Dolors Vidal

Serosurvey of Aujeszky’s disease virus infection in European wild boar in Spain

Serum samples from 693 hunted wild boar (Sus scrofa) were analysed by means of a blocking ELISA technique, and the mean (se) prevalence of antibodies to Aujeszky’s disease virus was 44 (4) per cent. All the seropositive wild boar were from south central Spain, except for one from central Spain, close to the main positive area. In this area, where large game species are increasingly managed for hunting, the seroprevalence was affected by the type of management. More intensively managed populations had a higher prevalence than wild boar living in natural situations, and the seroprevalence increased with the age of the animals; the seroprevalence was higher in females in all age groups. The seroprevalence in males more than one year old peaked after the breeding season, whereas females of the same age had a higher and constant seroprevalence throughout the year.

Detection and characterisation of O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in wild boars

The aim of this work was to determine the prevalence and characteristics of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) in free-ranging wild boars killed during the hunting season in southwest Spain. Faecal samples from 212 wild boars (Sus scrofa) were collected and examined for STEC. Characterisation of isolates was performed by PCR, serotyping, phage typing, and pulsed-field gel electrophoresis (PFGE). E. coli O157:H7 and non-O157 STEC were isolated from 7 (3.3%) and 11 (5.2%) animals, respectively, and the resulting 19 isolates were characterised. The PCR procedure indicated that 4 isolates carried the stx(1) gene, 12 carried the stx(2) gene, and 1 contained both of these genes. The ehxA, eae, and saa genes were detected in 13, 8, and 1 of the isolates, respectively. The eae-positive isolates comprised the types eae-gamma 1 and eae-zeta. The isolates belonged to 11 O:H serotypes, including 4 new serotypes not previously reported within STEC strains, and the majority of them were from serotypes previously associated with human infection. E. coli O157:H7 isolates belonged to phage types associated with severe human illness: PT14, PT34, and PT54. Indistinguishable PFGE types were found in E. coli O157:H7 isolates recovered from a wild boar and from a human patient with diarrhoea living in the same geographic area.

Methicillin resistant Staphylococcus aureus (MRSA) carriage in different free-living wild animal species in Spain

Methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening pathogen in humans and its presence in animals is a public health concern. The aim of this study was to measure the prevalence of MRSA in free-living wild animals. Samples from red deer (n=273), Iberian ibex (n=212), Eurasian Griffon vulture (n=40) and wild boar (n=817) taken from different areas in Spain between June 2008 and November 2011 were analyzed. Characterization of the isolates was performed by spa typing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing. A low prevalence of MRSA was found with 13 isolates obtained from 12 animals (0.89%; 95% CI: 0.46-1.56). All MRSA sequence types belonged to ST398 (t011 and t1451) and ST1 (t127). Genotypes and antimicrobial susceptibility patterns (tetracycline resistance in ST398 and clindamycin-erythromycin-tetracycline resistance in ST1) suggest that the MRSA found probably originated in livestock (ST398) or humans (ST1). This is the first report of MRSA carriers in free-living wild animals in Europe. Although our data showed that MRSA prevalence is currently low, free-living wild animals might act as reservoir and represent a potential risk for human health.

Possible interaction between a rodenticide treatment and a pathogen in common vole (Microtus arvalis) during a population peak

A common vole (Microtus arvalis) population peak in Northern Spain in 2007 was treated with large scale application of chlorophacinone, an anticoagulant rodenticide of the indandione family. Voles found dead and trapped alive were collected in treated and untreated areas. Residues of chlorophacinone were analyzed in liver of voles by HPLC-UV. Also, the presence of the pathogen Francisella tularensis was analyzed by PCR in samples of vole spleen. Chlorophacinone (82-3800 ng/g; wet weight liver) was only detected in voles found dead in treated areas (55.5%). The prevalence of F. tularensis in voles found dead in treated areas was also particularly high (66.7%). Moreover, chlorophacinone levels were lower in voles that were PCR-positive for F. tularensis (geometric mean [95% CI], 418 [143-1219] ng/g) than in those that were PCR-negative (1084 [581-2121] ng/g). Interactions between pathogens and rodenticides might be considered to reduce the doses used in baits or to avoid the use of the more toxic 2nd generation anticoagulant rodenticides.

Bioaccessibility of Pb from Ammunition in Game Meat Is Affected by Cooking Treatment

Background The presence of lead (Pb) ammunition residues in game meat has been widely documented, yet little information exists regarding the bioaccessibility of this Pb contamination. We study how cooking treatment (recipe) can affect Pb bioaccessibility in meat of animals hunted with Pb ammunition. Methodology/Principal Findings We used an in vitro gastrointestinal simulation to study bioaccessibility. The simulation was applied to meat from red-legged partridge (Alectoris rufa) hunted with Pb shot pellets and cooked using various traditional Spanish game recipes involving wine or vinegar. Total Pb concentrations in the meat were higher in samples with visible Pb ammunition by X-ray (mean±SE: 3.29±1.12 µg/g w.w.) than in samples without this evidence (1.28±0.61 µg/g). The percentage of Pb that was bioaccessible within the simulated intestine phase was far higher in meat cooked with vinegar (6.75%) and wine (4.51%) than in uncooked meat (0.7%). Risk assessment simulations using our results transformed to bioavailability and the Integrated Exposure Uptake Biokinetic model (IEUBK; US EPA) show that the use of wine instead of vinegar in cooking recipes may reduce the percentage of children that would be expected to have >10 µg/dl of Pb in blood from 2.08% to 0.26% when game meat represents 50% of the meat in diet. Conclusions/Significance Lead from ammunition in game meat is more bioaccessible after cooking, especially when using highly acidic recipes. These results are important because existing theoretical models regarding Pb uptake and subsequent risk in humans should take such factors into account.

Prevalence of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. in large game animals intended for consumption: Relationship with management practices and livestock influence

Although wild ruminants have been identified as reservoirs of Shiga-toxin producing Escherichia coli (STEC), little information is available concerning the role of Salmonella spp. and Campylobacter spp. in large game species. We evaluated the presence of these pathogens in faeces (N=574) and carcasses (N=585) sampled from red deer (N=295), wild boar (N=333) and other ungulates (fallow deer, mouflon) (N=9). Animal sampling was done in situ from 33 hunting estates during two hunting seasons. Salmonella spp. and Campylobacter spp. strains associated with human campylobacteriosis were infrequently detected indicating that both pathogens had a limited zoonotic risk in our study area. The overall STEC prevalence in animals was 21% (134/637), being significantly higher in faeces from red deer (90 out of 264). A total of 58 isolates were serotyped. Serotypes O146:H- and O27:H30 were the most frequent in red deer and the majority of isolates from red deer and wild boar were from serotypes previously found in STEC strains associated with human infection, including the serotype O157:H7. The STEC prevalence in red deer faeces was significantly higher with the presence of livestock (p<0, 01) where high densities of red deer (p<0.001) were present. To the best of our knowledge, this is the first study reporting the occurrence of Salmonella spp. and STEC in carcasses of large game animals. Furthermore, this study confirmed by pulsed-field gel electrophoresis (PFGE) that cross contamination of STEC during carcass dressing occurred, implying the likelihood of these pathogens entering into the food chain.

Environmental factors influencing the prevalence of a Clostridium botulinum type C/D mosaic strain in nonpermanent Mediterranean wetlands

Between 1978 and 2008, 13 avian botulism outbreaks were recorded in the wetlands of Mancha H�meda (central Spain). These outbreaks caused the deaths of around 20,000 birds from over 50 species, including globally endangered white-headed ducks (Oxyura leucoceophala). Here, a significant association was found between the number of dead birds recorded in each botulism outbreak and the mean temperature in July (always >26�C). The presence of Clostridium botulinum type C/D in wetland sediments was detected by real-time PCR (quantitative PCR [qPCR]) in 5.8% of 207 samples collected between 2005 and 2008. Low concentrations of Cl− and high organic matter content in sediments were significantly associated with the presence of C. botulinum. Seventy-five digestive tracts of birds found dead during botulism outbreaks were analyzed; C. botulinum was present in 38.7% of them. The prevalence of C. botulinum was 18.2% (n = 22 pools) in aquatic invertebrates (Chironomidae and Corixidae families) and 33.3% (n = 18 pools) in necrophagous invertebrates (Sarcophagidae and Calliphoridae families), including two pools of adult necrophagous flies collected around bird carcasses. The presence of the bacteria in the adult fly form opens up new perspectives in the epidemiology of avian botulism, since these flies may be transporting C. botulinum from one carcass to another.

Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2

Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection.

Sublethal Pb Exposure Produces Season-Dependent Effects on Immune Response, Oxidative Balance and Investment in Carotenoid-based Coloration in Red-Legged Partridges

Ingestion of lead (Pb) shot pellets constitutes the main cause of Pb poisoning in avifauna. We studied the effects of sublethal Pb exposure on immunity, carotenoid-based coloration, oxidative stress and trade-offs among these types of responses during spring and autumn in red-legged partridges (Alectoris rufa). We evaluated constitutive immunity testing lysozyme and natural antibody levels, and blood bactericidal and phagocytic activities. We studied induced immunity by testing PHA and humoral responses. We analyzed fecal parasite and bacterial abundance and oxidative stress biomarkers. Pb exposure in spring reduced natural antibody levels, whereas in autumn, it reduced lysozyme levels and increased phagocytic activity. Pb exposure increased PHA response in both seasons, and decreased T-independent humoral response in autumn. Pb exposure also increased noncoliform and decreased coliform Gram-negative gut bacteria. In spring, Pb exposure decreased antioxidant levels and increased coloration in males, whereas in autumn, it increased retinol levels but reduced coloration in both genders. Our results suggest that in spring, Pb-exposed females used antioxidants to cope with oxidative stress at the expense of coloration, whereas Pb-exposed males increased coloration, which may reflect an increased breeding investment. In autumn, both genders prioritized oxidative balance maintenance at the expense of coloration.

Serological survey of selected infectious diseases in mouflon ( Ovis aries musimon ) from south-central Spain

Serum samples from 101 mouflons (Ovis aries musimon) collected from July 2002 to January 2006 were tested for antibodies against Anaplasma spp., Brucella spp., bovine viral diarrhea virus, Chlamydophila abortus, Coxiella burnetii, Mycobacterium avium ssp. paratuberculosis, and Maedi-Visna virus. Mouflon came either from extensive farms or high ungulate density fenced hunting estates. Antibodies were detected against Anaplasma spp. (22.2%), C. burnetii (4.0%), M. avium ssp. paratuberculosis (1.0%) and C. abortus (1.0%). According to our results, mouflons could participate as a wild reservoir in the epidemiology of Anaplasma spp. infection and maybe Q fever, but they do not seem to contribute in the epidemiology of the rest of the studied infectious diseases in south-central Spain.