Large falcon species seem to be susceptible to WNV infection although detailed data on pathogenicity in these species were lacking. We wanted to evaluate susceptibility, viraemia levels, course of infection, clinical syndromes, organ viral loads and pathology of WNV infected large falcons (Falco rusticolus, Falco cherrug, Falco peregrinus and hybrids of these species). Therefore we chose the WNV lineage 1 NY99 isolate, which was associated with high wild bird mortality, and the WNV lineage 2 isolate goshawk Austria 2009, which was introduced into Europe in 2004 and thereafter was associated with numerous avian deaths amongst raptor species (Lanciotti et al. 1999; Bakonyi et al. 2013). After confirmation of high
Introduction 13
susceptibility of large falcons to both WNV strains and after establishment of the WNV live viral challenge model, we evaluated the safety, immunogenicity and efficacy of two commercial equine WNV vaccines (inactivated vaccine Duvaxyn® WNV and recombinant canarypoxvirus based live vaccine Recombitek®- Equine rWNV vaccine) and two plasmid DNA vaccines at research level (against lineage 1 and 2 respectively; DNA-1, DNA-2) using different vaccination protocols in the mentioned large falcon species. Furthermore the WNV lineage 2 isolate goshawk Austria 2009 was compared to known high virulent lineage 1 isolates (NY99 and Dakar) and low virulent lineage 2 isolate Uganda in a newly established rodent model using immunocompetent wild type mice (C57/Bl6) and interferon type I receptor knock out mice (IFNAR -/-).
14 Manuscript I
2 Manuscript I
Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons
Ute Zieglera, Joke Angenvoorta, Dominik Fischerb, Christine Fasta, Martin Eidena, Ariel V.
Rodrigueza, Sandra Revilla-Fernándezc, Norbert Nowotnyd,e, Jorge García de la Fuentef, Michael Lierzb, Martin H. Groschupa,*
aFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Riems, Germany,
bClinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Str. 91-93, 35392 Giessen, Germany, cInstitute for Veterinary Disease Control Mödling, Austrian Agency for Health and Food Safety (AGES), Robert Koch-Gasse 17, 2340
Mödling, Austria, dZoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Veterinärplatz 1, 1210 Vienna,
Austria, eDepartment of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman, fRoc Falcon S.L., Finca Caballera Alta,
Odèn, Lleida, Spain, *corresponding author
West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle).
Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain
Manuscript I 15
Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viraemia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viraemia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.
Published 2013 in Veterinary Microbiology
Volume 161, Issues 3 -4, pages 263 – 273 doi: 10.1016/j.vetmic.2012.07.041
link: http://www.sciencedirect.com/science/article/pii/S0378113512004439
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3 Manuscript II
Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)
Joke Angenvoort1†, Dominik Fischer2†, Christine Fast1, Ute Ziegler1, Martin Eiden1, Jorge Garcia de la Fuente3, Michael Lierz2 and Martin H Groschup1*
†Equal contributors, 1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald,
Insel Riems, Germany, 2Clinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Str. 91-93, 35392 Giessen, Germany, 3Roc Falcon S.L., Finca
Caballera Alta, 25283 Odèn, Lleida, Spain, *corresponding author
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
Manuscript II 17
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.
Published 2014 in Veterinary Research Volume 45, Page 41 doi: 10.1186/1297-9716-45-41
link: http://www.veterinaryresearch.org/content/45/1/41
18 Manuscript III
4 Manuscript III
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.)
Dominik Fischer1*†, Joke Angenvoort2†, Ute Ziegler2, Christine Fast2, Kristina Maier1, Stefan Chabierski3, Martin Eiden2, Sebastian Ulbert3, Martin H. Groschup2 and Michael Lierz1
†Equal contributors, 1Clinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Str. 91-93, 35392 Giessen, Germany, 2
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Riems, Germany, 3Fraunhofer Institute for Cell Therapy and Immunology, Perlickstraße 1, 04103 Leipzig, Germany,
*corresponding author
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.
Manuscript III 19
Plasmid shedding was not detected at any time. Viraemia, 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 viraemia, but full protection was not achieved in all groups.
Published 2015 in Veterinary Research Volume 46, Page 87 doi: 10.1186/s13567-015-0220-1
link: http://www.veterinaryresearch.org/content/46/1/87
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5 Manuscript IV
Differences in pathogenicity of West Nile virus strains in experimental infected mice
Joke Angenvoort1, Ute Ziegler1, Christine Fast1, Markus Keller1, Martin Eiden1, Martin H.
Groschup1
1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
to be submitted
Manuscript IV 21