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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

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3–20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency set

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

Microsporidia comprises a diverse group of obligate intracellular parasites that infect a broad range of invertebrates and vertebrates. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen able to infect many domestic and wild mammals that could act as reservoir of infection for humans and other animals, only few studies have documented its occurrence in wild carnivores. To determine the occurrence of E. bieneusi in wild carnivores, we examined 190 wild carnivores collected from different locations in Spain. Twenty-five fecal samples (13.2%) from three host species (European badger, beech marten, and red fox) were E. bieneusi-positive by PCR. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity with a total of eight distinct genotypes including four known (PtEbIX, S5, S9, and WildBoar3) and four novel (EbCar1-EbCar4) genotypes identified. Phylogenetic analysis showed that the four novel genotypes (EbCar1-EbCar4), S5, S9, and WildBoar3 clustered within the previously designated zoonotic Group 1. Our results demonstrate that human-pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination., Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

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

CRediT authorship contribution statement All authors contributed equally to the idea, literature review and writing of this manuscript., Biosafety measures (BSMs) often aim at reducing the likelihood of cross-species interactions at the wildlife-livestock interface. Examples include means to segregate wild ungulates from cattle at waterholes or at feeders. Subsidies or incentives for BSM implementation are expected to contribute to improved BSM acceptance. However, several recent experiences led us to write a cautionary commentary on the variable success of incentives in farm biosafety promotion. We list examples where, after offering farm-specific biosecurity action plans for free or subsidizing 100% of the cost of a given BSM, 25-40% of the farmers remained unwilling to invest efforts in farm biosafety and BSM maintenance. We suggest seeking a better understanding of farmers' motivations through social science research, to train farm veterinarians on biosecurity and on how to tailor biosafety communication, and to set up formal regional risk mitigation programs including financial, logistical, and educational assistance, as well as monitoring plans, through public-private collaboration, Depto. de Sanidad Animal, Centro de Vigilancia Sanitaria Veterinaria (VISAVET), TRUE, pub

Canine distemper virus (CDV) affects a huge diversity of domestic and wild carnivores, with increasing numbers of mortality events worldwide. The local cell-mediated immune response elicited against a natural infection is an important factor in determining the outcome of CDV infection. Therefore, the purposes of this study were to describe the local immune response within the central nervous systems (CNSs) of seven badgers naturally infected with CDV in Asturias (Atlantic Spain) and to determine the phenotype and distribution of microglial cells, T and B lymphocytes, and astrocytes in the foci of gliosis located in the thalamus and cerebellum using immunohistochemistry. The immunohistochemical assessment demonstrated the presence of Iba1-positive microglia and GFAP-positive astrocytes in the foci of gliosis, whereas T (CD3-negative) or B (CD20-negative) lymphocytes in those same lesions were absent. Our results also revealed that the badgers with natural CDV encephalitis presented lesions mostly located in the white matter of the thalamus and cerebellum, suggesting a CDV-specific tropism for the white matter of badger brains in those locations. The knowledge gained in the field of the immunopathogenesis of distemper disease affecting the CNSs of badgers could help to clarify CDV disease patterns in this species.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1–3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2–6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3–1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulateadapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate

Biosafety measures (BSMs) often aim at reducing the likelihood of cross-species interactions at the wildlifelivestock interface. Examples include means to segregate wild ungulates from cattle at waterholes or at feeders. Subsidies or incentives for BSM implementation are expected to contribute to improved BSM acceptance. However, several recent experiences led us to write a cautionary commentary on the variable success of incentives in farm biosafety promotion. We list examples where, after offering farm-specific biosecurity action plans for free or subsidizing 100% of the cost of a given BSM, 25–40% of the farmers remained unwilling to invest efforts in farm biosafety and BSM maintenance. We suggest seeking a better understanding of farmers’ motivations through social science research, to train farm veterinarians on biosecurity and on how to tailor biosafety communication, and to set up formal regional risk mitigation programs including financial, logistical, and educational assistance, as well as monitoring plans, through public-private collaboration.

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.

In autumn 2011, a disease outbreak caused by Spanish goat encephalitis virus (SGEV) was reported in a herd of goats from Asturias (north-western Spain), expanding the known geographic distribution of tick-borne encephalitis in Europe. The virus was classified as a new subtype (subspecies) within the Louping-ill virus species of the mammalian tick-borne flavivirus group. The aims of the present study were to describe the pathology in goats naturally infected with SGEV, as well as discuss the pathogenesis of the disease in that outbreak. A total of 22/85 (25.88%) goats (20 adults and 2 kids) died between October 2011 and June 2012, showing neurological clinical signs. Over three years, the mortality rate in the herd reached 100%. Neuropathological lesions caused by SGEV were severe and widespread throughout the central nervous system but were more severe and numerous in the proximal cervical spinal cord, medulla oblongata, pons and cerebellar cortex. They consisted of neuron necrosis, neuronophagia, mononuclear inflammatory cell perivascular cuffs (lymphocytes, plasma cells and macrophages) and gliosis. The distribution of viral antigens was restricted to the cytoplasm of neurons in several brain areas but not associated with inflammatory foci nor inflammatory cells. SGEV should be considered a significant pathogen of goats that results in severe neurological clinical disease and high mortality.

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.