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Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01-1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9-22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1-12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems., Ministerio de Economía y Competitividad, Consorcio Centro de Investigacion Biomédica en Red, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovacion, Unión Europea, Universidad de Córdoba, Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

Author Contributions: All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication., Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01-1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9-22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1-12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems., Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

The global rise of drug resistant tuberculosis has highlighted the need for improved diagnostic technologies that provide rapid and reliable drug resistance results. Here, we develop and validate a whole genome sequencing (WGS)-based test for identification of mycobacterium tuberculosis complex (MTB) drug resistance to rifampin, isoniazid, pyrazinamide, ethambutol, and streptomycin. Through comparative analysis of drug resistance results from WGS-based testing and phenotypic drug susceptibility testing (DST) of 38 clinical MTB isolates from patients receiving care in Los Angeles, CA, we found an overall concordance between methods of 97.4% with equivalent performance across culture media. Critically, prospective analysis of 11 isolates showed that WGS-based testing provides results an average of 36 days faster than phenotypic culture-based methods. We showcase the additional benefits of WGS data by investigating a suspected laboratory contamination event and using phylogenetic analysis to search for cryptic local transmission, finding no evidence of community spread amongst our patient population in the past six years. WGS-based testing for MTB drug resistance has the potential to greatly improve diagnosis of drug resistant MTB by accelerating turnaround time while maintaining accuracy and providing additional benefits for infection control, lab safety, and public health applications.

The Mycobacterium tuberculosis complex (MTBC) is a successful human pathogen. In 2022, approximately 10.6 million people have developed the active phase of the disease, and around 1.6 million people died due to tuberculosis (TB). The deciphering of the genome of M. tb has helped us to understand its evolutionary history and the causes underlying its worldwide spread. Using various genotyping methods and whole-genome sequencing techniques indicate that the M. tb complex is grouped in nine lineages, some are specifically adapted to humans, while others are animal-adapted lineages. The presence of most of the lineages in Africa makes this continent the most likely place of MTBC origin. Studies of the genome of M. tb have also assisted in the identification of genes involved in its virulence, pathogenicity, and the long co-evolutionary process with its main host, the human being. In this review of selected literature, we describe the genotypic and phenotypic diversity of the MTBC and its clinical and epidemiological implications., El complejo de Mycobacterium tuberculosis (CMTB) es uno de los patógenos con mayor éxito evolutivo por su alta transmisión en las poblaciones humanas. En el 2022, la OMS estimó que aproximadamente 10.6 millones de personas desarrollaron la fase activa de la enfermedad, principalmente la forma pulmonar (tuberculosis pulmonar, TBP), y alrededor de 1.6 millones, más, fallecieron debido a la tuberculosis (TB). Diversas técnicas con base en el genotipo y la secuencia del genoma de Mycobacterium tuberculosis (M. tb) han permitido entender su historia evolutiva y comprender las causas de su propagación mundial. El análisis y las comparaciones de la secuencia de los genomas completos indican que el CMTB se compone de nueve linajes, algunos están específicamente adaptados a los humanos y otros a los animales. La presencia de la mayoría de los linajes en África, hace de este continente el origen del CMTB. Los estudios del genoma del CMTB también han hecho posible la identificación de los genes implicados en su virulencia y patogenicidad, así como en el largo proceso co-evolutivo con su principal hospedero, el ser humano. En esta revisión se describe la diversidad genotípica y fenotípica del CMTB y sus implicaciones clínicas y epidemiológicas.

The Mycobacterium tuberculosis complex (MTBC) is a successful human pathogen. In 2022, approximately 10.6 million people have developed the active phase of the disease, and around 1.6 million people died due to tuberculosis (TB). The deciphering of the genome of M. tb has helped us to understand its evolutionary history and the causes underlying its worldwide spread. Using various genotyping methods and whole-genome sequencing techniques indicate that the M. tb complex is grouped in nine lineages, some are specifically adapted to humans, while others are animal-adapted lineages. The presence of most of the lineages in Africa makes this continent the most likely place of MTBC origin. Studies of the genome of M. tb have also assisted in the identification of genes involved in its virulence, pathogenicity, and the long co-evolutionary process with its main host, the human being. In this review of selected literature, we describe the genotypic and phenotypic diversity of the MTBC and its clinical and epidemiological implications., El complejo de Mycobacterium tuberculosis (CMTB) es uno de los patógenos con mayor éxito evolutivo por su alta transmisión en las poblaciones humanas. En el 2022, la OMS estimó que aproximadamente 10.6 millones de personas desarrollaron la fase activa de la enfermedad, principalmente la forma pulmonar (tuberculosis pulmonar, TBP), y alrededor de 1.6 millones, más, fallecieron debido a la tuberculosis (TB). Diversas técnicas con base en el genotipo y la secuencia del genoma de Mycobacterium tuberculosis (M. tb) han permitido entender su historia evolutiva y comprender las causas de su propagación mundial. El análisis y las comparaciones de la secuencia de los genomas completos indican que el CMTB se compone de nueve linajes, algunos están específicamente adaptados a los humanos y otros a los animales. La presencia de la mayoría de los linajes en África, hace de este continente el origen del CMTB. Los estudios del genoma del CMTB también han hecho posible la identificación de los genes implicados en su virulencia y patogenicidad, así como en el largo proceso co-evolutivo con su principal hospedero, el ser humano. En esta revisión se describe la diversidad genotípica y fenotípica del CMTB y sus implicaciones clínicas y epidemiológicas.

SignificancePrevious attempts to identify the action of natural selection in the Mycobacterium tuberculosis complex (MTBC) were limited by sample size and averaging across time and lineages. We investigate changes in selective pressures across time for every single gene of the MTBC. We developed a methodology to analyze temporal signals of selection in a large dataset (∼5,000 complete genomes) and showed that 1) almost half of the genes seem to have been under positive selection at some point in time; 2) experimentally confirmed epitopes tend to accumulate more mutations in deeper branches than in external branches; and 3) temporal signals identify genes that were conserved in the past but under positive selection in the present, suggesting ongoing adaptation to the host.

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.

The present work investigated the prevalence, spatial distribution, and temporal distribution of tuberculosis (TB) in free-ranging Eurasian badgers (Meles meles) and cattle in Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the role of badgers as a TB reservoir for cattle and other sympatric wild species in the region. Between 2008 and 2020, 673 badgers (98 trapped and 575 killed in road traffic accidents) in Asturias were necropsied, and their tissue samples were cultured for the Mycobacterium tuberculosis complex (MTC) isolation. Serum samples were tested in an in-house indirect P22 ELISA to detect antibodies against the MTC. In parallel, data on MTC isolation and single intradermal tuberculin test results were extracted for cattle that were tested and culled as part of the Spanish National Program for the Eradication of Bovine TB. A total of 27/639 badgers (4.23%) were positive for MTC based on bacterial isolation, while 160/673 badgers (23.77%) were found to be positive with the P22 ELISA. The rate of seropositivity was higher among adult badgers than subadults. Badger TB status was spatially and temporally associated with cattle TB status. Our results cannot determine the direction of possible interspecies transmission, but they are consistent with the idea that the two hosts may exert infection pressure on each other. This study highlights the importance of the wildlife monitoring of infection and disease during epidemiological interventions in order to optimize outcomes., Ministerio de Ciencia e Innovación (MICINN)/FEDER, Depto. de Producción Animal, Fac. de Veterinaria, TRUE, pub

Recently, the survival of Mycobacterium bovis on livestock mineral blocks has been confrmed, but little is known about its implication in the transmission of animal tuberculosis (TB) under feld conditions. The objective of this study was to describe the shared use of mineral supplements in four extensive beef cattle farms from a high TB prevalence area in South Central Spain, to identify the main factors explaining their use, and characterize its potential role for the transmission of Mycobac terium tuberculosis Complex (MTC). This is relevant to design control measures at the wildlife-livestock interface. Animal activity was monitored by camera-trapping at 12 mineral supplementation points during spring and fall. Additionally, swabs were periodically taken from the mineral substrates and analyzed by PCR searching for MTC DNA. Cattle, pig, goat, sheep, wild boar, and red deer were all recorded licking on mineral supplementation points. Livestock species were the main users and presented a diurnal use pattern. Wild ungulates presented a nocturnal-crepuscular use pattern, with scarce overlapping with livestock. Wild boar presence was positively related to cattle presence at mineral supplementation points, whereas red deer presence was higher in supplemental points closer to forested areas and in farms without hunting pressure. We recorded 266 indirect wildlife-livestock interactions (i.e., two consecutive visits that occurred within 78 h), all of them derived from 21 unique wildlife visits. All the analyzed swabs resulted negative to MTC DNA. Comparing to other environmental sources of MTC in our study area, mainly water ponds, this research evidenced that mineral blocks are less attractive to wildlife. However, the potential for interspecifc transmission of MTC or other pathogens cannot be discarded. The risk for interac tion at mineral supplementation points and further transmission can be prevented by implementing specifc measures in the context of integral biosecu

Recently, the survival of Mycobacterium bovis on livestock mineral blocks has been confrmed, but little is known about its implication in the transmission of animal tuberculosis (TB) under feld conditions. The objective of this study was to describe the shared use of mineral supplements in four extensive beef cattle farms from a high TB prevalence area in South Central Spain, to identify the main factors explaining their use, and characterize its potential role for the transmission of Mycobac terium tuberculosis Complex (MTC). This is relevant to design control measures at the wildlife-livestock interface. Animal activity was monitored by camera-trapping at 12 mineral supplementation points during spring and fall. Additionally, swabs were periodically taken from the mineral substrates and analyzed by PCR searching for MTC DNA. Cattle, pig, goat, sheep, wild boar, and red deer were all recorded licking on mineral supplementation points. Livestock species were the main users and presented a diurnal use pattern. Wild ungulates presented a nocturnal-crepuscular use pattern, with scarce overlapping with livestock. Wild boar presence was positively related to cattle presence at mineral supplementation points, whereas red deer presence was higher in supplemental points closer to forested areas and in farms without hunting pressure. We recorded 266 indirect wildlife-livestock interactions (i.e., two consecutive visits that occurred within 78 h), all of them derived from 21 unique wildlife visits. All the analyzed swabs resulted negative to MTC DNA. Comparing to other environmental sources of MTC in our study area, mainly water ponds, this research evidenced that mineral blocks are less attractive to wildlife. However, the potential for interspecifc transmission of MTC or other pathogens cannot be discarded. The risk for interac tion at mineral supplementation points and further transmission can be prevented by implementing specifc measures in the context of integral biosecu

Recently, the survival of Mycobacterium bovis on livestock mineral blocks has been confrmed, but little is known about its implication in the transmission of animal tuberculosis (TB) under feld conditions. The objective of this study was to describe the shared use of mineral supplements in four extensive beef cattle farms from a high TB prevalence area in South Central Spain, to identify the main factors explaining their use, and characterize its potential role for the transmission of Mycobac terium tuberculosis Complex (MTC). This is relevant to design control measures at the wildlife-livestock interface. Animal activity was monitored by camera-trapping at 12 mineral supplementation points during spring and fall. Additionally, swabs were periodically taken from the mineral substrates and analyzed by PCR searching for MTC DNA. Cattle, pig, goat, sheep, wild boar, and red deer were all recorded licking on mineral supplementation points. Livestock species were the main users and presented a diurnal use pattern. Wild ungulates presented a nocturnal-crepuscular use pattern, with scarce overlapping with livestock. Wild boar presence was positively related to cattle presence at mineral supplementation points, whereas red deer presence was higher in supplemental points closer to forested areas and in farms without hunting pressure. We recorded 266 indirect wildlife-livestock interactions (i.e., two consecutive visits that occurred within 78 h), all of them derived from 21 unique wildlife visits. All the analyzed swabs resulted negative to MTC DNA. Comparing to other environmental sources of MTC in our study area, mainly water ponds, this research evidenced that mineral blocks are less attractive to wildlife. However, the potential for interspecifc transmission of MTC or other pathogens cannot be discarded. The risk for interac tion at mineral supplementation points and further transmission can be prevented by implementing specifc measures in the context of integral biosecu

The present work investigated the prevalence, spatial distribution, and temporal distribution of tuberculosis (TB) in free-ranging Eurasian badgers (Meles meles) and cattle in Asturias (Atlantic Spain) during a 13-year follow-up. The study objective was to assess the role of badgers as a TB reservoir for cattle and other sympatric wild species in the region. Between 2008 and 2020, 673 badgers (98 trapped and 575 killed in road traffic accidents) in Asturias were necropsied, and their tissue samples were cultured for the Mycobacterium tuberculosis complex (MTC) isolation. Serum samples were tested in an in-house indirect P22 ELISA to detect antibodies against the MTC. In parallel, data on MTC isolation and single intradermal tuberculin test results were extracted for cattle that were tested and culled as part of the Spanish National Program for the Eradication of Bovine TB. A total of 27/639 badgers (4.23%) were positive for MTC based on bacterial isolation, while 160/673 badgers (23.77%) were found to be positive with the P22 ELISA. The rate of seropositivity was higher among adult badgers than subadults. Badger TB status was spatially and temporally associated with cattle TB status. Our results cannot determine the direction of possible interspecies transmission, but they are consistent with the idea that the two hosts may exert infection pressure on each other. This study highlights the importance of the wildlife monitoring of infection and disease during epidemiological interventions in order to optimize outcomes.

Animal tuberculosis (TB) is a multi-host disease caused by members of the Mycobacterium tuberculosis complex (MTC). Due to its impact on economy, sanitary standards of milk and meat industry, public health and conservation, TB control is an actively ongoing research subject. Several wildlife species are involved in the maintenance and transmission of TB, so that new approaches to wildlife TB diagnosis have gained relevance in recent years. Diagnosis is a paramount step for screening, epidemiological investigation, as well as for ensuring the success of control strategies such as vaccination trials. This is the frst review that systematically addresses data available for the diagnosis of TB in wildlife following the Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The article also gives an overview of the factors related to host, environment, sampling, and diagnostic techniques which can afect test performance. After three screenings, 124 articles were considered for systematic review. Literature indicates that post-mortem examination and culture are useful methods for disease surveillance, but immunological diagnostic tests based on cellular and humoral immune response detection are gaining importance in wildlife TB diagnosis. Among them, serological tests are especially useful in wildlife because they are relatively inexpensive and easy to perform, facilitate large-scale surveillance and can be used both ante- and post-mortem. Currently available studies assessed test performance mostly in cervids, European badgers, wild suids and wild bovids. Research to improve diagnostic tests for wildlife TB diagnosis is still needed in order to reach accurate, rapid and cost-efective diagnostic techniques adequate to a broad range of target species and consistent over space and time to allow proper disease monitoring.

The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donana National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12-24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an alpha value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.

The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donana National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12-24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an alpha value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.

The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donana National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12-24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an alpha value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.

The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donana National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12-24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an alpha value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.

The wild pig population on Molokai, Hawaii, USA is a possible reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and has been implicated in decades past as the source of disease for the island’s domestic cattle. Heat-inactivated vaccines have been effective for reducing disease prevalence in wild boar in Spain and could prove useful for managing M. bovis in Molokai wild pigs. We designed an experiment to test this vaccine in wild pigs of Molokai genetics. Fifteen 3–4-month-old pigs were orally administered 106–107 colony forming units (cfu) of heat-inactivated M. bovis (Vaccinates; n = 8; 0.2 mL) or phosphate buffered saline (Controls; n = 7; 0.2 mL). Each dose was administered in a 0.5 mL tube embedded in a fruit candy/cracked corn mix. Boosters were given seven weeks post-prime in the same manner and dose. Nineteen weeks post-prime, pigs were orally challenged with 1 × 106 cfu of virulent M. bovis. Twelve weeks post-challenge, pigs were euthanized and necropsied, at which time 23 different tissues from the head, thorax, and abdomen were collected and examined. Each tissue was assigned a lesion score. Ordinal lesion score data were analyzed using non-parametric Wilcoxon Signed Rank test. Effect size was calculated using Cohen’s d. Four of eight Vaccinates and four of seven Controls had gross and microscopic lesions, as well as culture-positive tissues. Vaccinates had statistically lower lesion scores than Controls in the following areas: gross thoracic lesion scores (p = 0.013 Cohen’s d = 0.33) and microscopic thoracic lesion scores (p = 0.002, Cohen’s d = 0.39). There were no differences in head lesion scores alone, both gross and microscopic, nor were there differences when comparing combined gross and microscopic head and thoracic lesion scores. These results are indicative that this vaccination protocol affords a modest degree of infection containment with this vaccine in Molokai wild pigs.

The presence of Mycobacterium tuberculosis complex (MTBC) in wild swine, such as in wild boar (Sus scrofa) in Eurasia, is cause for serious concern. Development of accurate, efficient, and noninvasive methods to detect MTBC in wild swine would be highly beneficial to surveillance and disease management efforts in affected populations. Here, we describe the first report of identification of volatile organic compounds (VOC) obtained from the breath and feces of wild boar to distinguish between MTBC-positive and MTBC-negative boar. We analyzed breath and fecal VOC collected from 15 MTBC-positive and 18 MTBC-negative wild boar in Donana National Park in Southeast Spain. Analyses were divided into three age classes, namely, adults (>2 years), sub-adults (12-24 months), and juveniles (<12 months). We identified significant compounds by applying the two-tailed statistical t-test for two samples assuming unequal variance, with an alpha value of 0.05. One statistically significant VOC was identified in breath samples from adult wild boar and 14 were identified in breath samples from juvenile wild boar. One statistically significant VOC was identified in fecal samples collected from sub-adult wild boar and three were identified in fecal samples from juvenile wild boar. In addition, discriminant function analysis (DFA) was used to build classification models for MTBC prediction in juvenile animals. Using DFA, we were able to distinguish between MTBC-positive juvenile wild boar and MTBC-negative juvenile wild boar using breath VOC or fecal VOC. Based on our results, further research is warranted and should be performed using larger sample sizes, as well as wild boar from various geographic locations, to verify these compounds as biomarkers for MTBC infection in this species. This new approach to detect MTBC infection in free-ranging wild boar potentially comprises a reliable and efficient screening tool for surveillance in animal populations.

Two strains of Mycobacterium tuberculosis complex can be separated as much as 2500 single nucleotide differences (Coscolla and Gagneux, 2014). In that limited amount of diversity, we find an astonishing range of clinical, epidemiological and biological phenotypes. The most striking is the strong host preferences depending on the infecting strain while more subtle differences can be found looking at different human tuberculosis isolates. Those subtle differences are the most difficult to spot given that analysis methods for so little diversity are limited and phenotypes like virulence are difficult to define and measure. Recent genomics advances allow to study the pathogen diversity at a resolution not available before from comparative species level, to global diversity to transmission in local settings. Here, we will review some of these recent results to highlight how population genomics approaches can aid not only to understand how MTBC evolved but also to identify relevant biomedical targets.

In recent decades, habitat change and the intensive management of wild ungulates for hunting have led to an increase in their populations in south-central Spain. This implies a higher generation of hunting waste, which can favour the transmission of infectious diseases, including tuberculosis (TB). The aim of this study was to assess the usefulness of the proper disposal of hunting waste as TB control measure in wild boar (Sus scrofa) and red deer (Cervus elaphus) during the 2008/2009 to 2016/2017 hunting seasons. Blood samples from 664 wild boar and 934 red deer were obtained in 14 game estates in two provinces in Andalusia (Area 1), where the disposal of hunting waste was implemented since the 2012/2013 hunting season. Besides, six game estates in the province of Ciudad Real, in Castilla-La Mancha (Area 2), an adjacent region where this management measure was not implemented during the studied period, were used as controls, sampling 277 wild boar and 427 red deer sera. The Mycobacterium tuberculosis complex (MTC), seroprevalence detected in wild boar from Area 1, was significantly higher before the disposal of big game hunting by-products (82.8%; 2008/2009–2012/2013) compared to the second period (61.8%; 2013/2014–2016/2017) (p <.001), after this control measure became established. By contrast, no significant differences between periods were found in wild boar (41.3% versus 44.8%; p =.33) and red deer (14.9% versus 11.6%; p =.19) from Area 2 as well as in red deer (10.8% versus 10.5%; p =.48) from Area 1. The proper disposal of hunting waste contributed to achieve a 25% reduction in MTC seroprevalence in wild boar. These results are of particular relevance regarding wild boar in the current context of re-emerging and emerging diseases such as TB and African Swine Fever in Europe. Further studies are needed to assess the effect of this measure on the health status of livestock and other wildlife species.

Currently, there are no European data about the frequency and clinical significance of nontuberculous mycobacteria (NTM) grown from respiratory samples during the treatment of tuberculosis (TB). We determined the frequency and clinical significance of NTM isolated before or during pulmonary tuberculosis treatment in Belgian laboratories., info:eu-repo/semantics/published

[Introduction]: In countries in which bovine tuberculosis (bTB) is still prevalent or is re-emerging the contact among different animal species in extensive systems may contribute to the circulation of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTC) and the spread of this disease. Thus, free-range pigs may be infected by MTC, developing subclinical infections, which are not detected until meat inspection procedures at slaughterhouse. Serodiagnosis has been recently proposed as a reliable screening tool for detecting infected herds. In this study four ELISA assays using different M. bovis peptides/proteins (MPB70+MPB83, INGENASA; treated bovine purified protein derivative, t-bPPD; bPPD1; and bPPD2 VACUNEK) as coating antigens were evaluated to diagnose MTC infection in pigs. [Materials and Methods]: Submandibular lymph nodes (SLN) and blood samples from 129 free-range pigs raised on Southern Spain farms with a history of condemnation due to tuberculosis-like lesions were sampled at slaughterhouse. SLN were tested by gross examination, histopathology, bacteriological culture and qPCR. Ninety-seven out of these animals were classified as bTB positive cases (compatible lesions and MTC detection by means of culture and qPCR) or bTB negative cases (absence of compatible lesions and negative MTC detection) and used as reference method. When necessary different cut-off values were evaluated. [Results]: All assays had a very good concordance between them (k ≥ 0.82). The MPB70+MPB83 based ELISA had the best sensitivity (Se) (78%, CI95 67.4%>88.5%) and a good concordance with the reference method (k=0.69). The t-bPPD and the bPPD1 in-house assays presented a slightly reduced Se (71.2%, CI95 59.6%>82.7%; and 66.1%, CI95 54%>78.2%; respectively) and a moderate concordance with the reference method (k=0.57 and 0.52, respectively). When the bPPD2 based ELISA was evaluated, similar Se to the previous ones was obtained using a cut-off of 0.35 (S

Eurasian wild boar (Sus scrofa) is widespread in the northern third of Morocco, Northern Africa. We aimed to investigate the presence of Mycobacterium bovis infection in wild boar and to generate information on the possible host status in this region. M. bovis was isolated in six of 43 wild boars. Culture-positive samples yielded spoligotype number SB1627. Sixty percent of lung tissue specimens or tracheobronchial lymph node samples yielded positive culture. Two thirds (64 %) of the wild boar submitted for histopathology revealed tuberculosis-compatible lesions. These lesions were particularly frequent in lymph nodes (63 %) and in lung tissue samples (43 %). Ziehl-Neelsen staining did not identify acid-fast bacilli, indicating paucibacillary infections. Our results confirm the presence of Mycobacterium tuberculosis complex (MTC) infection in Moroccan wild boar. Regarding the host status of wild boar in this region, the often generalized lesion pattern suggests a possible role of wild boar as a component of the MTC maintenance host community. However, our information was limited to 43 wild boars from one specific region. Thus, further research is needed to assess the host status of wild boar in Morocco.

Jedan od najvećih problema sa kojim se suočavaju programi kontrole tuberkuloze (TB) je širenje multirezistentnih (MDR) sojeva Mycobacterium tuberculosis kompleksa (MTK), odnosno sojeva rezistentnih najmanje na rifampicin i izoniazid, dva najvažnija antituberkulotika (ATL). Molekularno genetička karakterizacija MDR sojeva MTK prisutnih u datoj populaciji predstavlja važan korak u uspostavljanju adekvatnih mera kontrole MDR TB, ali ciljana istraživanja genoma MDR sojeva izolovanih u Srbiji do sada nisu rađena. Stoga su ovim istraživanjem obuhvaćeni svi MDR sojevi MTK izolovani u Srbiji tokom poslednjih šest godina i izvršena je molekularna identifikacija do nivoa vrste, genotipizacija izolata u cilju sagledavanja njihove filogenetske strukture i karakteristika transmisije, kao i detekcija mutacija odgovornih za rezistenciju na ATL. Za istraživanje su formirane dve kolekcije sojeva: svi MDR sojevi MTK izolovani u Srbiji u periodu 01.01.2008.-31.05.2014. godina (115 sojeva) i svi sojevi MTK izolovani od pacijenata obolelih od plućne TB u Srbiji tokom perioda 01.12.2010.-30.11.2011. godina (971 soj). Molekularna identifikacija ispitivanih sojeva MTK izvršena je primenom komercijalnog eseja zasnovanog na reakciji reverzne hibridizacije. Za genotipizaciju MDR sojeva MTK korišćene su konvencionalne tehnike spoligotipizacija i MIRU-VNTR tipizacija, a sekvenciranje genoma nove generacije (NGS) izvršeno je na Illumina MiSeq sekvenatoru, tehnikom sekvenciranja putem sinteze i reverzibilnih terminatora. Klasteri MDR sojeva MTK identifikovani primenom klasičnih tehnika genotipizacije definisani su kao grupe od najmanje dva soja sa identičnim genotipovima, a klasteri definisani na osnovu rezultata NGS kao grupe sojeva čiji su se genomi razlikovali u ≤12 polimorfizama pojedinačnih nukleotida (SNP). Identifikacija faktora rizika za transmisiju MDR TB u Srbiji izvedena je na osnovu poređenja demografskih, kliničkih, epidemioloških i laboratorijskih karakteristika pacijenata čiji su i, The spread of multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains, defined as resistant to at least the two most important antituberculous drugs isoniazid and rifampicin, is a major threat for tuberculosis (TB) control programmes. Molecular genetic characterization of MDR MTBC strains circulating in a population is essential to guide establishment of efficient MDR TB control strategies, but no such analysis of MDR MTBC strains isolated in Serbia has been performed so far. Therefore, this research included a representative collection of MDR MTBC strains isolated in Serbia, and molecular identification to the species level, comprehensive genotyping, and detection of mutations in genes conferring resistance to the first- and second-line anti-TB drugs were performed. The research included two collections of MTBC strains: all MDR MTBC strains isolated in Serbia from January 1st 2008 to May 31st 2014 (115 strains), and all MTBC strains isolated from patients diagnosed with pulmonary TB in Serbia from December 1st 2010 to November 30th 2011 (971 strains). Molecular identification of all MTBC strains was performed by using a commercial assay based on reverse hybridization. Spoligotyping and MIRU-VNTR methods were used as classical genotyping techniques, while next generation sequencing (NGS) by synthesis and reversible terminators was performed using the Illumina MiSeq sequencer. In classical genotyping analyses, a cluster was defined as a minimum of two strains harbouring identical genotype pattern. Genomic clusters were defined where no more than 12 single-nucleotide polymorphisms (SNPs) separated a patient isolate from that of at least one other patient in the cluster. Risk factors for active transmission of MDR TB in Serbia were identified by comparison of demographic, clinical, epidemiologic, and laboratory characteristics of patients whose strains were grouped in clusters and patients whose strains exhibited unique genotypes. Comprehensive anal

Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (. Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (. n=. 53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account.

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis and closely related mycobacteria of the Mycobacterium tuberculosis complex. They have an extensive host range and may cause zoonotic TB. A major obstacle to bTB eradication in livestock is the implication of wildlife in the natural cycle of the pathogen. The identification of wildlife reservoir hosts is crucial for the implementation of effective control measures. The European wild boar (Sus scrofa) is frequently considered a spillover or dead end host rather than a true reservoir, and scientific evidence is conflicting outside Mediterranean Spain. The aim of this review is to update current scientific evidence of the wild boar as a TB reservoir and to underline those aspects that need further research. Evidences supporting that wild boar is a TB reservoir host include: (i) presence of common M. tuberculosis complex genotypes in wild boar, domestic and wild animals and humans, (ii) high prevalence of M. bovis among wild boar in estates fenced for decades in complete absence of contact with domestic livestock, and other wild ungulates (iii) TB lesions are frequently seen in thoracic lymph nodes and lungs, suggesting that respiratory infection and excretion may occur, and (iv) extensive tuberculous lesions in more than one anatomical region occur in a high proportion of juvenile wild boar that probably represents the main source of mycobacterial excretion. Hence, epidemiological, pathological and microbiological evidence strongly suggests that, at least in Spanish Mediterranean ecosystems, wild boar are able to maintain TB infection in the wild and are most probably able to transmit the disease to other species, acting as a true wildlife reservoir. These results expand the list of wildlife species that act as natural reservoirs of TB in different parts of the world and suggest the need to control the infection in wild boar populations for the complete eradication of the disease 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.

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.