West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities. METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time. FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways. CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.

West Nile and Usutu viruses are efficiently inactivated in platelet concentrates by UVC light using the THERAFLEX UV-Platelets system.

BACKGROUND AND OBJECTIVES: West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses (Flaviviridae) that originated in Africa, have expanded their geographical range during the last decades and caused documented infections in Europe in the last years. Acute WNV and USUV infections have been detected in asymptomatic blood donors by nucleic acid testing. Thus, inactivation of both viral pathogens before blood transfusion is necessary to ensure blood product safety. This study aimed to investigate the efficacy of the THERAFLEX UV-Platelets system to inactivate WNV and USUV in platelet concentrates (PCs). MATERIALS AND METHODS: Plasma-reduced PCs were spiked with the virus suspension. Spiked PC samples were taken after spiking (load and hold sample) and after UVC illumination on the Macotronic UV illumination machine with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) J/cm2). Virus loads of WNV and USUV before and after illumination were measured by titration. RESULTS: Infectivity assays showed that UVC illumination inactivated WNV and USUV in a dose-dependent manner. At a UVC dose of 0.2 J/cm2, the WNV titre was reduced by a log10 factor of 3.59 ± 0.43 for NY99 (lineage 1) and 4.40 ± 0.29 for strain ED-I-33/18 (lineage 2). USUV titres were reduced at the same UVC dose by a log10 factor of 5.20 ± 0.70. CONCLUSIONS: Our results demonstrate that the THERAFLEX UV-Platelets procedure is an effective technology to inactivate WNV and USUV in contaminated PCs.

Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics.

The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.

West Nile Virus in Germany: An Emerging Infection and Its Relevance for Transfusion Safety.

West Nile virus (WNV) is an arthropod-borne virus (arbovirus). It circulates in an enzootic cycle between ornithophilic mosquitoes as vectors and reservoirs and avian host species for amplification, but humans can be infected as accidental hosts. In most individuals, WNV infection remains silent, while 20% develop mild symptoms of West Nile fever, and only 1% develop neuroinvasive disease (WNND). Human WNV cases have been identified in Southern and Eastern Europe for more than 20 years, but until 2018, Germany was considered to be a non-endemic country. This changed when in the exceptionally warm summer of 2018, conditions for viral replication in mosquitoes were ideal, and the first WNV cases among birds and horses were identified. The widespread domestic Culex mosquitoes are efficient vectors for WNV. Autochthonous mosquito-borne WNV infections in humans were reported in all following years, indicating a continuous circulation in the affected areas of Central-East Germany. So far, no clear expansion of the affected areas is discernible but may develop. WNV is a transfusion-transmissible-infection, and donor deferral or testing of donations after a stay in an affected area are effective means to ensure transfusion safety. WNV transmissions via blood products often result in WNND due to the predisposing underlying medical conditions of transfusion recipients. From 2020 onwards, roughly 80% of all blood establishments in Germany tested their donations for WNV using nucleic acid amplification techniques in the transmission season. Altogether, 19 confirmed WNV infections were identified from 2020-2021. As long as effective and affordable pathogen reduction is not available for all blood components, WNV testing or donor deferral will be essential. In order to timely identify affected areas, combined results of human and veterinary surveillance are needed. Partnerships between public health experts, transfusion medicine specialists, veterinarians, and entomologists should be strengthened to ensure a One Health approach.

Absence of classical and atypical (H- and L-) BSE infectivity in the blood of bovines in the clinical end stage of disease as confirmed by intraspecies blood transfusion.

While the presence of bovine spongiform encephalopathy (BSE) infectivity in the blood of clinically affected sheep has been proven by intraspecies blood-transfusion experiments, this question has remained open in the case of BSE-affected cattle. Although the absence of infectivity can be anticipated from the restriction of the agent to neuronal tissues in this species, evidence for this was still lacking. This particularly concerns the production and use of medicinal products and other applications containing bovine blood or preparations thereof. We therefore performed a blood-transfusion experiment from cattle in the clinical end stage of disease after experimental challenge with either classical (C-BSE) or atypical (H- and l-) BSE into calves at 4-6 months of age. The animals were kept in a free-ranging group for 10 years. Starting from 24 months post-transfusion, a thorough clinical examination was performed every 6 weeks in order to detect early symptoms of a BSE infection. Throughout the experiment, the clinical picture of all animals gave no indication of a BSE infection. Upon necropsy, the brainstem samples were analysed by BSE rapid test as well as by the highly sensitive Protein Misfolding Cyclic Amplification (PMCA), all with negative results. These results add resilient data to confirm the absence of BSE infectivity in the donor blood collected from C-, H- and l-BSE-affected cattle even in the final clinical phase of the disease. This finding has important implications for the risk assessment of bovine blood and blood products in the production of medicinal products and other preparations.

Usutu virus infection in aviary birds during the cold season.

The mosquito-borne flavivirus Usutu virus (USUV) is responsible for countless deaths in both resident populations and birds kept in outdoor aviaries. Since 2001, USUV outbreaks have attracted increased attention due to the rapid geographical spread of the virus and its close relationship to West Nile virus (WNV), an emerging pathogen in humans and animals. Similar to WNV, the USUV enzootic transmission cycle predominantly involves Culex spp. as vectors, whereas birds serve as amplifying reservoir hosts. In Europe, USUV-associated disease outbreaks in birds are almost exclusively described during late spring and early autumn (early April to late October). Contagiousness of virus particles excreted by infected birds has not yet been proven, so that the role of non-vector-borne transmission, as it is known for the closely related WNV, remains unclear. Here we report the diagnosis of USUV infection in 15 of 24 birds from mortality outbreaks that occurred during the cold season between late October 2018 and early April 2019, in eight different aviaries located in Germany. Detection of USUV was performed using standardized molecular biological methods and immunohistochemistry for verification of the infection. USUV infection in a parrot species, a tropical finch and two estrildid finches are reported for the first time. Further research on the occurrence of USUV infection during the cold season is key to understanding the dynamics of viral transmission as well as for a profound health risk assessment for aviary birds as well as humans.

Tick-borne encephalitis virus (TBEV) antibodies in animal sera - occurrence in goat flocks in Germany, longevity and ability to recall immunological information after more than six years.

BACKGROUND: TBE is an important tick-borne viral zoonosis in Europe and some parts of Asia. Humans can become infected by tick bite and in some cases also by consumption of nonpasteurized raw milk and raw milk products from ruminants. Serological investigations of milking flocks can help to assess the risk of TBEV infection for humans. 735 blood samples from 50 goat flocks from four federal states of Germany were tested by TBEV-VNT to assess a potential risk for TBEV infection. There are some gaps in the knowledge about immunity in animals, for example with regard to the longevity of TBEV immunity. Two goats and two sheep were immunized and TBEV antibody titers could be detected for up to 7 years. Furthermore, nothing is known about a possible long-lasting immunological memory that could quickly be reactivated by an additional contact to TBEV. Seven years after the first immunization two goats and two sheep as well as two naïve goats and two sheep were boostered and TBEV antibody titers followed. RESULTS: Only one sample in each of the three states was TBEV-antibody positive (VNT), albeit with low titers. However, in Baden-Württemberg seven samples were positive, among them four goats of the same flock. TBEV-antibody positive titers were detected in goats for up to 6 years and 10 months, in sheep for up to 4 years and 7 months. Seven years after immunization a clear immunological recall occurred in response to administration of one dose of vaccine in two goats and two sheep. CONCLUSION: It can be concluded that in the tested flocks the risk of an alimentary TBEV infection was low. However, in one single flock a considerably higher risk must be assumed. Antibody titers in goats and sheep can last very long after contact to TBEV, albeit at a low level. This should be taken into consideration in cases where the risk of an alimentary infection is assessed in a flock by serological investigations. The immunological recall gives rise to the suspicion that the immunological memory after a first contact to TBEV lasts for many years, probably lifelong.

Low levels of classical BSE infectivity in rendered fat tissue.

BSE infectivity in mesentery fat is most likely associated with embedded nervous tissue. To prove this mesentery containing celiac ganglion was taken from oral BSE infected cattle in different stages of the disease and from one control animal. Fat was rendered according to standard tallow production methods and the prion infectivity therein analysed in transgenic mouse bioassay. Rendered fat of the clinical animal revealed low infectivity levels, whereas preclinical and control animals remained negative. This study, although not representative, provides a proof of principle, indicating the potential contamination of melted mesenteric fat by embedded nervous structures during standard tallow production.

Evaluating the risk for Usutu virus circulation in Europe: comparison of environmental niche models and epidemiological models.

BACKGROUND: Usutu virus (USUV) is a mosquito-borne flavivirus, reported in many countries of Africa and Europe, with an increasing spatial distribution and host range. Recent outbreaks leading to regional declines of European common blackbird (Turdus merula) populations and a rising number of human cases emphasize the need for increased awareness and spatial risk assessment. METHODS: Modelling approaches in ecology and epidemiology differ substantially in their algorithms, potentially resulting in diverging model outputs. Therefore, we implemented a parallel approach incorporating two commonly applied modelling techniques: (1) Maxent, a correlation-based environmental niche model and (2) a mechanistic epidemiological susceptible-exposed-infected-removed (SEIR) model. Across Europe, surveillance data of USUV-positive birds from 2003 to 2016 was acquired to train the environmental niche model and to serve as test cases for the SEIR model. The SEIR model is mainly driven by daily mean temperature and calculates the basic reproduction number R0. The environmental niche model was run with long-term bio-climatic variables derived from the same source in order to estimate climatic suitability. RESULTS: Large areas across Europe are currently suitable for USUV transmission. Both models show patterns of high risk for USUV in parts of France, in the Pannonian Basin as well as northern Italy. The environmental niche model depicts the current situation better, but with USUV still being in an invasive stage there is a chance for under-estimation of risk. Areas where transmission occurred are mostly predicted correctly by the SEIR model, but it mostly fails to resolve the temporal dynamics of USUV events. High R0 values predicted by the SEIR model in areas without evidence for real-life transmission suggest that it may tend towards over-estimation of risk. CONCLUSIONS: The results from our parallel-model approach highlight that relying on a single model for assessing vector-borne disease risk may lead to incomplete conclusions. Utilizing different modelling approaches is thus crucial for risk-assessment of under-studied emerging pathogens like USUV.

INVESTIGATIONS INTO CAUSES OF NEUROLOGIC SIGNS AND MORTALITY AND THE FIRST IDENTIFICATION OF SARCOCYSTIS CALCHASI IN FREE-RANGING WOODPECKERS IN GERMANY.

Between June and November 2015, 25 woodpeckers (Picidae) with neurologic signs or unknown cause of death were admitted to a veterinary clinic. Alive birds were clinically examined. Birds that were found dead or died despite intensive care treatment were forwarded to a pathologic examination. Necropsy and subsequent tests included screening for several infectious agents and toxins. Three birds tested positive for Sarcocystis calchasi. Toxoplasma gondii was detected in one bird demonstrating intracerebral cysts. Mycoplasma gypis was detected in one woodpecker in the absence of respiratory signs. Several microbial pathogens (eg, Aspergillus fumigatus, Clostridium perfringens, and Escherichia coli) were isolated from single individuals. However, there was no consistent finding in all birds that could explain nervous signs and mortality of the woodpeckers examined. To the authors' knowledge, M. gypis and S. calchasi were detected in a woodpecker for the first time in this study.

Distribution of Usutu Virus in Germany and Its Effect on Breeding Bird Populations.

Usutu virus (USUV) is an emerging mosquitoborne flavivirus with an increasing number of reports from several countries in Europe, where USUV infection has caused high avian mortality rates. However, 20 years after the first observed outbreak of USUV in Europe, there is still no reliable assessment of the large-scale impact of USUV outbreaks on bird populations. In this study, we identified the areas suitable for USUV circulation in Germany and analyzed the effects of USUV on breeding bird populations. We calculated the USUV-associated additional decline of common blackbird (Turdus merula) populations as 15.7% inside USUV-suitable areas but found no significant effect for the other 14 common bird species investigated. Our results show that the emergence of USUV is a further threat for birds in Europe and that the large-scale impact on population levels, at least for common blackbirds, must be considered.

Tick-borne encephalitis in a naturally infected sheep.

BACKGROUND: Tick-borne encephalitis (TBE) is the most important viral tick borne zoonosis in Europe. In Germany, about 250 human cases are registered annually, with the highest incidence reported in the last years coming from the federal states Bavaria and Baden-Wuerttemberg. In veterinary medicine, only sporadic cases in wild and domestic animals have been reported; however, a high number of wild and domestic animals have tested positive for the tick-borne encephalitis virus (TBEV) antibody. CASE PRESENTATION: In May 2015, a five-month-old lamb from a farm with 15 Merino Land sheep and offspring in Nersingen/Bavaria, a TBEV risk area, showed impaired general health with pyrexia and acute neurological signs. The sheep suffered from ataxia, torticollis, tremor, nystagmus, salivation and finally somnolence with inappetence and recumbency. After euthanasia, pathological, histopathological, immunohistochemical, bacteriological, parasitological and virological analyses were performed. Additionally, blood samples from the remaining, healthy sheep in the herd were taken for detection of TBEV antibody titres. At necropsy and accompanying parasitology, the sheep showed a moderate to severe infection with Trichostrongylids, Moniezia and Eimeria species. Histopathology revealed mild to moderate necrotising, lymphohistiocytic and granulocytic meningoencephalitis with gliosis and neuronophagia. Immunohistochemistry for TBEV was negative. RNA of a TBEV strain, closely related to the Kumlinge A52 strain, was detected in the brain by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and subsequent PCR product sequencing. A phylogenetic analysis revealed a close relationship to the TBEV of central Europe. TBEV was cultured from brain tissue. Serologically, one of blood samples from the other sheep in the herd was positive for TBEV in an enzyme-linked immunosorbent assay (ELISA) and in a serum neutralisation test (SNT), and one was borderline in an ELISA. CONCLUSION: To the authors' knowledge this is the first report of a natural TBEV infection in a sheep in Europe with clinical manifestation, which describes the clinical presentation and the histopathology of TBEV infection.

Seroprevalence of Rift Valley fever virus in livestock during inter-epidemic period in Egypt, 2014/15.

BACKGROUND: Rift Valley fever virus (RVFV) caused several outbreaks throughout the African continent and the Arabian Peninsula posing significant threat to human and animal health. In Egypt the first and most important Rift Valley fever epidemic occurred during 1977/78 with a multitude of infected humans and huge economic losses in livestock. After this major outbreak, RVF epidemics re-occurred in irregular intervals between 1993 and 2003. Seroprevalence of anti-RVFV antibodies in livestock during inter-epidemic periods can be used for supporting the evaluation of the present risk exposure for animal and public health. A serosurvey was conducted during 2014/2015 in non-vaccinated livestock including camels, sheep, goats and buffalos in different areas of the Nile River Delta as well as the furthermost southeast of Egypt to investigate the presence of anti-RVFV antibodies for further evaluating of the risk exposure for animal and human health. All animals integrated in this study were born after the last Egyptian RVF epidemic in 2003 and sampled buffalos and small ruminants were not imported from other endemic countries. RESULTS: A total of 873 serum samples from apparently healthy animals from different host species (camels: n = 221; sheep: n = 438; goats: n = 26; buffalo: n = 188) were tested serologically using RVFV competition ELISA, virus neutralization test and/or an indirect immunofluorescence assay, depending on available serum volume. Sera were assessed positive when virus neutralization test alone or least two assays produced consistent positive results. The overall seroprevalence was 2.29% (95%CI: 1.51-3.07) ranging from 0% in goats, 0.46% in sheep (95%CI: 0.41-0.5), and 3.17% in camels (95%CI: 0.86-5.48) up to 5.85% in buffalos (95%CI: 2.75-8.95). CONCLUSION: Our findings assume currently low level of circulating virus in the investigated areas and suggest minor indication for a new RVF epidemic. Further the results may indicate that during long inter-epidemic periods, maintenance of the virus occur in vectors and also most probably in buffaloes within cryptic cycle where sporadic, small and local epidemics may occur. Therefore, comprehensive and well-designed surveillance activities are urgently needed to detect first evidence for transition from endemic to epidemic cycle.

Widespread activity of multiple lineages of Usutu virus, western Europe, 2016.

In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak.

Tick infestation in birds and prevalence of pathogens in ticks collected from different places in Germany.

The importance of ticks and tick-borne pathogens for human and animal health has been increasing over the past decades. For their transportation and dissemination, birds may play a more important role than wingless hosts. In this study, tick infestation of birds in Germany was examined. Eight hundred ninety-two captured birds were infested with ticks and belonged to 48 different species, of which blackbirds (Turdus merula) and song thrushes (Turdus philomelos) were most strongly infested. Ground feeders were more strongly infested than non-ground feeders, sedentary birds more strongly than migratory birds, and short-distance migratory birds more strongly than long-distance migratory birds. Mean tick infestation per bird ranged between 2 (long-distance migratory bird) and 4.7 (sedentary bird), in some single cases up to 55 ticks per bird were found. With the exception of three nymphs of Haemaphysalis spp., all ticks belonged to Ixodes spp., the most frequently detected tick species was Ixodes ricinus. Birds were mostly infested by nymphs (65.1 %), followed by larvae (32.96 %). Additionally, ticks collected from birds were examined for several pathogens: Tick-borne encephalitis virus (TBEV) and Sindbisvirus with real-time RT-PCR, Flaviviruses, Simbuviruses and Lyssaviruses with broad-range standard RT-PCR-assays, and Borrelia spp. with a Pan-Borrelia real-time PCR. Interestingly, no viral pathogens could be detected, but Borrelia spp. positive ticks were collected from 76 birds. Borrelia (B.) garinii, B. valaisiaina, B. burgdorferi s.s. and B. afzelii were determined. The screening of ticks and birds for viral pathogens with broad range PCR-assays was tested and the use as an "early warning system" is discussed.

DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly, via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.).

As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks. Plasmid shedding was not detected at any time. Viremia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups. Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viremia, but full protection was not achieved in all groups.

Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.).

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.

Tick-borne encephalitis virus (TBEV) - findings on cross reactivity and longevity of TBEV antibodies in animal sera.

BACKGROUND: By using animal sera as sentinels, natural TBEV foci could be identified and further analyses including investigations of ticks could be initiated. However, antibody response against TBEV-related flaviviruses might adversely affect the readout of such a monitoring. Therefore, the cross-reactivity of the applied TBEV serology test systems - enzyme linked immunosorbent assay (ELISA) and virus neutralization test (VNT) - as well as the longevity of TBEV antibody titres in sheep and goats were investigated in this study. RESULTS: Cross-reactivity of the TBEV antibody test systems with defined antibody-positive samples against selected members of the Flaviviridae family (e.g. Louping ill virus, West Nile virus) was observed for Louping-ill-positive sera only. In contrast, the commercial West Nile virus (WNV) competitive ELISA showed a high level of cross-reactivity with TBEV-specific positive sera.To assess the longevity of TBEV antibody titres, sera from two sheep and two goats, which had been immunized four times with a commercially available TBEV vaccine, were tested routinely over 28 months. In three of the four animals, TBEV-specific antibody titres could be detected over the whole test period.In addition, sera from the years 2010 and 2011 were collected in flocks in different villages of Baden-Württemberg and Thuringia to allow re-examination two to four years after the initial analysis. Interestingly, in most cases the results of the former investigations were confirmed, which may be caused by steadily existing natural TBEV foci. CONCLUSION: Cross-reactivity must be taken into consideration, particularly for TBEV serology in regions with a prevalence of Louping ill virus and for serological testing of WNV by cross-reactive ELISAs. Furthermore, over-interpretation of single TBEV-positive serological results should be avoided, especially in areas without a TBEV history.

Laryngeal and oropharyngeal adenocarcinoma with pulmonary metastases in a common raven (Corvus corax).

A captive 15-year-old male common raven (Corvus corax) was presented for post-mortem examination. It had been previously presented to a local veterinarian due to a 3-4 weeks long history of abnormal respiratory sounds. Upon admission, the bird demonstrated severe dyspnea and a massive amount of mucous in the oropharynx. After symptomatic treatment, dyspnea deteriorated dramatically, and euthanasia was elicited because of poor prognosis. The necropsy revealed a 2.65 x 2.15 x 2.18 cm expansile and poorly delineated cauliflower-shaped mass around the glottis and extending inside the tracheal lumen. Additionally, a dilated salivary gland in the adjacent tissue and multifocal reddish-fleshy areas in the lung parenchyma were detected. Histopathological examination identified the mass as moderately differentiated, tubular adenocarcinoma with invasive growth and moderate to marked cellular atypia and numerous mitoses. The presumptive origin of the neoplasia was one of the salivary glands. Multiple metastases were identified in the lung both macroscopically and histologically. Bacterial culture and molecular testing for West Nile and Usutu viruses were negative. To the authors' knowledge, this is the first report of metastatic laryngeal and oropharyngeal adenocarcinoma in a common raven.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>102 TCID50). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.