Bottle Aging and Storage of Wines: A Review.

Wine is perhaps the most ancient and popular alcoholic beverage worldwide. Winemaking practices involve careful vineyard management alongside controlled alcoholic fermentation and potential aging of the wine in barrels. Afterwards, the wine is placed in bottles and stored or distributed in retail. Yet, it is considered that wine achieves its optimum properties after a certain storage time in the bottle. The main outcome of bottle storage is a decrease of astringency and bitterness, improvement of aroma and a lighter and more stable color. This is due to a series of complex chemical changes of its components revolving around the minimized and controlled passage of oxygen into the bottle. For this matter, antioxidants like sulfur oxide are added to avoid excessive oxidation and consequent degradation of the wine. In the same sense, bottles must be closed with appropriate stoppers and stored in adequate, stable conditions, as the wine may develop unappealing color, aromas and flavors otherwise. In this review, features of bottle aging, relevance of stoppers, involved chemical reactions and storage conditions affecting wine quality will be addressed.

Blastocystis sp. Subtype Diversity in Wild Carnivore Species from Spain.

The occurrence and molecular diversity of the stramenopile eukaryote Blastocystis sp. was investigated by PCR and sequencing (Sanger and NGS) methods in 380 faecal specimens of free-living carnivores in Spain. Blastocystis sp. was confirmed in 1.6% (6/380) of the specimens analysed. Two samples from a common genet and a fox were successfully subtyped as ST7 by Sanger. Using NGS, ST14 was found in a fox and a European polecat, ST7 in a fox, and two additional foxes presented mixed infections of ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Wild carnivore species could act as carriers of zoonotic Blastocystis subtypes.

Cost-effectiveness of public health interventions against human influenza pandemics in France: a methodological contribution from the FLURESP European Commission project.

BACKGROUND: The FLURESP project is a public health project funded by the European Commission with the objective to design a methodological approach in order to compare the cost-effectiveness of existing public health measures against human influenza pandemics in four target countries: France, Italy, Poland and Romania. This article presents the results relevant to the French health system using a data set specifically collected for this purpose. METHODS: Eighteen public health interventions against human influenza pandemics were selected. Additionally, two public-health criteria were considered: 'achieving mortality reduction ≥40%' and 'achieving morbidity reduction ≥30%'. Costs and effectiveness data sources include existing reports, publications and expert opinions. Cost distributions were taken into account using a uniform distribution, according to the French health system. RESULTS: Using reduction of mortality as an effectiveness criterion, the most cost-effective options was 'implementation of new equipment of Extracorporeal membrane oxygenation (ECMO) equipment'. Targeting vaccination to health professionals appeared more cost-effective than vaccination programs targeting at risk populations. Concerning antiviral distribution programs, curative programs appeared more cost-effective than preventive programs. Using reduction of morbidity as effectiveness criterion, the most cost-effective option was 'implementation of new equipment ECMO'. Vaccination programs targeting the general population appeared more cost-effective than both vaccination programs of health professionals or at-risk populations. Curative antiviral programs appeared more cost-effective than preventive distribution programs, whatever the pandemic scenario. CONCLUSION: Intervention strategies against human influenza pandemics impose a substantial economic burden, suggesting a need to develop public-health cost-effectiveness assessments across countries.

Long-term avian influenza virus epidemiology in a small Spanish wetland ecosystem is driven by the breeding Anseriformes community.

During 2007-2009 and 2012-2014, avian influenza virus (AIV) was studied in a wild avian community of a northern Spanish wetland using non-invasive sampling methods and host identification by COI barcoding. The aim of this longitudinal study was to evaluate AIV dynamics in a natural wetland ecosystem, taking into account both virological aspects and ecological traits of hosts. Global AIV prevalence decreased significantly during the second sampling period (0.3%) compared to the first (6.6%). Circulating subtype distributions were also different between periods, with a noteworthy H5 and H7 subtype richness during the first sampling period. Mallard Anas platyrhynchos was identified as the main AIV host, although not all positive samples could be ascribed to the host. We modelled AIV prevalence with regard to the avian host community composition and meteorological data from the wetland. Statistical analysis revealed seasonal differences in AIV detection, with higher prevalence during the breeding season compared to other phenological events. The model also shows that the lower AIV prevalence during the second study period was associated with a significant reduction of breeding Anseriformes in the wetland, revealing a long-term fluctuation of AIV prevalence driven by the breeding Anseriformes community. This longitudinal study on AIV epidemiology in a natural ecosystem reveals that although prevalence follows seasonal and annual patterns, long-term prevalence fluctuation is linked to the breeding community composition and size. These results are relevant to understanding the influence of host ecology on pathogen transmission for preventing and managing influenza emergence.

Molecular Characterization of Enterocytozoon bieneusi in Wild Carnivores in Spain.

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.

Host and Environmental Factors Modulate the Exposure of Free-Ranging and Farmed Red Deer (Cervus elaphus) to Coxiella burnetii.

The control of multihost pathogens, such as Coxiella burnetii, should rely on accurate information about the roles played by the main hosts. We aimed to determine the involvement of the red deer (Cervus elaphus) in the ecology of C. burnetii. We predicted that red deer populations from broad geographic areas within a European context would be exposed to C. burnetii, and therefore, we hypothesized that a series of factors would modulate the exposure of red deer to C. burnetii. To test this hypothesis, we designed a retrospective survey of 47 Iberian red deer populations from which 1,751 serum samples and 489 spleen samples were collected. Sera were analyzed by enzyme-linked immunosorbent assays (ELISA) in order to estimate exposure to C. burnetii, and spleen samples were analyzed by PCR in order to estimate the prevalence of systemic infections. Thereafter, we gathered 23 variables-within environmental, host, and management factors-potentially modulating the risk of exposure of deer to C. burnetii, and we performed multivariate statistical analyses to identify the main risk factors. Twenty-three populations were seropositive (48.9%), and C. burnetii DNA in the spleen was detected in 50% of the populations analyzed. The statistical analyses reflect the complexity of C. burnetii ecology and suggest that although red deer may maintain the circulation of C. burnetii without third species, the most frequent scenario probably includes other wild and domestic host species. These findings, taken together with previous evidence of C. burnetii shedding by naturally infected red deer, point at this wild ungulate as a true reservoir for C. burnetii and an important node in the life cycle of C. burnetii, at least in the Iberian Peninsula.

Evaluation of different enrichment methods for pathogenic Yersinia species detection by real time PCR.

BACKGROUND: Yersiniosis is a zoonotic disease reported worldwide. Culture and PCR based protocols are the most common used methods for detection of pathogenic Yersinia species in animal samples. PCR sensitivity could be increased by an initial enrichment step. This step is particularly useful in surveillance programs, where PCR is applied to samples from asymptomatic animals. The aim of this study was to evaluate the improvement in pathogenic Yersinia species detection using a suitable enrichment method prior to the real time PCR (rtPCR). Nine different enrichment protocols were evaluated including six different broth mediums (CASO, ITC, PSB, PBS, PBSMSB and PBSSSB). RESULTS: The analysis of variance showed significant differences in Yersinia detection by rtPCR according to the enrichment protocol used. These differences were higher for Y. pseudotuberculosis than for Y. enterocolitica. In general, samples incubated at lower temperatures yielded the highest detection rates. The best results were obtained with PBSMSB and PBS2. Application of PBSMSB protocol to free-ranging wild board samples improved the detection of Y. enterocolitica by 21.2% when compared with direct rtPCR. Y. pseudotuberculosis detection was improved by 10.6% when results obtained by direct rtPCR and by PBSMSB enrichment before rtPCR were analyzed in combination. CONCLUSIONS: The data obtained in the present study indicate a difference in Yersinia detection by rtPCR related to the enrichment protocol used, being PBSMSB enrichment during 15 days at 4°C and PBS during 7 days at 4°C the most efficient. The use of direct rtPCR in combination with PBSMSB enrichment prior to rtPCR resulted in an improvement in the detection rates of pathogenic Yersinia in wild boar and could be useful for application in other animal samples.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

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 settings.

Efficacy of heat-inactivated Mycobacterium bovis vaccine delivered to European badgers (Meles meles) through edible bait.

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.

Presence of Bartonella species in wild carnivores of northern Spain.

The genus Bartonella was detected by PCR in 5.7% (12/212) of wild carnivores from Northern Spain. Based on hybridization and sequence analyses, Bartonella henselae was identified in a wildcat (Felis silvestris), Bartonella rochalimae in a red fox (Vulpes vulpes) and in a wolf (Canis lupus), and Bartonella sp. in badgers (Meles meles).

Beyond tuberculosis: Diversity and implications of non-tuberculous mycobacteria at the wildlife-livestock interface.

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

Stable prevalence of Coxiella burnetii in wildlife after a decade of surveillance in northern Spain.

Coxiella burnetii is an obligate intracellular zoonotic bacterium widespread in nature that causes Q fever in animals and humans. The most common sources of human infection are domestic ruminants, but wildlife can also act as reservoir. Here, spleen samples from 652 wild ungulates and 218 wild birds collected in 2011-2019 in the Basque Country (northern Spain) were analysed by real-time PCR (IS1111 gene) and the results compared with data from a past study in 2001-2006. Among wild ungulates, C. burnetii DNA was detected in 7.0% (6/86) of roe deer (Capreolus capreolus), 1.9% (9/484) of wild boar (Sus scrofa) and 2.4% (2/82) of red deer (Cervus elaphus). The prevalence in roe deer was significantly higher compared to wild boar (p = 0.006). Among wild birds, only one white stork (Ciconia ciconia) tested positive. SNP-typing of C. burnetii-positive samples showed that wild ungulates shared SNP 2, SNP 6 and SNP 8 genotypes with domestic ruminants of the region. However, the white stork harboured a C. burnetii genotype (SNP 3) never identified in the studied area before. Comparing these results with those obtained in the same area a decade before (2001-2006), no significant differences were observed in the prevalence of C. burnetii in any of the wildlife species, indicating stability in C. burnetii prevalence. Nevertheless, continuous surveillance is needed to monitor any future changes in the reservoir role of roe deer and wild boar considering the increase in density of both species observed in Europe in the last decades.

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa).

An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.

Interaction Patterns between Wildlife and Cattle Reveal Opportunities for Mycobacteria Transmission in Farms from North-Eastern Atlantic Iberian Peninsula.

Interactions taking place between sympatric wildlife and livestock may contribute to interspecies transmission of the Mycobacterium tuberculosis complex or non-tuberculous mycobacteria, leading to the spread of relevant mycobacterioses or to interferences with the diagnosis of tuberculosis. The aim of this study was to characterize the spatiotemporal patterns of interactions between wildlife and cattle in a low bovine tuberculosis prevalence Atlantic region. Camera traps were set during a one-year period in cattle farms with a history of tuberculosis and/or non-tuberculous mycobacterioses. The frequency and duration of wildlife visits, and the number of individuals per visit, were analysed through generalized linear mixed models. The seasons, type of place, type of point, and period of the day were the explanatory variables. A total of 1293 visits were recorded during 2741 days of camera observation. Only 23 visits showed direct contacts with cattle, suggesting that mycobacteria transmission at the wildlife-livestock interface would occur mainly through indirect interactions. Cattle pastures represented the most appropriate habitat for interspecies transmission of mycobacteria, and badgers' latrines appear to be a potential hotspot for mycobacteria circulation between badgers, wild boars, foxes, and cattle. According to both previous epidemiological information and the interaction patterns observed, wild boars, badgers, foxes, and small rodents are the species or group most often in contact with livestock, and thus may be the most involved in the epidemiology of mycobacterioses in the wildlife-livestock interface in this area.

Traditional plants from Asteraceae family as potential candidates for functional food industry.

Traditional plants have been used in the treatment of disease and pain due to their beneficial properties such as antioxidant, antiinflammation, analgesic, and antibiotic activities. The Asteraceae family is one of the most common groups of plants used in folk medicine. The species Achillea millefolium, Arnica montana, Bellis perennis, Calendula officinalis, Chamaemelum nobile, Eupatorium cannabinum, Helichrysum stoechas, and Taraxacum officinale have been used in different remedies in Northwest Spain. Besides health benefits, some of them like C. nobile and H. stoechas are already employed in cooking and culinary uses, including cocktails, desserts, and savory dishes. This study aimed to review the current information on nutritive and beneficial properties and bioactive compounds of these plants, which are not mainly used as foods but are possible candidates for this purpose. The report highlights their current uses and suitability for the development of new functional food industrial applications. Phenolic compounds, essential oils, and sesquiterpene lactones are some of the most important compounds, being related to different bioactivities. Hence, they could be interesting for the development of new functional foods.

Molecular survey of Besnoitia spp. (Apicomplexa) in faeces from European wild mesocarnivores in Spain.

Numerous studies have unsuccessfully tried to unravel the definitive host of the coccidian parasite Besnoitia besnoiti. Cattle infections by B. besnoiti cause a chronic and debilitating condition called bovine besnoitiosis that has emerged in Europe during the last two decades, mainly due to limitations in its control associated with the absence of vaccines and therapeutical tools. Although the exact transmission pathways of B. besnoiti is currently unknown, it is assumed that the parasite might have an indirect life cycle with a carnivore as definitive host. Current lack of studies in wildlife might underestimate the importance of free-living species in the epidemiology of B. besnoiti. Thus, the aim of the present study is to assess the presence of Besnoitia spp. in free-ranging mesocarnivores in Spain. DNA was searched by PCR on faeces collected from wild carnivores as a first approach to determine which species could be considered as potential definitive host candidates in further research. For this purpose, a total of 352 faecal samples from 12 free-living wild carnivore species belonging to the Canidae, Felidae, Herpestidae, Mustelidae, Procyonidae and Viverridae families were collected in seven Spanish regions. PCR testing showed that Besnoitia spp. DNA was present in four faecal samples from red foxes collected in western Spain, an area with the greatest density of extensively reared cattle and associated with high incidence of bovine besnoitiosis in the country. To date, this is the first report of a B. besnoiti-like sequence (99.57% homology) from carnivore faeces in a worldwide context. Red foxes might contribute to the epidemiology of B. besnoiti, although further studies, mostly based on bioassay, would be needed to elucidate the accuracy and extent of these interesting findings.

Spatial distribution and risk factors of Brucellosis in Iberian wild ungulates.

BACKGROUND: The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates. METHODS: A multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar (Sus scrofa), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs. RESULTS: Mean apparent prevalence below 0.5% was identified in chamois (Rupicapra pyrenaica), Iberian wild goat (Capra pyrenaica), and red deer (Cervus elaphus). Roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries) and Barbary sheep (Ammotragus lervia) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs. CONCLUSIONS: In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

Risk factors associated to a high Mycobacterium tuberculosis complex seroprevalence in wild boar (Sus scrofa) from a low bovine tuberculosis prevalence area.

Animal tuberculosis is a worldwide zoonotic disease caused principally by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). In southern Iberian Peninsula, wild reservoirs such as the wild boar, among other factors, have prevented the eradication of bovine tuberculosis. However, most of the studies have been focused on south-central Spain, where the prevalence of tuberculosis is high among wild ungulates and cattle herds. In northern regions, where wild boar density and bovine tuberculosis prevalence are lower, fewer studies have been carried out and the role of this species is still under debate. The aim of this study was to describe the temporal and spatial distribution of antibodies against MTC in wild boar from the Basque Country, northern Spain. Sera from 1902 animals were collected between 2010 and 2016. The seroprevalence was determined with an in house enzyme-linked immunosorbent assay and the search of risk factors was assessed by Generalized Linear Models. Overall, 17% of wild boars (326/1902; 95%CI, [15.5%-18.9%]) showed antibodies against MTC. Risk factors associated with seropositivity were the year and location of sampling, the number of MTC positive cattle, the distance to positive farms and the percentage of shrub cover. Younger age classes were associated with increased antibody titres among seropositive individuals. The seroprevalence detected was higher than those previously reported in neighbouring regions. Hence, further studies are needed to better understand the role of wild boar in the epidemiology of tuberculosis in low tuberculosis prevalence areas and consequently, its relevance when developing control strategies.

Detection of Wood Mice (Apodemus sylvaticus) Carrying Non-Tuberculous Mycobacteria Able to Infect Cattle and Interfere with the Diagnosis of Bovine Tuberculosis.

Mycobacterial infections caused by the Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacteria (NTM) are of great medical and veterinary relevance. The aim of this research was to study whether small mammals play a role in the epidemiology of mycobacterioses. Four samplings of 100 traps were performed in each of three cattle farms with previous history of tuberculosis or NTM between 2017 and 2018. A total of 108 animals belonging to seven species were trapped, classified, and necropsied, and tissues were submitted to microbiological and molecular methods for mycobacteria identification. The wood mouse (Apodemus sylvaticus) was the most abundant species (87%). No MTC was detected but six different NTM were identified (M. intracellulare, M. avium subsp. paratuberculosis, M. gordonae, M. celatum, M. fortuitum, and a not determined Mycobacterium sp.), showing a prevalence of 6.5%. No significant association was found between mycobacteria prevalence and the analyzed factors. Although a role in the epidemiology of MTC could not be attributed to small mammals, A. sylvaticus carries NTM that could be pathogenic or interfere with the diagnosis of tuberculosis. According to our results, there is a risk of NTM transmission at the wildlife-livestock interface through potential indirect contacts between small mammals and cattle.

Local Lung Immune Response to Mycobacterium bovis Challenge after BCG and M. bovis Heat-Inactivated Vaccination in European Badger (Meles meles).

Tuberculosis (TB) vaccination could be used as a key part of integrated strategies for the disease's control if an effective and safe vaccine under field conditions is obtained. Recent studies in Spain have evaluated the protective efficacy of two oral vaccines against experimental challenge with live intra-bronchial Mycobacterium bovis in captive badgers: the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. With the objective of increasing the knowledge of the cellular development progress of infection and generating further tools to discriminate between mild and severe TB lesions between and within animals, the immunopathology of tuberculous lesions was studied to characterize the local immune response (cell type profile) within lung granulomas from control (non-vaccinated), BCG vaccinated and HIMB-vaccinated experimentally infected badgers with M. bovis. Four immunohistochemical protocols, for the specific detection of macrophages, T lymphocytes, B lymphocytes and plasma cells within TB granulomas in formalin fixed sections of the right middle lung lobe (lobe targeted for the M. bovis delivery), were performed. Immunolabelled sections were scanned and five randomly selected areas were analyzed with digital image analysis software. The results were expressed as the proportion of the positively immunolabelled area within the total area of the selected site. Data was analyzed using the statistical analysis software (SAS). In the three treatment groups, macrophages were the most abundant inflammatory cells within the granulomas, followed by B lymphocytes and plasma cells. T lymphocyes were absent in those granulomas. This would suggest a predominance of a non-specific innate response mediated by phagocytic cells over an adaptative humoral immune response. The proportion of macrophages and plasma cells was higher in BCG and HIMB-vaccinated badgers, respectively, suggesting the establishment of an adaptative humoral response in HIMB-vaccinated badgers. The lower bacterial load at the lung level, as well as the volume of lesions in lungs using magnetic resonance imaging in badgers with the HIMB vaccine in relation with local immune response presented, must be highlighted, since it would be an advantage in favor of its use under field conditions in terms of reducing TB transmission and environmental contamination.