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Mycobacterium bovis (Mb) is the causative agent of bovine tuberculosis (bTb). Genetic selection aiming to identify less susceptible animals has been proposed as a complementary measure in ongoing programs toward controlling Mb infection. However, individual animal phenotypes for bTb based on interferon-gamma (IFNɣ) and its use in bovine selective breeding programs have not been explored. In the current study, IFNɣ production was measured using a specific IFNɣ ELISA kit in bovine purified protein derivative (bPPD)-stimulated blood samples collected from Holstein cattle. DNA isolated from the peripheral blood samples collected from the animals included in the study was genotyped with the EuroG Medium Density bead Chip, and the genotypes were imputed to whole-genome sequences. A genome-wide association analysis (GWAS) revealed that the IFNɣ in response to bPPD was associated with a specific genetic profile (heritability = 0.23) and allowed the identification of 163 SNPs, 72 quantitative trait loci (QTLs), 197 candidate genes, and 8 microRNAs (miRNAs) associated with this phenotype. No negative correlations between this phenotype and other phenotypes and traits included in the Spanish breeding program were observed. Taken together, our results define a heritable and distinct immunogenetic profile associated with strong production of IFNɣ in response to Mb.

The mechanisms underlying host resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are largely unknown. In the current study, we hypothesize that cows with an ability to produce higher levels of interferon-gamma (IFNɣ) might control MAP infection more successfully. To test this hypothesis, IFNɣ production was measured using a specific IFNɣ ELISA kit in avian purified protein derivative (aPPD)-stimulated blood samples collected from 152 Holstein cattle. DNA isolated from peripheral blood samples of the animals included in the study was genotyped with the EuroG Medium-Density Bead Chip, and the genotypes were imputed to whole-genome sequencing. A genome-wide association analysis (GWAS) revealed that high levels of IFNɣ in response to the aPPD were associated with a specific genetic profile (heritability = 0.64) and allowed the identification of 71 SNPs, 40 quantitative trait loci (QTL), and 104 candidate genes. A functional analysis using the 104 candidate genes revealed a significant enrichment of genes involved in the innate immune response and, more specifically, in necroptosis. Taken together, our results define a heritable and distinct immunogenetic profile associated with the production of high IFNɣ levels and with the capacity of the host to lyse MAP-infected macrophages by necroptosis.

Badgers (Meles meles) are a major tuberculosis (TB) reservoir in Europe, with the potential to transmit infection to cattle. Here we assessed whether a recently described oral tuberculosis vaccine based on heat-inactivated Mycobacterium bovis (HIMB), delivered as edible baits, can protect badgers from infection. Eight badgers were given individually five baits, each one consisting of a ball of peanut butter, natural peanut and oat flakes including a dose of the vaccine containing 5 × 107 colony-forming units. In parallel, a control group of seven badgers did not receive the vaccine. One month and a half later a second dose of the vaccine was offered to the vaccinated group. Ninety-four days after the second dose, all badgers were challenged with M. bovis (103 colony-forming units per animal) delivered endobronchially to the right middle lung lobe. Clinical, immunological, pathological and bacteriological variables were measured throughout the whole study to assess the efficacy of the vaccine. Two vaccinated animals showed high bacterial load of M. bovis and worsening of pathological lesions of TB. Conversely, the other six vaccinated animals showed slight improvement in bacterial load and pathology with respect to the control group. These results suggest that delivering the TB vaccine via food bait can partially protect wild badger populations, although vaccination can lead to either protection or tolerization, likely depending on the animal's immune status and general condition at the time of vaccination. Further optimization of the vaccination trial/strategy is needed to reduce the rate of tolerization, such as altering vaccine dose, number of doses, type of bait, use of adjuvants or route of administration.

The Preprint section in the original version of this Article was omitted. The Preprint section now reads: “This article was submitted to an online preprint archive58.” As a result Reference 58 was omitted, which is listed below. 58. Canive, M., et al. Host Genetics Underlying Pathological Outcomes to Mycobacterium Avium Subsp. Paratuberculosis Infection is Governed by Distinct Genetic Variants. Preprint at https:// doi. org/ 10. 21203/ rs.3. rs- 668666/ v1 (2021). The original Article has been corrected.

Table S1. Summary of the distributions of the proportion of the population harvested each year and supporting data. Figure S1. Histogram and probability density of the estimated proportion of the population harvested each year, autocorrelation and trace plots. Table S2. Summary of the estimated distributions of abundance and supporting data at each site. The Geweke test was -0.557 (P=0.577) for the wild boar and 1.085 (P=0.278) for the red deer models. Figure S2. Histogram and probability density of the abundance estimated at each site, autocorrelation and trace plots of the Markov Chain Monte Carlo simulations. Table S3. Summary of the estimated distributions and supporting references for the sensitivity and specificity of the diagnostic tests. Figure S3. Histogram and probability density of the estimated sensitivity and specificity of the diagnostic tests employed, autocorrelation and trace plots of the Markov Chain Monte Carlo simulations. Table S4. Summary of the estimated distributions of the true prevalence by host species and supporting data at each study site. Figure S4. Histogram and probability density of the estimated true prevalence at each site, autocorrelation and trace plots of the Markov Chain Monte Carlo simulations. Figure S5. Histogram and probability density of the estimated probability of Mycobacterium tuberculosis complex excretion from infected wild boar and red deer, autocorrelation and trace plots of the Markov Chain Monte Carlo simulations. Table S5. Summary of the estimated distributions of the R0 by host species at each study site. Figure S6. Histogram and probability density of the estimated R0 by host species at each study site, autocorrelation and trace plots of the Markov Chain Monte Carlo simulations. For intelligibility, only R0 estimates <200 were plotted. Table S6. Probability of animal tuberculosis being a single-host or multi-host disease at each site. Probabilities derived from the 100,000 estimates of the R0 of Mycobacterium t

Non-tuberculous mycobacteria (NTM) circulate between the environment, animals and humans entailing a double concern: their ability to interfere with tuberculosis diagnosis and their potential to cause infections in their hosts. However, published records on NTM infections in animals are still scarce. The aims of the present study were to describe the diversity of NTM circulating among wild and domestic species from Spain and to analyze their implications as potential pathogenic microorganisms or as sources of interferences in the diagnosis of bovine tuberculosis. Overall, 293 NTM isolates of 277 animals were obtained from tissue samples collected between 2012 and 2019, and analyzed through a multigene approach for mycobacteria identification. Thirty-one species were identified, being Mycobacterium avium subsp. avium (Maa) and M. avium subsp. hominissuis (Mah), but also M. bouchedurhonense, M. nonchromogenicum and M. lentiflavum, the most abundant ones. Maa and M. lentiflavum were isolated in several animals showing tuberculosis-like lesions. Maa, Mah and M. nonchromogenicum were recovered from many cattle that had reacted to the tuberculin skin test. Other NTM were also associated to these phenomena. These four mycobacterial species were geographically associated between wild boar and other hosts. The findings of the present study suggest that a high diversity of NTM circulates among wildlife and livestock. Wild boar and M. avium seem to play a relevant role in this epidemiological scenario.

The contribution of wildlife species to pathogen maintenance in multi-host communities has seldom been quantified. To assess the relative contribution of the main wildlife hosts of animal tuberculosis (TB) to its maintenance, we estimated the basic reproduction number (R0) of Mycobacterium tuberculosis complex in wild boar and red deer at 29 sites in the Iberian Peninsula. Host abundance and true TB prevalence were estimated for each species at each site by sampling from distributions incorporating the uncertainty in the proportion of the population harvested each year, sensitivity, and specificity of the diagnostic methods, while excretion of mycobacteria was estimated using site-occupancy models. The distributions of these parameters were then used to estimate, at each site, the R0,wild boar (range 0.1 – 55.9, average 8.7, standard deviation 11.8), and the R0,red deer (0.1 – 18.9, 2.2, 3.9). Animal TB is maintained in epidemiological scenarios ranging from any single species acting as a maintenance host (the wild boar in 18 sites and the red deer in 5), to facultative multi-host disease (6 sites). The prevalence of TB in the red deer is likely an important driver of the epidemiology in multi-host communities. The wild boar was the main maintenance host of TB in most of the study sites and could have an epidemiological role linking the wildlife multi-host community and livestock.

Tick vaccines are necessary as part of a One Health approach for the control of tick infestations and tick-borne diseases. Subolesin (SUB, also known as 4D8) is a tick protective antigen that has shown efficacy in vaccine formulations for the control of ectoparasite infestations and pathogen infection/transmission. A recent proof-of-concept study reported oral vaccination combining Rhipicephalus microplus SUB with heat inactivated Mycobacterium bovis (IV) as an immunostimulant for the control of cattle tick infestations. Based on the efficacy of Rhipicephalus decoloratus SUB for the control of multiple cattle tick species in Uganda, herein we design a controlled pen trial using an oral formulation combining R. decoloratus SUB with IV for the control of R. decoloratus and Rhipicephalus appendiculatus cattle tick infestations. Vaccine efficacy (E) of SUB + IV on tick life cycle was compared with IV and SUB alone and with PBS as control. The IgG antibody titers against SUB and M. bovis P22 and the serum levels of selected protein immune biomarkers (IL-1beta, TNF-alpha, C3) were determined and analyzed as possible correlates of protection. Oral immunization with IV and SUB alone and in SUB + IV combination were effective for the control of tick infestations (E = 71–96% for R. decoloratus and 87–99% for R. appendiculatus) with highest E (higher than 95%) for SUB + IV. The results demonstrated that oral immunization with the SUB + IV formulation resulted in effective control of cattle tick infestations through the activation of multiple immune mechanisms. These results support the application of oral vaccine formulations with SUB + IV for the control of cattle infestations with Rhipicephalus species towards improving animal health.