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Tuberculosis BCG vaccination induced non-specific protective effects in humans led to postulate the concept of trained immunity (TRAIM) as an innate type of immune mechanism that triggered by a pathogen, protects against others. Killed vaccines have been considered not to be effective. However, field efficacy of a commercial vaccine against paratuberculosis, as well as of a recently developed M. bovis heat-inactivated vaccine (HIMB) prompted to test whether it could also induce TRAIM. To this, we used a sarcoptic mange rabbit model. Twenty-four weaned rabbits were treated orally or subcutaneously with a suspension of either HIMB (10 7 UFC) or placebo. Eighty-four days later the animals were challenged with approximately 5000 S. scabiei mites on the left hind limb. Skin lesion extension was measured every 2 weeks until 92 days post-infection (dpi). Two animals were killed at 77 dpi because of extensive skin damage. The rest were euthanized and necropsied and the lesion area and the mite burden per squared cm were estimated. Specific humoral immune responses to S. scabiei and to M. bovis were investigated with the corresponding specific ELISA tests. Subcutaneously and orally HIMB vaccinated animals compared with placebo showed reduced lesion scores (up to 74% and 62%, respectively) and mite counts (-170% and 39%, respectively). This, together with a significant positive correlation (r = 0.6276, p = 0.0031) between tuberculosis-specific antibodies and mite count at 92 dpi supported the hypothesis of non-specific effects of killed mycobacterial vaccination. Further research is needed to better understand this mechanism to maximize cross protection., (© 2024. The Author(s).)

This study aimed to assess the efficacy of a heat-inactivated Mycobacterium caprae (HIMC) vaccine in goats experimentally challenged with the same strain of M. caprae. Twenty-one goats were divided into three groups of seven: vaccinated with heat-inactivated Mycobacterium bovis (HIMB), with HIMC and unvaccinated. At 7 weeks post-vaccination all animals were endobronchially challenged with M. caprae. Blood samples were collected for immunological assays and clinical signs were recorded throughout the experiment. All goats were euthanized at 9 weeks post-challenge. Gross pathological examination, analysis of lung pathology using computed tomography, and bacterial load quantification in pulmonary lymph nodes (LN) by qPCR were carried out. Only HIMC vaccinated goats showed a significant reduction of lung lesions volume and mycobacterial DNA load in LN compared to unvaccinated controls. Both vaccinated groups showed also a significant reduction of the other pathological parameters, an improved clinical outcome and a higher proportion of IFN-γ-producing central memory T cells after vaccination. The results indicated that homologous vaccination of goats with HIMC induced enhanced protection against M. caprae challenge by reducing lung pathology and bacterial load compared to the heterologous vaccine (HIMB). Further large-scale trials are necessary to assess the efficacy of autovaccines under field conditions., (© 2022. The Author(s).)

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 (R 0 ) 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 R 0,wild boar (range 0.1 - 55.9, average 8.7, standard deviation 11.8), and the R 0,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., Competing Interests: Conflict of interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2022 Wiley-VCH GmbH.)

Bovine paratuberculosis (PTB) is a chronic inflammatory disease caused by Mycobacterium avium susbp. paratuberculosis (MAP). Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) significantly associated with susceptibility to bovine PTB. The main objective of this study was to identify quantitative trait loci (QTLs) associated with MAP infection in Spanish Holstein cows (N = 983) using combinations of diagnostic tests and imputed whole-genome sequence (WGS) data. The infection status of these animals was defined by three diagnostic methods including ELISA for MAP-antibodies detection, and tissue culture and PCR for MAP detection. The 983 cows included in this study were genotyped with the Bovine MD SNP50 Bead Chip, and the corresponding genotypes were imputed to WGS using the 1,000 Bull genomes reference population. In total, 33.77 million SNP variants per animal were identified across the genome. Linear mixed models were used to calculate the heritability (h2) estimates for each diagnostic test and test combinations. Next, we performed a case-control GWAS using the imputed WGS datasets and the phenotypes and combinations of phenotypes with h2 estimates > 0.080. After performing the GWAS, the test combinations that showed SNPs with a significant association (PFDR ≤ 0.05), were the ELISA-tissue PCR-tissue culture, ELISA-tissue culture, and ELISA-tissue PCR. A total of twelve quantitative trait loci (QTLs) highly associated with MAP infection status were identified on the Bos taurus autosomes (BTA) 4, BTA5, BTA11, BTA12, BTA14, BTA23, BTA24, and BTA28, and some of these QTLs were linked to immune-modulating genes. The identified QTLs on BTA23 spanning from 18.81 to 22.95 Mb of the Bos taurus genome overlapped with several QTLs previously found to be associated with PTB susceptibility, bovine tuberculosis susceptibility, and clinical mastitis. The results from this study provide more clues regarding the molecular mechanisms underlying susceptibility to PTB infection in cattle and might be used to develop national genetic evaluations for PTB in Spain., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl- myo -inositol mannosides (PIMs) are glycolipids considered to be not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane-associated acyltransferase that transfers a palmitoyl moiety from palmitoyl coenzyme A (palmitoyl-CoA) to the 6-position of the mannose ring linked to the 2-position of inositol in PIM 1 /PIM 2 We validate that the function of PatA is vital for M. tuberculosis in vitro and in vivo We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significantly reduced levels of Ac 1 PIM 2 , an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo Altogether, the experimental data highlight the significance of the early steps of the PIM biosynthetic pathway for M. tuberculosis Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis , the etiologic agent of TB, emphasizes the need to identify new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined that PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis These results highlight the importance of early steps of the PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target., (Copyright © 2021 American Society for Microbiology.)

Animal tuberculosis remains a great source of socioeconomic and health concern worldwide. Its main causative agents, Mycobacterium bovis and Mycobacterium caprae, have been isolated from many different domestic and wild animals. Naturally, occurring tuberculosis is extremely rare in rabbits, and implication of M. caprae has never been reported earlier. This study describes a severe tuberculosis outbreak caused by M. caprae in a Spanish farm of rabbits raised for meat for human consumption. The disease was first identified in a cachectic dam, and then it was confirmed in ten does with similar clinical signs. Subsequently, a depopulation operation was ordered for public health, animal welfare and environmental reasons. To broaden knowledge of spontaneous tuberculosis in rabbits, a study focused on pathological, epidemiological and diagnostic aspects was carried out on 51 does and 16 kittens after receiving the necessary authorizations. These animals were subjected to a modified intradermal test. After being euthanized, rabbits were examined for the presence of visible tuberculosis-compatible lesions. Lung, kidney, caecal appendix and sacculus rotundus samples underwent microbiological and anatomopathological analysis. Infection was revealed by at least one of the methods used in 71% of dams and in 44% of kittens. The intradermal test was shown to be a good indicator of infection. Lung was the tissue for which more animals were positive but renal and intestinal tissues were also affected in many cases. Apparently, M. caprae spread mainly through the aerogenous route. Infection was pathologically characterized by the absence of evident fibrous capsules surrounding granulomas. A spoligotype (SB0415) frequently found in this area was considered responsible for the outbreak but the source could not be established. Regardless of the exceptional nature of animal tuberculosis in this host, rabbit industry might not escape from its effects and therefore, current biosafety and surveillance strategies should also consider this disease., (© 2019 Blackwell Verlag GmbH.)

The Eurasian wild boar (Sus scrofa) is a reservoir for tuberculosis (TB) in which vaccination is a valuable tool for control. We evaluated the protection and immune response achieved by homologous and heterologous regimes administering BCG and heat-inactivated Mycobacterium bovis (IV). Twenty-one wild boar piglets were randomly allocated in five groups: Control, homologous BCG, homologous IV, heterologous IV-BCG, heterologous BCG-IV. Significant 67% and 66% total lesion score reductions were detected in homologous IV (IVx2) and heterologous IV-BCG groups when compared with Control group (F 4,16  = 6.393, p = 0.003; Bonferroni Control vs IVx2 p = 0.026, Tukey Control vs IV-BCG p = 0.021). No significant differences were found for homologous BCG (although a 48% reduction in total lesion score was recorded) and BCG-IV (3% reduction). Heterologous regimes did not improve protection over homologous regimes in the wild boar model and showed variable results from no protection to similar protection as homologous regimes. Therefore, homologous regimes remain the best option to vaccinate wild boar against TB. Moreover, vaccine sequence dramatically influenced the outcome underlining the relevance of studying the effects of prior sensitization in the outcome of vaccination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Eurasian wild boar (Sus scrofa) is the main wild reservoir of the Mycobacterium tuberculosis complex in Mediterranean woodlands and a key risk factor for cattle tuberculosis (TB) breakdowns. Wild boar vaccination therefore has the potential to be a valuable tool for TB control. We tested two orally delivered vaccines, heat-inactivated Mycobacterium bovis (IV) and BCG, in four sites (two per vaccine type: one Managed and one Natural or unmanaged) during four years. TB was also monitored in 15 unvaccinated sites (spatial control), as well as in all sites from one year prior to intervention (temporal control). The rationale is that by vaccinating 2-6 month old wild boar piglets we can reduce disease at the population level during the study period. This is achievable due to the fast turnover of wild boar populations. Vaccine baits were deployed using selective piglet feeders and this method proved highly successful with uptake rates of 50 to 74% in Natural sites and 89 to 92% in Managed sites. This is relevant for the potential delivery of vaccines to control other diseases, too. Local wild boar TB prevalence at the beginning of the study was already high ranging from 50 to 100%. TB prevalence increased in unvaccinated sites (6%), while a significant decline occurred in the Managed IV site (34%). Changes recorded in the remaining sites were not significant. The short-term impact of vaccination observed in the field was complemented by mathematical modelling, representative of the field system, which examined the long-term impact and showed that vaccination of piglets reduced prevalence and increased abundance at the population level. We conclude that IV could become part of integrated TB control schemes, although its application must be tailored for each specific site., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background/aims: Animal tuberculosis (TB) is a complex animal health problem that causes disruption to trade and significant economic losses. TB involves a multi-host system where sheep, traditionally considered a rare host of this infection, have been recently included. The aims of this study were to develop an experimental TB infection model in sheep with a Mycobacterium caprae field strain isolated from a tuberculous diseased ewe, and to use this to evaluate the safety and efficacy of two vaccines against TB in sheep, the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine., Methods: Eighteen 2 month-old lambs were experimentally challenged with M. caprae by the endotracheal route (1.5 × 103 CFU). They were separated per treatment group into parenterally vaccinated with a live BCG Danish strain vaccine (n = 6), orally vaccinated with a suspension of HIMB (n = 6) and unvaccinated controls (n = 6). Clinical, immunological, pathological and bacteriological parameters of infection were measured., Results: All lambs were successfully infected and developed gross TB lesions in the respiratory system. The BCG vaccine conferred considerable protection against experimental TB in lambs, as measured by a reduction of the gross lesion volumes and bacterial load. However, HIMB vaccinated animals did not show protection., Conclusions: This study proposes a reliable new experimental model for a better understanding of tuberculosis in sheep. BCG vaccination offers an effective prospect for controlling the disease. Moreover alternative doses and/or routes of administration should be considered to evaluate the efficacy of the HIMB vaccine candidate.

Ticks are responsible for the transmission of pathogens of veterinary importance, including those affecting sheep. The current study was designed to investigate co-infections with tick-borne and other pathogens in a naturally infected sheep flock with poor health condition using serology and PCR. Infection with Anaplasma ovis was detected by serology and PCR in 56% of the animals. The presence of Rickettsia spp. of the Spotted Fever Group (SFG) was detected by PCR and sequence analysis in 31% of the animals. All the animals were negative for Anaplasma phagocytophilum either by serology or PCR. Twelve sheep were randomly selected for anatomopathological studies. Five of these animals presented lesions consistent with Mycobacterium tuberculosis complex (MTBC) infection and spoligotyping confirmed infection with Mycobacterium bovis spoligotype SB0339. Co-infection with tick-borne pathogens and MTBC could contribute to the poor health condition observed in these animals but other uncontrolled factors may also be responsible. The differential expression of immune response genes supported previous findings in ruminants and suggested that infection with tick-borne pathogens and M. bovis may results in unique gene expression patterns in sheep. The results underline the need for further research into the possible role of sheep in the epidemiology of animal tuberculosis., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Rabbits are susceptible to infection by different species of the genus Mycobacterium. Particularly, development of specific lesions and isolation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis, both subspecies of the M. avium complex, has been reported in wildlife conditions. Although, rabbit meat production worldwide is 200 million tons per year, microbiological data on this source of meat is lacking and more specifically reports of mycobacterial presence in industrially reared rabbit for human consumption have not been published. To this end, we sought mycobacteria by microbiological and histopathological methods paying special attention to Mycobacterium avium subsp. paratuberculosis in rabbits from commercial rabbitries from the North East of Spain., Results: M. avium subsp. paratuberculosis was not detected either by culture or PCR. However, Mycobacterium avium subsp. avium was detected in 15.15% (10/66) and Mycobacterium avium subsp. hominissuis was detected in 1.51% (1/66) of gut associated lymphoid tissue of sampled animals by PCR, whereas caecal contents were negative. 9% (6/66) of the animals presented gross lesions suggestive of lymphoid activation, 6% (4/66) presented granulomatous lesions and 3% (2/66) contained acid fast bacilli. Mycobacterial isolation from samples was not achieved, although colonies of Thermoactinomycetes sp. were identified by 16s rRNA sequencing in 6% (4/66) of sampled animals., Conclusions: Apparently healthy farmed rabbits that go to slaughter may carry M. avium subspecies in gut associated lymphoid tissue.

Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar.

Background: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain., Results: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge., Conclusions: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions.

Individuals in natural populations are exposed to a diversity of pathogens which results in coinfections. The aim of this study was to investigate the relation between natural infection with tuberculosis (TB) due to infection by bacteria of the Mycobacterium tuberculosis complex and porcine circovirus type 2 (PCV2) in free-ranging Eurasian wild boar (Sus scrofa). Apparent prevalence for TB lesions and PCV2 infection was extremely high in all age classes, including piglets (51% for TB; 85.7% for PCV2). Modeling results revealed that the relative risk of young (less than 2 years old) wild boar to test positive to PCV2 PCR was negatively associated with TB lesion presence. Also, an interaction between TB, PCV2, and body condition was evidenced: in wild boar with TB lesions probability of being PCV2 PCR positive increased with body condition, whereas this relation was negative for wild boar without TB lesions. This study provides insight into the coinfections occurring in free-ranging host populations that are naturally exposed to several pathogens at an early age. Using TB and PCV2 as a case study, we showed that coinfection is a frequent event among natural populations that takes place early in life with complex effects on the infections and the hosts.

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.

Background: Most countries carrying out campaigns of bovine tuberculosis (TB) eradication impose a ban on the use of mycobacterial vaccines in cattle. However, vaccination against paratuberculosis (PTB) in goats is often allowed even when its effect on TB diagnosis has not been fully evaluated. To address this issue, goat kids previously vaccinated against PTB were experimentally infected with TB., Results: Evaluation of interferon-γ (IFN-γ) secretion induced by avian and bovine tuberculins (PPD) showed a predominant avian PPD-biased response in the vaccinated group from week 4 post-vaccination onward. Although 60% of the animals were bovine reactors at week 14, avian PPD-biased responses returned at week 16. After challenge with M. caprae, the IFN-γ responses radically changed to show predominant bovine PPD-biased responses from week 18 onward. In addition, cross-reactions with bovine PPD that had been observed in the vaccinated group at week 14 were reduced when using the M. tuberculosis complex-specific antigens ESAT-6/CFP-10 and Rv3615c as new DIVA (differentiation of infected and vaccinated animals) reagents, which further maintained sensitivity post-challenge. Ninety percent of the animals reacted positively to the tuberculin cervical comparative intradermal test performed at 12 weeks post-infection. Furthermore, post-mortem analysis showed reductions in tuberculous lesions and bacterial burden in some vaccinated animals, particularly expressed in terms of the degree of extrapulmonary dissemination of TB infection., Conclusions: Our results suggest a degree of interference of PTB vaccination with current TB diagnostics that can be fully mitigated when using new DIVA reagents. A partial protective effect associated with vaccination was also observed in some vaccinated animals.

Information on lesion distribution and characteristics is essential to determine the significance of a species as a reservoir host for tuberculosis (TB). Herein, we describe the extension and distribution of lesions in 127 Mycobacterium tuberculosis Complex culture positive European wild boars (Sus scrofa), and use this information to discuss the role of this wildlife species in TB epidemiology in Mediterranean Spain. Macroscopic TB-compatible lesions were detected in 105 of 127 wild boars (82.68%). Only microscopic lesions were found in 11 wild boars (8.66%). Lesions were not evident in 11 wild boars (8.66%). A total of 49 wild boars had lesions confined to one anatomical region (42.2%, localized TB), while 67 animals had lesions in more than one anatomical region (57.8%, generalized TB). Head lymph nodes (LNs), particularly the mandibular LNs, were most frequently affected (107/116, 92.24%), and 43 wild boar had only mandibular LN lesions. Histopathology evidenced TB lesions in 38.1% of the lungs, 23% of the livers and 13% of the spleens examined. Mammary gland lesions were observed in three cases. When TB lesions were localized, granulomas characterized by a mixed inflammatory cell population were more predominant, whereas strongly necrotic-calcified granulomas were more prevalent in generalized cases of TB infection. Large lesions in more than one anatomical region were more frequent among juveniles. The histopathological characteristics of the tuberculous reaction and the associated tissue damage in various organs, together with the gross pathology, indicate that at least those wild boar with large lesions and generalized infections have the potential to excrete mycobacteria by several routes. This finding, in the context of unusually high densities of wild boar and fencing and feeding, reinforces the suggestion that wild boar can act as a true TB reservoir under the particular circumstances of Mediterranean Spain. Further studies on the routes of excretion as well as the effect of altering management methods would be of interest to confirm the role of wild boar in TB epidemiology in Spain.

In recent decades the management of large game mammals has become increasingly intensive in south central Spain (SCS), resulting in complex epidemiological scenarios for disease maintenance, and has probably impeded schemes to eradicate tuberculosis (TB) in domestic livestock. We conducted an analysis of risk factors which investigated associations between the pattern of tuberculosis-like lesions (TBL) in wild boar (Sus scrofa) and red deer (Cervus elaphus) across 19 hunting estates from SCS and an extensive set of variables related to game management, land use and habitat structure. The aggregation of wild boar at artificial watering sites was significantly associated with an increasing risk of detecting TBL in both species, which probably relates to enhanced opportunities for transmission. Aggregation of wild boar at feeding sites was also associated with increased risks of TBL in red deer. Hardwood Quercus spp. forest availability was marginally associated with an increased risk of TB in both species, whereas scrubland cover was associated with a reduced individual risk of TBL in the wild boar. It is concluded that management practices that encourage the aggregation of hosts, and some characteristics of Mediterranean habitats could increase the frequency and probability of both direct and indirect transmission of TB. These findings are of concern for both veterinary and public health authorities, and reveal tuberculosis itself as a potential limiting factor for the development and sustainability of such intensive game management systems in Spanish Mediterranean habitats.

Background: Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied., Results: All bovines, 4 and 26% of Spanish sheep and goats, respectively, and the deer and wild boar studied, carried IS1311-Cattle type strains. IS1311-Sheep type encompassed 96% and 74% of Spanish sheep and goats, and all three Portuguese sheep. Thirty-seven distinct multiplex PFGE profiles were found, giving 32 novel profiles. Profiles 2-1 and 1-1 accounted for the 85% of cattle isolates. Ten distinct profiles were detected in Spanish sheep, none of them with an incidence higher than 25%. Profile 16-11 (43%) and another three profiles were identified in Spanish caprine cultures. The hierarchical analysis, clustered all profiles found in cattle, "wild" hosts and some small ruminants within the same group. The other group included 11 profiles only found in Spanish sheep and goats, including Spanish pigmented profiles. Differences in growth requirements associated with isolate genotype were observed., Conclusion: Cattle in Spain are infected with cattle type strains, while sheep and goats are mainly infected with sheep type strains. Although 7H9 broth based culture media seem to broadly cover the growth requirements of most Map strains, the use of various solid media is recommended to reduce any recovery biases. High genetic homogeneity of isolates from cattle, and heterogeneity of those from sheep and goats have been detected.

We describe the distribution of tuberculosis-like lesions (TBL) in wild boar (Sus scrofa) and red deer (Cervus elaphus) in Spain. Animals with TBL were confirmed in 84.21% of mixed populations (n=57) of red deer and wild boar and in 75% of populations of wild boar alone (n=8) in central and southern Spain (core area). The prevalence of TBL declined towards the periphery of this region. In the core area, the prevalence ranged up to 100% in local populations of wild boar (mean estate prevalence 42.51%) and up to 50% in red deer (mean estate prevalence 13.70%). We carried out exploratory statistical analyses to describe the epidemiology of TBL in both species throughout the core area. Prevalence of TBL increased with age in both species. Wild boar and red deer mean TBL prevalence at the estate level were positively associated, and lesion scores were consistently higher in wild boars than in red deer. The wild boar prevalence of TBL in wild boar did not differ between populations that were or were not cohabiting with red deer. Amongst the wild boars with TBL, 61.19% presented generalized lesions, and the proportion of generalized cases was similar between sex and age classes. In red deer, 57.14% of TBL-positive individuals presented generalized lesions, and the percentage of generalized cases increased with age class, but did not differ between the sexes. These results highlight the potential importance of wild boar and red deer in the maintenance of tuberculosis in south central Spain.

The role of European wild ungulates in the epidemiology of tuberculosis (TB) is still under discussion. This study describes the geographical distribution and molecular typing of 77 Mycobacterium tuberculosis complex isolates belonging either to M. bovis or to M. caprae, cultivated from hunter harvested red deer (Cervus elaphus) and European wild boar (Sus scrofa) in 24 Spanish localities, and compares them with spoligotypes detected previously in humans, livestock or wild animals, as described in the literature. The distribution of the molecular type patterns suggests that the population of M. tuberculosis complex strains isolated from Spanish wild ungulates is spatially structured despite the lack of important geographical barriers and despite the increasingly frequent wildlife translocations. Red deer and the European wild boar can share the same molecular types in localities in which the M. tuberculosis complex was isolated from both species. Strains of bovine and caprine origin do circulate in the same local wildlife populations. Six out of 11 spoligotypes were similar to types described in human cases. The isolation of TB strains in fenced estates from wild animals that have not had contact with domestic livestock for at least the past two decades, strongly suggests that the M. tuberculosis complex is able to survive in these populations. Therefore, wildlife including cervids and the wild boar need to be considered in the epidemiology and control of tuberculosis.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity. [ABSTRACT FROM AUTHOR]

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non‐tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high‐field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low‐field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB‐vaccinated healthy control (n = 10) and healthy PTB‐unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77–1). In summary, plasma fingerprinting using HF and LF‐NMR differentiated TB subjects from uninfected animals, and PTB and PTB‐vaccinated subjects who may provide a TB‐false positive, highlighting the use of LF‐NMR‐based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods. [ABSTRACT FROM AUTHOR]

Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis that affects cattle worldwide. According to their severity and extension, PTB-associated histological lesions have been classified into the following groups; focal, multifocal, and diffuse. It is unknown whether these lesions represent sequential stages or divergent outcomes. In the current study, the associations between host genetic and pathology were explored by genotyping 813 Spanish Holstein cows with no visible lesions (N = 373) and with focal (N = 371), multifocal (N = 33), and diffuse (N = 33) lesions in gut tissues and regional lymph nodes. DNA from peripheral blood samples of these animals was genotyped with the bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data using the 1000 Bull genomes reference population. A genome-wide association study (GWAS) was performed using the WGS data and the presence or absence of each type of histological lesion in a case–control approach. A total of 192 and 92 single nucleotide polymorphisms (SNPs) defining 13 and 9 distinct quantitative trait loci (QTLs) were highly-associated (P ≤ 5 × 10−7) with the multifocal (heritability = 0.075) and the diffuse (heritability = 0.189) lesions, respectively. No overlap was seen in the SNPs controlling these distinct pathological outcomes. The identified QTLs overlapped with some QTLs previously associated with PTB susceptibility, bovine tuberculosis susceptibility, clinical mastitis, somatic cell score, bovine respiratory disease susceptibility, tick resistance, IgG level, and length of productive life. Pathway analysis with candidate genes overlapping the identified QTLs revealed a significant enrichment of the keratinization pathway and cholesterol metabolism in the animals with multifocal and diffuse lesions, respectively. To test whether the enrichment of SNP variants in candidate genes involved in the cholesterol metabolism was associated with the diffuse lesions; the levels of total cholesterol were measured in plasma samples of cattle with focal, multifocal, or diffuse lesions or with no visible lesions. Our results showed reduced levels of plasma cholesterol in cattle with diffuse lesions. Taken together, our findings suggested that the variation in MAP-associated pathological outcomes might be, in part, genetically determined and indicative of distinct host responses. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar. [ABSTRACT FROM AUTHOR]

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that seldom causes disease in livestock and humans. This study evaluated the effects on immunodiagnosis and the pathological findings in goats after experimental exposure by different routes and doses to M. microti. In a first experiment goats were challenged orally (PO, n = 7) or intranasally (IN, n = 7) with 104 CFU. In a second experiment, the endobronchial route was assessed, with a low dose of 102 CFU (EB-LD, n = 7) and a high dose of 105 CFU (EB-HD, n = 7) as well as the subcutaneous route (SC, n = 5). Temperature, body weight, clinical signs and immunological responses were monitored. Pathological evaluation was carried out and samples were processed for mycobacterial detection. demonstrated the induction of a subclinical pulmonary infection in all the EB-HD challenged animals. Infection was also confirmed in one animal of the SC group, but not in the EB-LD, PO or IN groups. Two animals belonging to the EB-HD and SC groups, respectively, showed positive results to the single intradermal tuberculin test, and another two animals of the EB-HD and EB-LD groups showed doubtful (inconclusive) results, indicating that M. microti can induce mild responses to tuberculin skin testing. No positive results were observed when defined antigens absent in M. microti (ESAT-6 and CPF-10) were used. Our results indicate that animals exposed to M. microti can yield positive results to the skin tests currently performed in livestock tuberculosis eradication campaigns and reinforce the need to use specific antigens in antemortem tests to avoid interference with M. bovis/M. caprae diagnosis. • M. microti induces subclinical tuberculosis in experimentally challenged goats. • M. microti infection can induce positive reactions to tuberculin skin tests. • The use of antigens absent in M. microti overcomes these diagnostic interferences. [ABSTRACT FROM AUTHOR]