Antibodies to West Nile virus and related flaviviruses in wild boar, red foxes and other mesomammals from Spain

Red foxes (Vulpes vulpes), wild boar (Sus scrofa) and Iberian pigs (Sus scrofa domestica) that are raised extensively outdoors, as well as other wild mesomammals from south central Spain and wild boar from Doñana National Park (DNP), were tested for antibodies against related flaviviruses by ELISA and for antibodies against WNV by VNT. Mean flavivirus seroprevalence according to ELISA was 20.4 ± 7.8% (21 out of 103) in red foxes, 12.6 ± 2.8% (69 out of 545) in wild boars, and 3.3 ± 2.7% (6 out of 177) in Iberian pigs. A stone marten (Martes foina) also tested positive. Flavivirus seroprevalence in wild boar was significantly higher in DNP, and increased with age. Haemolysis of the serum samples limited interpretation of VNT to 28 samples, confirming WNV seroprevalence in one red fox, four Iberian pigs and nine wild boars. ELISA positive, microVNT negative samples suggest presence of non-neutralizing antibodies against WNV or antibodies to other antigenically related flaviviruses. Despite the importance of wetlands for flavivirus maintenance and amplification, WNV/flavivirus seroprevalence in wild boar and red foxes was not associated to wetland habitats. This is the first report of exposure of red foxes to WNV. With view to use of the tested species as sentinels for flavivirus activity, limited exposure of Iberian pigs that would be available for regular sampling, low numbers of foxes collected and concentration of wild boar harvest in the winter season are major drawbacks.This study has been supported by projects PAC08-0296-7771 (JCCM), and AG2008-02504GAN. A.V. Gutierrez-Guzman is a JCCM fellow (PAC08-029).Peer Reviewe

Ecological factors driving avian influenza virus dynamics in Spanish wetland ecosystems

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007-2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor - independently to other factors - explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity.This work was financially supported by INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria), project FAU2006-00019-C03-00 and by Department of Environment, Territorial Planning, Agriculture and Fisheries of the Basque Government. P. Acevedo was supported by a Beatriu de Pinós fellowship funded by Comissionat per a Universitats i Recerca del Departament d'Innovació, Universitats i Empresa, of the Generalitat de Catalunya and the COFUND Programme-Marie Curie Actions under 7th Marc Programme of the European Community.Peer Reviewe

Pathogenicity of two recent Western Mediterranean West Nile virus isolates in a wild bird species indigenous to Southern Europe: the red-legged partridge

West Nile virus (WNV) is an emerging zoonotic pathogen whose geographic spread and incidence in humans, horses and birds has increased significantly in recent years. WNV has long been considered a mild pathogen causing self-limiting outbreaks. This notion has changed as WNV is causing large epidemics with a high impact on human and animal health. This has been particularly noteworthy since its introduction into North America in 1999. There, native bird species have been shown to be highly susceptible to WNV infection and disease with high mortalities. For this reason, the effect of WNV infection in North American bird species has been thoroughly studied by means of experimental inoculations in controlled trials. To a lesser extent, European wild birds have been shown to be affected clinically by WNV infection. Yet experimental studies on European wild bird species are lacking. The red-legged partridge (Alectoris rufa) is a gallinaceous bird indigenous to the Iberian Peninsula, widely distributed in South Western Europe. It plays a key role in the Mediterranean ecosystem and constitutes an economically important game species. As such it is raised intensively in outdoor facilities. In this work, red-legged partridges were experimentally infected with two recent WNV isolates from the Western Mediterranean area: Morocco/2003 and Spain/2007. All inoculated birds became viremic and showed clinical disease, with mortality rates of 70% and 30%, respectively. These results show that Western Mediterranean WNV variants can be pathogenic for some European bird species, such as the red-legged partridge

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

Ecological Factors Driving Avian Influenza Virus Dynamics in Spanish Wetland Ecosystems

Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007-2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor - independently to other factors - explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity

Detection of low pathogenic avian influenza viruses in wild birds in Castilla-La Mancha (south central Spain)

The Iberian Peninsula is located along the East Atlantic and Black Sea/Mediterranean flyways and is the third ranking European country as wintering quarter for wild migrating birds after Turkey and Rumania. For these reasons, Spanish wetlands are of importance in AIV surveillance, and of great interest for the study of the epidemiology of LPAIV under Mediterranean climate conditions. Nevertheless, information on prevalence of LPAIV viruses in Spain is still scarce and is restricted to two serological surveys carried out in the south of the country during 1990 and 1994 and one virological study performed recently in North East Spain. In the present study we analysed the prevalence of AIV circulating in wild birds in continental wetlands in central Spain and determined temporal, spatial and species variation. Real time RTPCR was performed on 1435 faecal samples and cloacal swabs from 54 species. An overall AIV prevalence of 2.6% was detected with a peak during November and December, when thousands of migrating wild birds arrive to Spain for wintering. Highest prevalence rates were detected in Phoenicopteriformes and Anseriformes. AIV prevalence obtained from cloacal swabs and fresh faeces did not vary significantly, which supports faecal sampling as an appropriate method for large scale LPAIV surveillance programs. Viral culture was achieved in samples obtained from two Mallards and a White stork, in which subtypes H7N9 and H11N9, respectively, were identified. Our results reflect a similar scenario in AIV epidemiology in small continental wetlands as compared to large coastal humid areas in Europe and underline the importance of including species such as flamingos and storks in surveillance programs, since their role in AIV ecology in these areas could be more important than previously considered.We are grateful to all the personnel of the Wildlife Rehabilitation Centres in Castilla-La Mancha for their collaboration and to all the people that helped in the field work. We also thank JCCM for support, Tomás Velasco for sharing his knowledge of wetlands and waterbirds in Central Spain and Vega Álvarez (NEIKER) for technical support. We wish to thank the “Spanish National Reference Laboratory” for official confirmation of isolates. This work is a contribution to grant INIA RTA2005-111-00, grant JCCM PA298-05 and grant MEC/INIA FAU2006-019-C03. E. Pérez-Ramírez currently has an I3p fellowship from the Spanish Science Council (CSIC).Peer Reviewe

Antibodies to West Nile virus and related flaviviruses in wild boar, red foxes and other mesomammals from Spain

Red foxes (Vulpes vulpes), wild boar (Sus scrofa) and Iberian pigs (Sus scrofa domestica) that are raised extensively outdoors, as well as other wild mesomammals from south central Spain and wild boar from Doñana National Park (DNP), were tested for antibodies against related flaviviruses by ELISA and for antibodies against WNV by VNT. Mean flavivirus seroprevalence according to ELISA was 20.4 ± 7.8% (21 out of 103) in red foxes, 12.6 ± 2.8% (69 out of 545) in wild boars, and 3.3 ± 2.7% (6 out of 177) in Iberian pigs. A stone marten (Martes foina) also tested positive. Flavivirus seroprevalence in wild boar was significantly higher in DNP, and increased with age. Haemolysis of the serum samples limited interpretation of VNT to 28 samples, confirming WNV seroprevalence in one red fox, four Iberian pigs and nine wild boars. ELISA positive, microVNT negative samples suggest presence of non-neutralizing antibodies against WNV or antibodies to other antigenically related flaviviruses. Despite the importance of wetlands for flavivirus maintenance and amplification, WNV/flavivirus seroprevalence in wild boar and red foxes was not associated to wetland habitats. This is the first report of exposure of red foxes to WNV. With view to use of the tested species as sentinels for flavivirus activity, limited exposure of Iberian pigs that would be available for regular sampling, low numbers of foxes collected and concentration of wild boar harvest in the winter season are major drawbacks.This study has been supported by projects PAC08-0296-7771 (JCCM), and AG2008-02504GAN. A.V. Gutierrez-Guzman is a JCCM fellow (PAC08-029).Peer Reviewe

Análisis serológico para virus de influenza aviar en aves silvestres: comparación de dos pruebas comerciales de ensayo por inmunoabsorción ligado a enzimas competitivo

Serologic testing of wild birds for avian influenza virus (AIV) surveillance poses problems due to species differences and nonspecific inhibitors that may be present in sera of wild birds. Recently available competitive enzyme-linked immunosorbent assay (cELISA) kits offer a new species-independent approach. In this study we compare two commercial competitive cELISAs, using a total of 184 serum and plasma samples from 23 species of wild birds belonging to 10 orders. Thirteen samples were from experimentally high pathogenicity AI and low pathogenicity AI infected red-legged partridges (Alectoris rufa), 77 samples were from a flock of sentinel hybrid ducks confirmed infected by AI by real-time PCR, and 94 samples were from wild birds admitted to a rehabilitation center. Both ELISAs detected AI antibodies in the experimentally infected partridges, whereas hemagglutination inhibition (HI) was negative. Concordance in results between the two ELISAs was 51.5%. When specific subtype-H5/H7 HI-positive samples were considered for comparison, ELISA 1 appeared to perform better on ducks, whereas ELISA 2 appeared to perform better in other wild bird species. Overall, 68.2% of H5/H7 positive samples tested positive by ELISA 1 and 36% by ELISA 2. Both ELISAs detected AIV-antibody-positive samples negative by specific HI against 9 of the 16 existing hemagglutinin (HA) subtypes. Presumably this reflects either higher sensitivity of cELISA when compared to HI, presence of antibodies against HA subtypes not tested, or unspecific reactions. Performance of ELISA 1 on ducks appears to be comparable to in-house cELISA previously used by other authors in wild birds, but requires a relatively large sample volume. Alternatively, although ELISA 2 required a smaller sample volume, it was less effective at identifying HI-positive samples. The results reflect the necessity of validation of cELISA tests for individual species or at least families, as required by the OIE.E. Pérez-Ramírez has an I3p fellowship from the Spanish Science Council (CSIC). P. Acevedo is currently funded by a Juan de la Cierva research contract awarded by the Ministerio de Ciencia e Innovación (MICINN) and is also supported by the project CGL2006-09567/BOS. This work was funded by project RTA2005-0111-00 and FAU2006-019-C03. We thank the JCCM for support, and the personnel of Wildlife Rehabilitation Centers of Castilla-La Mancha for their collaboration.Peer Reviewe

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 húmeda (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.This study was supported by the Spanish Ministry of Environment (grants OAPN 099/2003 and OAPN 035/2009). Dolors Vidal was supported with a JAE DOC contract from the Spanish Council of Research (CSIC). Mark A. Taggart was supported by a Juan de la Cierva Fellowship from the Spanish Ministry of Science and Innovation (MICINN). Ibone Anza was supported by a JAE PRE grant from CSIC, and Elisa PérezRamírez was supported by a I3p grant from CSIC.Peer Reviewe

Pathogenesis of two western Mediterranean west Nile virus lineage 1 isolates in experimentally infected red-legged partridges (Alectoris rufa)

This article belongs to the Special Issue West Nile Virus Infection.West Nile virus (WNV) is the most widespread flavivirus in the world with a wide vertebrate host range. Its geographic expansion and activity continue to increase with important human and equine outbreaks and local bird mortality. In a previous experiment, we demonstrated the susceptibility of 7-week-old red-legged partridges (Alectoris rufa) to Mediterranean WNV isolates Morocco/2003 and Spain/2007, which varied in virulence for this gallinaceous species. Here we study the pathogenesis of the infection with these two strains to explain the different course of infection and mortality. Day six post-inoculation was critical in the course of infection, with the highest viral load in tissues, the most widespread virus antigen, and more severe lesions. The most affected organs were the heart, liver, and spleen. Comparing infections with Morocco/2003 and Spain/2007, differences were observed in the viral load, virus antigen distribution, and lesion nature and severity. A more acute and marked inflammatory reaction (characterized by participation of microglia and CD3+ T cells) as well as neuronal necrosis in the brain were observed in partridges infected with Morocco/2003 as compared to those infected with Spain/2007. This suggests a higher neurovirulence of Morocco/2003, probably related to one or more specific molecular determinants of virulence different from Spain/2007.This study has been supported by the project PAC08-0296-7771 (JCCM), and from INIA-MARM funds (INIA CC08-020). Elena Sotelo was a fellow from INIA. Elisa Perez Ramirez was a fellow of the National Research Council (CSIC) and Ana Valeria Gutierrez was a fellow of the JCCM.Peer reviewe