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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.

A total of 289 cloacal swabs from pigeons from 29 different breeders in Germany were collected. In addition, samples from pigeons exhibited at shows were collected. The detailed health status of the pigeon flocks was recorded. Samples were analysed for the presence of the recently discovered pigeon rotavirus and pigeon circovirus. Pigeon rotavirus was found in 10.3% and pigeon circoviruses was found in 65.5% of sampled pigeon lofts. The study revealed a strong relationship between the attendance of shows and the occurrence of different clinical signs. The higher prevalence of pigeon rotavirus in exhibited animals indicates that exhibitions are a risk factor for the transmission of this pathogen. Electronic supplementary material The online version of this article (10.1007/s00705-020-04834-w) contains supplementary material, which is available to authorized users.

The current prevalence of avian leukosis virus (ALV) in fancy chickens in Germany is unknown. Therefore, 537 cloacal swabs from 50 purebred fancy-chicken flocks in Saxony were tested for the presence of the ALV p27 protein using a commercial antigen-capture ELISA. The detection rate was 28.7% at the individual-animal level and 56.0% at the flock level. Phylogenetic analysis of PCR products obtained from 22 different flocks revealed the highest similarity to ALV subtype K. When classifying breeds by their origin, ALV detection rates differed significantly. Evaluation of questionnaire data revealed no significant differences between ALV-positive and negative flocks regarding mortality.

Avian pathogens such as bornaviruses, circoviruses and polyomaviruses are widely distributed in captive collections of psittacine birds worldwide and can cause fatal diseases. In contrast, only lit...

Pigeon rotavirus A (RVA) isolates of genotype G18P[17] are causing disease outbreaks and fatalities in pigeon lofts in Australia, Germany, Belgium, Denmark and USA since 2016. Most disease outbreaks have been reported from juvenile pigeons (Overall 18 pigeons (16 fancy pigeons and one racing pigeon from 9 pigeon fanciers, as well as one feral pigeon from a rescue center) were sent in for routine diagnostic necropsy including histopathologic, parasitologic and microbiologic examinations. Molecular biologic examinations for detection of RVA, circovirus, Usutu virus, West Nile virus andDisease outbreaks in juvenile and adult pigeons were reported 7-14 days after fancy pigeon shows. One fancier who had previously vaccinated his pigeons with an autogenous pigeon RVA vaccine, noted no morbidity and mortality among his pigeons and thus sent in a healthy pigeon for diagnostic purposes. Reported clinical signs in the other pigeons were regurgitation, green slimy diarrhea, anorexia, apathy and death after 24 hours. Hepatic necrosis and detection of pigeon RVA isolates of genotype G18P[17] confirmed disease outbreaks caused by pigeon RVA in all pigeons, except for the vaccinated pigeon. Besides pigeon circovirus, which was detected in 15 of 18 pigeons, all other pathogens were singular findings.In disease outbreaks following fancy pigeon shows in juvenile and adult pigeons diagnostics should include pigeon RVA of genotype G18P[17].Tauben-Rotavirus-A-Isolate des Genotyps G18P[17] verursachen in Australien, Deutschland, Belgien, Dänemark und den USA seit 2016 Krankheitsausbrüche und Todesfälle überwiegend bei Jungtauben. Berichte über Rotavirus-A-assoziierte Krankheiten bei Rassetauben im Zusammenhang mit Rassetaubenausstellungen in Deutschland sind jedoch selten.Insgesamt 18 Tauben (16 Rassetauben und eine Brieftaube von 9 Rassetaubenzüchtern sowie eine Stadttaube aus einer Auffangstation) wurden zur routinemäßigen diagnostischen Sektion einschließlich histopathologischer, parasitologischer und mikrobiologischer Untersuchungen eingeschickt. Bei allen Tauben erfolgte zudem eine molekularbiologische Untersuchung zum Nachweis von Rotavirus A, Circovirus, Usutu-Virus, West-Nil-Virus undKrankheitsausbrüche bei Jung- und Alttauben wurden 7–14 Tage nach Rassetaubenausstellungen beschrieben. Ein Züchter hatte seine Tauben zuvor mit einer autogenen Tauben-Rotavirus-A-Vakzine geimpft, berichtete über keine Morbidität und Mortalität bei seinen Tieren und hatte daher zu Diagnostikzwecken eine gesunde Taube eingesandt. Klinische Symptome bei den anderen Tauben waren Regurgitieren, grün-schleimige Diarrhö, Anorexie, Apathie und Tod nach 24 Stunden. Lebernekrosen und der Nachweis von Tauben-Rotavirus-A-Isolaten des Genotyps G18P[17] bestätigten Tauben-Rotavirus A als Krankheitsursache bei den Tieren mit Ausnahme der Taube aus dem geimpften Bestand. Neben dem Tauben-Circovirus, das bei 15 von 18 Tauben nachgewiesen wurde, waren alle anderen Krankheitserreger singuläre Befunde.Kommt es nach Rassetaubenausstellungen zu Krankheitsausbrüchen bei Jung- und Alttauben, sollte die Diagnostik den Nachweis von Tauben-Rotavirus A des Genotyps G18P[17] beinhalten.

Precise health monitoring of laboratory animals is a critical factor for surveillance and accuracy of animal experiments. Rotavirus epizootic diarrhea of infant mice (EDIM) leads to infections in mice that can influence animal studies, e.g., by altering the intestinal physiology. Thus, the aim of this study was establishing a highly sensitive and specific ELISA for the serological detection of EDIM infections in rodents. First, virus proteins were separated by SDS-PAGE and immunogenic proteins were visualized by immunoblotting and identified after in-gel digestion by tandem mass spectrometry. Subsequently, the major immunogen VP6 (virus protein 6) was expressed in Escherichia coli in high yields, purified by affinity chromatography, and used to establish an indirect ELISA. The diagnostic sensitivity and specificity were both above 99 % and the selectivity better than 98.7 % for animals infected by other pathogens listed by the Federation of Laboratory Animal Science Associations. Importantly, the Strep-rVP6-His-ELISA was more sensitive than a commercial virus-based ELISA and is a time- and cost-efficient complement to EDIM-specific immune-fluorescence assays. In conclusion, the assay can improve health monitoring by reducing the risk of missed EDIM infections in animal housing facilities, thereby improving animal welfare, reliability of animal studies, and protection of precious mice breeds.

Enzootic bovine leukosis (EBL) is caused by the bovine leukemia virus (BLV). The disease is spread worldwide and causes severe economic impact in cattle production. Different molecular methods are applied for the diagnosis of BLV infections. The most common methods for the direct detection of proviral BLV-DNA are real-time polymerase chain reactions.

ObjectivesFeline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting cats worldwide. The objectives of the study were to estimate the prevalence of these retroviruses in domestic cats in Hungary and to characterise the phylogenetic relationships of FIV strains.MethodsA total of 335 anticoagulated whole-blood samples obtained from both a healthy and ill cat population were examined for the presence of FIV and FeLV with two methods: ELISA and PCR. Statistical analysis was carried out to analyse the data obtained. Sequencing and phylogenetic analysis of partial polymerase ( pol) gene sequences was performed to describe circulating FIV subtypes.ResultsStatistical analysis showed 11.8% and 9.9% true prevalence of FeLV and FIV, respectively, with ELISA. The apparent prevalence calculated from the PCR results were 17.3% for FeLV and 13.1% for FIV. Phylogenetic analysis of partial pol gene sequences obtained from 22 FIV strains showed that all observed Hungarian strains belonged to FIV subtype B. The strains were grouped into several monophyletic subgroups reflecting the geographic locations of the origin of the samples. The overall mean genetic similarity between the analysed strains was 98.2%.Conclusions and relevanceWe report the first thorough overview of the prevalence of FeLV and FIV in Hungary, which is relatively high, and give insight into the genetic diversity of Hungarian strains of FIV.

Avian pathogens such as bornaviruses, circoviruses and polyomaviruses are widely distributed in captive collections of psittacine birds worldwide and can cause fatal diseases. In contrast, only little is known about their presence in free-ranging psittacines and their impact on these populations. Rose-ringed parakeets (

Die Gesichter der FHV-1-bedingten Erkrankungen am Katzenauge sind sehr verschieden. Fur die Auswahl des geeigneten Therapieregimes ist die Erkennung des jeweils vorliegenden Stadiums essenziell.

Quantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement. Fil: Jaworski, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Pluta, A.. National Veterinary Research Institute; Polonia Fil: Rola Luszczak, M.. National Veterinary Research Institute; Polonia. Animal and Plant Health Agency; Reino Unido Fil: McGowan, S.L.. Animal and Plant Health Agency; Reino Unido Fil: Finnegan, C.. Animal and Plant Health Agency; Reino Unido Fil: Heenemann, K.. University of Leipzig; Alemania Fil: Carignano, Hugo Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Alvarez, Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Murakami, K.. Iwate University; Japón Fil: Willems, L.. Université de Liège; Bélgica Fil: Vahlenkamp, T.W.. University of Leipzig; Alemania Fil: Trono, Karina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Choudhury, B.. Animal and Plant Health Agency; Reino Unido Fil: Kuzmak, J.. National Veterinary Research Institute; Polonia

Low pathogenic avian influenza virus of subtype H9N2 is panzootic in multiple avian species causing respiratory manifestations and severe economic losses. H9N2 co-circulate simultaneously with high pathogenic avian influenza virus subtype H5N1 in Egyptian chicken farms suggesting the possibility of reassortment. The aim of the present study was to isolate and characterize H9N2 from the recent outbreaks in chicken farms. Also the diversity of amantadine-resistant mutants among these isolates was tested by in situ ELISA and sequence analysis. Three influenza H9N2 viruses, designated A/chicken/Egypt/SCU8/2014, A/chicken/Egypt/SCU9/2014 and A/chicken/Egypt/SCU20/2014 were isolated from commercial broiler and broiler breeder chickens in specific pathogen free embryonated chicken eggs. The eight gene segments were amplified by RT-PCR, cloned, and subjected to full length sequencing. Phylogenetic analysis of these viruses revealed a close relationship between Egyptian, Middle Eastern and Israel isolates with an average of 96-99 % nucleotide homology and identified an ancestor relationship to low pathogenic H9N2 Quail/HK/G1/1997 prototype. The internal segments of the currently isolated viruses were derived from the same sub-lineage with no new evidence of reassortment. The three isolates were sensitive to amantadine as suggested by absence of mutations of M2 and confirmed by a phenotypic assay. In conclusion, avian influenza H9N2 virus is circulating in Egyptian chicken farms causing respiratory manifestations. Continuous monitoring of the molecular epidemiology and its impact on the virulence as well as emergence of new strains are necessary.

Usutu virus (USUV) is a mosquito-borne flavivirus accounting for large-scale deaths in resident bird populations. In this study, we show the introduction of USUV to Eastern Germany resulting in massive death of birds, particularly blackbirds (Turdus merula). We found that three diverse USUV lineages (“Europe 3,” “Africa 2,” and “Africa 3-like”) circulated simultaneously. Moreover, we detected USUV in Culex pipiens in a region where no dead birds were reported, strengthening the need for mosquito monitoring to uncover the spread of arboviruses. Furthermore, phylogenetic analyses revealed that mutations accumulated, in particular, in the NS3 region within short time periods. In addition, comparison of whole-genome sequences showed that diverse isolates of the cluster “Africa 3-like” are cocirculating in Germany due to independent introduction events.

Low-pathogenic avian influenza viruses (LPAIVs) of subtype H9N2 have become widespread in poultry in many Asian countries with relevance to respiratory diseases of multifactorial origin. In Bangladesh, LPAIVs of subtype H9N2 co-circulate simultaneously with highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in commercial and backyard poultry. The aim of this study was to characterize LPAIVs of subtype H9N2 currently circulating in Bangladesh. The selected isolate A/Chicken/Bangladesh/VP01/2006 (H9N2) was propagated in chicken embryos. All eight gene segments were amplified by RT-PCR, cloned, and subjected to full-length sequencing. The sequence data obtained were compared with reference strains available in GenBank. Phylogenetic analysis of LPAIV H9N2 from Bangladesh revealed a close relationship to Indian, Pakistani and Middle Eastern isolates and identified an ancestor relationship to LPAIV H9N2 Quail/HK/G1/1997. The internal genes M and NP belong to lineage G1, whereas NS, PA, PB1 and PB2 belong to the prototype virus A/Chicken/Korea/38349-p96323/96. The internal genes showed high sequence homology to an HPAIV of subtype H7N3 from Pakistan, whereas the PB1 gene showed similarly high nucleotide homologies to recently circulating HPAIV H5N1 from Bangladesh, revealing two independent reassortment events. Examination of the hemagglutinin cleavage site of LPAIV H9N2 confirmed its low pathogenicity. The receptor-binding sites indicated a binding preference for human-type receptors. Several mutations in internal proteins are associated with increased virulence and altered host range, while other amino acids were found to be highly conserved among LPAIV H9N2 isolates.

Clinical observations and diagnostic procedures carried out to elucidate the cause of high mortality in 2-8-wk-old ornamental ducks (mandarin, wood, falcated, and silver teal ducks) are described. At necropsy, ducklings showed general pallor of skeletal and heart muscles, subcutaneous gelatinous transudates, pericarditis, ascites, and severe edema and hyperemia of lungs. Histopathologic examination revealed that the most important changes were located in the crop, bursa of Fabricius, and lungs with presence of amorphic basic intracytoplasmic inclusions. No bacteria or fungi could be detected from affected organs and ascitic fluid. Viral diagnosis included molecular detection for the presence of goose parvovirus (GPV), circovirus, avian influenza, herpesviruses, paramyxovirus, reovirus, and polyomavirus. Both GPV and circovirus could be detected by real-time PCR and nested broad-spectrum PCR, respectively. Phylogenetically, full-length nucleotide sequence of GPV showed a close similarity ranging from 95.6% to 97.9% with European and Asian pathogenic GPV. On the other hand, the detected circovirus showed nucleotide identity of 90% to 98% with goose circoviruses (GoCVs). This is the first report of GoCVs and GPV in ornamental ducks. The concurrence of GPV and GoCV infections is thought to contribute to the high mortality.

A novel polyomavirus was identified in a fatally diseased Gouldian finch (Erythrura gouldiae). The new polyomavirus, strain VL 1209, was detected using a broad-spectrum nested PCR.

To investigate the prevalence of the recently described genogroup VI canine noroviruses (CNVs) in dogs in Europe, we tested 510 serum samples from dogs in 14 European countries for anti-IgG CNV antibodies. Seropositive dogs were found throughout Europe. Dogs with antibodies against human noroviruses were also found.

Bovine leukemia virus (BLV) causes a persistent infection with provirus formation in B-lymphocytes. A real- time polymerase chain reaction (PCR) based on the conserved BLV polymerase (BLV pol) gene sequences was developed. Dually labeled probes were used to permit detection by the 5' exonuclease assay. The assay was validated with 350 samples of bovine peripheral blood mononuclear cells including 144 samples from BLV-seropositive animals worldwide (South America, Europe, Middle East, Australia) representing 5 of the recently described 7 BLV envelope-based genotypes. The BLV pol real- time PCR proved to be highly specific and sensitive with the detection of up to 1 copy of an internal control plasmid. The 95% confidence intervals for assay sensitivity and specificity were ≥98.27% and ≥98.33%, respectively. Restriction fragment length polymorphism and phylogenetic BLV pol-based sequence analysis of the investigated samples were performed and compared with the previous described BLV env-based genotypes. Grouping of the sequences based on the pol gene yielded similar results as the env gene-based assay.