Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5–6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.     

Pathology and tissue tropism of natural West Nile virus infection in birds: a review

West Nile virus (WNV) is a globally distributed arthropod-borne flavivirus capable of infecting a wide variety of vertebrates, with birds as its natural reservoir. Although it had been considered a pathogen of little importance for birds, from the 1990’s, and especially after its introduction in the North American continent in 1999, thousands of birds have succumbed to West Nile infection. This review summarizes the pathogenesis and pathology of WNV infection in birds highlighting differences in lesion and antigen distribution and severity among bird orders and families. Despite significant species differences in susceptibility to infection, WNV associated lesions and viral antigen are present in the majority of organs of infected birds. The non-progressive, acute or more prolonged course of the disease accounts for part of the differences in lesion and viral antigen distribution and lesion severity. Most likely a combination of host variables and environmental factors in addition to the intrinsic virulence and pathogenicity of the infecting WNV strain influence the pathogenesis of the infection.     

Prevalence and relevance of avian Toxoplasma gondii infections in Europe

Toxoplasma (T.) gondii is a parasitic protozoon whose life cycle includes a definite and an intermediate host. Besides a wide range of mammals also birds are suitable intermediate hosts. There are a high number of case reports, prevalence data and experimental studies regarding avian toxoplasmosis. In many domestic, wild and zoo bird species which are relevant in Europe the susceptibility for T. gondii infection could be demonstrated though susceptibility for clinical disease often seems to be low, especially in gallinaceous birds. Comprehensive Europe-wide studies concerning the occurrence of avian toxoplasmosis have not been conducted so far. The general infestation ratio of birds on European free-range poultry farms seems to be relatively high. The European wild bird population represents a significant reservoir for the pathogen. When evaluating the seroprevalence it has to be considered that sensitivity and specificity of the detection methods for T. gondii specific antigens which are used frequently in mammals often are not satisfying in birds.     

Monitoring of West Nile virus infections in Germany

West Nile virus (WNV) is a flavivirus that is maintained in an enzootic cycle between ornithophilic mosquitoes, mainly of the Culex genus, and certain wild bird species. Other bird species like ravens, jays and raptors are highly susceptible to the infection and may develop deadly encephalitis, while further species of birds are only going through subclinical infection. The objective of this study was to continue in years 2009-2011 the serological and molecular surveillance in wild birds in Germany (see Vector Borne Zoonotic Dis. 10, 639) and to expand these investigations for the first time also to sera from domestic poultry and horses collected between 2005 and 2009. All three cohorts function as indicators for the endemic circulation of WNV. The presence of WNV-specific antibodies was detected in all samples by virus neutralization test (VNT), indirect immunofluorescence test (IFT) and/or enzyme-linked immunosorbent assay (ELISA). The presence of WNV genomes was monitored in relevant sera using two qRT-PCRs that amplify lineage 1 and 2 strains. A total of 364 migratory and resident wild bird serum samples (with emphasis on Passeriformes and Falconiformes) as well as 1119 serum samples from domestic poultry and 1282 sera from horses were analysed. With the exception of one hooded crow, antibody carriers were exclusively found in migratory birds, but not in resident birds/domestic poultry or in local horses. Crows are facultative, short-distance winter migrants in Germany. WNV-specific nucleic acids could not be demonstrated in any of the samples. According to these data, there is no convincing evidence for indigenous WNV infections in equines and in wild/domestic birds in Germany. However, since a few years, WNV infections are endemic in other European countries such as Austria, Hungary, Greece and Italy, a state-of-the-art surveillance system for the detection of incursions of WNV into Germany deems mandatory.     

West Nile virus infection of horses

West Nile virus (WNV) is a flavivirus closely related to Japanese encephalitis and St. Louis encephalitis viruses that is primarily maintained in nature by transmission cycles between mosquitoes and birds. Occasionally, WNV infects and causes disease in other vertebrates, including humans and horses. West Nile virus has re-emerged as an important pathogen as several recent outbreaks of encephalomyelitis have been reported from different parts of Europe in addition to the large epidemic that has swept across North America. This review summarises the main features of WNV infection in the horse, with reference to complementary information from other species, highlighting the most recent scientific findings and identifying areas that require further research.     

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa)

ABSTRACT: 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.     

West Nile virus: lessons from the 21st century

Introduction: West Nile virus (WNV) first appeared in the United States in 1999, causing illness and death in birds, horses, and humans. While the initial outbreak of this sometimes deadly viral disease was limited to the northeastern United States, the virus had an inexorable migration across the continental United States over the next 3 years, causing huge losses among the affected species. The purpose of this review is to present currently available information regarding the epi‐demiology, diagnosis, treatment, and prevention of WNV infection. Veterinarians, particularly those in an emergency practice, serve as an important source of reliable information regarding this disease for animal owners and the public in general. Data sources: Data sources used for the preparation of this review include computer‐based searches of PubMed and Commonwealth Agricultural Bureaux (CAB) abstracts. A search in PubMed using ‘West Nile’ retrieved 1468 ‘hits’ or references, while a similar search in CAB abstracts produced 815 references. Additional information was obtained from various meeting proceedings, particularly data presented in abstract form, and from the Centers for Disease Control (CDC) website dedicated to WNV. Human data synthesis: Prior to the mid‐1990s, reported large‐scale epidemics of WNV infection in humans predominantly presented as acute, mild, febrile disease, sometimes associated with lymphadenopathy and skin rash. The recent large epidemic in the United States, in contrast, has prominently featured encephalitis, particularly among the elderly. Additionally, polio‐encephalomyelitis‐like complications resulting in long‐term neurologic sequelae have been reported. There are many WNV‐permissive native avian and mosquito hosts in the Unites States and there appear to be few limitations to the spread of the disease in the United States. It is expected that the virus will be identified in all 48 continental states, Mexico, and Canada by the end of 2003. Veterinary data synthesis: The horse is the animal species most affected by the recent WNV epidemic in the United States, and losses to the equine industry have been large and unprecedented. A United States Department of Agriculture (USDA)‐approved vaccine against WNV has been in use in horses since 2001 and appears to be effective in limiting the incidence of disease in well‐vaccinated populations. WNV infection has been documented in other species of mammals, including camelids (alpaca/llamas) and dogs, and veterinarians should include WNV as a differential diagnosis for animals presenting with clinical signs consistent with central nervous system infection. A large concern exists for endangered bird populations, particularly birds of prey, whether in zoos or in the wild.     

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.     

Ecological Surveillance for West Nile in Catalonia (Spain), Learning from a Five‐Year Period of Follow‐up

To enhance early detection of West Nile virus (WNV) transmission, an integrated ecological surveillance system was implemented in Catalonia (north‐eastern Spain) from 2007 to 2011. This system incorporated passive and active equine surveillance, periodical testing of chicken sentinels in wetland areas, serosurveillance wild birds and testing of adult mosquitoes. Samples from 298 equines, 100 sentinel chickens, 1086 wild birds and 39 599 mosquitoes were analysed. During these 5 years, no acute WNV infection was detected in humans or domestic animal populations in Catalonia. WNV was not detected in mosquitoes either. Nevertheless, several seroconversions in resident and migrant wild birds indicate that local WNV or other closely related flaviviruses transmission was occurring among bird populations. These data indicate that bird and mosquito surveillance can detect otherwise silent transmission of flaviviruses and give some insights regarding possible avian hosts and vectors in a European setting.     

Anatomopathological aspects of avian aspergillosis

Aspergillosis is a fungal disease caused by fungi of the genus Aspergillus, in particular A. fumigatus and A. flavus. This paper focuses on anatomopathological aspects resulting from a chronic infection from Aspergillus spp in the chicken (Gallus domesticus), in the herring gull (Larus cachinnans micaelli) and in the red-legged partridge (Alectoris rufa rufa). Microscopically, we observed some histological lesions that are related to the two typical forms of Aspergillosis: a deep nodular form, typical of organs with a non-aerated parenchyma, and a non-encapsulated superficial diffuse form typical of the serosae and the lung. The observed forms of aspergillosis have been found in animals raised in poor hygienic environmental conditions or malnourished animals (chicken); in wild birds from wildlife recovery centres (herring gull), which underwent some forms of stress, such as traumas, detention, starvation, extended antibiotic treatments; in game birds (red-legged partridge) used for restocking natural areas that had been negatively affected by such stressors as captivity in aviaries, containment and transport in cages, release in unsuitable environments and malnutrition. The observed anatomopathological and istopathological aspects can therefore be regarded as the outcome of a number of factors that have reduced the typical resistance of the species and impaired the efficiency of their immune systems.     

The effect of exogenous corticosterone on West Nile virus infection in Northern Cardinals (Cardinalis cardinalis)

ABSTRACT: The relationship between stress and disease is thought to be unambiguous: chronic stress induces immunosuppression, which likely increases the risk of infection. However, this link has not been firmly established in wild animals, particularly whether stress hormones affect host responses to zoonotic pathogens, which can be transmitted to domesticated animal, wildlife and human populations. Due to the dynamic effects of stress hormones on immune functions, stress hormones may make hosts better or poorer amplifying hosts for a pathogen contingent on context and the host species evaluated. Using an important zoonotic pathogen, West Nile virus (WNV) and a competent host, the Northern Cardinal (Cardinalis cardinalis), we tested the effects of exogenous corticosterone on response to WNV infection. Corticosterone was administered at levels that individuals enduring chronic stressors (i.e., long-term inclement weather, food shortage, anthropogenic pollution) might experience in the wild. Corticosterone greatly impacted mortality: half of the corticosterone-implanted cardinals died between five - 11 days post-inoculation whereas only one of nine empty-implanted (control) birds died. No differences were found in viral titer between corticosterone- and empty-implanted birds. However, cardinals that survived infections had significantly higher average body temperatures during peak infection than individuals that died. In sum, this study indicates that elevated corticosterone could affect the survival of WNV-infected wild birds, suggesting that populations may be disproportionately at-risk to disease in stressful environments.     

West Nile virus and its emergence in the United States of America

Zoonotic West Nile virus (WNV) circulates in natural transmission cycles involving certain mosquitoes and birds, horses, humans, and a range of other vertebrates are incidental hosts. Clinical infections in humans can range in severity from uncomplicated WNV fever to fatal meningoencephalitis. Since its introduction to the Western Hemisphere in 1999, WNV had spread across North America, Central and South America and the Caribbean, although the vast majority of severe human cases have occurred in the United States of America (USA) and Canada. By 2002-2003, the WNV outbreaks have involved thousands of patients causing severe neurologic disease (meningoencephalitis and poliomyelitis-like syndrome) and hundreds of associated fatalities in USA. The purpose of this review is to present recent information on the epidemiology and pathogenicity of WNV since its emergence in North America.     

Evidence of West Nile virus seropositivity in wild birds on the island of Cyprus

West Nile Virus (WNV)¹ is an emerging pathogen in Cyprus, with the first human case of infection reported in 2016, and another documented in 2018. A cluster of cases in humans was then reported in 2019. However, little is known regarding which avian species might bring WNV to Cyprus. Here, we investigated seroprevalence of WNV antibodies in migratory and resident birds, captured across Cyprus to assess to what extent human populations might be exposed to WNV. We used Enzyme-Linked Immunosorbent Assay (ELISA)² to test for the presence of WNV antibodies in 836 avian blood samples of 44 species captured between 2015 and 2020. A seropositivity rate of 1.3 % was found. The majority of seropositive wild birds belonged to the migratory species Sylvia atricapilla, a common and widespread migrant, implying a high risk of WNV being introduced throughout Cyprus.     

Bagaza virus is pathogenic and transmitted by direct contact in experimentally infected partridges,but is not infectious in house sparrows and adult mice

Bagaza virus (BAGV) is a mosquito-borne flavivirus belonging to the Ntaya serocomplex. In 2010, a disease outbreak was reported in Cádiz (Southern Spain) affecting game birds (red-legged partridges and common pheasants). In this work, red-legged partridges were inoculated experimentally with infectious BAGV isolated from this outbreak in order to make a complete clinical and analytical assessment of the disease caused by the pathogen in this species. Viral load (by real-time RT-PCR) in blood, oral and cloacal swabs, and feathers, and neutralizing antibody titres (by VNT) were measured. In order to determine direct contact transmission, non-inoculated partridges were caged together with the inoculated ones. To assess infectiousness in other species, house sparrows and mice were also inoculated with the virus. All the inoculated partridges were clinically affected, and 30% of them died. All the infected individuals lost weight, with larger losses being recorded in females. Conversely, no mortality or disease symptoms were observed in the sparrows or mice. Remarkably, all the contact partridges acquired the infection by direct (non-vectored) transmission. This study confirms that the red-legged partridge is a susceptible host for BAGV infection, and that this pathogen is transmitted by direct contact. Long-lasting viral loads detected in calami of immature feathers demonstrate that feather sampling could be a useful strategy in active surveillance programs for early detection of BAGV.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0233-9) contains supplementary material, which is available to authorized users.     

A metapopulation model to simulate West Nile virus circulation in Western Africa, Southern Europe and the Mediterranean basin

In Europe, virological and epidemiological data collected in wild birds and horses suggest that a recurrent circulation of West Nile virus (WNV) could exist in some areas. Whether this circulation is permanent (due to overwintering mechanisms) or not remains unknown. The current conception of WNV epidemiology suggests that it is not: this conception combines an enzootic WNV circulation in tropical Africa with seasonal introductions of the virus in Europe by migratory birds. The objectives of this work were to (i) model this conception of WNV global circulation; and (ii) evaluate whether the model could reproduce data and patterns observed in Europe and Africa in vectors, horses, and birds. The model was calibrated using published seroprevalence data obtained from African (Senegal) and European (Spain) wild birds, and validated using independent, published data: seroprevalence rates in migratory and resident wild birds, minimal infection rates in vectors, as well as seroprevalence and incidence rates in horses. According to this model, overwintering mechanisms are not needed to reproduce the observed data. However, the existence of such mechanisms cannot be ruled out.     

Isolation from predators: a key factor in the failed release of farmed red-legged partridges (Alectoris rufa) to the wild?

1. The effects of predation isolation were studied in two lines of 10 month-old farmed red-legged partridges (Alectoris rufa). Birds came from bred generations in captivity (F) and directly from wild parents (W). 2. Releases were carried out in March for two consecutive years on private farmland with a stable population of wild red-legged partridge using 68 tagged birds (36 F, 32 W). 3. No statistical differences were found in survival time (mean 18 d) or home range values (mean 7·38 ha); 49% of mortality was attributable to raptors, 36% to wild canids and 15% had doubtful causes of death. F birds suffered more predation by raptors and W partridges more by mammalian carnivores. 4. A statistical difference was found in post-release dispersion: the means were 243·4 m in F and 593·3 m in W. 5. The result of predation isolation was similar in both groups, which confirms the adverse effects of current production systems on anti-predator behaviour. Farmers should consider the introduction of new rearing techniques with the aim of improving survival after release.     

West Nile virus: North American experience

West Nile virus, a mosquito‐vectored flavivirus of the Japanese encephalitis serogroup, was first detected in North America following an epizootic in the New York City area in 1999. In the intervening 11 years since the arrival of the virus in North America, it has crossed the contiguous USA, entered the Canadian provinces bordering the USA, and has been reported in the Caribbean islands, Mexico, Central America and, more recently, South America. West Nile virus has been reported in over 300 species of birds in the USA and has caused the deaths of thousands of birds, local population declines of some avian species, the clinical illness and deaths of thousands of domestic horses, and the clinical disease in over 30 000 Americans and the deaths of over 1000. Prior to the emergence of West Nile virus in North America, St. Louis encephalitis virus and Dengue virus were the only other known mosquito‐transmitted flaviviruses in North America capable of causing human disease. This review will discuss the North American experience with mosquito‐borne flavivirus prior to the arrival of West Nile virus, the entry and spread of West Nile virus in North America, effects on wild bird populations, genetic changes in the virus, and the current state of West Nile virus transmission.     

Study of survival, dispersal and home range of autumn-released red-legged partridges (Alectoris rufa)

1. On a private property with a stable population of wild red-legged partridge (Alectoris rufa) and an appropriate habitat for the survival of the species, reinforcement repopulations were carried out in the months of October and November for two consecutive years using 5- to 6-month-old birds reared on a commercial game farm. 2. Of the 36 released birds, none was still alive by the following spring's breeding period. Mean survival time was 9.4 d in the first year and 7.6 d in the second year. 3. Seventy-two per cent of mortality was attributable to predation, 11% to hunting and 17% to doubtful causes of death, accidents and starvation. 4. Post-release mean dispersion was 377.8 m in the first year and 526.3 m in the second. Mean home range was 7.1 ha in the first year and 5.4 ha in the second. 5. The production systems and handling practices of commercial game farms may have modified some anti-predator ethological patterns and strategies, which might make it more difficult for the birds to adapt and integrate into the wild, resulting in reduced survival due to premature mortality.     

Detection and molecular analysis of West Nile virus infections in birds of prey in the eastern part of Austria in 2008 and 2009

The emergence of West Nile virus (WNV) was expected in Austria since the initial discovery of the infection in neighbouring Hungary in 2003/2004. In 2008 six cases of West Nile disease were diagnosed at the Institute for Veterinary Disease Control M?dling, Austrian Agency for Health and Food Safety (AGES), involving five goshawks (Accipiter gentilis) and one gyrfalcon (Falco rusticolus), which were found dead in the eastern Austrian federal states of Lower Austria, Vienna and Styria, respectively. Pathomorphological and immunohistochemical findings suggested a WNV infection. Virus was isolated in embryonated specific pathogen free chicken eggs and propagated in mouse neuroblastoma cells (NA), in which a cytopathic effect occurred. The virus was identified and characterised by electron microscopic examination and molecular detection using RT-PCR, sequencing, and phylogenetic analysis. The Austrian WNV sequences exhibited nucleotide identities of 99.9% to the lineage 2 WNV sequences described in Hungary since 2004. In addition, 71 sera of 14 different bird species were screened for the presence of WNV antibodies using a commercial ELISA: 43.7% of the tested samples showed antibody titers. Selected positive sera were also subjected to WNV neutralisation tests, in which the ELISA results were verified in 66%. The results of this study confirm unambiguously the presence of a lineage 2 WNV infection in birds of prey in the eastern part of Austria.     

Experimental West Nile virus infection in Eastern Screech Owls (Megascops asio)

Eastern Screech Owls (EASOs) were experimentally infected with the pathogenic New York 1999 strain of West Nile virus (WNV) by subcutaneous injection or per os. Two of nine subcutaneously inoculated birds died or were euthanatized on 8 or 9 days postinfection (DPI) after <24 hr of lethargy and recumbency. All subcutaneously inoculated birds developed levels of viremia that are likely infectious to mosquitoes, with peak viremia levels ranging from 10(5.0) to 10(9.6) plaque-forming units/ml. Despite the viremia, the remaining seven birds did not display signs of illness. All birds alive beyond 5 DPI seroconverted, although the morbid birds demonstrated significantly lower antibody titers than the clinically normal birds. Cagemates of infected birds did not become infected. One of five orally exposed EASOs became viremic and seroconverted, whereas WNV infection in the remaining four birds was not evident. All infected birds shed virus via the oral and cloacal route. Early during infection, WNV targeted skin, spleen, esophagus, and skeletal muscle. The two morbid owls had myocardial and skeletal muscle necrosis and mild encephalitis and nephritis, whereas some of the clinically healthy birds that were sacrificed on 14 DPI had myocardial arteritis and renal phlebitis. WNV is a significant pathogen of EASOs, causing pathologic lesions with varying clinical outcomes.