High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

A Comparison of Host Responses to Infection with Wild-Type Avian Influenza Viruses in Chickens and Tufted Ducks.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV. IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks show a limited immune response to infection with low pathogenic avian influenza viruses. These findings and future studies in different IAV hosts are important for our understanding of barriers to IAV transmission between species and ultimately from the wild reservoir to humans.

An oseltamivir-resistant avian H1N1 influenza A virus can transmit from mallards to chickens similarly to a wild-type strain: implications for the risk of resistance transmission to humans.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the risk of an OC-resistant pandemic strain.

Highly Pathogenic Avian Influenza A(H5N1) Virus in a Harbor Porpoise, Sweden.

We found highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b associated with meningoencephalitis in a stranded harbor porpoise (Phocoena phocoena). The virus was closely related to strains responsible for a concurrent avian influenza outbreak in wild birds. This case highlights the potential risk for virus spillover to mammalian hosts.

Tissue distribution of angiotensin-converting enzyme 2 (ACE2) receptor in wild animals with a focus on artiodactyls, mustelids and phocids.

Natural cases of zooanthroponotic transmission of SARS-CoV-2 to animals have been reported during the COVID-19 pandemic, including to free-ranging white-tailed deer (Odocoileus virginianus) in North America and farmed American mink (Neovison vison) on multiple continents. To understand the potential for angiotensin-converting enzyme 2 (ACE2)-mediated viral tropism we characterised the distribution of ACE2 receptors in the respiratory and intestinal tissues of a selection of wild and semi-domesticated mammals including artiodactyls (cervids, bovids, camelids, suids and hippopotamus), mustelid and phocid species using immunohistochemistry. Expression of the ACE2 receptor was detected in the bronchial or bronchiolar epithelium of several European and Asiatic deer species, Bactrian camel (Camelus bactrianus), European badger (Meles meles), stoat (Mustela erminea), hippopotamus (Hippopotamus amphibious), harbor seal (Phoca vitulina), and hooded seal (Cystophora cristata). Further receptor mapping in the nasal turbinates and trachea revealed sparse ACE2 receptor expression in the mucosal epithelial cells and occasional occurrence in the submucosal glandular epithelium of Western roe deer (Capreolus capreolus), moose (Alces alces alces), and alpaca (Vicunga pacos). Only the European badger and stoat expressed high levels of ACE2 receptor in the nasal mucosal epithelium, which could suggest high susceptibility to ACE2-mediated respiratory infection. Expression of ACE2 receptor in the intestinal cells was ubiquitous across multiple taxa examined. Our results demonstrate the potential for ACE2-mediated viral infection in a selection of wild mammals and highlight the intra-taxon variability of ACE2 receptor expression, which might influence host susceptibility and infection.

Highly Pathogenic Avian Influenza (HPAI H5Nx, Clade 2.3.4.4.b) in Poultry and Wild Birds in Sweden: Synopsis of the 2020-2021 Season.

Highly pathogenic avian influenza (HPAI, Gs/Gd lineage) was introduced to Europe in 2005 and has since caused numerous outbreaks in birds. The 2020-2021 season was the hitherto most devastating when considering bird numbers and duration in Europe. Surveillance data, virologic results and epidemiologic investigations from the 2020-2021 outbreaks in Sweden were analysed. Subtypes H5N8 and H5N5 were detected on 24 farms with poultry or other captive birds. In wild birds, subtypes H5N8, H5N5, H5N1, H5N4, H5Nx were detected in 130 out of 811 sampled birds. There was a spatiotemporal association between cases in wild birds and poultry. Based on phylogeny and epidemiology, most of the introductions of HPAI to commercial poultry were likely a result of indirect contact with wild birds. A definite route of introduction to poultry could not be established although some biosecurity breaches were observed. No spread between farms was identified but airborne spread between flocks on the same farm was suspected. Our findings exemplify the challenges posed by the continuously changing influenza viruses that seem to adapt to a broader species spectrum. This points to the importance of wild bird surveillance, compliance to biosecurity, and identification of risk factors for introduction on poultry farms.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

Alternate routes of influenza A virus infection in Mallard (Anas platyrhynchos).

The natural reservoir for all influenza A viruses (IAVs) is wild birds, particularly dabbling ducks. During the autumn, viral prevalence can be very high in dabbling ducks (> 30%) in the Northern Hemisphere, and individuals may be repeatedly infected. Transmission and infection is through the fecal-oral route, whereby birds shed viruses in feces and conspecifics are infected though feeding in virus-contaminated water. In this study we wanted to assess two alternative infection routes: cloacal drinking and preening. Using experimental infections, we assessed patterns of infection using a combination of virus shedding, as assessed by real-time PCR from cloacal swabs, and patterns of viral replication using virus-immunohistochemistry of gastrointestinal tissues. The cloacal drinking experiment consisted of two trials using cloacal inoculation at two different time points to account for age differences, as well as a trial whereby ducks were allowed to take up virus-laden water through the cloaca. All ducks became infected, and rather than the bursa of Fabricius being the main site of replication, the colon had the highest intensity of replication, as inferred through immunohistochemistry. In experiments assessing preening, feathers were contaminated with virus-laden water and all ducks became infected, regardless of whether they were kept individually or together. Further, naive contacts were infected by the individuals whose feathers were virus-contaminated. Overall, we reinforce that IAV transmission in dabbling ducks is multifactorial-if exposed to virus-contaminated water ducks may be infected through dabbling, preening of infected feathers, and cloacal drinking.

Post mortem findings and their relation to AA amyloidosis in free-ranging Herring gulls (Larus argentatus).

Since the late 1990s, high mortality and declining populations have been reported among sea birds including Herring gulls (Larus argentatus) from the Baltic Sea area in Northern Europe. Repeated BoNT type C/D botulism outbreaks have occurred, but it remains unclear whether this is the sole and primary cause of mortality. Thiamine deficiency has also been suggested as a causal or contributing factor. With this study, we aimed to investigate gross and microscopic pathology in Herring gulls from affected breeding sites in Sweden in search of contributing diseases. Herring gulls from Iceland served as controls. Necropsies and histopathology were performed on 75 birds, of which 12 showed signs of disease at the time of necropsy. Parasites of various classes and tissues were commonly observed independent of host age, e.g. oesophageal capillariosis and nematode infection in the proventriculus and gizzard with severe inflammation, air sac larid pentastomes and bursal trematodiasis in pre-fledglings. Gross and microscopic findings are described. Notably, amyloidosis was diagnosed in 93 and 33% of the adult birds from Sweden and Iceland, respectively (p<0.001), with more pronounced deposits in Swedish birds (p<0.001). Gastrointestinal deposits were observed in the walls of arteries or arterioles, and occasionally in villi near the mucosal surface. Amyloid was identified within the intestinal lumen in one severely affected gull suggesting the possibility of oral seeding and the existence of a primed state as previously described in some mammals and chickens. This could speculatively explain the high occurrence and previously reported rapid onset of amyloidosis upon inflammation or captivity in Herring gulls. Amyloid-induced malabsorbtion is also a possibility. The Herring gull SAA/AA protein sequence was shown to be highly conserved but differed at the N-terminus from other avian species.

Sublethal Lead Exposure Alters Movement Behavior in Free-Ranging Golden Eagles.

Lead poisoning of animals due to ingestion of fragments from lead-based ammunition in carcasses and offal of shot wildlife is acknowledged globally and raises great concerns about potential behavioral effects leading to increased mortality risks. Lead levels in blood were correlated with progress of the moose hunting season. Based on analyses of tracking data, we found that even sublethal lead concentrations in blood (25 ppb, wet weight), can likely negatively affect movement behavior (flight height and movement rate) of free-ranging scavenging Golden Eagles (Aquila chrysaetos). Lead levels in liver of recovered post-mortem analyzed eagles suggested that sublethal exposure increases the risk of mortality in eagles. Such adverse effects on animals are probably common worldwide and across species, where game hunting with lead-based ammunition is widespread. Our study highlights lead exposure as a considerably more serious threat to wildlife conservation than previously realized and suggests implementation of bans of lead ammunition for hunting.

Chewing lice Trichodectes pinguis pinguis in Scandinavian brown bears (Ursus arctos).

In April 2014 and 2015, we noted localized alopecia (neck, forelimbs, and chest) and hyperpigmentation on two adult brown bears (Ursus arctos) captured in central-south Sweden for ecological studies under the Scandinavian Brown Bear Research Project. In spring 2015, a brown bear was shot because of human-wildlife conflict in the same region. This bear also had extensive alopecia and hyperpigmentation. Ectoparasites were collected from the affected skin areas in all three individuals and preserved in ethanol for identification. Based on morphological characteristics, the lice were identified as Trichodectes spp. and Trichodectes pinguis pinguis. To our knowledge, these are the first reported cases of chewing lice in free-ranging brown bears in Scandinavia.

Pathogenicity and tissue tropism of currently circulating highly pathogenic avian influenza A virus (H5N1; clade 2.3.2) in tufted ducks (Aythya fuligula).

Reports describing the isolation of highly pathogenic avian influenza (HPAI) virus (H5N1) clade 2.3.2 in feces from apparently healthy wild birds and the seemingly lower pathogenicity of this clade compared to clade 2.2 in several experimentally infected species, caused concern that the new clade might be maintained in the wild bird population. To investigate whether the pathogenicity of a clade 2.3.2 virus was lower than that of clades previously occurring in free-living wild birds in Europe, four tufted ducks were inoculated with influenza A/duck/HongKong/1091/2011 (H5N1) clade 2.3.2 virus. The ducks were monitored and sampled for virus excretion daily during 4 days, followed by pathologic, immunohistochemical, and virological investigations. The virus produced severe disease as evidenced by clinical signs, presence of marked lesions and abundant viral antigen in several tissues, especially the central nervous system. The study shows that HPAI-H5N1 virus clade 2.3.2 is highly pathogenic for tufted ducks and thus, they are unlikely to maintain this clade in the free-living population or serve as long-distance vectors.

Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 µg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Resistance mutation R292K is induced in influenza A(H6N2) virus by exposure of infected mallards to low levels of oseltamivir.

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.

Pathobiology and virus shedding of low-pathogenic avian influenza virus (A/H1N1) infection in mallards exposed to oseltamivir.

Low-pathogenic avian influenza (LPAI) viruses in wild birds are important as they can constitute the basis for the development of highly pathogenic avian influenza viruses or form part of human-adapted strains with pandemic potential. However, the pathogenesis of LPAI viruses is not well characterized in dabbling ducks, one of the natural reservoirs of LPAI viruses. Between 21 September 2009 and 21 December 2009, we used real-time reverse transcriptase polymerase chain reaction (q-PCR), histopathology, and immunohistochemistry (IHC) to study Mallards (Anas platyrhynchos) infected with an influenza A/H1N1 virus isolated from a wild Mallard in Sweden. The ducks were either inoculated intraesophageally ("artificial infection") or infected by virus shed by other ducks in the experiment ("contact infection"). The ducks were subjected to three low concentrations (80 ng/L, 1 µg/L, and 80 µg/L) of the active metabolite of oseltamivir (Tamiflu(®)), oseltamivir carboxylate (OC), which resulted in the development of the viral resistance mutation H274Y at 1 and 80 µg/L. The LPAI virus infection was localized to the intestinal tract and cloacal bursa except in one Mallard. The exception was a duck euthanized 1 day postinoculation, whose infection was located solely in the lung, possibly due to intratracheal deposition of virus. The intestinal infection was characterized by occasional degenerating cells in the lamina propria and presence of viral antigen as detected by IHC, as well as positive q-PCR performed on samples from feces and intestinal contents. Histopathologic changes, IHC positivity, and viral shedding all indicated that the infection peaked early, around 2 days postinfection. Furthermore, more viral antigen and viral RNA were detected with IHC and q-PCR in the proximal parts early in the infection. There was no obvious difference in the course of the infection in artificial versus contact infection, when the level of OC was increased from 80 ng/L to 1 µg/L (based on IHC and q-PCR), when the level of OC was increased to 80 µg/L, or when the resistance mutation H274Y developed (based on q-PCR).

Characterization of encephalitis in wild birds naturally infected by highly pathogenic avian influenza H5N1.

During the outbreak of highly pathogenic avian influenza (HPAI) H5N1 in Sweden in 2006, disease and mortality were observed in a number of wild bird species. Encephalitis was one of the most consistent and severe findings in birds submitted for postmortem examination. However, the distribution and severity of the inflammation varied among individuals. This study characterized the encephalitis and the phenotype of the cellular infiltrate in brains of 40 birds of various species naturally infected with HPAI H5N1. Brain sections stained with hematoxylin and eosin and immunostained for influenza A viral antigen were evaluated in parallel to brain sections immunostained with antibodies against T lymphocytes (CD3+), B lymphocytes (CD79a+), macrophages (Lectin RCA-1+), and astrocytes expressing glial fibrillary acidic protein. The virus showed marked neurotropism, and the neuropathology included multifocal to diffuse areas of gliosis and inflammation in the gray matter, neuronal degeneration, neuronophagia, vacuolation of the neuropil, focal necrosis, perivascular cuffing, and meningitis. Broad ranges in severity, neuroanatomical distribution, and type of cellular infiltrate were observed among the different bird species. Since neurotropism is a key feature of HPAI H5N1 infection in birds and other species and because the clinical presentation can vary, the characterization of the inflammation in the brain is important in understanding the pathogenesis of the disease and also has important diagnostic implications for sample selection.

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards.

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008-2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2-4 nM in wild-type viruses to 400-700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58-293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Influenza virus in a natural host, the mallard: experimental infection data.

Wild waterfowl, particularly dabbling ducks such as mallards (Anas platyrhynchos), are considered the main reservoir of low-pathogenic avian influenza viruses (LPAIVs). They carry viruses that may evolve and become highly pathogenic for poultry or zoonotic. Understanding the ecology of LPAIVs in these natural hosts is therefore essential. We assessed the clinical response, viral shedding and antibody production of juvenile mallards after intra-esophageal inoculation of two LPAIV subtypes previously isolated from wild congeners. Six ducks, equipped with data loggers that continually monitored body temperature, heart rate and activity, were successively inoculated with an H7N7 LPAI isolate (day 0), the same H7N7 isolate again (day 21) and an H5N2 LPAI isolate (day 35). After the first H7N7 inoculation, the ducks remained alert with no modification of heart rate or activity. However, body temperature transiently increased in four individuals, suggesting that LPAIV strains may have minor clinical effects on their natural hosts. The excretion patterns observed after both re-inoculations differed strongly from those observed after the primary H7N7 inoculation, suggesting that not only homosubtypic but also heterosubtypic immunity exist. Our study suggests that LPAI infection has minor clinically measurable effects on mallards and that mallard ducks are able to mount immunological responses protective against heterologous infections. Because the transmission dynamics of LPAIVs in wild populations is greatly influenced by individual susceptibility and herd immunity, these findings are of high importance. Our study also shows the relevance of using telemetry to monitor disease in animals.

Pathology of natural highly pathogenic avian influenza H5N1 infection in wild tufted ducks (Aythya fuligula).

Highly pathogenic avian influenza (HPAI) subtype H5N1 is an infectious systemic viral disease that results in high morbidity and mortality in poultry, and has been reported in a wide range of wild bird species during the last few years. An outbreak of HPAI H5N1 occurred in wild birds in Sweden in 2006 that affected several duck species, geese, swans, gulls, and raptors. Tufted ducks (Aythya fuligula) accounted for the largest number of positive cases and, therefore, were selected for more in-depth histologic and immunohistochemical evaluations. The main histologic lesions associated with the presence of avian influenza antigen were found in the brain, pancreas, and upper respiratory tract. Other tissues in which influenza antigen was variably found included liver, lung, adrenal glands, kidneys, and peripheral nerve ganglia. The current study describes the pathology and viral tissue targeting of H5N1 by using histology, polymerase chain reaction, and immunohistochemistry, and highlights the range and variation in the presentation of the natural disease in tufted ducks.

Mycotic proventriculitis in gray partridges (Perdix perdix) on two game bird farms.

Proventriculitis and chronic respiratory disease were diagnosed in two flocks of gray partridges (Perdix perdix) on unrelated Swedish game bird farms. Affected birds showed loss of condition, respiratory signs, and flock mortality rates of 50 and 98%, respectively. The proventricular lesions were associated closely with fungal organisms that were microscopically indistinguishable from the ascomycetous yeast Macrorhabdus ornithogaster (former provisional name "megabacterium"). At necropsy, the proventriculi were swollen and hyperemic, and viscous mucus adhered to the mucosa. Proventricular hemorrhages were commonly detected, and one bird had proventricular rupture and peritonitis. Microscopically, mild to severe subacute to chronic lymphoplasmacytic proventriculitis, microabscesses, necrosis, epithelial metaplasia, disrupted koilin, ulcers, and hemorrhages were observed. Transmission electron microscopy of the proventricular microorganisms revealed a membrane-bound nucleus, vacuoles, ribosomes, microtubules in parallel arrays, and a two-layered cell wall but no mitochondria. Scanning electron microscopy of the proventricular epithelium demonstrated masses of organisms with occasional constrictions in parallel arrangement. Many of the birds also suffered from concurrent respiratory bacterial infections and/or gastrointestinal candidiasis. The clinical course and gross and microscopic proventricular lesions were similar to those described in psittacine and passerine pet birds colonized by M. ornithogaster-like microorganisms but differed from published case reports and experimental infections of chickens in which the clinical signs and lesions have been considerably milder. The findings presented in this paper suggest that mycotic proventriculitis, presumably associated with M. ornithogaster, may be a serious but possibly opportunistic, although unusual, disease problem in gray partridges on game farms.