021). R: A Language and environment for statistical computing. (Version 4.1) [Computer software]. Retrieved from https://cran.r-project.org. (R packages retrieved from MRAN snapshot 2022-01-01). Gallucci, M. (2019). Factor. Retrieved from https://cran.r-project.org/package=psych. GAMLj: General analyses for linear models. [jamovi module]. Retrieved from https://gamlj.github.io/.)(Gallucci, 2019; The jamovi project, 2022).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 IGRA\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e displays the dynamics of immune response in the IFNγ release assay with different antigens including the Nil control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePanel A in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the levels of IFNγ in unstimulated blood. Overall, only time of sampling had a statistically significant effect on the basal blood IFNγ levels (p\u0026thinsp;=\u0026thinsp;0.0009), while neither treatment (p\u0026thinsp;=\u0026thinsp;0.1963) or interaction effects (p\u0026thinsp;=\u0026thinsp;0.2851) were significant. The control group showed peaks at 15 DPI, 75 DPI and 90 DPI that were marginally significantly different from the initial mean (p\u0026thinsp;=\u0026thinsp;0.0654, p\u0026thinsp;=\u0026thinsp;0.0581 and p\u0026thinsp;=\u0026thinsp;0.0357) and the other groups means (p\u0026thinsp;=\u0026thinsp;0.1272, 0.0637, 0.1840, and 0.1236 for BCGO, CPV P, HIMB-O and HIMB-P at 15 DPI, p\u0026thinsp;=\u0026thinsp;0.3482, 0.1816, 0.6238 and 0.1349 on 75 DPI, and p\u0026thinsp;=\u0026thinsp;0.4616, p\u0026thinsp;=\u0026thinsp;0.0724, p\u0026thinsp;=\u0026thinsp;0.5153 and p\u0026thinsp;=\u0026thinsp;0.09621 on day 90 respectively). The vaccinated groups showed also some increase in the IFNγ means between at the 75 and 90 DPI that were significantly different from pre-vaccination controls (p\u0026thinsp;=\u0026thinsp;0.0113 BCGO, p\u0026thinsp;=\u0026thinsp;0.0237 CPVP, 0.0066 HIMBO, p\u0026thinsp;=\u0026thinsp;0.030 HIMBP). These raised levels of basal IFNγ in untreated blood would be suggestive of an active immune response following challenge in the animals at these time points. Regarding the diagnostic antigens, time of sampling had the main effect on the aviPPD IGRA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel B), followed by interaction (p\u0026thinsp;=\u0026thinsp;0.0293) and treatment (p\u0026thinsp;=\u0026thinsp;0.0401). No differences were found before challenge in any of the groups (BCG p\u0026thinsp;=\u0026thinsp;0.2342, CPVP p\u0026thinsp;=\u0026thinsp;0.0536, HIMBO p\u0026thinsp;=\u0026thinsp;0.2433, HIMBP p\u0026thinsp;=\u0026thinsp;0.6860, NoVac p\u0026thinsp;=\u0026thinsp;0.0528) compared to each group\u0026rsquo;s first control, nor compared to NoVac (BCGO p\u0026thinsp;=\u0026thinsp;0. 9378, CPVPE p\u0026thinsp;=\u0026thinsp;0.3296, HIMBO p\u0026thinsp;=\u0026thinsp;0.2043, HIMBP p\u0026thinsp;=\u0026thinsp;0.9866). After vaccination and challenge, all groups significantly increased their avPPD IGRA mean readings, that never differed between them. Both vaccination and time of control were highly significant effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as was their interaction (p\u0026thinsp;=\u0026thinsp;0.0014) regarding the boPPD IGRA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel C). No significant differences were observed in the two pre-challenge controls (BCG 0.3565, CPV p\u0026thinsp;=\u0026thinsp;0.2741, HIMBO p\u0026thinsp;=\u0026thinsp;0.6092, HIMBP p\u0026thinsp;=\u0026thinsp;0.9770. NoVac p\u0026thinsp;=\u0026thinsp;0.4409), nor relative to NoVac group (BCG p\u0026thinsp;=\u0026thinsp;0.3860, HIMBO p\u0026thinsp;=\u0026thinsp;0.5212, HIMBPE p\u0026thinsp;=\u0026thinsp;0.9896). Afterwards, all groups had statistically significant increased means with respect to their initial readings and no differences between groups were observed until the end of the experiment except for the CPV groups that, starting on 30 DPI, sustained decreased readings relative to the NoVac group (p\u0026thinsp;=\u0026thinsp;0.0118, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, p\u0026thinsp;=\u0026thinsp;0.0104, p\u0026thinsp;=\u0026thinsp;0.0538). The more specific antigens behaved similarly. For p22 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel D), there were no significant differences in the pre-challenge controls between BCG and HIMB (no CPVP group for this antigen) and NoVac group (0.7585\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;p\u0026thinsp;=\u0026thinsp;0.8153). At 15 DPI, the BCG group had larger values than the NoVac (p\u0026thinsp;=\u0026thinsp;0.0305), while the HIMB did not differ (p\u0026thinsp;=\u0026thinsp;0.2847). From then on, BCG 0.0038, HIMB 0.0288 30 DPI; 0.9005 BCG, 0.3908 HIMB 60 DPI; 0.0082 BCG 0.0180 HIMB 75 DPI; 0.4231 BCG, 0.4412 HIMB 105 DPI. For ESAT/CFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel E), no significant differences were observed in the first two controls (0.7216\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;p \u0026lt;=0.9827) between NoVac and the vaccinated groups. At 15 DPI, both BCG (p\u0026thinsp;=\u0026thinsp;0002) and HIMB (p\u0026thinsp;=\u0026thinsp;0.0140) groups had higher readings than the NoVac which was not different from CPVP (p\u0026thinsp;=\u0026thinsp;0.9089). Afterwards, both BCG and HIMB (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) had higher readings than NoVac which had higher ones than CPVP (p\u0026thinsp;=\u0026thinsp;0.0193) for 30 DPI. These differences were maintained\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both M. bovis vaccines and for the CPVP (p\u0026thinsp;=\u0026thinsp;0.0002) by 60 DPI. By 75 DPI, the differences started to diminish p\u0026thinsp;=\u0026thinsp;0.0248 and p\u0026thinsp;=\u0026thinsp;0.0537) until at the last control there were no differences between groups (p\u0026thinsp;=\u0026thinsp;0.3589 and p\u0026thinsp;=\u0026thinsp;0.7459 for BCG and HIMB). Differences with the initial IGRA values were observed as soon as at 15 DPI (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0009) for all antigens. No significant differences in Rv3020v IGRA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel F) response were observed between NoVac (0.1682\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;p \u0026lt;=0.9463) and the rest of the groups except at 60 DPI when HIMB showed a smaller value (p\u0026thinsp;=\u0026thinsp;0.0417) while BCG was not significant (p\u0026thinsp;=\u0026thinsp;0.1999). Concerning Rv3615c (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel G) no differences were observed during the first three controls (0.5095\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;p \u0026gt;=0.9803). By 30 DPI, the BCG group showed a larger IGRA response p\u0026thinsp;=\u0026thinsp;0.0054) and CPVP smaller (p\u0026thinsp;=\u0026thinsp;0.0390) than NoVac which did not differ from HIMB (p\u0026thinsp;=\u0026thinsp;0.2969). By 60 DPI, CPVP had an even larger difference with NoVac (p\u0026thinsp;=\u0026thinsp;0.0018), while the other groups had values closer to the NoVac (p\u0026thinsp;=\u0026thinsp;0.2846 and p\u0026thinsp;=\u0026thinsp;0.9332 for BCG and HIMB, respectively). At the last control, no differences were observed between groups (p\u0026thinsp;=\u0026thinsp;0.7733 and p\u0026thinsp;=\u0026thinsp;0.7939 for BCG and HIMB: there was 150 DPI control for the CPVP group). The NoVac group increased the IGRA response from 30 DPI (p\u0026thinsp;=\u0026thinsp;0.0172) onwards (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0100). The differences were even larger in the BCG, HIMB and CPVP groups that also started showing statistically different mean values from the same 30 DPI (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) until the end when some convergence occurred (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0500). Panel I in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the bovine tuberculosis diagnostic positive results according to the supplier. Only one animal of the CPVPE group at the first control and another different one in the second (p\u0026thinsp;=\u0026thinsp;0.5165 and p\u0026thinsp;=\u0026thinsp;0.6338, respectively) were classified as positive by the IGRA in the first experiment. After challenge, positivity frequencies ranged between 20 and 50 in the 15 DPI control (p\u0026thinsp;=\u0026thinsp;0.8087), while in the 30 DPI and 60 DPI controls the HIMBP reached 100% positivity but CPVPE decreased to 9.1% and 18.2% (p\u0026thinsp;=\u0026thinsp;0.0039 and p\u0026thinsp;=\u0026thinsp;0.0040, respectively). This would indicate that HIMBP experienced no diagnostic interference from the 30 DPI onwards, while the CPVPE seemed to inhibit the specific cell immune reactivity at least until 90 DPI to boPPD and even more to the more specific antigens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Skin test\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays the skin tests results at the last control. It shows that the HIMBO was the group with the least reactivity. However, differences between groups were not statistically significant. NoVac and BCGO groups showed the highest reactivity with in the comparative test (100%) and CVPE the lowest (36%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Clinical signs and post mortem lesions\u003c/h2\u003e \u003cp\u003eNo clinical signs were observed after vaccination or challenge. All but five animals presented gross lesions compatible with TB in at least one location (one each from BCGO, CPVPE and HIMBO and two from NoVac) and ten did not yield any \u003cem\u003eM. bovis\u003c/em\u003e isolation (one from BCGO, six from CPVPE, one from HIMBO and two from NoVac). The two negatives both to gross lesions and to isolation belonged to the BCGO and to the CPVPE groups. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the mean scores and SEM of pathological and microbiological variables for each group and tissue. Both main effects, treatment (p\u0026thinsp;=\u0026thinsp;0.0140) and tissue (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and their interaction were significantly different (p\u0026thinsp;=\u0026thinsp;0.0007) for isolation. For lesion score, treatment (p\u0026thinsp;=\u0026thinsp;0.3113) was not statistically significant, but tissue (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and their interaction (0.0796) were (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This implies vaccination would induce different effects depending on the tissue. Indeed, taken together, all vaccinated groups but the HIMBO had higher bacterial counts than the non-vaccinated controls (data not shown). However, there was a much lower mean count in lung for all vaccinated groups, accounting to between 84% and 95% reductions in lung but to 1019%, 144%, 276% and 855% increases (BCGO, CPVPE, HIMBO and HIMBP, respectively) in the lymphoid tissue. Right the opposite occurred with the lesion score that showed lower values in the NoVac groups than in any other ones in the non-lymphoid compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Correlation analyses\u003c/h2\u003e \u003cp\u003eNo clear pattern appeared after submitting to correlation analysis the individual immune test results with the post-mortem pathological and microbiological results (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Considering all groups together, only 9 correlations out of 224 fulfilled the selection criteria (both Pearson and Spearman of the same sign and with a p\u0026thinsp;\u0026lt;\u0026thinsp;0.1). All were negatively related to lesions except one that correlated early ESCF response at 15 DPI with bacterial counts in lung. Four related specific and generic \u003cem\u003eM. bovis\u003c/em\u003e antigen reactions at late samplings with lung lesions and some were lost when including other non-lymphoid locations. Treatment groups seemed to behave differently each one showing many correlations that were lost when treated together in the all-groups analysis. The non-vaccinated controls only showed a significant correlation between Rv3615 and bacterial isolation in the lymphoid tissues, while in the vaccinated groups IGRA post-challenge responses to different antigens tended to correlate with lesion scores. A notable exception were correlations of basal IFNγ, avPPD and boPPD at the first control of the BCGO group. Since no treatment had been applied at that time, this could be interpreted the other way round, that is, that non-specific reactivity at vaccination time might interact with bacterial multiplication at the end of the experiment. The CPVPE group showed few correlations and somewhat contradictory. Even though the correlation of the avPPD with bacterial load correlated negatively both in lung and in lymphoid tissues by 90 DPI, there was a positive correlation of basal IFNγ at 90 DPI with bacterial load in lung. This group did not show any correlation of the immune response with the lesion scores. The HIMBO vaccinated group showed a positive relation between one of the specific antigens and the lung bacterial load at 30 DPI. This correlation was even more stable regarding the basal IFNγ levels, since it appeared by 30 DPI and persisted until the end of the experiment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations between IGRA and IDR results and post-mortem isolations and lesions according to location and compartment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"25\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c20\" colnum=\"20\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c21\" colnum=\"21\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c22\" colnum=\"22\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c23\" colnum=\"23\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c24\" colnum=\"24\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c25\" colnum=\"25\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eLung\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c13\" namest=\"c8\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c19\" namest=\"c14\"\u003e \u003cp\u003eLymphoid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c25\" namest=\"c20\"\u003e \u003cp\u003eNon-Lymphoid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eLesion score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003eLesion score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c19\" namest=\"c17\"\u003e \u003cp\u003eLesion score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c22\" namest=\"c20\"\u003e \u003cp\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c25\" namest=\"c23\"\u003e \u003cp\u003eLesion score\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eA/T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003ep\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAll groups\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep22/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.8985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eESCF/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.3532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eESCF/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-0.3696\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e0.0224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003ep22/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.8236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRv3020/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.5333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eRv3615/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-0.3374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e0.0383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eRv3020/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.5803\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eboPPD/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.6752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e 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align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eRv3615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-0.8004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.0054\u003c/p\u003e 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colname=\"c2\"\u003e \u003cp\u003eNil/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.7834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eRv3615/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-0.7466\u003c/p\u003e \u003c/td\u003e 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align=\"left\" colname=\"c23\"\u003e \u003cp\u003eRv3615/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.6846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0419\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eavPPD/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eboPPD/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e0.9874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eboPPD/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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colname=\"c23\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNil/A75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.6965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e 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align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eavPPD/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-0.7214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e0.0122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIMBO\u003c/p\u003e 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colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eNil/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.6948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.0378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003eRv3020/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e0.753\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e0.0192\u003c/p\u003e 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colname=\"c8\"\u003e \u003cp\u003eNil/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.8181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eRv3615/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.7982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eRv3020/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.738\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0232\u003c/p\u003e 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align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eboPPD/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.9892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eboPPD/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNil/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.7616\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.0171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e 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align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eboPPD/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.9887\u003c/p\u003e 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\u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eavPPD/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.7598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0175\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eESCF/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.8624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eESCF/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-0.8839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIMBP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNil/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eESCF/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.9416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.0168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eboPPD/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-0.9924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.0008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003eavPPD/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e0.9234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e0.0251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eboPPD/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e0.8737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0529\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNil/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eRv3615/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.8452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.0714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eboPPD/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-0.9118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eboPPD/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e0.9479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e0.0141\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNil/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eRv3020/00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.8279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.0834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eboPPD/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eboPPD/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNil/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.9118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eRv3020/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.8147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.0931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eboPPD/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003eboPPD/150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003eMissing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRv3615/30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c23\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c24\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c25\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"25\" nameend=\"c25\" namest=\"c1\"\u003e \u003cp\u003eA/T: Antigen and time\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Principal Components Analysis\u003c/h2\u003e \u003cp\u003eThe factor analysis clearly shows that the specific immune response as assessed by the IGRA test with different antigens is strongly and negatively associated with the gross lesion scores defining the first component of data variability that accounts to 38.6% of it. The lymphoid bacterial load also aligns to this axis in positive association with pathology in both lung and lymphoid tissue. Strikingly, bacterial burden in lung and other tissues lay nearly at the 0 correlation with component 1, and define nearly completely the second component (15.3% of variability) along with gross lesions in other tissues and the basal IFNγ levels.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe most significant finding of this study is the substantial reduction in lung bacterial load achieved through vaccination. This result provides a mechanistic basis for epidemiological evidence that vaccination can reduce the basic reproduction rate (R₀) below 1, leading to a progressive decrease in tuberculosis prevalence in vaccinated populations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A similar observation has been reported for paratuberculosis, another relevant mycobacteriosis in ruminants [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. This conclusion is partly attributable to a refined vaccine efficacy assessment strategy that differentiates between lymphoid and environment-interface compartments[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], further elucidating the complex pathogenesis of mycobacteriosis.\u003c/p\u003e \u003cp\u003eThe experimental assays summarized here were designed to assess the immune mechanisms involved in the pathogenesis of \u003cem\u003eM. bovis\u003c/em\u003e infection. They also evaluated sensitizations caused by various TB vaccine candidates, their delivery routes, potential interference with standard bovine tuberculosis diagnostic tests, and the protection offered by inactivated vaccines. The study tested several types of antigens (live and heat-inactivated homologous, as well as heterologous inactivated antigens) and two delivery routes for the inactivated vaccines.\u003c/p\u003e \u003cp\u003eAll tested antigens and routes demonstrated potential to elicit protective immune responses compared to non-vaccinated controls when challenged with a field strain of \u003cem\u003eM. bovis\u003c/em\u003e. However, the nature of the response varied depending on the antigen, route, and the parameters measured, occasionally leading to higher bacterial loads or lesion scores in vaccinated groups. This variability underscores the need for tailored approaches to vaccine efficacy evaluation based on intervention goals.\u003c/p\u003e \u003cp\u003eUnstimulated IFN-γ levels in blood remained at baseline during the pre-challenge period but increased by day 15 post-infection (DPI) across all groups for generic PPD antigens. Conversely, responses to specific antigens remained unchanged until 30 DPI, only reaching comparable levels across all groups by 60 DPI, with exceptions in the NoVac and BCGO groups for ESAT-CFP and Rv3615 antigens, respectively. This indicates that some vaccines may not induce false positives without infection, while enhancing the detection capabilities of in vitro cellular immunity tests during early post-infection stages, particularly for PPD antigens. However, delayed responses to defined antigens may reduce the sensitivity of cell-immunity-based tests.\u003c/p\u003e \u003cp\u003eBCG and HIMBP vaccination showed minimal interference with standard diagnostic antigens but significantly reduced detectability with specific antigens. This aligns with previous findings in non-vaccinated animals [50\u0026ndash;52, 65, 66], highlighting their potential for \u003cem\u003eDifferentiating Infected from Vaccinated Animals\u003c/em\u003e (DIVA) strategies in BCG or parenterally vaccinated HIMB individuals.\u003c/p\u003e \u003cp\u003eSkin tests were evaluated only for sensitivity in this study, as they were performed solely at the final post-infection control. The standard boPPD appeared to benefit from vaccination, as significant differences relative to the NoVac control group were observed in all groups except HIMBO. The only difference observed with the defined antigen APHA1 was an increased mean thickening in the HIMBPE group, while no differences were noted with APHA2. The comparative test appeared to perform satisfactorily, particularly with respect to the MAP vaccine. In summary, vaccine interference either enhanced the detectability of true infections or was irrelevant when compared to the official skin test responses in non-vaccinated animals.\u003c/p\u003e \u003cp\u003eDifferentiating lung and lymphoid responses revealed distinct patterns of vaccination response possibly related to pathological and immune responses decoupling with bacterial load outside the lymphoid tissue according to the PCA. This might be an important observation that needs further attention as it could substantially change tuberculosis pathogenetic mechanism models. Vaccination appeared to influence bacterial load and lesion development in the lung but have less impact on lymphoid tissues. This supports the tuberculosis model of a lymphatic disease with entry and exit points in the lungs[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. While protecting the lungs may reduce transmission, bacteria may persist in lymph nodes in a latent form. This dual effect supports a shift from eradication goals toward coexistence and control of transmission, as long as R₀ remains below 1 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and economic impacts are manageable.\u003c/p\u003e \u003cp\u003eOral administration of HIMB was associated with lower IFN-γ responses compared to parenteral routes, potentially indicating an anti-inflammatory effect. However, specific immune responses varied based on antigen type and delivery route, suggesting the need for tailored strategies to optimize protective efficacy.\u003c/p\u003e \u003cp\u003eVaccination reduced lung bacterial loads but did not consistently impact gross lesion scores, challenging their reliability as a measure of vaccine efficacy. Instead, focusing on bacterial load reductions in transmission-critical tissues such as lung is may provide a more accurate evaluation for epidemiological purposes.\u003c/p\u003e \u003cp\u003eThe study confirms discrepancies between experimental and field outcomes observed in previous research [25, 67]. Field studies often report higher vaccine efficacy, likely due to lower infectious doses generated by lower transmission from vaccinated but infected animals and reduced animal-to-animal contact. This supports the need for revised evaluation criteria that better reflect real-world conditions.\u003c/p\u003e \u003cp\u003eThe results suggest vaccination induces epidemiological latency, confining bacteria to lymphoid tissues and reducing transmission. This aligns with recent findings by Fromsa et al. (2024)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], which demonstrated additive effects of vaccination in preventing dissemination and enhancing resistance in na\u0026iuml;ve individuals.\u003c/p\u003e \u003cp\u003eWider adoption of vaccines, particularly those offering higher lung protection and having lower biological risks, could significantly reduce control program costs. Additionally, the non-specific immune enhancement provided by these vaccines may reduce antibiotic usage and the associated risk of resistance development.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study demonstrates that vaccination significantly reduces lung bacterial loads while exerting minimal effects on lymphoid tissues. Gross lesion evaluations alone are insufficient to measure vaccine efficacy, particularly for transmission risk. The findings support the hypothesis that vaccination induces compartmentalized immune responses, providing a mechanistic explanation for its efficacy in reducing transmission.\u003c/p\u003e \u003cp\u003eEffective control of chronic, multi-host infections like tuberculosis may benefit from shifting from eradication to strategies that strengthen barriers to spread and enhance population-level resilience. Vaccination goals in animals should prioritize reducing transmission rather than total sterilization, especially in multi-host ecosystems, in the initial stages and in those that have shown to be refractory to eradication.\u003c/p\u003e \u003cp\u003eThese findings underscore the potential of inactivated vaccines as biosafe and cost-effective tools for improving bovine tuberculosis control. They also highlight the need for revised evaluation methods to accurately assess vaccine efficacy in both experimental and field settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving animal care and housing complied with European, national, and regional laws and ethical guidelines. The experimental design was reviewed and approved by NEIKER\u0026rsquo;s Animal Care and Use Committee (OEBA-NEIKER-2015-010) and the Department of Agriculture of Diputaci\u0026oacute;n Foral de Bizkaia (MYCOINVAC-39/2015-BFA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported with funds from the Spanish Ministry of Economy and Competitiveness (Research Project AGL2014-56305-C3-3-R) and the Department of Economic Development and Competitiveness of the Basque Government. MS held a fellowship from the Department of Education of the Basque Government (PRE_2017_2_0043).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAJ, JMG, ISE designed the experiments, obtained the funding and dealt with animal procurement, vaccination, sampling and general supervision. EMI, MFU, JMG and ISE carried out the necropsies and sample collection at post-mortem. MGE, EMO, AET, RAR, PVA and NEL helped in the samplings and laboratory processing. CGO helped in the manuscript drafting. RAJ summarized and standardized the data, performed the statistical analyses and wrote the manuscript. All authors read and approved the successive drafts. Then the manuscript was submitted to ChatGPT 4o for improving language and further changes were manually made. All authors read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Gareth Jones and Martin Vordermeier (Animal and Plant Health Agency, UK) for supply of the defined antigen peptide cocktails used in IGRA and protein cocktails used in skin tests. We are indebted to the NEIKER-BRTA high biosafety animal facilities for their careful handling of the experimental animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinal data used for evolution of immune response (EvolImmuneTests.xlsx) and post-mortem findings (Post-Mortem.xlsx) analyses are included as two Excel spreadsheet files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBezos J, S\u0026aacute;ez-Llorente JL, \u0026Aacute;lvarez J, et al (2023) Bovine tuberculosis in Spain, is it really the final countdown? 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Vet Immunol Immunopathol 181:10\u0026ndash;14. https://doi.org/10.1016/J.VETIMM.2016.02.003\u003c/li\u003e\n\u003cli\u003eJones GJ, Gordon S V., Hewinson RG, Vordermeier HM (2010) Screening of Predicted Secreted Antigens from Mycobacterium bovis Reveals the Immunodominance of the ESAT-6 Protein Family. Infect Immun 78:1326. https://doi.org/10.1128/IAI.01246-09\u003c/li\u003e\n\u003cli\u003eJones GJ, Konold T, Hurley S, et al (2022) Test performance data demonstrates utility of a cattle DIVA skin test reagent (DST-F) compatible with BCG vaccination. Sci Rep 12:1\u0026ndash;8. https://doi.org/10.1038/s41598-022-16092-8\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Veiga L, Fuertes M, Geijo M V., et al (2023) Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria. 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J Clin Microbiol 48:3176\u0026ndash;3181. https://doi.org/10.1128/JCM.00420-10\u003c/li\u003e\n\u003cli\u003eSevilla IA, Molina E, Elguezabal N, et al (2015) Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay. J Clin Microbiol 53:. https://doi.org/10.1128/JCM.03168-14\u003c/li\u003e\n\u003cli\u003eKamerbeek J, Schouls L, Kolk A, et al (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosisfor diagnosis and epidemiology. J Clin Microbiol 35:907\u0026ndash;914\u003c/li\u003e\n\u003cli\u003eLindsey J (1999) On the use of corrections for overdispersion. Appl Stat 48:553\u0026ndash;561\u003c/li\u003e\n\u003cli\u003ePagui EKC, Salvan A, Sartori N (2022) Improved estimation in negative binomial regression. https://doi.org/10.1002/sim.9361\u003c/li\u003e\n\u003cli\u003eGallucci M (2019) GAMLj: General Analyses for the Linear Model in Jamovi\u003c/li\u003e\n\u003cli\u003eThe jamovi project (2022) jamovi (Version 2.3) [Computer software]\u003c/li\u003e\n\u003cli\u003eGarrido J, Juste R (2023) 8. The whole story of the long-term vaccination trial in dairy cattle in the Basque Country. What about non-specific effects? In: Proceedings of the 7th and 8th IDF Paratuberculosis Fora. IDF/FIL\u003c/li\u003e\n\u003cli\u003eCoad M, Clifford DJ, Vordermeier HM, Whelan AO (2013) The consequences of vaccination with the Johne\u0026rsquo;s disease vaccine, Gudair, on diagnosis of bovine tuberculosis. Vet Rec 172:266. https://doi.org/10.1136/vr.101201\u003c/li\u003e\n\u003cli\u003eCockle PJ, Gordon S V., Hewinson RG, Vordermeier HM (2006) Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle. Clinical and Vaccine Immunology 13:1119\u0026ndash;1124. https://doi.org/10.1128/CVI.00209-06\u003c/li\u003e\n\u003cli\u003eGortazar C, Beltr\u0026aacute;n-Beck B, Garrido JM, et al (2014) Oral re-vaccination of Eurasian wild boar with Mycobacterium bovis BCG yields a strong protective response against challenge with a field strain. BMC Vet Res 10:. https://doi.org/10.1186/1746-6148-10-96\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tuberculosis, cattle, skin test, interferon gamma, infection, transmission, vaccine, inactivated vaccine, Interferon gamma, M. bovis, route","lastPublishedDoi":"10.21203/rs.3.rs-6032013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6032013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Bovine tuberculosis (TB) caused by \u003cem\u003eMycobacterium bovis\u003c/em\u003e remains a persistent zoonotic and agricultural challenge, despite advances in eradication programs targeting cattle. The complexity of TB epidemiology, especially in extensive livestock systems and wildlife reservoirs, necessitates novel control strategies. Vaccination has re-emerged as a promising tool, with growing interest in inactivated vaccines for both efficacy and diagnostic compatibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e: This study evaluates the comparative efficacy of homologous and heterologous inactivated vaccines against the standard live Bacille Calmette–Guérin (BCG) vaccine in calves, focusing on \u003cem\u003eM. bovis\u003c/em\u003e isolation, gross TB lesions, immune responses, and compatibility with Differentiating Infected from Vaccinated Animals (DIVA) strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Data from four controlled vaccination and challenge trials involving 41 calves were analyzed. Animals were vaccinated with live BCG or heat-inactivated \u003cem\u003eM. bovis\u003c/em\u003e via the oral or parenteral routes, with immunologic assays, skin tests, and post-mortem analyses conducted to evaluate vaccine performance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: All vaccination strategies significantly reduced \u003cem\u003eM. bovis \u003c/em\u003ebacterial loads in the lungs compared to non-vaccinated controls, achieving up to 99% reductions. However, bacterial loads in lymphoid tissues increased, underscoring tuberculosis as a primarily lymphatic disease. Diagnostic interference varied by vaccine type and administration route, with oral administration showing lower interference. Gross lesion scores were inconsistent across groups, suggesting limited utility as a measure of vaccine efficacy. Immune responses revealed enhanced detection of infection post-vaccination, particularly with inactivated vaccines, which showed promising compatibility with DIVA strategies. Lung bacterial load appeared to be decoupled from cellular immune responses and lymph node lesions and bacterial load which were negatively correlated among themselves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study demonstrates that inactivated vaccines offer a safe and effective means of reducing TB transmission by confining bacterial presence to lymphoid tissues and minimizing diagnostic interference. Vaccination programs should shift from eradication goals to transmission control, prioritizing reductions in reproductive rate (R₀) over total bacterial clearance. These findings highlight the need for revised evaluation criteria and support the integration of inactivated vaccines into TB control strategies.\u003c/p\u003e","manuscriptTitle":"Tuberculosis vaccination: Microbiological and immunological summary of a series of experimental challenge studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-11 07:51:13","doi":"10.21203/rs.3.rs-6032013/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2cd07f2-c253-4ecb-9ca1-a3f998312eae","owner":[],"postedDate":"March 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:42:28+00:00","versionOfRecord":{"articleIdentity":"rs-6032013","link":"https://doi.org/10.1186/s13567-025-01637-2","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2025-10-24 16:16:20","publishedOnDateReadable":"October 24th, 2025"},"versionCreatedAt":"2025-03-11 07:51:13","video":"","vorDoi":"10.1186/s13567-025-01637-2","vorDoiUrl":"https://doi.org/10.1186/s13567-025-01637-2","workflowStages":[]},"version":"v1","identity":"rs-6032013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6032013","identity":"rs-6032013","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}