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.

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.

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.

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.

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.

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.

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.

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.

Protective Effect of Oral BCG and Inactivated Mycobacterium bovis Vaccines in European Badgers (Meles meles) Experimentally Infected With M. bovis.

In Europe, badgers (Meles meles) are recognized as major tuberculosis (TB) reservoir hosts with the potential to transmit infection to associated cattle herds. Recent studies in Spain have demonstrated that vaccination with a heat-inactivated Mycobacterium bovis vaccine (HIMB) successfully protects captive wild boar and red deer against progressive disease. The aim of this study was to evaluate the efficacy of two oral vaccines against TB in a badger model: the live-attenuated M. bovis bacillus Calmette-Guérin BCG vaccine (Danish strain) and a HIMB vaccine. Twenty-four badgers were separated in three treatment groups: oral vaccinated with live BCG (108 CFU, n = 5), oral vaccinated with HIMB (107 CFU, n = 7), and unvaccinated controls (n = 12). All badgers were experimentally infected with M. bovis (103 CFU) by the endobronchial route targeting the right middle lung lobe. Throughout the study, clinical, immunological, pathological, and bacteriological parameters of infection were measured. Both vaccines conferred protection against experimental TB in badger, as measured by a reduction of the severity and lesion volumes. Based on these data, HIMB vaccination appears to be a promising TB oral vaccine candidate for badgers in endemic countries.

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.

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.

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.

Naturally Avian Influenza Virus-Infected Wild Birds Are More Likely to Test Positive for Mycobacterium spp. and Salmonella spp.

Wild birds often harbor infectious microorganisms. Some of these infectious microorganisms may present a risk to domestic animals and humans through spillover events. Detections of certain microorganisms have been shown to increase host susceptibility to infections by other microorganisms, leading to coinfections and altered host-to-host transmission patterns. However, little is known about the frequency of coinfections and its impact on wild bird populations. In order to verify whether avian influenza virus (AIV) natural infection in wild waterbirds was related to the excretion of other microorganisms, 73 AIV-positive samples (feces and cloacal swabs) were coupled with 73 AIV-negative samples of the same sampling characteristics and tested by real-time PCR specific for the following microorganisms: West Nile virus, avian avulavirus 1, Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Mycobacterium avium subspecies, Mycobacterium tuberculosis complex, and Mycobacterium spp. Concurrent detections were found in 47.9% (35/73) of the AIV-positive samples and in 23.3% (17/73) of the AIV-negative samples (P = 0.003). Mycobacterium spp. and Salmonella spp. were found to be significantly more prevalent among the AIV-positive samples than among the AIV-negative samples (42.9% vs. 22.8%; P = 0.024 and 15.2% vs. 0.0%; P = 0.0015, respectively). Prevalence of concurrent detections differed significantly among sampling years (P = 0.001), host families (P = 0.002), host species (P = 0.003), AIV subtypes (P = 0.003), and type of sample (P = 0.009). Multiple concurrent detections (more than one of the tested microorganisms excluding AIV) were found in 9.6% (7/73) of all the AIV-positive samples, accounting for 20% (7/35) of the concurrent detection cases. In contrast, in AIV-negative samples we never detected more than one of the selected microorganisms. These results show that AIV detection was associated with the detection of the monitored microorganisms. Further studies of a larger field sample set or under experimental conditions are necessary to infer causality in these trends.

Las aves silvestres frecuentemente albergan microorganismos infecciosos. Algunos suponen un riesgo por su posible transmisión a animales domésticos o representar un problema de salud pública si son zoonóticos. Se ha relacionado la detección de algunos microorganismos microbianos con una mayor susceptibilidad del hospedador a la infección por otros, llevando a una coinfección y a una alteración de los patrones de transmisión entre hospedadores. Sin embargo, poco se sabe sobre la frecuencia y el impacto de estas coinfecciones en la epidemiologia de las enfermedades en las aves silvestres. Con el ánimo de determinar si una infección natural con el virus de la influenza aviar (VIA) en aves acuáticas se relaciona con la excreción de otros microorganismos, se seleccionaron 73 muestras positivas a VIA y un número igual de negativas de similares características y se sometieron a análisis por PRC a tiempo real para la detección de los siguientes agentes: virus del Nilo occidental, avulavirus aviar de tipo 1, Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, subspecies de Mycobacterium avium, complejo Mycobacterium tuberculosis y Mycobacterium spp. Se detectaron otros agentes concurrentes en el 48% (35/73) de las muestras positivas a VIA frente al 23.3% (17/73) en las negativas (p=0.003). La prevalencia de Mycobacterium spp. y Salmonella spp. fue significativamente mayor entre las muestras positivas a VIA que entre las negativas (42.9% vs. 22.8%; p=0.024 y 15.2% vs. 0.0%; p=0.0015 respectivamente). La prevalencia de otros agentes difirió significativamente entre el año de recogida, la familia (p=0.002), la especie (p=0.003), los subtipos de VIA (p=0.003) y el tipo de muestra (p=0.009). Se detectaron múltiples microorganismos en el 9.6% (7/73) de las muestras positivas a VIA, lo que se correspondió con un 20% (7/35) de las detecciones concurrentes. Sin embargo en las muestras negativas a VIA no detectamos más de uno de los microorganismos estudiados. Estos resultados confirman que la detección de los agentes microbianos monitorizados se vio incrementada en presencia del VIA. Consideramos necesario la realización de estudios con un mayor número de muestras o en condiciones experimentales para inferir causalidad sobre estas tendencias.

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.

Avian Influenza Virus Surveillance in South-Central Spain Using Fecal Samples of Aquatic Birds Foraging at Landfills.

Aquatic wild birds have been intensively studied to better understand their role in avian influenza virus (AIV) maintenance and spread. To date, AIV surveillance has primarily focused on natural aquatic environments where different bird species aggregate and viral survival is enhanced. However, artificial habitats such as landfills are attracting substantial numbers of wild birds, AIV reservoir species included. The use of landfills as a predictable food source has significantly influenced population size, migratory traits, and feeding behavior of white storks (Ciconia ciconia) and black-headed gulls (Chroicocephalus ridibundus) among others. Considering the proximity of landfills to urban settlements and frequently poultry-farms, targeted monitoring of AIV in bird species that forage at landfills but are known to also frequent urban and agricultural habitats could be a useful means for monitoring of AIV, especially during periods of bird aggregation. During the wintering season 2014-2015, the prevalence of AIV in five avian species at two landfills in South-Central Spain was explored by rRT-PCR and species related temporal variation in AIV prevalence determined. We collected and tested 1,186 fresh fecal samples from white storks (N = 689), cattle egrets (Bubulcus ibis, N = 116) and mixed flocks of gulls (N = 381) as well as cloacal and oral swabs from five birds found dead. Seven samples contained AIV, five from gulls and one each from a stork and a cattle egret. Overall, AIV prevalence was 0.60%. No significant temporal variation was observed in AIV prevalence. Prevalence differed significantly among the sampled taxonomic groups, being highest in gulls (1.31%). H16N3 subtype was detected from a cattle egret and H11N9 subtype from a white stork, whereas gulls harbored both subtypes in addition to H11N3 subtype. H16 subtype detection in a cattle egret evidences its host range may not be restricted to gulls. Our results indicate that wild birds foraging at landfills may carry different LPAIV subtypes.

Occurrence and molecular genotyping of Giardia duodenalis and Cryptosporidium spp. in wild mesocarnivores in Spain.

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers (Meles meles, n=70), ferrets (Mustela putorius furo, n=2), genets (Genetta genetta, n=6), Iberian lynxes (Lynx pardinus, n=6), beech martens (Martes foina, n=8), mongooses (Herpestes ichneumon, n=2), otters (Lutra lutra, n=2), polecats (Mustela putorius, n=2), red foxes (Vulpes vulpes, n=87), wildcats (Felis silvestris, n=2), and wolves (Canis lupus, n=6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003-April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia (n=1), Asturias (n=69), Basque Country (n=49), Castile-La Mancha (n=38), and Extremadura (n=36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit (ssu) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase (G. duodenalis) and the 60-kDa glycoprotein (C. parvum and C. ubiquitum) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum. The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin.

Prevalence of Yersinia enterocolitica and Yersinia pseudotuberculosis in wild boars in the Basque Country, northern Spain.

BACKGROUND: Yersiniosis is a zoonosis widely distributed in Europe and swine carry different serotypes of Yersinia enterocolitica and Y. pseudotuberculosis. The aim of this study was to determine the prevalence of Y. enterocolitica and Y. pseudotuberculosis in wild boars in northern Spain. The blood of wild boars (n = 505) was sampled between 2001 and 2012. Seroprevalence was determined in 490 serum samples with an indirect enzyme-linked immunosorbent assay. Seventy-two of the animals were also examined for the presence of Y. enterocolitica or Y. pseudotuberculosis in the tonsils with real-time polymerase chain reaction. All the tonsils were analysed twice, directly and after cold enrichment in phosphate-buffered saline supplemented with 1 % mannitol and 0.15 % bile salts. RESULTS: Antibodies directed against Y. enterocolitica and Y. pseudotuberculosis were detected in 52.5 % of the animals. Yersinia enterocolitica was detected with real-time polymerase chain reaction in 33.3 % of the wild boars and Y. pseudotuberculosis in 25 %. Significant differences were observed according to the sampling year, and the highest prevalence was during winter and spring. The highest antibody levels and Y. enterocolitica prevalence were observed in mountainous areas at altitudes higher than 600 m, with very cold winters, and with the highest annual rainfall for each dominant climate. Areas with low and medium livestock populations were associated with the highest seroprevalence of Yersinia spp. in wild boars, whereas areas with high ovine populations had the highest prevalence of Y. enterocolitica. CONCLUSIONS: This study shows that Y. enterocolitica and Y. pseudotuberculosis are highly prevalent among wild boars in the Basque country, with Y. enterocolitica most prevalent. The risk of infection among wild boars is influenced by the season and the area in which they live.