Madalena Vieira-Pinto

Salmonella sp. in Game (Sus scrofa and Oryctolagus cuniculus)

The role of wildlife in the epidemiology of Salmonella sp.-induced diseases is a matter of increasing concern to public health. However, to date, reports on the occurrence of Salmonella sp. in game hunted for human consumption are very limited. The current study was designed to evaluate the prevalence of Salmonella sp. in fecal samples of wild boars and wild rabbits hunted in Northern Portugal. The results show that 22% of the (17/77) wild boar and 48% (38/80) of the wild rabbit presented Salmonella sp. in their feces. Two serovars were identified from samples of wild boars: Salmonella Typhimurium (65%) and Salmonella Rissen (35%). Five serovars were identified from wild rabbit samples: Salmonella Rissen (29%), Salmonella Enteritidis (26%), Salmonella Havana (24%), Salmonella Typhimurium (16%), and Salmonella Derby (5%). These results confirm the importance of wild boar and wild rabbit as carriers of pathogenic Salmonella serovars. Hence, they could represent sources of infection not only for animals (wild and domestic) but also for humans.

Implications and challenges of tuberculosis in wildlife ungulates in Portugal: A molecular epidemiology perspective

Mycobacterium bovis and, more rarely, Mycobacterium caprae, may cause zoonotic bovine tuberculosis (bTB) in an extensive range of animal species. In Portugal, during 2009, a remarkable raise of bTB incidence was registered in cattle along with an increase of new cases in wildlife. In this work, we reassess and update the molecular epidemiology of bTB in wild ungulates by including 83 new M. bovis and M. caprae isolates from wild boar and red deer obtained during 2008-2009. Spoligotyping identified 27 patterns in wild ungulates, including 11 patterns exclusive from deer and five from wild boar. The genetic relatedness of wildlife and livestock isolates is confirmed. However, the relative prevalence of the predominant genotypes is different between the two groups. Contrasting with the disease in livestock, which is widespread in the territory, the isolation of bTB in wildlife is, apparently, geographically localized and genotypic similarities of strains are observed at the Iberian level.

Hepatitis E Virus in Sylvatic and Captive Wild Boar from Portugal

Hepatitis E virus (HEV) is a zoonotic agent today considered a major Public Health issue in industrialized countries. HEV strains belonging to zoonotic genotype 3 are widely present in swine, being today considered important reservoirs for human disease. Unlike in swine, only scarce data are available on the circulation of HEV in wild boar. This study describes the detection and molecular characterization of HEV in livers from sylvatic wild boar hunted in Portugal and destined for consumption. Additionally, the detection of HEV in stools of a confined wild boar population also destined for consumption is also described. A total of 80 liver samples collected during the hunting season of 2011/2012 and 40 stools collected in February 2012 from a wild boar breeding farm in Portugal were tested by a nested broad-spectrum RT-PCR assay targeting open reading frame (ORF) 1. Twenty livers (25.0%) and 4 stools (10%) were positive for HEV. Phylogenetic analysis showed that all strains clustered with sequences classified as HEV genotype 3 subgenotype e. To our knowledge, this is the first report documenting the occurrence and molecular analysis of HEV in sylvatic and captive wild boar destined for human consumption in Portugal. This report demonstrates for the first time the circulation of HEV in wildlife reservoirs of Portugal adding knowledge to the epidemiology of HEV in wild boar populations.

Multiple Zoonotic Parasites Identified in Dog Feces Collected in Ponte de Lima, Portugal—A Potential Threat to Human Health

Dogs play many roles and their presence within people’s houses has increased. In rural settings dog faeces are not removed from the streets, representing an environmental pollution factor. Our aim was to evaluate the occurrence of environmental contamination with zoonotic intestinal parasites of three groups of dogs in Ponte de Lima, Portugal, with a particular emphasis on Echinococcus granulosus. We collected 592 dog faecal samples from the environment, farm and hunting dogs. Qualitative flotation coprological analysis was performed and the frequency in the positive samples ranged between 57.44% and 81.19% in different groups. We isolated up to four different parasites in one sample and detected seven intestinal parasitic species, genera or families overall. Ancylostomatidae was the most prevalent parasite, followed by Trichuris spp., Toxocara spp., Isospora spp., Dipylidium caninum, Taeniidae and Toxascaris leonina. Taeniidae eggs were analyzed with the PCR technique and revealed not to be from Echinococcus. The parasite prevalence and the diversity of zoonotic parasites found were high, which calls for a greater awareness of the problem among the population, especially hunters. Promoting research at the local level is important to plan control strategies. Health education should be developed with regard to farmers and hunters, and a closer collaboration between researchers, practitioners and public health authorities is needed.

Combined evaluation of bovine tuberculosis in wild boar (Sus scrofa) and red deer (Cervus elaphus) from Central-East Portugal

Bovine tuberculosis (bTB), widely considered a disease of livestock, can also affect large game species despite their poorly understood role in the epidemiology of the endemic state of the disease. The Idanha-a-Nova County, situated in the eastern region of central Portugal, is one of the most important regions for large game hunting activity. In order to investigate the role of large game and the presence of bTB in Idanha-a-Nova, 132 wild boar (Sus scrofa) and 339 red deer (Cervus elaphus) were inspected for bTB-compatible lesions (bTBCL) during the 2008–2009 hunting season. Tissue samples with bTBCL were collected and processed for bacteriological confirmation and spoligotyping of Mycobacterium bovis isolates. In wild boar, bTB lesions were single, mostly calcified (21/29, 72.4%) and situated in the mandibular lymph nodes. Lesions were more severe and largely non-calcified (36/40, 90%) in red deer. Wild boar presented a consistently higher bTB infection rate than the red deer; however, the difference was only significant in the southern B area. This area was significantly affected by bTB for both animal species (up to 72.7% in wild boar and 34.8% in red deer) and requires the urgent implementation of intervention measures to contain the disease. Wild boar may assume an important role in the emergence of new disease foci in distant areas that infected red deer cannot reach. Spoligotyping of M. bovis isolates grouped the strains into three clusters, revealing patterns common to wild boar, red deer, and also to local cattle, indicating possible transmission among different animal species.

Genomic and proteomic evaluation of antibiotic resistance in Salmonella strains

Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence. Through 2-DE we obtained a total of 229 spots from both strains. All were suitable for MALDI-TOF/TOF and, in correlation with bioinformatic databases, allowed accurate identification and characterization of proteins. S. Enteritidis recovered from wild rabbits was sensitive to all the antibiotics tested in contrast to S. Typhimurium isolated from wild boars which presented a resistance phenotype to ampicillin, streptomycin and chloramphenicol. Nevertheless, despite the different ratio of proteins observed in each proteome according to their biological function, no significant difference was observed in the involvement of these proteins in pathogenicity. Bearing in mind that serotypes are related to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.

Paratuberculosis in European wild rabbits from the Iberian Peninsula.

Of the non-ruminant wildlife species known to harbor Mycobacterium avium paratuberculosis (MAP), the rabbit (Oryctolagus cuniculus) is thought to pose the greatest risk of transmission to cattle. We analyzed 80 hunter-harvested wild rabbits from a core study area in southern Spain, and sera from 157 wild rabbits sampled opportunistically on seven additional sites. Gross lesions compatible with paratuberculosis were observed in two of 80 necropsied rabbits. Histopathology revealed focal to diffuse multibacillary MAP-compatible lesions in 8 of 10 rabbits examined. Presence of MAP was confirmed in one rabbit with gross lesions by positive amplification curves for both IS900 and ISMAP02. However, no isolate was obtained from 47 samples by culture. We adapted an indirect ELISA for the detection of MAP antibodies. At the established cut-off of 0.5, 6 of 237 wild rabbit sera (2.5%) yielded a positive ELISA result. Antibodies were detected in rabbits from 3 of 8 sampling sites. Considering the increasing relevance of MAP infection for animal health, these results open a challenging field for future research.

Serological evidence of Toxoplasma gondii in hunted wild boar from Portugal

Toxoplasmosis is an important parasitic zoonosis with a worldwide distribution, being the parasitic disease with the highest occurrence in Europe. Wild boar has an important role in the epidemiological cycle of Toxoplasma gondii as an intermediate host, that can potentially infect humans when the meat is consumed raw or undercooked. The purpose of this work was to determine the presence of antibodies to T. gondii in serum of hunted wild boar. During the hunting season 2011/2012, sera samples were collected from 97 wild boar and tested for IgG antibodies to T. gondii, using the modified agglutination test. Twenty out of the 97 wild boar (20.6%) were seropositive for T. gondii IgG antibodies. Multivariate logistic regression analysis showed that males and older animals were associated with T. gondii seropositivity. These results show that T. gondii has an important presence in wild boar population from Portugal, suggesting a potential zoonotic risk for humans when wild boar meat or meat products are consumed raw or undercooked.

European Rabbits as Reservoir for Coxiella burnetii

We studied the role of European rabbits (Oryctolagus cuniculus) as a reservoir for Coxiella burnetii in the Iberian region. High individual and population seroprevalences observed in wild and farmed rabbits, evidence of systemic infections, and vaginal shedding support the reservoir role of the European rabbit for C. burnetii.

Epidemiology of taeniosis/cysticercosis in Europe, a systematic review: Western Europe

BackgroundTaenia solium and Taenia saginata are zoonotic parasites of public health importance. Data on their occurrence in humans and animals in western Europe are incomplete and fragmented. In this study, we aimed to update the current knowledge on the epidemiology of these parasites in this region.MethodsWe conducted a systematic review of scientific and grey literature published from 1990 to 2015 on the epidemiology of T. saginata and T. solium in humans and animals. Additionally, data about disease occurrence were actively sought by contacting local experts in the different countries.ResultsTaeniosis cases were found in twelve out of eighteen countries in western Europe. No cases were identified in Iceland, Ireland, Luxembourg, Norway, Sweden and Switzerland. For Denmark, Netherlands, Portugal, Slovenia, Spain and the UK, annual taeniosis cases were reported and the number of detected cases per year ranged between 1 and 114. Detected prevalences ranged from 0.05 to 0.27%, whereas estimated prevalences ranged from 0.02 to 0.67%. Most taeniosis cases were reported as Taenia spp. or T. saginata, although T. solium was reported in Denmark, France, Italy, Spain, Slovenia, Portugal and the UK. Human cysticercosis cases were reported in all western European countries except for Iceland, with the highest number originating from Portugal and Spain. Most human cysticercosis cases were suspected to have acquired the infection outside western Europe. Cases of T. solium in pigs were found in Austria and Portugal, but only the two cases from Portugal were confirmed with molecular methods. Germany, Spain and Slovenia reported porcine cysticercosis, but made no Taenia species distinction. Bovine cysticercosis was detected in all countries except for Iceland, with a prevalence based on meat inspection of 0.0002–7.82%.ConclusionsDetection and reporting of taeniosis in western Europe should be improved. The existence of T. solium tapeworm carriers, of suspected autochthonous cases of human cysticercosis and the lack of confirmation of porcine cysticercosis cases deserve further attention. Suspected cases of T. solium in pigs should be confirmed by molecular methods. Both taeniosis and human cysticercosis should be notifiable and surveillance in animals should be improved.