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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.

As a response to the need for improved and cost-efficient West Nile virus (WNV) and other flavivirus surveillance tools, we tested 887 juvenile free-living red deer, 742 free-living juvenile wild boar, and 327 farmed deer, to detect temporal variability in exposure to these viruses. Thirty of 742 wild boar samples (4%; 95% CI 2.8,5.7) yielded a positive ELISA result. Antibody-positive individuals had been sampled between 2003 and 2011 in localities from central and southern Spain. No wild boar from the northern half of Spain (n=120) tested positive. Regarding juvenile wild red deer, only two out of 887 samples yielded a positive ELISA result (0.2%; 95% CI 0.1,0.8). These two samples came from the same site and sampling year. The likelihood of detecting contact with WNV or cross-reacting flaviviruses was 18 times higher among juvenile wild boar than among juvenile red deer. ELISA positivity among farmed deer increased 10-fold after local flavivirus outbreaks recorded in the summer and autumn of 2010. This survey demonstrated the potential usefulness of juvenile wild ungulates, particularly wild boar, as suitable flavivirus sentinels in southwestern Europe, and that systematic serum banking of samples from hunter-harvested wildlife or from individual farmed ungulates provides valuable material for retrospective epidemiological surveys and future disease monitoring., This study was supported by POII09-0141-8176 (Castilla-La Mancha) and by JCCM PPIC10-0226-0243. The authors also acknowledge the support of the Ministerio de Medio Ambiente, Rural y Marino (MARM).

After the introduction of West Nile virus (WNV) into North America, bird mortalities associated with West Nile disease have dramatically increased in this continent and, to a lesser extent, in Europe. The different West Nile disease incidence in birds in these 2 continents demands an explanation, and experimental studies can provide important information. The authors inoculated thirteen 9-week-old red-legged partridges (Alectoris rufa) with 10 plaque-forming units of a WNV strain isolated in New York in 1999. The objective was to study the pathogenesis of the infection in a native Euro-Mediterranean bird species with a WNV strain known to be highly pathogenic for numerous native American bird species. Additionally, the authors evaluated the dynamics of inflammatory cell activation and recruitment into the brain. WNV was detected in tissues 3 days postinoculation (dpi), and the birds developed macroscopic and microscopic lesions. Two partridges succumbed to the disease. The most affected tissues were the heart, brain, and spinal cord. The main microscopic findings were the presence of mononuclear infiltrates in the heart and brain, gliosis, and degeneration and necrosis of cardiomyocytes and neurons. These lesions were aggravated in the birds that died or were euthanized 7 dpi or later. In the brain, there was an upregulation of microglial cells and astrocytes and an increase in the number of T cells, especially after 7 dpi. These results show that this WNV strain is of moderate virulence for the red-legged partridge and that WNV-infected red-legged partridges develop an immune cell response in the brain similar to that of mammals., This study was supported by grant SAF2008-04232, funded by the Spanish Ministry for Science and Innovation, and grants FAU2008-00006, RTA2011-00036, and RTA2013-0013-C04, from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. V. Gamino (323/09) was a research fellow supported by the Regional Government of Castilla–La Mancha.

Using eye samples of nine 9-week-old experimentally West Nile virus (WNV)–infected red-legged partridges (Alectoris rufa), time course of lesions and WNV antigen appearance in ocular structures were examined. In addition, eye samples of 6 red-legged partridges and 3 common pheasants (Phasianus colchicus) naturally infected with Bagaza virus (BAGV) were used to study lesions and flavivirus antigen distribution in relation to apparent blindness in the former. The rapid onset of microscopic lesions and early presence of viral antigen in the eye of experimentally WNV-infected partridges, prior to the central nervous system involvement, suggested hematogenous spread of the virus into the eye. BAGV-infected partridges had a more pronunced inflammatory reaction and more widespread flavivirus antigen distribution in the retina compared with pheasants and experimentally fatally WNV-infected partridges. Our results suggest that flavivirus replication and development of lesions in ocular structures of gallinaceous game birds vary with the specific virus and host species involved., This study was supported by grants AG2008-02504GAN and SAF-2008-04232 funded by the Spanish Ministry for Science and Innovation, grants FAU2008-00006 and RTA2011-00036 from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), and The Network of Animal Disease Infectiology and Research Facilities, NADIR-EU-228394, funded by the EU.

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n=28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n=29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread., This work was supported by grants FAU2008-00006 and RTA2011-00036 from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and by the Network of Animal Disease Infectiology and Research-European Union (NADIR-EU-228394). M.A.M.A. is a recipient of a “Junta de Ampliación de Estudios (JAE)” doctoral fellowship from the Spanish Research Council (CSIC). T.M.R. is a recipient of a “Formación de Personal Investigador (FPI)” pre-doctoral fellowship from INIA.

In late summer 2010 a mosquito born flavivirus not previously reported in Europe called Bagaza virus (BAGV) caused high mortality in red-legged partridges (Alectoris rufa) and ring-necked pheasants (Phasianus colchicus). We studied clinical findings, lesions and viral antigen distribution in naturally BAGV infected game birds in order to understand the apparently higher impact on red-legged partridges. The disease induced neurologic signs in the two galliform species and, to a lesser extent, in common wood pigeons (Columba palumbus). In red-legged partridges infection by BAGV caused severe haemosiderosis in the liver and spleen that was absent in pheasants and less evident in common wood pigeons. Also, BAGV antigen was present in vascular endothelium in multiple organs in red-legged partridges, and in the spleen in common wood pigeons, while in ring-necked pheasants it was only detected in neurons and glial cells in the brain. These findings indicate tropism of BAGV for endothelial cells and a severe haemolytic process in red-legged partridges in addition to the central nervous lesions that were found in all three species., This study was supported by grant AG2008-02504GAN funded by the Spanish Ministry for Science and Innovation. A.V. Gutiérrez-Guzmán (PAC08-0296-7771) and V. Gamino (323/09) are research fellows supported by the regional government of Castilla – La Mancha (JCCM) and I.G. Fernández-de-Mera was funded by MCINN Program Juan de la Cierva, JCI-2009-04518, 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).

Resumen del póster presentado al Joint Meeting of 30th Meeting of the European Society of Veterinary Pathology-Annual Meeting of the European College of Veterinary Pathology and 24th Annual Meeting of the Spanish Society of Veterinary Pathology, celebrado en León (España) del 5 al 8 de septiembre de 2012., [Introduction]: Recent flavivirus outbreaks in avian species have shown the potential need of vaccination of birds with economic and/or ecologic value. During the study of the immunogenicity induced by two different vaccines against West Nile virus (WNV) that were being experimentally tested in red-legged partridges (Alectoris rufa), we analyzed the macroscopic and microscopic changes produced after intramuscular injection in the pectoral muscle. [Material and methods]: A commercial inactivated vaccine (Duvaxyn®, Pfizer, Spain) and an E protein based recombinant vaccine candidate with different commercial adjuvants (Stimune® and MetaStim®) were tested. They were applied intramuscularly in the pectoral muscle in two experiments. While local injection volume increased, injection frequency decreased between experiments. During necropsies, pectoral muscle samples were collected for microscopic examination. [Results]: No mortality or clinical signs were observed related to vaccinations. At necropsy, white or yellowish nodules were seen in the pectoral muscle. Microscopic changes included myofibril degeneration, inflammation and oil cysts (the latter only with the recombinant vaccine). Severity of lesions increased with injection volume and decreased as number of injection sites increased. [Discussion]: Local reactions with no apparent clinical effect are observed with both vaccines and are modified by vaccine application schemes. The implication of the adjuvants and/or other vaccine components should be studied further.

8 páginas, 4 figuras, 1 tabla.-- et al., 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., We would like to acknowledge the Junta de Comunidades de Castilla - La Mancha (JCCM) for their support. This study is a contribution of the epidemiological network of rehabilitation centers in the community of Castilla - La Mancha, and the REVISA network, and has been supported by the project PAC08-0296-7771 (JCCM), and from INIA-MARM funds (INIA CC08-020). Elena Sotelo is a fellow from INIA. Elisa Perez Ramirez is a fellow from the National Research Council (CSIC) and Ana Valeria Gutierrez is a fellow from JCCM.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- et al., [Background]: During the last decade, the spread of many flaviviruses (Genus Flavivirus) has been reported, representing an emerging threat for both animal and human health. To further study utility of wild ruminant samples in West Nile virus (WNV) surveillance, we assessed spatio-temporal trends and factors associated with WNV and cross-reacting flaviviruses exposure, particularly Usutu virus (USUV) and Meaban virus (MBV), in wild ruminants in Spain. Serum samples from 4693 wild ruminants, including 3073 free-living red deer (Cervus elaphus), 201 fallow deer (Dama dama), 125 mouflon (Ovis aries musimon), 32 roe deer (Capreolus capreolus) and 1262 farmed red deer collected in 2003-2014, were screened for WNV and antigenically-related flavivirus antibodies using a blocking ELISA (bELISA). Positive samples were tested for neutralizing antibodies against WNV, USUV and MBV by virus micro-neutralization tests. [Results]: Mean flavivirus seroprevalence according to bELISA was 3.4 ± 0.5 % in red deer, 1.0 ± 1.4 % in fallow deer, 2.4 ± 2.7 % in mouflon and 0 % in roe deer. A multivariate logistic regression model revealed as main risk factors for seropositivity in red deer; year (2011), the specific south-coastal bioregion (bioregion 5) and presence of wetlands. Red deer had neutralizing antibodies against WNV, USUV and MBV. [Conclusions]: The results indicate endemic circulation of WNV, USUV and MBV in Spanish red deer, even in areas without known flavivirus outbreaks. WNV antibodies detected in a free-living red deer yearling sampled in 2010, confirmed circulation this year. Co-circulation of WNV and USUV was detected in bioregions 3 and 5, and of WNV and MBV in bioregion 3. Sampling of hunted and farmed wild ruminants, specifically of red deer yearlings, could be a complementary way to national surveillance programs to monitor the activity of emerging flaviviruses., This study has been supported by projects PAC08-0296-7771 (JCCM), AG2008-02504GAN, FAU2008-00019-C03-01 and AGL2013-49159-C2-2-R. Gutierrez-Guzman AV, was a JCCM fellow (PAC08-0296-7771).