EAZA Usutu and West Nile Virus Management Guidelines

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EAZA Usutu and West Nile virus Management Guidelines

EAZA Raptors TAG

Edition 1 (Version 1.0) - September 19, 2019 Editors

Dominik Fischer | Dr. med. vet., DipECZM(WPH) | Veterinary Advisor to the EAZA Raptors TAG

Allan Muir | EAZA Executive Office, EU Policy coordinator

David Aparici Plaza | EAZA Executive Office, Animal programmes and Conservation coordinator

Kirsi Pynnonen-Oudman | Dr (Animal Physiology) | Helsinki Zoo, chair EAZA Raptors TAG

Katharina Herrmann | EAZA Executive Office, Animal programmes and Conservation coordinator

Citation

Fischer, D., Muir, A., Aparici Plaza, D., Herrmann, K., Pynnonen-Oudman, K. (eds.) 2019. EAZA Usutu and West Nile virus management guidelines – Edition One. EAZA Executive Office, Amsterdam: 1-27.

Cover photo credit: Dr. Kirsi Pynnonen-Oudman | Helsinki Zoo

Acknowledgements

This EAZA guidelines for the Usutu and West Nile virus are the result of a collaboration of many people. It would not have been achieved without the input of the EAZA Raptors TAG, the EAZA Veterinary Committee, veterinary advisors, external veterinary experts (namely Dr. Ute Ziegler and Dr. Christine Fast from the Institute of Novel and Emerging Infectious Diseases at the Federal Research Institute for Animal Health (Friedrich-Loeffler Institute) in Germany and Dr. Helene Pendl from PendlLab in Switzerland), the participants of the EAZA Usutu and West Nile virus workshop and the staff of the EAZA Executive Office. By adding information from personal experiences of colleagues (namely Dr. Helena Vaidlová (Prague Zoo), Dr. Viktória Sós-Koroknai, Dr. Károly Erdélyi, and Dr. Endre Sós (Budapest Zoo), Dr. Martin Kaiser and Dr. Andreas Pauly (Tierpark Berlin), Dr. Pavel Kvapil (Zoo Ljubljana), and Dr. Tobias Knauf-Witzens (Zoo Wilhelma Stuttgart) this manuscript was improved significantly. The editors thank Stephanie Sanderson (EAZWV) and Luisa Ziegler (JLU Giessen) for critical revision and correction of the manuscript and all people who added pictures for a better illustration. The content is mainly based on the EAZA Usutu and West Nile virus workshop, which took place on 18th May 2019 in Berlin Zoo, Germany and was organised by the EAZA Raptors TAG, the EAZA Veterinary Committee and the EAZA Executive Office as part of Life funding programme. Special thanks to the event hosts Tobias Rahde and Berlin Zoo and to all people who contributed to the workshop and these guidelines.

Table of contents

Common introduction to Usutu virus and West Nile virus ...... 3 Specific facts about Usutu virus ...... 6 Specific facts about West Nile virus ...... 10 Usutu virus and West Nile virus management plan ...... 13 Conclusion ...... 15 References ...... 16 ANNEX 1: The potential value and role of a veterinary advisors for avian Taxon Advisory Groups..... 20 ANNEX 2: Quick guide fact sheets for Usutu virus and West Nile virus...... 22 ANNEX 3: List of known affected bird species for Usutu virus ...... 24 ANNEX 4: List of known affected bird species for West Nile virus ...... 25

Common introduction to Usutu virus and West Nile virus

Usutu virus (USUV) and West Nile virus (WNV) are single-stranded RNA viruses and members of the Flaviviridae family. They belong to the Japanese encephalitis virus (JEV) serogroup and may cause infection in birds, mammals, amphibians and reptiles as well as in humans (zoonosis). These viruses are enveloped by viral glycoproteins and portions of the host cell membranes. As enveloped viruses they are adaptable and modifiable in a short time to elude the animal’s immune system and to cause long lasting (persistent) infection.

USUV and WNV are classified as arboviruses which is the abbreviation for arthropod-borne viruses. This means that these viruses are amplified and transmitted via arthropod vectors (e.g. mosquitoes, ticks, and flies) following consumption of a vertebrate host’s blood. Both viruses are maintained in an enzootic cycle between birds and arthropods, predominantly mosquitoes of the genus Culex (Ciota, 2017). Viral transmission occurs when an infected mosquito (with the virus present in the mosquito salivary glands) takes a blood meal from a naïve animal host. Once inside this host, the virus starts the amplification process within the bloodstream (viraemia), replicating itself at large quantities to transmit to further hosts via this vector cycle. Birds play a major role as amplifying hosts, developing a high titre and long viraemia capable of infecting the biologic arthropod vector (Komar et al., 2003; Michel et al., 2018). Some host species are “dead-end hosts” as the viral load does not reach sufficient levels to allow the virus to be transmitted back to the mosquito (see picture 1).

Picture 1. Arbovirus transmission cycle using West Nile virus as example

Both viruses originated from Africa. Migratory avian species and the accidental transport of infected vectors helped to spread the infectious agents over long distances to other countries and continents (Hubálek et al., 2004). As many migratory birds fly between Europe and Africa, this may explain the increased number of WNV cases in European countries, and particularly in Southern Europe, observed in the last few years (Calistri et al., 2010; David & Abraham, 2016; Papa, 2017).

3 EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Additionally, overwintering mechanisms such as transovarial infection of mosquitos, hibernation of infected mosquitos or of chronically infected amphibians helped to establish the infection agents in southern Europe (Farajollahi et al., 2005; Monaco et al., 2011).

USUV was firstly discovered in 1959 in South Africa and named after Usutu river in Swaziland. It was spread to Europe in the last decades. The first European clinical cases were detected in blackbirds (Turdus merula) in Italy in the 1990s. In 2001, avian infections were reported in captive birds in Vienna, Austria and in 2004 in birds in Switzerland, Spain and Hungary. In 2009, the first human infection occurred outside Africa in two immunocompromised patients in Italy. Since 2010/2011 the virus is distributed through Germany and the BeNeLux countries. Great grey owls (Strix nebulosa) (see picture 2) have been frequently affected by this virus recently.

Picture 2. Great grey owl (Strix nebulosa) © Peter Kottmann, Germany

WNV was first isolated in 1937 from a woman in Uganda (formerly called West Nile District). Since then, the virus has gradually dispersed via migratory birds out of Africa. Initially in spread to the more southern regions of Europe, Asia, and Australasia. In 1999, the virus was spread to New York, USA and has caused multiple infections and deaths in humans and animals in the Americas thereafter. Currently, WNV is considered to be the most widely spread arbovirus in the world and infections have occurred in multiple European countries including Italy, Greece, Turkey, Hungary, Czech Republic, Slovenia, Spain, Portugal, France, Austria and Germany. Northern goshawks (Accipiter gentilis) (see picture 3) have been frequently affected by this virus recently.

Picture 3. Northern goshawk (Accipiter gentilis) © Kai Siebert, German Falconers Association (Deutscher Falkenorden)

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Specific facts about Usutu virus

Background Usutu virus (USUV) infection is considered to be an emerging disease with zoonotic potential (may affect humans). USUV poses a threat to both in situ and ex situ bird populations and USUV has spread across Europe in the last few years (see picture 4).

Picture 4. Spread of Usutu virus across Europe. From open access publication: Saiz, Juan-Carlos & Blázquez, Ana-Belén. (2017). Usutu virus: Current knowledge and future perspectives. Virus Adaptation and Treatment. Volume 9. 27-40. 10.2147/VAAT.S123619.

In 2016, four different lineages of USUV were reported, spread across Europe (see picture 5).

Picture 5. Distribution of four different lineages of USUV in central Europe from 2011 to 2016. (https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2017.22.4.30452)

Clinical signs in birds Most birds do not show any clinical symptoms associated to USUV infection (subclinical or asymptomatic infection). Therefore, detecting the disease in most avian species is challenging. Highly susceptible birds such as blackbirds (Turdus merula), song thrushes (Turdus philomelos), and closely related songbirds (Turdus spp. and sparrows), kingfishers, and owls (especially great grey owls (Strix nebulosa), snowy owls (Bubo scandiaca) and northern hawk-owls (Surnia ulula)) are regarded highly susceptible to USUV and an infection may cause clinical signs and impacts negatively on their populations.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Clinical signs associated with USUV infection (see picture 6) in susceptible birds may include: . Apathy, weakness . Reduced appetite and weight loss . Inability to fly, drooping wings . Ruffled feathers and/or feather loss . Neurologic signs (uncoordinated, staggered movements, torticollis (twisted neck), ataxia, seizures) . Death (mass die-offs in naïve populations)

Picture 6. Blackbird (Turdus merula) with apathy and dropping wings (left) and central nervous signs (seizures) (right) ©Sylvia Urbaniak, Birds of Prey Rehabilitation Centre Rhineland, Germany

Pathological findings in birds At necropsy, affected animals mainly display an enlarged and necrotic liver and spleen besides seromucous (catarrhal) enteritis as gross-pathological findings (see picture 7).

Picture 5. Necropsy of a great grey owl (Strix nebulosa) died from USUV infection. Left: Necropsy overview. Right: Close-up of swollen liver and spleen. ©Clinic for Birds, Reptiles, Amphibians and Fish of Justus Liebig University of Giessen, Germany

Histopathological findings in birds On histopathological examination various degrees of multifocal acute necrosis in liver, kidney, heart and spleen may be detected (see pictures 6 - 9) besides lesions in the intestines. Myocardial lesions may include infiltration by plasma cells and lymphocytes. Additionally, an acute brain inflammation (non-suppurative encephalitis) can frequently be seen with multifocal areas of neurophagia and microgliosis. However, not all lesions are always present and may vary between species.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Picture 6. Histology slide of the cerebrum of a great grey owl (Strix nebulosa) infected with Usutu virus (HE, 100x): Left: Glianodule, focal necrosis; Right: Perivascular haemorrhages and mononuclear cuffing, multifocal haemorrhagic, partially necrotizing, lymphoplasmacellular to mixed cell encephalitis and meningitis ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

Picture 7. Histology slide of the cerebrum and the meninges of a great grey owl (Strix nebulosa) infected with Usutu virus (HE, 100x left and 1000x right): Left: Prominent perivascular mononuclear (lymphoplasmacytic) infiltrates; multifocal haemorrhagic, partially necrotizing, lymphoplasmacellular to mixed cell encephalitis and menigitis; Right: progressed stage of hemorrhage with extravascular erythrocytes in stage of disintegration mixed with mononuclear cells (lymphocytes and plasmacells suspected, two degenerating granulocytes extravascularly at center to 9 o'clock, free iron pigment. ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

Picture 8. Histology slides: Left: Heart of a great grey owl (Strix nebulosa) infected with Usutu virus (HE, 1000x): Degeneration and necrosis of myofibrils in myocardium; Right: Liver of a common hill mynah (Gracula religiosa) (HE, 400x): Diffuse, coalescing, hemorrhagic necrosis of liver parenchyma, perivascular mononuclear cuffs. ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Picture 9.Histology slides of the intestines of a great grey owl (Strix nebulosa) infected with Usutu virus (HE, 400x): Viable tissue area, clear signs of enteritis intra vitam with intravascular granulocytosis, lymphoid necrosis within mononuclear infiltrates into the lamina propria mucosae, and villous oedema. ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

Further diagnostics Since many of these findings are not unique to USUV infection, specific diagnostic procedures are required for the confirmation of this viral infection. In living animals, serology (testing for viral antibodies in the host’s blood) is suitable using haemagglutination inhibition, plaque reduction neutralization tests or virus neutralization tests. As there is the potential for cross-reactivity with other members of the Japanese encephalitis virus antigen complex (serogroup) within the family of Flaviviridae, blood should be collected using serum tubes without anticoagulant. Then blood cells in the coagulum may be used for viral genome identification using polymerase chain reaction (PCR) parallel to the assessment of antibodies. This increases the likelihood of pathogen identification.

In dead animals, after a gross pathological examination (necropsy), immunohistochemistry or in-situ hybridization may be used on histological fixed tissues. Moreover, a real-time PCR may be performed using organs of dead animals, especially brain, liver and spleen to detect the viral genome.

Therapy in birds As there is no known effective antiviral treatment available, supportive and symptomatic treatment (e.g. intravenous fluid support and nutrition in affected individuals unable to eat, sling support for severely ataxic individuals, protection from and treatment of traumatic injuries occurring due to ataxia and recumbency) is recommended. Therapy includes anti-inflammatory, anti-infective (antibiosis and antimycosis against secondary infections), and analgesic treatment, fluid therapy and supplementation of vitamins and thermal support. The infected birds should be isolated from the rest of the bird collection and strict hygiene and vector control to prevent spreading of the pathogen should be applied.

Relevance USUV is of significant impact to captive bird collections. In 2018, 25% of the great grey owl population in German EAZA zoos died from USUV infections. This emphasizes the importance of testing and monitoring and the implementation of prophylactic measures (see below). This applies especially to birds of the orders Passeriformes (blackbirds, sparrows), Coraciiformes (kingfishers) and Strigiform (especially northern species such as great grey owls, snowy owls, hawk owls, and long-eared owls). However, other species might be at risk as well. Unfortunately, it is expected that the rate of USUV infections will rise in the coming years due to an increase of global temperatures and the growing number of biological vectors (especially mosquitos) linked to climate change.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Specific facts about West Nile virus

Background West Nile virus (WNV) is a virus with high diversity levels in its sequence and there are currently 9 lineages known for WNV worldwide, with lineages 1, 2, 3, and 8 being reported in Europe (Fall et al., 2017; Bakonyi et al., 2006). However, most cases in Europe are associated to lineage 1 (clade 1a) and lineage 2. At the present time, WNV is expanding its geographical range in Europe and causing increasing numbers of outbreaks linked to high bird mortality, but also to diseases in mammals (mainly equids) and humans (see picture 10). In 2018, the WNV transmission season started earlier and a higher number of WNV infections were reported in humans and equids as compared to previous years (Haussig et al., 2018). In particular, 1503 human cases (= 7.2-fold increase compared to 2017) with 181 deaths in humans and 285 outbreaks in horses were recorded in 2018 by the European Union (EU) Member States (ECDC, 2018). Recently it was discovered, that artificial light at night extends the infectious-to-vector period of avian reservoir host of WWNV in urban regions and enables a prolonged maintenance of transmissible viral titres (Kernbach et al., 2019).

Picture 7. Map of human WNV cases in Europe 2011-2019 https://atlas.ecdc.europa.eu/public/index.aspx?config=config-map-table&Header=None&Navigation=Embedded&Dataset= 138&HealthTopic=60&GeoResolution=1&TimeResolution=Week&TimeSeriesRepresentation=T&FixDataset=1&FixHealthTopic=1

Affected species and clinical signs in humans, mammals, reptiles and amphibians Most humans (~ 80 % of infected persons) show no sign of infection (subclinical infection). Approximately 20 % of infected persons exhibit symptoms including fever and flu-like symptoms (headache, malaise, rash, ocular pain, myalgia, sore throat, chills, conjunctival congestion, gastrointestinal signs) and, in some rare cases, encephalitis and meningitis may develop, which can be fatal (< 1 % of infected persons).

Horses remain asymptomatic in most cases and only rarely develop febrile disease. Neurologic signs are more common than in humans (~ 8 % of infected horses) and often persist throughout life. In 22- 44 % of the horses with central nervous signs (lameness, weakness, paresis, paralysis, ataxia, gait abnormalities, recumbency, hypermetria, knuckling, falling to knees, blindness, tremors) WNV infection is fatal.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Other mammalian species which can be affected besides equids include marine mammals, rodents, rabbits, camelids, ruminants, and carnivores (e.g. polar bears, wolves, canids, skunks, cats).

Reptiles and amphibians may be infected by WNV as well. Infections have been reported in several reptilian species (e.g. Alligator mississippiensis and Varana salvidorii) and amphibian species (e.g. anurans such as Rana ridibunda), but clinical signs have been rarely described.

Clinical signs in birds In general, most birds are subclinically infected. However, WNV infection may cause severe and fatal clinical disease in more than 200 avian species including geese, corvids, raptors (several species of eagles, hawks, falcons, and owls), psittacines, passerines, flamingos, rheas and many other avian species (Chambers & Monath 2003)

Clinical signs of WNV infection in birds may include (severity may increase with disease progression): . Depression (mental dullness), generalized lack of awareness (apathy) (see picture 11) . Reduced appetite (anorexia, inappetence), dysphagia (difficulty eating), regurgitation . Weakness, reduced stamina, inability to fly . Disorientation, bad posture, recumbency . Central nervous signs (head tremors, stumbling, circling, uncoordinated movements (ataxia), abnormal head and neck posture (torticollis), seizures (see picture 11) . Reduced vision or blindness due to ocular lesions (anterior uveitis and fundic lesions such as fibrin adhesions, chorioretinal lesions and retinal atrophy) . Shedding of greenish urine/urates . Ruffled feathers, abnormal feather development, pinching of blood feathers, feather loss (may be a prolonged issue (chronic) if birds survive the acute disease) . Sudden death (especially in immunocompromised and very susceptible individuals)

Picture 11. Clinical signs in two juvenile Northern goshawks (Accipiter gentilis) infected with West Nile virus. Left: Incoordination and central nervous disorders. ©Georgios Vergatos, Germany; Right: Depression and weakness. © Thomas Schön, Wildvogelhilfe Saalekreis (rehabilitation centre for wild birds), Germany

Pathological findings in birds In necropsy, heart (myocardial) lesions are varied and may include myocarditis, myocardial necrosis, degeneration, mineralization, fibrosis and haemorrhage (see picture 12). Lesions in the kidney may include tubular epithelial and glomerular cell degeneration and necrosis. Acute brain haemorrhages and enlarged spleen (splenomegaly) may be additional findings.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Picture 12. Heart of a hybrid falcon (Falco cherrug X Falco rusticolus) with a necrotizing myocarditis © Christine Fast, Friedrich-Loeffler Insitut (FLI), Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Greifswald, Germany

Histopathological findings in birds On histopathology a mild to severe brain and meningeal inflammation (acute non-suppurative (meningo)-encephalitis) with infiltrates and perivascular cuffing - mainly consisting of lymphocytes and plasma cells - has been described. Inflammatory lesions in the heart muscle (myocardium, mild to severe, necrotizing myocarditis), kidneys (nephritis), liver (hepatitis), lung (pneumonitis) and rarely lesions in pancreas and adrenals may develop besides more common eye lesions (endophthalmitis).

Picture 13. Histology slides of the cerebrum of a scarlet breasted lorikeet (Trichoglossus forsteni) infected with West Nile virus (HE; 100x): Hyperaemia, multifocal haemorrhages, fibrinthrombi, miliary necroses with glianodule formation, perivascular mononuclear cuffing spurious to absent. ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

Picture 14.Histology slides of the cerebellum of birds infected with West Nile virus (HE; 100x): Left: Purple naped lory (Lorius domicella): mild hyperaemia, signs of degeneration, (astro-)gliosis in the molecular layer, interphase oedema in the Purkinjecell layer, suspicion of Purkinje cell loss, some with pycnotic nuclei. Right: Scarlet breasted lorikeet (Trichoglossus forsteni): hyperaemia, mild perivascular cuffing, multifocal miliary haemorrhages into the arbor vitae. ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Picture 15.Histology slides. Left: Skeletal muscle of a purple naped lory (Lorius domicella) infected with West Nile virus (HE; 100x) with degeneration and necrosis of myofibrils and lymphohistiocytic infiltrates; Right: Liver of a scaly-breasted Lorikeet (Trichoglossus chlorolepidotus) infected with West Nile virus (HE, 400x): perivascular lymphoplasmacytic cuff and vacuolation in the liver, signs of lymphoid necrosis (several very dark pycnotic nuclei), prominent hyperaemia, diffuse vacuoling degeneration and disseminated necrosis of hepatocytes. ©Dr. Helene Pendl, PendlLab, Zug, Switzerland.

Further diagnostics A complete blood count and blood chemistry evaluation is needed to assess the current health status and organ function in a living patient.

For specific WNV diagnostic in live birds, serum and blood clot, preferably paired (acute and convalescent) samples, or oral swabs, or feather pulp may be used for further diagnostics. For the detection of antibodies within the blood the plaque reduction neutralization test (PRNT) and the virus neutralization test (VNT) are available as well as the indirect ELISA. However, cross- reactions with other flaviviruses are possible. A blocking ELISA using monoclonal antibody can detect WNV-specific antibodies in serum from any vertebrate species.

Moreover, the detection of virus or virus antigen may be performed by virus isolation, or demonstration of WN viral antigen or genomic sequences in tissue, blood, cerebrospinal fluid, or other body fluids. Realtime RT-PCR is a sensitive test for virus RNA detection; NASBA and VecTest are also used. Virus isolation may be performed from oral and cloacal swabs, liver, kidney, heart and brain samples. Immunohistochemistry may be performed in this organs as well, offering nearly equivalent sensitivity compared to virus isolation.

Therapy in birds For therapy supportive and symptomatic treatment (e.g. intravenous fluid support and nutrition in affected individuals unable to eat, sling support for severely ataxic individuals, protection from and treatment of traumatic injuries occurring due to ataxia and recumbency) is recommended, as there are no confirmed effective antiviral treatments available. Therapy includes anti-infective (antibiosis and antimycosis against secondary infections), anti-inflammatory and analgesic treatment (e.g. meloxicam or celecoxib), fluid therapy (e.g. Lactated Ringers Solution), supplementation of vitamins (especially vitamins B and E) and thermal support. Ivermectin has been anecdotally reported to have the ability to suppress virus replication and may be added to the treatment for West Nile virus in raptors (Robinson, 2018). A separation of infected birds from the flock and strict hygiene to prevent spreading of the pathogen is valid.

Usutu virus and West Nile virus Management Plan

Which are the affected species? These viruses are known to affect a wide range of species. At the 2019 Bird TAG meeting holders were asked to list species in their institutions that had shown clinical disease. These included raptors (Strigiformes, Accipitriformes and Falconiformes), nutcrackers, gulls, herons, storks, mergansers, geese, flamingos, rheas, psittacines, passerines, corvids and others. Please see annex 3 for a list of known affected bird species.

How to manage the disease in endemic areas? a) Monitoring and Surveillance: Given the clinical significance of these diseases, all bird holders are encouraged to put in place a robust disease surveillance protocol. Different European countries have different surveillance and monitoring regimes in place. Holders should therefore contact the government veterinary authority in their country and organize a sampling scheme. These programmes usually facilitate transport of samples to the testing laboratory and provide sampling equipment. Quarantine and disease screening of birds coming to a collection are strongly recommended prior to including the animal in the collection. As well as undertaking necropsies and screening in dead zoo birds (as per zoo licencing regulations), it is important that free ranging birds found dead near the zoo should also be screened. This will provide an early warning system that active disease transmission is occurring locally. It is also recommended that zoos keep in contact with other raptor holders locally so that they can keep each other informed as to when active virus transmission is occurring in their area. Therefore, a contact list of institutions keeping raptors should be created and constantly updated. b) General prevention inside the facilities/institutions To reduce the transmission of these viruses, it is essential to apply vector control measures, especially focused on mosquito control.

Control measures to be considered include: - draining and/or removal of pools of stagnant water that may serve as breeding grounds for mosquitos. - Adding fish to ponds to reduce the number of mosquito larvae is another option. - Insecticides can also be applied as they are effective for controlling mosquito plagues, although its use may be controversial. - Bacillus thuringiensis, as used in agriculture, may be another option, but the effects on other organisms are not fully understood. - The use of detergents to fight mosquitos can be harmful to the environment and this may cause more complications. - Instead, it is recommended to use live traps (light or pheromone based) and to mosquito proof aviaries using mosquito netting. - The use of repellents may be beneficial, but many repellents do have strong side effect in birds and therefore should be used with caution.

Given the high mortality rate in some species, and the difficulty in keeping them safely in endemic areas, population managers should consider holding valuable EAZA breeding birds only in areas where the viruses are not yet recorded (e.g. north Scandinavia).

3 EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

c) Specific prophylaxis (vaccination) There are vaccines developed against Japan encephalitis virus for use in humans and against WNV for use in horses. Whether these vaccines may offer a cross-protection against USUV is not known.

For the protection of horses against WNV associated disease different types of vaccines are available (EU-licensed for use in equids): 1. Killed vaccines: a) Duvaxyn® WNV, Fort Dodge Animal Health b) Equilis West Nile®, Intervet c) West Nile - Innovator® and Equip WNV®, Zoetis 2. Recombinant vaccine in a canarypox vector: a) RECOMBITEK® Equine West Nile vaccine, Merial Animal Health b) Proteq West Nile®, Merial / Boehringer Ingelheim 3. DNA vaccines: West Nile-Innovator® DNA, Fort Dodge Animal Health; licensed by USDA 2005, not commercially available); 4. Chimera vaccines: PreveNile®, Intervet

Currently there are no licenced vaccinations for birds. Several have been used experimentally with varialbe results. The DNA vaccine reduced mortality in fish crows (Corvus ossifragus) and has been used successfully in raptors (e.g. red-tailed hawks and Californian condors) while in penguins there was better response to the killed vaccine than to a plasmid DNA vaccine. A modified live vaccine has been used in domestic geese in Israel. The recombinant vaccine RECOMBITEK®, a killed vaccine (Duvaxyn®) and two DNA-vaccines (designed by Fraunhofer Institute, Leipzig, Germany) have been used successfully and without side effects in large falcons for vaccination. RECOMBITEK® and Duvaxyn® decreased viremia, mortality and viral shedding if boosted twice, while DNA vaccines needed electroporation to induce partial protection in the falcons. Therefore, vaccination against WNV may be considered in endemic areas to protect diurnal raptors within the collection. In owls, detailed studies are lacking and therefore the support and funding of vaccination studies against WNV and USUV in owls is suggested. So far vaccination in owls has only been attempted anecdotally against WNV in snowy owls in Slovenia using 0.5 ml of recombinant horse vaccine per bird, repeated twice after 4 and 8 weeks (Pavel Kvapil personal communication). However, seroconversion has not been evaluated and virus challenge is lacking after vaccination in owls. Therefore, tolerance and safety of the vaccine in snowy owls seems sufficient, but efficacy is unknown as well as the situation in other owl species. d) Treatment of affected birds As treatment, antibiotics are not working against these viruses and there are no antiviral drugs developed yet. Therefore, treatment is limited to supportive and symptomatic care (e.g. intravenous fluid support, assisted feeding, protection from and treatment of traumatic injuries occurring due to ataxia and recumbency). This includes anti-inflammatory, anti-infective (antibiosis and antimycosis against secondary infection), and analgesic treatment as well as the supplementation of vitamins and thermal support. e) Disinfection after flavivirus contact Disinfection may be useful if there is concern about virus being spread in excretions and secretions from infected animals. Disinfection and virus inactivation may be archived by using low pH, high temperature and/or ultraviolet light. Temperatures above 56°C for over 30 minutes have been tested sufficient to inactivate WNV and the thermal inactivation point (TIP) has been set to 40°C. Hence, the use of boiling water is recommended for thermal inactivation.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

WNV is inactivated in acid environment of pH 1–3, but stable in alkaline environment of pH 7–9.

As disinfectants organic and lipid solvents, common detergents, 1 % iodine, phenol iodophors, 70 % ethanol, 2 % glutaraldehyde, 3 - 8 % formaldehyde, 1 % sodium hypochlorite, 2 - 3 % hydrogen peroxide, 500 - 5000 ppm available chlorine are named to offer a sufficient disinfection.

Moreover, flaviviruses are reported sensitive to ultraviolet light and gamma irradiation, but these are difficult to quantify and assess.

Timeline? Best as soon as possible, to be prepared for the next mosquito season.

Do you need assistance from (other) wildlife veterinarians? The EAZA Veterinary Committee is able to put you directly in touch with the relevant Veterinary Advisor and the experts in the Taxon Advisory Group for Raptors. Any information regarding the virus and its management in your institution supports the further fine-tuning of the management guidelines in the future.

Can we think of mechanism to count animals and create a system to alert people? Annual reports from institutions in winter are recommended. There is certainly a need for creating a standardized system to report and to document cases.

How to secure population of affected areas? Space/capacity in northern countries to keep animals from affected areas is certainly a problem. Therefore, it is imperative to find the most valuable individuals and to focus on saving those. Conclusion Usutu virus (USUV) and West Nile virus (WNV) are a serious threat to many species of birds as well as mammals, reptiles and amphibians. Humans may develop disease as well (zoonotic potential) which needs to be considered for zoo staff and visitors. It is has to be preconceived that the situation may worsen due to climate change, increased availability of vectors and migration of avian hosts.

It is important for all stakeholders to understand the basics of these important and emerging viral diseases and for institutions to have clear surveillance and monitoring measures. Control in zoos should also rely on surveillance of feral avifauna roaming in and around the zoo. Any unexplained mortality of key species such as owls, sparrows or blackbirds should be investigated and positive cases should be reported. Note that there are existing disease monitoring programs in Europe for both viruses where experts may assist in sampling and shipment of samples.

For prevention, vector control efforts should be maximized and all zoo water-management systems should be closely monitored for mosquitos. In this regard, drainage systems should be improved to avoid standing water and thus reduce availability of larval habitats. Specific vaccines are developed against WNV, but these have as yet only been tested in a limited number of avian species.

In the event of a suspected case of WNV and USUV infection, it is recommended to collect blood (serum tubes without anticoagulant) to enable the investigation of direct virus shedding and the indirect detection of antibodies. All carcasses should be submitted for pathological examination and further diagnostics to specialized institutions.

In summary, preventive methods including vector and habitat control are currently the best strategies to fight against WNV and USUV associated diseases.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

References and further reading Angenvoort, J. et al. (2014) Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.); Vet Res 45: 41. Austin, R.J. et al. (2004) An outbreak of West Nile virus-associated disease in domestic geese (Anser anser domesticus) upon initial introduction to a geographic region, with evidence of bird to bird transmission. Can Vet J 45(2): 117-23. Bakonyi, T. (2006) Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerg Infect Dis [serial on the Internet]. 2006 Apr [date cited]. http://dx.doi.org/10.3201/eid1204.051379 Becker, N. et al. (2012) Epizootic emergence of Usutu virus in wild and captive birds in Germany. PLoS One 7, e32604. Buckley, A. et al. (2003) Serological evidence of West Nile virus, Usutu virus and Sindbis virus infection of birds in the UK. J Gen Virol 84: 2807-2817. Bunning, M.L. et al. (2007) DNA vaccination of the American crow (Corvus brachyrhynchos) provides partial protection against lethal challenge with West Nile virus. Avian Dis 51(2): 573-7. Busquets, N. et al. (2012) Experimental West Nile virus infection in Gyr-Saker hybrid falcons. Vector Borne Zoonotic Dis 12(6): 482-9. Busquets, N. et al. (2019) Detection of West Nile virus lineage 2 in North ‐ Eastern Spain (Catalonia). Transbound Emerg Dis 66: 617-621. Cadar, D. (2017) Widespread activity of multiple lineages of Usutu virus, western Europe, 2016. Euro Surveill 22(4): pii=30452. https://doi.org/10.2807/1560-7917.ES.2017.22.4.30452 Calistri, P. et al. (2010) Epidemiology of West Nile in Europe and in the Mediterranean Basin. The Open Virology Journal 4: 29-37. Chambers, T.J. & Monath, T.P. (2003) The flaviviruses: detection, diagnosis and vaccine development. Advances in virus research 61: 3-577 Campbell, G.L. et al. (2002) West Nile virus. The Lancet Infectious Diseases 2(9): 519-29. Chancey, C. et al. (2015) The global ecology and epidemiology of West Nile virus. Biomed Res Int. 2015: 376230. Doi: 10.1155/2015/376230. Chang, G.J. et al. (2007) Prospective immunization of the endangered California condors (Gymnogyps californianus) protects this species from lethal West Nile virus infection. Vaccine 25(12): 2325-30. Ciota, A.T. (2017) West Nile virus and its vectors. Current Opinion in Insect Science 22: 28-36. D'Agostino, J.J. & Isaza, R. (2004) Clinical signs and results of specific diagnostic testing among captive birds housed at zoological institutions and infected with West Nile virus. J Am Vet Med Assoc 224(10): 1640-1643. David, S. (2016) Epidemiological and clinical aspects on West Nile virus, a globally emerging pathogen pathogen. Infect Dis, 48: 571–586. Davis, M.R. et al. (2008) West Nile virus seroconversion in penguins after vaccination with a killed virus vaccine or a DNA vaccine. J Zoo Wildl Med 39(4): 582-589. Duff, P. et al. (2018) West Nile virus. EAZWV Transmissible Disease Fact Sheet No. 65, 5th EAZWV Transmissible Disease Handbook, 65. Durand, B. (2017) Geographic variations of the bird-borne structural risk of West Nile virus circulation in Europe. PLoS ONE 12(10): e0185962. https://doi.org/10.1371/journal.pone.0185962 ECDC (2018) Epidemiological update: West Nile virus transmission season in Europe. Eiden, M. (2010) Two new real-time quantitative reverse transcription polymerase chain reaction assays with unique target sites for the specific and sensitive detection of lineages 1 and 2 West Nile virus strains. J Vet Diagn Invest 22(5): 748-53. Ellis, A.E. et al. (2007) Pathology and epidemiology of natural West Nile viral infection of raptors in Georgia. J Wildl Dis 43(2): 214-223.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Erdelyi, K. et al. (2007) Clinical and pathologic features of lineage 2 West Nile virus infections in birds of prey in Hungary. Vector Borne Zoonotic Diseases 7(2): 181-188. Fall, G. et al. (2017) Biological and phylogenetic characteristics of West African lineages of West Nile virus. PLoS Negl Trop Dis 11(11): e0006078. https://doi.org/10.1371/journal.pntd.0006078 Farajollahi, A.R.Y. et al. (2005) Detection of West Nile Viral RNA from an overwintering Pool of Culex pipens pipiens (Diptera: Culicidae) in New Jersey, 2003. J Med Entomol 42(3): 490-494. Fassbinder-Orth, C.A. et al. (2009) Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein. Avian Dis 53(4): 502-509. Fischer, D. et al. (2015) DNA vaccines encoding the envelop 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.). Vet Res 46: 87. doi: 10.1186/s13567-015-0220-1 Fitzgerald, S.D. et al. (2003) Clinical and Pathologic Features of West Nile Virus Infection in Native North American Owls (Family Strigidae). Avian Dis 47(3): 602-610. Food Safety Risks from Wildlife: Challenges in Agriculture, Conservation, and Public Health. Eds.: Michele Jay-Russell, Michael P. Doyle. Springer, 2015. Gamino, V. & Höfle, U. (2013). Pathology and tissue tropism of natural West Nile virus infection in birds: a review. Vet Res 44(1): 39. Gancz, A.Y. et al. (2006) Pathology and tissue distribution of West Nile virus in North American owls (family: Strigidae). Avian Pathol 35(1): 17-29. Gray, T.J. & Webb, C.E. (2014) A review of the epidemiological and clinical aspects of West Nile virus. Int J Gen Med 7: 193. Haussig, J.M. et al. (2018) Early start of the West Nile fever transmission season 2018 in Europe. Euro Surveill 23(32). Hofle, U. et al. (2008) West Nile virus in the endangered Spanish imperial eagle. Vet Microbiol 129(1- 2): 171-178. Hubálek, Z. (2004) An annotated checklist of pathogenic microorganisms associated with migratory birds. J Wildl Dis 40(4): 639-659. Ip, H.S. et al. (2014) West Nile virus transmission in winter: the 2013 Great Salt Lake bald eagle and eared grebes mortality event. PLoS Currents 6. Jarvi, S.I. et al. (2008) Protective efficacy of a recombinant subunit West Nile virus vaccine in domestic geese (Anser anser). Vaccine 26(42): 5338-5344. Jimenez-Clavero, M.A. et al. (2008) West Nile virus in golden eagles, Spain, 2007. Emerg Infect Dis 14(9): 1489-1491. Kernbach, M.E. et al. (2019) Light pollution increases West Nile virus competence of a ubiquitous passerine reservoir species. Proceedings of the Royal Society B 286.1907: 20191051. Kilpatrick, A.M. et al. (2010) DNA vaccination of American robins (Turdus migratorius) against West Nile virus. Vector Borne Zoonotic Diseases 10(4): 377-380. Klenk, K. et al. (2004) Alligators as West Nile virus amplifiers. Emerg Infect Dis; 10(12):2150-5. Komar, N. et al. (2003) Experimental Infection of North American Birds with the New York 1999 Strain of West Nile Virus. Emerg Infect Dis 9(3): 311–322. Kramer, L.D. et al. (2008) A global perspective on the epidemiology of West Nile virus. Annual review of entomology 53: 61-81. Lécu, A. et al. (2018) Usutu virus. EAZWV Transmissible Disease Fact Sheet No. 136, 5th EAZWV Transmissible Disease Handbook, 136. Lühken, R. et al. (2019) West Nile virus epizootic in Germany, 2018. Antiviral Research 162: 39-43. Malkinson M. & Banet C. (2002) The role of birds in the ecology of West Nile virus in Europe and Africa. Curr Top Microbiol Immunol 267: 309-322. Marra P.P. et al. (2004) West Nile Virus and Wildlife. BioScience 2004/05/01 54(5): 393-402.

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McLean R.G. et al. (2001) West Nile Virus Transmission and Ecology in Birds. Annals of the New York Academy of Sciences 951(1): 54-57. Michel, F. et al. (2018) West Nile Virus and Usutu Virus Monitoring of Wild Birds in Germany. Int J Environ Res Publ Health, 15. Michel, F. et al. (2019) Evidence for West Nile Virus and Usutu Virus infections in wild and resident birds in Germany, 2017 and 2018. Viruses 11(7): 674; https://doi.org/10.3390/v11070674. Monaco, F. et al. (2011) West Nile disease epidemic in Italy: First evidence of overwintering in Western Europe? Research in Veterinary Science 91(2): 321-326. Nemeth, N. et al. (2006) Natural and experimental West Nile virus infection in five raptor species. J Wildl Dis 42(1): 1-13. Nemeth, N. et al. (2006) Experimental West Nile Virus Infection in Eastern Screech Owls (Megascops asio). Avian Dis 50(2): 252-258. Okeson, D.M. et al. (2007) Antibody response of five bird species after vaccination with a killed West Nile virus vaccine. J Zoo Wildl Med 38(2): 240-244. Olsen, G.H. et al. (2009) Pathogenicity of West Nile virus and response to vaccination in Sandhill cranes (Grus canadensis) using a killed vaccine. J Zoo Wildl Med 40(2): 263-271. Palmieri, C. et al. (2011) Pathology and immunohistochemical findings of West Nile virus infection in psittaciformes. Vet Pathol 48(5): 975-984. doi: 10.1177/0300985810391112 Papa, A. (2013) West Nile virus infections in humans—Focus on Greece. Journal of Clinical Virology 58(2): 351-353. Papa, A. (2017) Emerging arboviral human diseases in Southern Europe. J Med Virol 89: 1315-1322. Papa, A. et al. (2019) Emergence of West Nile virus lineage 2 belonging to the Eastern European subclade, Greece. Archives of Virology 164(6): 1673-1675. Peterson, A.T. et al. (2003) Migratory birds modeled as critical transport agents for West Nile Virus in North America. Vector Borne Zoonotic Diseases 3(1): 27-37. Rappole, J.H. et al. (2000) Migratory birds and spread of West Nile virus in the Western Hemisphere. Emerg Infect Dis 6(4): 319-328. Redig, P.T. et al. (2011) Effect of West Nile virus DNA-plasmid vaccination on response to live virus challenge in red-tailed hawks (Buteo jamaicensis). Am J Vet Res 72(8): 1065-1070. Reiter, P. (2010) West Nile virus in Europe: understanding the present to gauge the future. Euro surveillance bulletin 15(10): 19508. Robinson, M. (2018) Ivermectin as a possible treatment for West Nile virus in raptors. HawkChalk 58(2): 80-82. Sá e Silva, M. et al. (2013) Domestic goose as a model for West Nile virus vaccine efficacy. Vaccine 31(7): 1045-1050. Saiz, J.-C. & Blázquez, A.-B. (2017) Usutu virus: Current knowledge and future perspectives. Virus Adaptation and Treatment 9: 27-40. doi:10.2147/VAAT.S123619 Sambri, V. et al. (2013) West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention. Clin Microbiol Infect 19: 699-704. Samina, I. et al. (2007) Safety and efficacy in geese of a PER.C6-based inactivated West Nile virus vaccine. Vaccine 25(49): 8338-8345. Schneeweiss, A. et al. (2011) A DNA vaccine encoding the E protein of West Nile Virus is protective and can be boosted by recombinant domain DIII. Vaccine 29(37): 6352-6357. Seidowski, D. et al. (2010) West Nile Virus Monitoring of Migratory and Resident Birds in Germany, Vector Borne and Zoonotic Diseases 10(7): 639-647. Steele, K.E. et al. (2000) Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet Pathol 37(3): 208-224.

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Turell, M.J. et al. (2003) DNA vaccine for West Nile virus infection in fish crows (Corvus ossifragus). Emerg Infect Dis 9(9): 1077-1081. Van der Meulen, K.M. et al. (2005) West Nile virus in the vertebrate world. Arch Virol 150(4): 637-657. Vilibic‐Cavlek, T. et al. (2019) Prevalence and molecular epidemiology of West Nile and Usutu virus infections in Croatia in the “One health” context, 2018. Transbound Emerg Dis. Weissenböck, H. et al. (2002) Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, Central Europe. Emerg Infect Dis 8: 7. Weissenböck H. et al. (2010) Zoonotic mosquito-borne flaviviruses: Worldwide presence of agents with proven pathogenicity and potential candidates of future emerging diseases. Vet Microbiol 140(3–4): 271-280. Wheeler, S.S. et al. (2011) Efficacy of three vaccines in protecting Western Scrub-Jays (Aphelocoma californica) from experimental infection with West Nile virus: implications for vaccination of Island Scrub-Jays (Aphelocoma insularis). Vector Borne Zoonotic Diseases 11(8): 1069-1080. Wodak, E. et al. (2010) Detection and molecular analysis of West Nile virus infections in birds of prey in the eastern part of Austria in 2008 and 2009. Vet Microbiol 149(3-4): 358-366. Wunschmann, A. et al. (2004) Pathologic findings in red-tailed hawks (Buteo jamaicensis) and Cooper's hawks (Accipiter cooper) naturally infected with West Nile virus. Avian Dis 48(3): 570-580. Wunschmann, A. et al. (2005) Pathologic and immunohistochemical findings in goshawks (Accipiter gentilis) and great horned owls (Bubo virginianus) naturally infected with West Nile virus. Avian Dis 49(2): 252-259. Wunschmann, A. et al. (2014) Clinical, pathological, and immunohistochemical findings in bald eagles (Haliaeetus leucocephalus) and golden eagles (Aquila chrysaetos) naturally infected with West Nile virus. J Vet Diagn Invest 26(5): 599-609. Ziegler, U. et al. (2012) Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons. Vet Microbiol 161(3-4): 263-273. Ziegler, U. et al. (2015) Epidemic spread of Usutu virus in southwest Germany in 2011 to 2013 and monitoring of wild birds for Usutu and West Nile viruses. Vector Borne and Zoonotic Diseases 15 (8): 481-488; doi: 10.1089/vbz.2014.1746. Ziegler, U. et al. (2019). West Nile virus epizootic in Germany, 2018. Antiviral Res 162: 39-43.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

ANNEX 1: The potential value and role of a veterinary advisors for avian Taxon Advisory Groups

Dominik Fischer, EAZA TAG vet advisor of TAG Falconiformes and Strigiformes, Raptor Center & Wildlife Zoo Hellenthal & Clinic for Birds, Reptiles, Amphibians & Fish of Justus Liebig University Giessen, Germany Kirsi Pynnonen-Oudman, EAZA TAG Vice-Chair of TAG Falconiformes & Strigiformes, Helsinki Zoo, Finland

Ten years ago the situation of veterinary advisors in bird Taxon Advisory Groups (TAGs) was quite different: only a few TAGs had specific advisors and certainly not many had a nominated veterinary expert to assist with the TAG work. At the moment, 26 of the 39 TAGs have at least one veterinary advisor (TAG vet advisor). In addition, 98 of the 400 programs (EEP and ESB) have veterinary advisors.

What is the potential value and the role of TAG vet advisors for the different bird orders? TAG vet advisors have an important role in overseeing problems and diseases, addressing husbandry and dietary requirements and in the coordination of diagnostic, prophylactic and therapeutic measures. In this regard, the independent point of view of the TAG vet advisor may assist the local veterinary colleagues by solving specific problems in zoological institutions. However, TAG vet advisors depend on a good cooperation with skilled local veterinarians, who may be employed by the zoos or consulted from private practice. Furthermore, a continuous exchange about avian medicine with universities, research institutions and specific veterinary associations, such as the European Association for Avian Medicine (EAAV), the European Association for Zoo and Wildlife Veterinarians (EAZWV) or the European College for Zoological Medicine (ECZM), are valuable for TAG vet advisors to be updated anytime. Therefore, continuous research in infectious diseases, animal welfare and species conservation (e.g. development and improvement of assisted reproduction in birds) is essential. The TAG vet advisor needs to communicate the results of such research projects to zoos to enable the evaluation and the implementation in the zoological management of avian species. Another task of TAG vet advisors is the veterinary supervision of species conservation projects, considering the release of captive born individuals into the wild. In particular, prior to release the health situation of captive and wild populations need to be assessed using a throughout risk assessment procedure with a specific focus on potential disease transmission from captive to wild populations (1).

To give an example of such actions, the vet advisors of TAG Falconiformes and Strigiformes supported the TAG chairs in conception and edition of the “EAZA Falconiformes and Strigiformes Taxon Advisory Group Husbandry and Management Guidelines For Demonstration Birds” (2). Moreover, they achieved consultancy of colleagues at the involved EAZA institutions and discussion about different diseases of raptors in the TAG. In this regards, diagnostic measures and treatment options of avian malaria caused by Plasmodium spp. and other haemoparasites were reviewed in Northern owls. Necessary information was spread via the chairpersons of the TAG at the TAG meeting in Belfast and an overview-sheet about treatment options was issued. Recently, the increasing number of infections by the arthropod-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) in goshawks (Accipiter gentilis) and owls required action. In this regard, the fact sheets of the 5th EAZWV Transmissible Disease Handbook about WNV (No. 65) and USUV (No. 136) were updated and information were distributed by the TAG chairs to EAZA institutions. As Usutu virus infections seem to pose a risk especially in great grey owls (Strix nebulosa) in Central and Southern Europe, preventive health care and improvement of owl husbandry are currently prioritized. Vector management and prevention, are discussed besides vaccination. Moreover, the current situation and spread of Usutu virus and West Nile virus are discussed during the Falconiformes and Strigiformes TAG meeting at the Mid-year TAG meeting in Berlin to elaborate recommendations for the raptor holders. In preparation

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

to this meeting the TAG vet advisor exchanged with leading national and private research institutions to provide the latest updates on these emerging viral pathogens. In conclusion, a good cooperation of TAG vet advisors with local veterinarians, veterinary associations, research institutions, zoos, private practitioners and involved stakeholders is essential to enable a state of the art medical management of birds and avian collections. The veterinary advisor should act as a liaison between all parties to spread knowledge across the different stakeholders, to maintain healthy captive populations, and to improve training and husbandry conditions. From our personal point of view voluntary commitment for the EAZA TAG is sometimes challenging, but we appreciate being involved in a continuous process of improvement and further development that is beneficial for the kept animals, the zoological institutions, the involved staff and the visitors. Therefore, we encourage to support the TAGs and to get involved in TAG work according to personal interest, skills and experience, specific training and education.

References 1 Jakob-Hoff RM, MacDiarmid SC, Lees C, Miller PS, Travis D, Kock R (2014). Manual of Procedures for Wildlife Disease Risk Analysis, World Organisation for Animal Health, Paris. Published in association with the International Union for Conservation of Nature and the Species Survival Commission. 2 Habben M, Parry-Jones J, Fischer D (2016) EAZA Falconiformes and Strigiformes Taxon Advisory Group Husbandry and Management Guidelines For Demonstration Birds. European Association for Zoos and Aquaria - Falconiformes and Strigiformes TAG E-book. Approved by the EEP Committee as Best Practice Guidelines, available online (https://www.eaza.net/assets/Uploads/CCC/EAZA- BPG-Husbandry-and-Management-Guidelines-for-Demonstration-Birds.pdf): 1-52. 3 Duff P, McLean RG, Fischer D (2018) West Nile virus. EAZWV Transmissible Disease Fact Sheet No. 65, 5th EAZWV Transmissible Disease Handbook, 65 4 Lécu, A, Beck C, Fischer D (2018) Usutu virus. EAZWV Transmissible Disease Fact Sheet No. 136, 5th EAZWV Transmissible Disease Handbook, 136.

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES USUTU VIRUS (USUV) ANNEX 2: Quick guide fact sheets for USUV and WNV

SPECIES AFFECTED: Birds

SIGNS & SYMPTOMS: apathy, lack of flight-response, ruffled feathers, weakness, staggering and uncoordinated movements (ataxia), seizures, torticollis, enlarged liver and spleen (post-mortem)

PREVENTION METHODS: apply vector control measures, especially focused on mosquito control. Moreover, apply habitat control such as draining and/or removal of pools of stagnant water that may serve as breeding grounds for mosquitoes. Adding fish, eating mosquitoes in ponds. Careful application of insecticides and Bacillus thuringiensis, but the effects on other organisms are not fully understood. Use of livetraps (light or pheromone based) and sealing of aviaries by placing mosquito nets around the aviaries. The use of repellents may be beneficial, but strong side effects are reported in some birds

AVAILABLE VACCINES: There are currently no specific USUV vaccines available and it is not clear whether vaccines against other flaviviruses offer cross-protection.

Distribution of USUV in Europe OTHER RECOMMENDED ACTIONS: Report case, participate in surveillance & monitoring programmes European Countries affected:

RECOMMENDED CLINICS/LABS: Spain, Italy, France, Greece, Germany,

Italy: Istituto G. Caporale Teramo, Via Campo Boario, 64100 - Teramo – Italy, Tel: +390861 332440, 28 Austria, Switzerland, The Netherlands, Fax: +390861 332251 Belgium, Luxembourg, Andorra, Germany: Friedrich-Loeffler-Institut, Institut für neue und neuartige Tierseuchenerreger, Südufer 10, D-17493 Lichtenstein, Croatia,Hungary, Serbia, Greifswald, Tel: +49383517-1163/-1287. Fax: +49 38351 7-1226 Czech Republic, Poland France: Anses, UMR 1161 Virologie , 94700 Maisons Alfort, France. Belgium: CODA CERVA : Sciensano, Rue Juliette Wytsman 14, 1050 Bruxelles, Tel: +32 2 642 51 11. Email : [email protected] Netherlands: Dr. van Haeringen Laboratorium B.V., Agro Business Park 100, 6708 PW Wageningen , Tel: (+31) 0317 416 402. Email: [email protected] 22

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES WEST NILE

SPECIES AFFECTED: Birds VIRUS (WNV) SIGNS & SYMPTOMS: Depression, weakness, reduced stamina, inability to fly, generalized lack of awareness (apathy), bad posture, recumbency, reduced appetite (anorexia, inappetence), dysphagia, disorientation, central nervous signs (head tremors, stumbling, circling, uncoordinated movements (ataxia), abnormal head and neck posture (torticollis), seizures, ocular lesions, reduced vision or blindness, ruffled feathers, abnormal feather development, sudden death

AVAILABLE VACCINES: Killed vaccines, recombinant vaccine in a canarypox vector and DNA vaccines (all licensed for use in equids) have been used in birds successfully (please review specific literature)

OTHER RECOMMENDED ACTIONS: Report case, participate in surveillance & monitoring

RECOMMENDED CLINICS/LABS: OIE Reference Laboratory: Diagnostic Virology Laboratory, National Veterinary Services Laboratories, Dr E.N. Ostlund, P.O. Box 844, Ames, IA 50010 UNITED STATES OF AMERICA; Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48; Email: [email protected] Germany: Friedrich-Loeffler-Institut, Institut für neue und neuartige Tierseuchenerreger, Südufer 10, D-17493 Greifswald, Tel: +49383517-1163/-1287. Fax: +49 38351 7-1226 Italy: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale" - Campo Boario - 64100 Teramo, Tel: +39-0861 3321, Fax: +39-0861 332251 France: Anses, UMR 1161 Virologie , 94700 Maisons Alfort, France. Belgium: CODA CERVA : Sciensano, Rue Juliette Wytsman 14, 1050 Bruxelles, Tel: +32 2 642 51 11. Email : [email protected] Netherlands: Dr. van Haeringen Laboratorium B.V., Agro Business Park 100, 6708 PW Wageningen , Tel: (+31) 0317 416 402. Email: [email protected]

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

ANNEX 3: List of known affected bird species for Usutu Virus (positive in isolation/IHC/PCR)* * This list is intended for orientation, but most likely incomplete. Daily updates are required.

Strigiformes  Great grey owls (Strix nebulosa)  Snowy owls (Bubo scandiaca)  Brown owl (Strix aluco)  Northern long-eared owl (Asio otus)  Northern hawk owl (Surnia ulula)  Little owl (Athene noctua)

Falconiformes  European kestrel (Falco tinnunculus)

Passeriformes  Eurasian blackbird (Turdus merula)  Song thrush (Turdus philomelos)  Fieldfare (Turdus pilaris)  Common starling (Sturnus vulgaris)  House sparrow (Passer domesticus)  Eurasian bullfinch (Pyrrhula pyrrhula)  Grey-Headed Bullfinch (Pyrrhula erythaca)  Great tits (Parus major)  Domestic canary bird (Serinus canaria forma domestica)  Blue tits (Cyanistes caeruleus)  Golden-breasted starling (Cosmopsarus regius)  Carrion Crow (Corvus corone)  Western Jackdaw (Coloeus monedula)

Charadriiformes  Inca tern (Larosterna inca)

Coraciiformes  Common kingfisher (Alcedo atthis)

Piciformes  European green woodpecker (Picus viridis)  Great Spottet Woodpecker (Dendrocopos major)

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

ANNEX 4: List of known affected bird species for West Nile Virus (positive in isolation/IHC/PCR)* * This list is intended for orientation, but most likely incomplete. Daily updates are required.

See also a list of 198 fatally affected avian species published by Chambers, T.J. & Monath, T.P. (2003) entitled “The flaviviruses: detection, diagnosis and vaccine development” in the journal “Advances in virus research”, volume 61, pages 3-577.

Strigiformes  Great grey owls (Strix nebulosa)  Snowy owls (Bubo scandiaca)  Brown owl (Strix aluco)  Eurasian pygmy owl (Glaucidium passerinum)

Rheiformes  Lesser rhea (Rhea pennata)

Anseriformes  Anser spp.

Ciconiformes  Abdim's stork (Ciconia abdimii)

Psittaciformes  lorikeets (Trichoglossus spp.)  Coconut lorikeet (Trichoglossus haematodus)  Sunset lorikeet (Trichoglossus forsteni)  Scaly-breasted lorikeet (Trichoglossus chlorolepidotus)  Purple-naped lory (Lorius domicellus)  Vernal hanging parrot (Loriculus vernalis)  Kea (Nestor notabilis)  Rosellas (Platycercus spp)  Conures (Enicognathus, Aratinga, and Nandayus spp.)

Phoenicopteriformes  American flamingo (Phoenicopterus ruber)  Chilean flamingo (Phoenicopterus chilensis

Passeriformes  Common blackbirds (Turdus merula)  Song thrush (Turdus philomelos)  White-crested laughingthrush (Garrulax leucolophus)  Sumatran laughingthrush (Garrulax bicolor)

EAZA USUTU AND WEST NILE VIRUS MANAGEMENT GUIDELINES

Accipitriformes  Sparrow hawk (Accipiter nisus)  Northern goshawk (Accipiter gentilis) (Wünschmann et al. 2005, Wodak et al. 2011, Erdélyi et al. 2007, Richter et al. 2009)  Bearded vulture (Gypaetus barbatus)  Stella sea eagle (Halaeetus pelagicus)  Bald eagle (Halaeetus leucocephalus) (Steele et al. 2000)  American kestrel (Falco spaverius) (Komar et al. 2003, Nemeth et al. 2006, 2009)  Golden Eagle (Aquila chrysaetus)(D`Agostino et al. 2004, Jiménez-Clavero et al. 2008)  Cooper's hawk (Accipiter cooperii) (Wünschmann et al. 2004)  California condors (Gymnogyps californianus) (Chang et al. 2006)  Red-tailed hawks (Buteo jamaicensis) (Wünschmann et al. 2004, Ellis et al. 2007)  Spanish imperial eagle (Aquila adalberti) (Höfle et al. 2008)  Bonelli's eagle (Aquila fasciata) (Jiménez-Clavero et al. 2008)  Peregrine falcon (Falco peregrinus) (Nemeth et al. 2009)  Gyrfalcons (Falco ruticolus) / Hybridfalcons (Falco spp.) (Wodak et al. 2011)