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Gloria Herrero-García is funded by Junta de Castilla y León and FSE (contract ref LE036-20); Rita Vaz-Rodrigues is funded by Universidad de Castilla-La Mancha (UCLM) and ESF (contract ref. 2022/20675); Pilar Pozo is funded by Juan de la Cierva Formación 2021, funded by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and NextGenerationEU/PRTR (contract ref FJC2021-046805-I); Patricia Barroso is funded by a Juan de la Cierva post-doc fellowship (contract ref DC2022-049103-I); and Alberto García-Rodríguez is funded by the EU-NextGenerationEU funds through the 2021–2023 Margarita Salas call for the requalification of the Spanish university system, convened by the Universidad de León, Despite enormous efforts, complete animal tuberculosis (TB) eradication has only been achieved in few regions. Epidemiological analyses are key to identify TB risk factors and set up targeted biosecurity measures. Here, we conducted an in-depth characterization of 84 extensive beef cattle farms from a high TB prevalence region in Western Spain, and assessed how farm management and wildlife presence on farms contribute to cattle TB risk. Twenty-six out of 84 variables were associated with cattle farm TB positivity. Farm management variables associated with TB positivity included older cattle, larger herd size, highly fragmented farm structure and greater connectivity between farms. TB-positive farms provided supplemental feed over a higher number of months, used calf feeders, and had higher number of waterholes. Detecting Eurasian wild boar (Sus scrofa), red fox (Vulpes vulpes), European badger (Meles meles), roe deer (Capreolus capreolus), or Egyptian mongoose (Herpestes ichneumon) on cattle farms was also associated with farm TB positivity. The best ordinal regression model indicated that in farms with a large herd size (> 167 animals) the odds of being positive or recurrently positive (versus negative) was 7.34 (95% CI = 2.43–23.51) times higher that of farms with small herd size. Further, for every additional host species detected in the farm premises, the odds of being TB-positive increased 56%. We conclude that both cattle management and wildlife need to be targeted for successful TB control in grazing-based farming systems., A pesar de los enormes esfuerzos, la erradicación completa de la tuberculosis (TB) animal solo se ha logrado en algunas regiones. Los estudios epidemiológicos son fundamentales para identificar factores de riesgo de TB y establecer medidas de bioseguridad específicas. Aquí, realizamos una caracterización exhaustiva de 84 explotaciones de ganado bovino extensivo en una provincia occidental de España con alta prevalencia de TB bovina, y evaluamos cómo el manejo del ganado y la presencia de fauna silvestre contribuían al riesgo de esta enfermedad. De las 84 variables estudiadas, 26 mostraron asociación con la positividad a TB bovina. Entre las variables de manejo destacaron: ganado de mayor edad, rebaños de mayor tamaño, explotaciones altamente fragmentadas y mayor conectividad entre ganaderías. Asimismo, las explotaciones positivas a TB prolongaban la alimentación suplementaria, utilizaban cebaderos para terneros y tenían un mayor número de charcas. La detección de jabalí (Sus scrofa), zorro (Vulpes vulpes), tejón (Meles meles), corzo (Capreolus capreolus) o meloncillo (Herpestes ichneumon) en las ganaderías también se asoció con la positividad a TB. El mejor modelo de regresión ordinal mostró que en explotaciones con un tamaño de rebaño grande (> 167 animales) las probabilidades de ser positivo o recurrentemente positivo (frente a negativo) eran 7,34 (95 % IC = 2,43–23,51) veces superiores, a las de las explotaciones con un tamaño de rebaño pequeño. Además, por cada especie hospedadora susceptible detectada adicionalmente, la probabilidad de que la ganadería fuese positiva a TB aumentaba un 56 %. Concluimos que tanto el manejo del ganado como la interacción con fauna silvestre deben ser considerados para un control exitoso de la TB en explotaciones con sistemas de pastoreo extensivo., European Commission, Ministerio de Ciencia, Innovación y Universidades (España), Depto. de Sanidad Animal, Fac. de Veterinaria, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

In dry climates, livestock farming contributes to waterhole creation and maintenance, thereby contributing to biodiversity conservation. However, these lentic water bodies also represent a critical environmental connection between microorganisms and their vertebrate hosts since the water can facilitate pathogen persistence and transmission. Therefore, interventions for tuberculosis (TB) risk mitigation at the wildlife-livestock interface often focus on segregating host species at water points. We hypothesized that waterhole characteristics modulate their use by vertebrates and subsequent pathogen exposure risk. We visited 298 waterholes on 80 TB-positive and 40 TB-negative cattle farms in Spain to assess differences in waterhole characteristics and identify possible management implications. There was an average of 2.7 waterholes per farm. This represents 0.02 waterholes per km2 of farmland and 3.5 m2 of lentic waterbodies per km2 of farmland. Among the studied waterholes, 95% were man-made. Waterholes on TB-positive farms were 42% closer to covering vegetation than waterholes located on TB-negative ones. Farms with man-made waterholes showed a higher risk of TB than those with natural ones. The density of waterholes per surface unit was negatively associated with the farm TB risk. Waterholes placed on TB-positive farms were more intensively trampled by livestock. The best model explained farm positivity to TB as a function of the distance from the waterholes to the nearest cover vegetation, the aquatic vegetation richness found in the waterhole, the intensity of livestock use (trampling), the surrounding waterhole density, and the interaction between waterhole perimeter and the total number of signs of potential wildlife TB hosts per waterhole. Identifying the key waterhole features related to infection risk might allow designing One Health−inspired biosecurity measures such as increasing the number of waterholes, placing new waterholes farther away from cover, or f

2023 Acuerdos transformativos CRUE, Tuberculosis (TB) is a contagious chronic disease due to infection with Mycobacterium tuberculosis complex (MTC) bacteria. Monitoring of wildlife, especially potential reservoirs, is important for detecting changes in disease occurrence and assessing the impact of interventions. Here, we examined whether wild boar (Sus scrofa) may contribute to the re-emergence of TB in Asturias (10,604 km2), northern Spain. Although this province was declared free of TB in cattle in November 2021, MTC bacteria remain prevalent in several “hotspots,” with the European badger (Meles meles) suggested as a TB potential wild reservoir. Drawing on data from the Spanish National Bovine Tuberculosis Eradication Program and the Government of the Principality of Asturias covering the period 2014–2020, we analyzed the prevalence of TB in cattle and wild boar in this region. In hotspots (592 km2), we also investigated the ranging behavior and habitat use of five cows that belonged to farms with a history of TB and six trapped sympatric wild boar. During the observation period, TB prevalence was 0.14% among cattle overall and 0.13–0.41% in hotspots, which was much lower than the prevalence in wild boar, which was 3.15% overall and 5.23–5.96% in hotspots. Infected cattle and infected wild boar in hotspots shared the same strains of M. bovis, and GPS tracking showed spatiotemporal overlap between the species, mainly around pastures during sunrise (06:00–07:00 h) and sunset (19:00–20:00 h). Our results suggest that in addition to cattle and badgers, wild boar possibly help maintain TB in northern Spain, increasing the host richness that influences TB transmission risk in the area, which should be taken into account in monitoring and eradication efforts., Ministerio de Ciencia e Innovación (España), Principado de Asturias, Ministerio de Agricultura, Pesca y Alimentacion (España), Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Fac. de Veterinaria, TRUE, pub, APC financiada por la UCM

Tuberculosis (TB) is a contagious chronic disease due to infection with Mycobacterium tuberculosis complex (MTC) bacteria. Monitoring of wildlife, especially potential reservoirs, is important for detecting changes in disease occurrence and assessing the impact of interventions. Here, we examined whether wild boar (Sus scrofa) may contribute to the re-emergence of TB in Asturias (10,604 km2), northern Spain. Although this province was declared free of TB in cattle in November 2021, MTC bacteria remain prevalent in several “hotspots,” with the European badger (Meles meles) suggested as a TB potential wild reservoir. Drawing on data from the Spanish National Bovine Tuberculosis Eradication Program and the Government of the Principality of Asturias covering the period 2014–2020, we analyzed the prevalence of TB in cattle and wild boar in this region. In hotspots (592 km2), we also investigated the ranging behavior and habitat use of five cows that belonged to farms with a history of TB and six trapped sympatric wild boar. During the observation period, TB prevalence was 0.14% among cattle overall and 0.13–0.41% in hotspots, which was much lower than the prevalence in wild boar, which was 3.15% overall and 5.23–5.96% in hotspots. Infected cattle and infected wild boar in hotspots shared the same strains of M. bovis, and GPS tracking showed spatiotemporal overlap between the species, mainly around pastures during sunrise (06:00–07:00 h) and sunset (19:00–20:00 h). Our results suggest that in addition to cattle and badgers, wild boar possibly help maintain TB in northern Spain, increasing the host richness that influences TB transmission risk in the area, which should be taken into account in monitoring and eradication efforts.

The Cantabrian capercaillie (Tetrao urogallus cantabricus) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli, Clostridium perfringens, or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.

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

Dolphins are marine mammals that often live in coastal habitats. Common causes of severe skeletal disorders among wild dolphins are congenital vertebral anomalities, collisions with sea vessels, trauma, hunting-related injury, infectious diseases, environmental pollution, and tumors. A free-ranging male, 3-year-old common dolphin (Delphinus delphis) was found dead in the coast of Asturias in northern Spain. Postmortem examination revealed lordosis in the caudal vertebral column, while X-ray imaging and computer tomography showed well-organized palisade-like periosteal proliferation, appearing as florid-like accretions, along the spinous apophysis of 26 lumbar-caudal vertebrae. The transverse apophysis was affected on only a few caudal vertebrae. The cortical layer remained intact. Histology of vertebra tissue showed periosteal proliferation of cancellous bone. The animal was diagnosed with hypertrophic osteopathy. The lungs showed diffuse parasitic granulomatous bronchointerstitial pneumonia caused by Halocercus delphini, consolidation of the pulmonary tissue, congestion, and alveolar edema. The animal was also afflicted by parasitic granulomatous gastritis caused by Anisakis simplex sensu lato and tattoo skin disease. The dolphin suffered from hypertrophic osteopathy associated with pulmonary Halocercus delphini infestation. This syndrome, known as hypertrophic pulmonary osteopathy, has been described in diverse terrestrial mammals, including domestic animals, wildlife and humans, but not in dolphins. This case reports the first description of hypertrophic osteopathy associated to a pulmonary disorder in dolphin, and it provides insights into factors that can induce column malformation in dolphins, suggesting the importance of taking thoracic lesions into account during differential diagnosis.

Crimean–Congo haemorrhagic fever (CCHF) is an emerging tick-borne human disease in Spain. Understanding the spatiotemporal dynamics and exposure risk determinants of CCHF virus (CCHFV) in animal models is essential to predict the time and areas of highest transmission risk. With this goal, we designed a longitudinal survey of two wild ungulate species, the red deer (Cervus elaphus) and the Eurasian wild boar (Sus scrofa), in Doñana National Park, a protected Mediterranean biodiversity hotspot with high ungulate and CCHFV vector abundance, and which is also one of the main stopover sites for migratory birds between Africa and western Europe. Both ungulates are hosts to the principal CCHFV vector in Spain, Hyalomma lusitanicum. We sampled wild ungulates annually from 2005 to 2020 and analysed the frequency of exposure to CCHFV by a double-antigen ELISA. The annual exposure risk was modelled as a function of environmental traits in an approach to understanding exposure risk determinants that allow us to predict the most likely places and years for CCHFV transmission. The main findings show that H. lusitanicum abundance is a fundamental driver of the fine-scale spatial CCHFV transmission risk, while inter-annual risk variation is conditioned by virus/vector hosts, host community structure and weather variations. The most relevant conclusion of the study is that the emergence of CCHF in Spain might be associated with recent wild ungulate population changes promoting higher vector abundance. This work provides relevant insights into the transmission dynamics of CCHFV in enzootic scenarios that would allow deepening the understanding of the ecology of CCHFV and its major determinants.

[ES]: La fiebre hemorrágica de Crimea Congo (FHCC) es la enfermedad vírica transmitida por garrapatas (Orden Ixodida) más extendida mundialmente. Está causada por un Orthonairovirus, cuyo ciclo de vida incluye garrapatas (principalmente Hyalomma), como vectores y reservorios, y animales como amplificadores del agente infeccioso u hospedadores/dispersores de los vectores. Los humanos son los únicos que padecen la enfermedad. Es posible que, siendo la abundancia de garrapatas variable en espacio y tiempo, la transmisión del virus de la fiebre hemorrágica de Crimea Congo (VFHCC) también esté sujeta a esta variación espaciotemporal y, por ello, ser un proceso dinámico. España, y más concretamente el Parque Nacional de Doñana (PND), es una zona con gran riesgo de entrada del virus debido a las condiciones adecuadas que presenta (ambiente, amplia presencia de garrapatas, gran variedad de mamíferos silvestres y zona esencial de parada de millones de aves migratorias), y por tanto ideal para la realización de este estudio, donde se buscó comprender la dinámica espaciotemporal del VFHCC y se trató de estimar la relación entre la abundancia de garrapatas vectores y el riesgo de exposición/infección al virus. El 60% de los ungulados silvestres analizados entre 2006 y 2019 resultaron seropositivos al VFHCC, mientras que el 12,8% de las garrapatas alimentadas analizadas entre 2015 y 2019 resultaron positivas a la PCR, detectándose tanto genotipo III (africano) como V (europeo), y observándose una variación temporal entre las muestras positivas. Se encontró además un efecto potencial de la abundancia predicha de Hyalomma lusitanicum sobre el riesgo de exposición al virus de los ungulados silvestres. Sin embargo, no se pudo confirmar tal efecto sobre la prevalencia de infección al VFHCC en garrapatas alimentadas., [EN]: Crimean-Congo hemorrhagic fever (CCHF) is the most widespread viral disease transmitted by ticks (Order Ixodida). It is caused by an Orthonairovirus, which life cycle includes ticks (mainly Hyalomma), as vectors and reservoirs, and animals as amplifiers of the infectious agent or as hosts/dispersers of the vectors. Humans are the only ones who suffer from the disease. Since tick abundance varies in space and time, it is possible that the transmission of CrimeanCongo hemorrhagic fever virus (CCHFV) also follows this spatiotemporal variation, and, therefore, it is a dynamic process. Spain, and more specifically Doñana National Park (PND), is considered and area under high risk of CCHFV circulation due to the favourable conditions it displays (environment, wide presence of ticks, a wide variety of wild mammals, and being an essential stopover for millions of migrating birds), and therefore ideal for the realization of this study. We tried to understand the spatiotemporal dynamics of the virus and we attempted to estimate the relationship between the abundance of the tick vectors and the risk of exposure/infection to the virus, all within the PND. The 60% of the wild ungulates analyzed between 2006 and 2019 were seropositive to CCFHV, while 12,8% of the fed ticks analyzed between 2015 and 2019 were PCR positive, detecting both genotype III (African) and genotype V (European), and observing a temporal variation among the positive samples. An effect of the predicted abundance of Hyalomma lusitanicum over the risk of exposure of the wild ungulates to the virus was also found, however, an effect over the prevalence of infection to VFHCC in fed ticks could not be confirmed.

[EN] The Cantabrian capercaillie (Tetrao urogallus cantabricus) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli, Clostridium perfringens, or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.

[EN] Tuberculosis (TB) is a contagious chronic disease due to infection with Mycobacterium tuberculosis complex (MTC) bacteria. Monitoring of wildlife, especially potential reservoirs, is important for detecting changes in disease occurrence and assessing the impact of interventions. Here, we examined whether wild boar (Sus scrofa) may contribute to the re-emergence of TB in Asturias (10,604 km2), northern Spain. Although this province was declared free of TB in cattle in November 2021, MTC bacteria remain prevalent in several “hotspots,” with the European badger (Meles meles) suggested as a TB potential wild reservoir. Drawing on data from the Spanish National Bovine Tuberculosis Eradication Program and the Government of the Principality of Asturias covering the period 2014–2020, we analyzed the prevalence of TB in cattle and wild boar in this region. In hotspots (592 km2), we also investigated the ranging behavior and habitat use of five cows that belonged to farms with a history of TB and six trapped sympatric wild boar. During the observation period, TB prevalence was 0.14% among cattle overall and 0.13–0.41% in hotspots, which was much lower than the prevalence in wild boar, which was 3.15% overall and 5.23–5.96% in hotspots. Infected cattle and infected wild boar in hotspots shared the same strains of M. bovis, and GPS tracking showed spatiotemporal overlap between the species, mainly around pastures during sunrise (06:00–07:00 h) and sunset (19:00–20:00 h). Our results suggest that in addition to cattle and badgers, wild boar possibly help maintain TB in northern Spain, increasing the host richness that influences TB transmission risk in the area, which should be taken into account in monitoring and eradication efforts.

[EN] Dolphins are marine mammals that often live in coastal habitats. Common causes of severe skeletal disorders among wild dolphins are congenital vertebral anomalities, collisions with sea vessels, trauma, hunting-related injury, infectious diseases, environmental pollution, and tumors. A free-ranging male, 3-year-old common dolphin (Delphinus delphis) was found dead in the coast of Asturias in northern Spain. Postmortem examination revealed lordosis in the caudal vertebral column, while X-ray imaging and computer tomography showed well-organized palisade-like periosteal proliferation, appearing as florid-like accretions, along the spinous apophysis of 26 lumbar-caudal vertebrae. The transverse apophysis was affected on only a few caudal vertebrae. The cortical layer remained intact. Histology of vertebra tissue showed periosteal proliferation of cancellous bone. The animal was diagnosed with hypertrophic osteopathy. The lungs showed diffuse parasitic granulomatous bronchointerstitial pneumonia caused by Halocercus delphini, consolidation of the pulmonary tissue, congestion, and alveolar edema. The animal was also afflicted by parasitic granulomatous gastritis caused by Anisakis simplex sensu lato and tattoo skin disease. The dolphin suffered from hypertrophic osteopathy associated with pulmonary Halocercus delphini infestation. This syndrome, known as hypertrophic pulmonary osteopathy, has been described in diverse terrestrial mammals, including domestic animals, wildlife and humans, but not in dolphins. This case reports the first description of hypertrophic osteopathy associated to a pulmonary disorder in dolphin, and it provides insights into factors that can induce column malformation in dolphins, suggesting the importance of taking thoracic lesions into account during differential diagnosis.

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