Outbreak of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cats, Poland, June to July 2023.

BackgroundOver a 3-week period in late June/early July 2023, Poland experienced an outbreak caused by highly pathogenic avian influenza (HPAI) A(H5N1) virus in cats.AimThis study aimed to characterise the identified virus and investigate possible sources of infection.MethodsWe performed next generation sequencing and phylogenetic analysis of detected viruses in cats.ResultsWe sampled 46 cats, and 25 tested positive for avian influenza virus. The identified viruses belong to clade 2.3.4.4b, genotype CH (H5N1 A/Eurasian wigeon/Netherlands/3/2022-like). In Poland, this genotype was responsible for several poultry outbreaks between December 2022 and January 2023 and has been identified only sporadically since February 2023. Viruses from cats were very similar to each other, indicating one common source of infection. In addition, the most closely related virus was detected in a dead white stork in early June. Influenza A(H5N1) viruses from cats possessed two amino acid substitutions in the PB2 protein (526R and 627K) which are two molecular markers of virus adaptation in mammals. The virus detected in the white stork presented one of those mutations (627K), which suggests that the virus that had spilled over to cats was already partially adapted to mammalian species.ConclusionThe scale of HPAI H5N1 virus infection in cats in Poland is worrying. One of the possible sources seems to be poultry meat, but to date no such meat has been identified with certainty. Surveillance should be stepped up on poultry, but also on certain species of farmed mammals kept close to infected poultry farms.

Molecular analysis Polish isolates of goose hemorrhagic polyomavirus from geese and free-living birds.

Goose haemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis and enteritis of geese (HNEG), a lethal disease of goose. The study describes the results of a molecular analysis Polish isolates of GHPV from geese and free-living birds based on complete VP1 gene and VP2 gene sequences. The sequences were analyzed and aligned with different GHPV isolates sequences accessible in the GenBank database. This study indicates affiliation GHPV isolates from fee-living birds and GHPV isolates circulating in Polish goose flocks and around the world to the same genetic groups, which proves their evolutionary relationship and indicates the potential role of free-living birds as a source of infections for poultry.

Highly Pathogenic Avian Influenza H5Nx in Poland in 2020/2021: a Descriptive Epidemiological Study of a Large-scale Epidemic.

Introduction: Highly pathogenic avian influenza (HPAI) outbreaks caused by the Gs/Gd lineage of H5Nx viruses occur in Poland with increased frequency. The article provides an update on the HPAI situation in the 2020/2021 season and studies the possible factors that caused the exceptionally fast spread of the virus. Material and Methods: Samples from poultry and wild birds delivered for HPAI diagnosis were tested by real-time RT-PCR and a representative number of detected viruses were submitted for partial or full-genome characterisation. Information yielded by veterinary inspection was used for descriptive analysis of the epidemiological situation. Results: The scale of the epidemic in the 2020/2021 season was unprecedented in terms of duration (November 2020-August 2021), number of outbreaks in poultry (n = 357), wild bird events (n = 92) and total number of affected domestic birds (approximately ~14 million). The major drivers of the virus spread were the harsh winter conditions in February 2020 followed by the introduction of the virus to high-density poultry areas in March 2021. All tested viruses belonged to H5 clade 2.3.4.4b with significant intra-clade diversity and in some cases clearly distinguished clusters. Conclusion: The HPAI epidemic in 2020/2021 in Poland struck with unprecedented force. The conventional control measures may have limited effectiveness to break the transmission chain in areas with high concentrations of poultry.

Fowl adenovirus strains 1/A and 11/D isolated from birds with reovirus infection.

Inclusion body hepatitis (IBH) is, in some cases, a fatal disease affecting fowl by adenovirus strains which are subdivided into 5 species (A-E). In the current study, we investigated sequences from the Loop L1 region of the hexon gene of sequences of adenovirus field stains 1/A and 11/D isolated from a poultry flock co-infected with IBH and avian reoviruses ARVs. In early 2021, an epidemiologic survey highlighted the coinfection adenoviruses with other viruses (orthoreovirus infection) as being particularly deleterious within the poultry industry. Here, we investigated the Loop L1 HVR1-4 region of the hexon gene with relative synonymous codon usage (RSCU) designation and RSCU inclusive of all the mutations. These are the first results that have been presented on fowl adenovirus species A and D with simultaneous reovirus infection in 38-days old broiler chickens in Poland.

Characterisation of adenovirus strains represented species B and E isolated from broiler chicken flocks in eastern Poland.

Fowl adenovirus strains were isolated from the internal organs of 3-wk-old broiler flocks exhibited clinical signs associated with inclusion body hepatitis (IBH). The isolated strains were molecularly characterised and sequencing revealed three distinct clusters. One cluster showed close proximity at the nucleotide level with adenovirus type/species - 6/E, 7/E, 8a/E, and 8b/E. The second cluster contained five reference sequences belonging to the species FAdV-D and E. A third cluster contained one field and four reference sequences belonging to the FAdV-5/B, FAdV-4/C, FAdV-2/D, and FAdV-1/A type/species respectively. The heterogenicity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses, were carried out. The maximum likelihoods for the examined sequences were estimated. The data indicated that correlation between isolated of adenovirus type/species 5/B, and E in Poland have been presented. Indicated adenovirus types and their combinations with locally circulating FAdVs strains could have implications for current detection methods and pathogenicity on infected chickens.

Occurrence of Marek's Disease in Poland on the Basis of Diagnostic Examination in 2015-2018.

INTRODUCTION: Marek's disease (MD) is a tumourous disease caused by Marek's disease virus (MDV) and most commonly described in poultry. The aim of the study was to determine the occurrence of Marek's disease virus infections in Poland and analyse clinical cases in the years 2015-2018. MATERIAL AND METHODS: The birds for diagnostic examination originated from 71 poultry flocks of various types of production. Birds were subjected to anatomopathological examination post mortem, during which liver and spleen sections and other pathologically changed internal organs were taken. These sections were homogenised with generally accepted methods, then total DNA was isolated and amplified with a real-time PCR. A pair of primers complementary to the MDV genome region encoding the meq gene were used. RESULTS: MDV infection was found predominantly in broiler chicken flocks (69.01%), and also in layer breeder (9.85%) and commercial layer flocks (7.04% each). CONCLUSION: The results of research conducted in the years 2015-2018 clearly indicate that the problem of MDV infections is still current.

Correction to: Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

An amendment to this paper has been published and can be accessed via the original article.

Isolation and molecular characterization of Fowl adenovirus strains in Black grouse: First reported case in Poland.

This article describes the isolation, molecular characterization, and genotyping of two fowl adenovirus (FAdVs) strains with GenBank Accession numbers (MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B) obtained from the internal organs of black grouse (Lyrurus tetrix). This study also reveals the first confirmation of fowl adenovirus in Poland, supporting one of the hypotheses about the probability of fowl adenovirus interspecies transmission. The adenovirus strain sequences were investigated via phylogenetic analysis and were found to have an overall mean pairwise distance of 2.189. The heterogeneity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses and Tajima's test for the examined sequences were carried out. The Maximum Likelihood for the examined sequences substitutions was performed. The results of the sequence analysis identified MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B as strains of fowl adenovirus 2/11/D, with the Fowl adenovirus D complete sequence showing a 93% match. Wild birds may act as a natural reservoir for FAdVs and likely play an important role in the spreading of these viruses in the environment. The findings reported here suggest horizontal transmission within and between avian species.

Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

BACKGROUND: The present study on the role of strains of adenovirus in wildlife reservoirs, and their prevalence is under exploration. In several previous studies, the presence of adenovirus strains in wild birds has been investigated. Worldwide distribution and outbreaks of adenovirus infections have been reported by many authors. The present study investigated the prevalence of FAdVs in 317 samples of different bird species from the northwestern region of Poland. An applied specific, sensitive, and efficient, without cross-reactivity loop-mediated isothermal amplification (LAMP) method to gauge the prevalence of fowl adenovirus strains in wild birds was developed and used. RESULTS: The method was based on the sequence of the loop L1 HVR1-4 region of the hexon gene of the FAdV genome reference strains FAdV-2 KT862805 (ANJ02325), FAdV-3 KT862807 (ANJ02399) and FAdV-11 KC750784 (AGK29904). The results obtained by LAMP were confirmed by real-time PCR. Among 317 samples obtained from wild birds, eight FAdV isolates (2.52%) were identified and produced a cytopathic effect (CPE) in chicken embryo kidney cells (CEK). Three FAdV types belonging to species Fowl adenovirus D were detected, which were isolated from three adenovirus types 2/3/11, and have been confirmed in three mute swans (Cygnus olor), three wild ducks (Anas platyrhynchos), one owl (Strigiformes), and one common wood pigeon (Columba palumbus). CONCLUSIONS: This study provides the first accurate quantitative data for the replication of fowl adenovirus strains in wild birds in Poland, indicating adenovirus interspecies transmission, and demonstrating the circulation of FAdVs in wild birds.

Elaboration of Triplex PCR for Detection of Selected Viral Infections in Waterfowl.

INTRODUCTION: Viral infections are the greatest threat to waterfowl and cause significant economic losses. Diagnosis and differentiation of three goose viruses is difficult in the field and often requires laboratory confirmation. Therefore, the aim of the study was to develop a triplex PCR and optimise its parameters for simultaneous detection of DNA of goose parvovirus (GPV), goose polyomavirus (GHPV), and goose circovirus (GoCV). MATERIAL AND METHODS: The DNA of viruses isolated from field cases from the National Veterinary Research Institute's own collection was used for the study. The primer attachment temperature, the number of reaction cycles, and the Taq DNA polymerase and Mg2+ concentrations were optimised. The sensitivity and specificity of this triplex PCR was also determined. RESULTS: Based on the obtained results, triplex PCR parameters were optimised for simultaneous detection of DNA of GPV, GHPV, and GoCV in one sample. The following PCR products of the expected size were obtained: GPV DNA of 806 bp, GoCV DNA of 571 bp, and GHPV DNA of 180 bp. CONCLUSION: The developed triplex PCR method proved to be useful for simultaneous detection of infections with three waterfowl viruses and will be used in relevant laboratory diagnostics.

Occurrence of Reovirus (ARV) Infections in Poultry Flocks in Poland in 2010-2017.

INTRODUCTION: Avian reovirus (ARV) infections in poultry populations are reported worldwide. The reovirus belongs to the genus Orthoreovirus, family Reoviridae. The aim of the study was to evaluate the incidence of ARV infections in the poultry population based on diagnostic tests performed in 2010-2017. MATERIAL AND METHODS: Samples of the liver and spleen were collected from sick birds suspected of ARV infection and sent for diagnostics. Isolation was performed in 5-7-day-old SPF chicken embryos infected into the yolk sac with homogenates of internal organs of sick birds. Four primer pairs were used to detect the σNS, σC, σA, and µA ARV RNA gene fragments. A nested PCR was used for the detection of the σNS and σC genes. RESULTS: In 2010-2017, ARV infection was found in birds from 81 flocks of broiler chickens and/or layers, 8 flocks of slaughter turkeys, and in 4 hatchery embryos at 17-20 days of incubation. The primers used in RT-PCR and nested PCR did not allow effective detection of ARV RNA in all virus-positive samples. CONCLUSION: The problem of ARV infections in the poultry population in Poland still persist. The primers used for various ARV segments in RT-PCR and nested PCR did not allow effective detection of RNA in the visceral organs of sick birds. The presented results confirm the necessity of using classical diagnostic methods (isolation in chicken embryos, AGID).

Avian Poxvirus Infection in Polish Great Tits (Parus Major).

INTRODUCTION: Avian poxvirus infections are widespread in the domestic poultry population but are also reported in wild birds. In poultry, these infections cause significant economic losses, while wild birds may be a reservoir for poxvirus which affects breeding poultry. However, wild birds may also exhibit characteristic anatomopathological changes. This study concerns the infection of wild-living great tits (Parus major) with the avian poxvirus in Poland. MATERIAL AND METHODS: Samples of internal organs and skin collected from great tits were homogenised and total cellular DNA was isolated. In PCR, the primers complementary to gene encoding the core protein 4b of the HP44 strain of fowl poxvirus (FPV) were used. RESULTS: After electrophoresis in 2% agarose gel, the PCR product of 578 bp characteristic for FPV was obtained in DNA samples isolated from skin lesions and the heart. The analysis of the nucleotide sequence of the virus strain showed 99% similarity to many poxviruses previously isolated from great tits and other free birds at various sites in the world. CONCLUSIONS: This paper is the first clinically documented evidence obtained in laboratory conditions of avian poxvirus cases in great tits in Poland.

Detection of Avian Reoviruses in Wild Birds in Poland.

INTRODUCTION: The purpose of this study was to determine the occurrence of avian reovirus (ARV) infections in wild birds in Poland and attempt to propagate the selected ARV strains in chicken embryo kidney (CEK) cells or chicken SPF embryos. MATERIAL AND METHODS: The study included 192 wild birds representing 32 species, collected between 2014 and 2016. A part of the S4 segment encoding the σNS protein of avian reoviruses (ARVs) isolated from different species of wild birds from that period was amplified. RESULTS: The presence of ARV was demonstrated in 58 (30.2%) wild birds belonging to nine orders. The isolated strains were propagated in chicken embryos by yolk sac inoculation, and CPE was induced in the infected CEK monolayer. Agar gel precipitation showed that two ARV isolates from rock pigeon and mute swan shared a common group-specific antigen with chicken reovirus S1133. Specific products of predicted size were found in two ARV isolates from the chicken embryo passage and 13 ARVs isolated from CEK cells. CONCLUSION: The study indicates the high prevalence of ARV among wild birds in Poland and its possible transmission to farmed birds.