Elisa Pérez-Ramírez

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.

Experimental infections of wild birds with West Nile virus.

Avian models of West Nile virus (WNV) disease have become pivotal in the study of infection pathogenesis and transmission, despite the intrinsic constraints that represents this type of experimental research that needs to be conducted in biosecurity level 3 (BSL3) facilities. This review summarizes the main achievements of WNV experimental research carried out in wild birds, highlighting advantages and limitations of this model. Viral and host factors that determine the infection outcome are analyzed in detail, as well as recent discoveries about avian immunity, viral transmission, and persistence achieved through experimental research. Studies of laboratory infections in the natural host will help to understand variations in susceptibility and reservoir competence among bird species, as well as in the epidemiological patterns found in different affected areas.

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.

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.

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.

Flaviviruses in game birds, southern Spain, 2011-2012.

To the Editor: Certain arthropod-borne epornitic flaviviruses, namely, West Nile virus (WNV) and Usutu virus (USUV), have spread recently in parts of Europe (1,2). In southern Spain, the emergence of a third virus of this type, known as Bagaza virus (BAGV), is of concern (3). Because of the outbreaks in 2010 in Cadiz (southern Spain) of WNV infection, which affected birds, horses, and humans, and of BAGV infection, which affected game birds (partridges and pheasants), and the reported presence of USUV in mosquitoes in this area (4), a surveillance program was implemented in partridges and pheasants during the next hunting season (October 2011–February 2012) to assess the possible circulation of these 3 flaviviruses in the area. Serum samples and brain tissue from 159 hunted-harvested wild red-legged partridges (Alectoris rufa) and 13 common pheasants (Phasianus colchicus) were collected on 12 hunting properties from Cadiz (Technical Appendix Figure). All sampled birds were reared and shot in the wild. The age of the partridges was determined according to plumage characteristics. Presence of antibodies against WNV was tested with a commercial epitope-blocking ELISA (Ingezym West Nile Compac, INGENASA, Madrid, Spain) (5). Virus-neutralization titers against WNV (strain Eg-101), BAGV (strain Spain/2010), and USUV (strain SAAR1776) were determined by micro virus neutralization test (VNT) as described (6). Viral genome in brain tissue samples was examined by heminested pan-flaviviral reverse transcription PCR (7). All 172 tissue homogenates examined were negative by this test. Overall seroprevalence for WNV by epitope-blocking ELISA was 29%. Prevalence of neutralizing antibodies measured by VNT was 23% for WNV, 15% for BAGV, and 10% for USUV. Seroprevalence rates were higher for pheasants than for partridges for WNV (Fisher exact test, p = 0.0003), BAGV (p<0.0001), and USUV (p<0.0001) (Table). The significance of this result is uncertain, given that just 2 hunting areas were sampled for pheasants. Table Results of serologic studies in red-legged partridges and common pheasants, southern Spain, 2011–2012* Neutralizing antibodies to >1 flavivirus were detected in 15 of the 45 VNT-positive partridges and in 6 of the 12 VNT-positive pheasants (Table). Specificity, as determined by neutralizing antibodies titer comparisons (8), showed virus-specific neutralizing antibodies to WNV, BAGV, and USUV in 19 partridges, 9 partridges, and 1 partridge, respectively, in 3 pheasants to WNV and in another 3 pheasants to BAGV (0 to USUV). Serum from 9 partridges and 6 pheasants remained inconclusive (neutralizing antibodies titer differences <4-fold [8]). WNV-reacting antibodies by ELISA were shown in 11 of 12 hunting properties (Technical Appendix Figure). In all locations but 1, ELISA-positive results were confirmed by VNT for NT-Abs to WNV, BAGV, or USUV. Of them, neutralizing antibodies to only WNV were detected in 2 locations, whereas neutralizing antibodies to at least 2 (WNV/USUV or WNV/BAGV) of the 3 flaviviruses were detected in 8 locations. Within these locations, flavivirus-specific NT-Ab responses were differentiated in several samples: neutralizing antibodies to either WNV or BAGV were detected in samples from 6 locations, whereas samples with neutralizing antibodies to either WNV or USUV were detected in 1 location. Analysis of VNT results in juvenile partridges showed specific neutralizing antibodies to WNV (13%) or BAGV (9%); 4% of these samples were positive for flavivirus but inconclusive for any of the flaviviruses tested (Table). Overall, these results indicated recent circulation of 3 different epornitic flaviviruses—WNV, USUV, and BAGV—in resident game birds in Cadiz, the southernmost province in Spain. A high proportion of birds showed neutralizing antibodies to >1 flavivirus. Some are likely to be attributable to cross-neutralization, although co-infection cannot be ruled out because the results showed co-circulation of >1 flavivirus in game birds in most locations studied. Furthermore, the presence of specific neutralizing antibodies in juvenile partridges indicated that WNV and BAGV circulated in the area 1 year after the outbreaks of 2010. For USUV, specific neutralizing antibodies were detected only in an adult partridge, indicating infection acquired during the previous years. Nevertheless, this finding does not rule out recent co-circulation of USUV together with the other 2 viruses in the same game bird populations, considering that USUV has been repeatedly detected in mosquitoes in nearby areas (4). Evidence of infection by at least WNV and BAGV in 2 consecutive seasons strongly supports the premise that these viruses overwintered in the area. Capability of WNV to overwinter in southern Europe was shown in Italy during 2008–2009 (9) and in Spain during 2007–2008 (10). Overwintering of BAGV after its introduction into Spain could indicate a risk for its expansion in areas with similar climates (Mediterranean basin). The risk for dissemination of WNV, BAGV, or USUV infections not only to game birds, but also to other wildlife, domestic animals, and humans, calls for improvements in surveillance programs, particularly those that monitor susceptible hosts, such as game birds. Technical Appendix: Study area in Spain (province of Cadiz) showing the position of hunting properties analyzed. Click here to view.(101K, pdf)

Serologic Testing for Avian Influenza Viruses in Wild Birds: Comparison of Two Commercial Competition Enzyme-Linked Immunosorbent Assays

Abstract 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.

Experimental infection of house sparrows (Passer domesticus) with West Nile virus strains of lineages 1 and 2

West Nile virus (WNV) is a zoonotic pathogen which is maintained in an enzootic cycle between mosquitoes and birds; humans, equines, other mammals and some bird species are dead-end hosts. Lineage 1 WNV strains have predominated in Europe since the 1960s. However, in 2004 lineage 2 strains emerged in Hungary and Russia, respectively, spreading since then to a number of neighbouring countries (e.g., Austria, Greece, Italy, Serbia and Romania). Wild bird mortality is a hallmark of North American WNV outbreaks, a feature uncommon in Europe. This study aimed to compare the course of infection of lineage 1 (NY99) and lineage 2 (Austria/2008) WNV strains in the house sparrow, a bird species common in Europe and North America. House sparrows were inoculated with either NY99 or Austria/2008 WNV strains, or sham-inoculated, and clinical and analytic parameters (viraemia, viral load, antibodies) were examined until 14 days after inoculation. Although all inoculated sparrows became infected, no mortality or clinical signs were observed due to the infection. However, the magnitude and duration of viraemia were higher for NY99 - than for Austria/2008 - infected birds. The house sparrow proved to be a competent host for both strains, although the competence index calculated for NY99 was higher than for Austria/2008. Viral load in tissues and swabs was also higher in NY99-inoculated sparrows. In conclusion, the house sparrow is a convenient avian model for studying host competence of WNV strains. The observed differences between NY99 and Austria/2008 strains might have important epidemiological consequences for disease incidence and dispersal capacity.

Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests

Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.

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.