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Aim. To conduct a virological, PCR, PCR-RFLP and sequencing study of infectious laryngotracheitis virus (ILTV) isolates obtained from sick and dead chickens at industrial and backyard poultry farms in the eastern region of Ukraine collected over the years 2010–2019 and to establish their pathotype and relationship with internationally occurring strains. Methods. Material for virological studies was collected in the framework of research program of the NSC IEСVM during 2010-2019 in the poultry farms in the North-Eastern region of Ukraine, where the birds with the respiratory clinical signs were found. In total, 28 poultry farms were observed. ILTV isolates were obtained with conventional methods, using 10–12-day-old chicken embryos. A 0,2 ml of 10–20 % suspension of pathological material in PBS was used for inoculation. For in-depth studies, we used 4 isolates of ILTV obtained from sick and dead chickens from industrial and backyard poultry farms in Kharkiv, Luhansk, Donetsk, and Sumy regions from 2010–2019. The identification of ILTV isolates was performed via conventional PCR. The pathotype of ILTV strains was determined using PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) analysis. The PCR-RFLP was performed at Royal GD, the Netherlands. The (partial) sequencing of the US8 gene was performed using Sanger sequencing method. The phylogenetic analysis, using sequences of 2 Ukrainian strains (MZ323228, MZ333273) and 17 international gene sequences present in GenBank, was performed using the Maximum Likelihood method. For comparative analysis, sequences of vaccine ILT virus strains were used. Results. Over the years 2010-2019, 7 isolates of ILTV were obtained from sick and dead poultry with typical clinical signs and internal lesions at industrial and backyard farms of the Kharkiv, Donetsk, Luhansk and Sumy regions, and the Autonomous Republic of Crimea. Other avian respiratory viral and bacterial pathogens were not detected. Five isolates were obtained from poultry of industrial holdings where vaccination against ILT is carried out. Using PCR-RFLP analysis of 4 isolates, we found that three of them (Sumy 6-11/19, A 04-12, B 2-10) to belong to vaccine-type ILTV strains and only one, B 59-11strain, belongs to wild-type ILTV. Vaccine-type ILTV strains circulated and possibly still circulate in Ukraine in industrial and backyard poultry farms among both vaccinated and non- vaccinated poultry. An ILTV wild-type strain was obtained from non-vaccinated chickens from a backyard farm, which may indicate an important role of backyard farms in maintaining the circulation of the virus. After partial sequencing and phylogenetic analysis of the ILTV US8 gene the two Ukrainian strains studied were placed into two different clusters: The vaccine-type B 2-10 strain, obtained from sick vaccinated chickens from an industrial farm, was close to vaccine-type strains circulating in, China, Italy and the USA. The wild-type B 59-11strain, obtained from sick non-vaccinated backyard chickens, was located in another cluster and closest to a the wild-type B 59-11 ILTV strain from Brazil. Conclusions. In this article we describe for the first time the characterization of vaccine-type and wild-type isolates of ILTV in industrial and backyard poultry farms, proving their relevance for the poultry production in Ukraine. The results obtained show the need and prospects for further monitoring of ILTV circulation in small backyard poultry farms and in industrial poultry farms, especially following the frequent use for vaccination of live attenuated wild-type ILTV strains in Ukraine. Further molecular, phylogenetic and epidemiological characterization of the strains obtained should be performed in the near future to further precise their attributes, epidemiology and origin.

The first and the main link in the system of prevention of the occurrence and distribution of avian influenza and Newcastle disease is monitoring and the effective prophylaxis of the above diseases. At the same time the conducting of disinfection of the objects of veterinary control is an important stage in the system of veterinary and sanitary measures. A number of disinfectants that contain different classes of chemical compounds as active substances have been developed and proposed for practical use. The large-scale production of disinfectants and their introduction into practice is impossible without the preliminary laboratory assessment of their antimicrobial properties, the determination of the spectrum of their biocidal effect and physical, chemical and toxicological properties. The aim of our work was to study the virucidal properties of a new aldehyde disinfectant using the test models of the viruses of Newcastle disease and avian influenza. The experiments to study virucidal properties of the disinfectant regarding the viruses of avian influenza and Newcastle disease have been carried out at the Department for Avian Diseases Study of the National Scientific Center ‘Institute of Experimental and Clinical Veterinary Medicine’ in accordance with the guidelines ‘Methods for determining and evaluating the safety and quality of disinfectants and disinfecting detergents used in the production, storage, transportation and sale of products of animal origin’ (Kotsiumbas et al., 2010). The determination of the virucidal properties of the disinfectant has been conducted in two stages: stage 1 — the determination of the virucidal activity of the product by the suspension method; stage 2 — determination of the virucidal activity of the product on test objects. As a result of the research conducted by the suspension method the presence of the virucidal properties of the innovative disinfectant (the mixture of quaternary ammonium compounds — 25%, glutar aldehyde — 11%, isopropanol, non-ionic surfactants) for the viruses of avian influenza and Newcastle disease has been determined. It has been found that the above preparation completely inactivated the infective properties of viruses when used in the concentration of 0.1%, with the interval of 30 minutes and in the concentration of 0.5% — 15-minute interval. It has been proved that the use of the disinfectant in 0.1% concentration for 30 minutes disinfected the test objects (wood, metal, tile, textile) that were contaminated by the pathogenic agents of avian influenza and Newcastle disease

Avian orthoavulavirus 13 (AOAV-13), also named avian paramyxovirus 13 (APMV-13), has been found sporadically in wild birds around the world ever since the discovery of AOAV-13 (AOAV-13/wild goose/Shimane/67/2000) in a wild goose from Japan in 2000. However, there are no reports of AOAV-13 in China. In the present study, a novel AOAV-13 virus (AOAV-13/wild goose/China/Hubei/V93-1/2015), isolated from a wild migratory waterfowl in a wetland of Hubei province of China, during active surveillance from 2013 to 2018, was biologically and genetically characterized. Phylogenetic analyses demonstrated a very close genetic relationship among all AOAV-13 strains, as revealed by very few genetic variations. Moreover, pathogenicity tests indicated that the V93-1 strain is a low virulent virus for chickens. However, the genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, which is 12 nt or 162 nt longer than the other AOAV-13 strains that have been reported to date. The length difference of 12 nt in strain V93-1 is due to the existence of three repeats of the conserved sequence, “AAAAAT”, in the 5′-end trailer of the genome. Moreover, the HN gene of the V93-1 virus is 2070 nt in size, encoding 610 aa, which is the same size as the AOAV-13 strain from Japan, whereas that of two strains from Ukraine and Kazakhstan are 2080 nt in length, encoding 579 aa. We describe a novel AOAV-13 in migratory waterfowl in China, which suggests that diversified trailer region sequences and HN gene lengths exist within serotype AOAV-13, and highlight the need for its constant surveillance in poultry from live animal markets, and especially migratory birds.

Prevention measures are crucial in actual production, whereas the outbreak of a disease requires immediate detection and elimination of the source of infection as well as complex veterinary and sanitary measures. Here the critical role is attributed to disinfection, which breaks the epizootic chain due to the elimination of pathogenic microorganisms on the objects under veterinary surveillance and involves the application of various disinfectants. Aim. The study of virucidal properties of the novel disinfectant DZPT-2. Methods. The culture technique was used at the fi rst stage of studies to determine the embryotoxic effect of the preparation on chicken embryos and the cytotoxic effect of the disinfectant on BHK-21 cell culture. The second stage of experiments envisaged the study of virucidal effect of the disinfectant using both the suspension method, test- objects (batiste, wood, glazed tile, metal, glass) and the bioburden. Results. The experiments using the agent of bovine viral diarrhea demonstrated that the disinfectant DZPT-2 in the concentration of 1.0 % of the active ingredient (AI) when exposed for at least 30 min and in the concentration of 1.5 % of AI when exposed for 15– 60 min disinfects all the test-objects, contaminated by the virus, completely. When exposed for up to 15 min, the disinfectant DZPT-2 does not demonstrate its virucidal effect on Newcastle disease virus, but it disinfects all the test-objects, contaminated by the mentioned virus, when used in the concentration of 0.5–1.0 % of AI and exposed for at least 30 min. Conclusions. It was determined that the preparation DZPT-2 demonstrates its virucidal properties in the concentration of 0.5 % of AI (Newcastle disease virus) and 1.0 % of AI (agent of bovine viral diarrhea) when exposed for 30 min. The new disinfectant DZPT-2 is a promising preparation to be used in practical veterinary medicine to prevent and fi ght viral diseases of farm livestock and poultry.

Background The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. Results Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014–2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian (n = 2) and South Korean (n = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. Conclusions We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions. Electronic supplementary material The online version of this article (10.1186/s12917-017-1211-4) contains supplementary material, which is available to authorized users.

Objective To study the immune response in chicken on the administration of LPAIV isolated from the natural reservoir. Introduction Influenza is a serious problem for the health of people, animals and birds. Therefore, comprehensive study of influenza virus, its natural reservoir, pathogenesis and immune response will provide further opportunity to ensure protection for animals, birds and people from this infection. Methods Four-week-old commercial chickens were intranasally inoculated with a H4N6 LPAIV A/Garganey/Chervonooskilske/4-11/2009 (H4N6), isolated from the cloacal swab of clinically healthy garganey in 2009 in Ukraine. Cecum, spleen, lung, and trachea samples were collected from infected chickens on 1 - 14 dpi and examined by immunohistochemical and virology techniques. On these days, we collected blood samples for serological analysis. Detection of antibodies to avian influenza virus subtype H4 was performed with chicken serum samples by HI test and ELISA. The studies were done according to IACUC. Results Upon intravenous and intranasal infection with this virus (A/Garganey/Chervonooskilske/4-11/2009), no clinical signs were observed in chickens and no pathological changes were found at necropsy. Infection of poultry with this virus provoked an antibody response at 10 days after intranasal inoculation which ranged from 1:8 to 1:32 serum antibody titers. Only 2 of 5 chickens were positive by the HI test and 3 of 5 were positive by ELISA at intranasal inoculation. All 10 chickens were positive both by HI test and by ELISA after intravenous inoculation. Specific antibodies (HI test) to influenza virus H4 were detected in titer ranges of 1:128 to 1:1024. In immunohistological studies, the respiratory tract organs (lungs and trachea) showed higher level of humoral immunity (IgM, IgG, IgA-expressing cells) in the lung compared to the trachea. Also, indicators of cell mediated immunity as measured by the CD4 and macrophage markers were higher in LPAIV-infected chickens in the lungs at 14 days post infection compared to uninfected chickens. Lymphocytes expressing CD8 were increased starting 7dpi. The chickens in the infected group showed 2 times higher levels of CD8 cells compared to the control chickens. IFN- γ transcripts were observed in the AI-infected chickens starting at 7dpi that coincides with the increasing level of CD4 cells. The number of lymphocytes which secrete IL-2 and IL-15 in AI-infected chickens were in general 1.5 to 2 times higher compared to the uninfected chickens. In AIV-infected chickens, the level of cells expressing IFN- γ , IL-2, and IL-15 increased at 7-days after infection. The peak time coincided with a period of increasing CD8 cells. However, there was no significant difference in these cytokine levels between the AIV-infected and uninfected groups. In the cecum, lower levels of CD4 cells were seen on 5 dpi but levels slowly increased from 7 dpi to 14 dpi following AIV infection. In the ceca, a significant increase in the number of cells expressing IgM and IgG was found. LPAIV infection induced an increase in macrophages and lymphocytes expressing CD4 and CD8 in the spleen throughout the period examined in this study indicating their role in host response to viral infection. The levels of macrophages in chickens of AIV- infected group were 2 times higher than the control after 1 dpi. Conclusions Although infection with a LPAIV did not cause obvious clinical disease, viral replication was detected in the trachea and spleen and both local and systemic cellular and humoral immune responses were elicited in these LPAIV-infected chickens. Our results indicate the potential possibility for infection of poultry with viruses isolated from wild birds. But currently it is not completely known why some viruses from wild birds can cause infection in poultry, while others can not. Further study of the immune response will enable us to determine the features of the pathogenesis of low pathogenic avian influenza.

Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.

Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13.

Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site (“113RQKR↓F117”), and selected ones have intracerebral pathogenicity index values ranging from 1.61 to 1.96. These isolates are most closely related to viruses circulating in Eastern Europe, followed by viruses isolated in Asia during the same period of time. Interestingly, the majority of the viruses were isolated from backyard poultry, suggesting the possibility of a “domestic” or “urban” cycle of maintenance. The molecular characterization of the nucleotide sequence of the complete fusion protein gene of the studied viruses suggests continued circulation of virulent NDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. The continuous monitoring of backyard poultry flocks for the presence of circulating virulent NDV strains will allow early identification of Newcastle disease outbreaks.

Avian paramyxovirus serotype 4 (APMV-4) is a single stranded RNA virus that has most often been isolated from waterfowl. Limited information has been reported regarding the prevalence, pathogenicity, and genetic diversity of AMPV-4. To assess the intercontinental dispersal of this viral agent, we sequenced the fusion gene of 58 APMV-4 isolates collected in the United States, Japan and the Ukraine and compared them to all available sequences on GenBank. With only a single exception the phylogenetic clades of APMV-4 sequences were monophyletic with respect to their continents of origin (North America, Asia and Europe). Thus, we detected limited evidence for recent intercontinental dispersal of APMV-4 in this study.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

The abstract is devoted to monitoring studies of circulation of the AIV subtypes H5 and H7 in wild waterfowl and shorebirds around the Azov-Black Sea in Ukraine

Since the first detection of H15 avian influenza viruses (AIVs) in Australia in 1979, only seven H15 strains have been reported. A new H15 AIV was detected in Ukraine in 2010, carrying the unique HA-NA subtype combination H15N7. This virus replicated efficiently in chicken eggs, and antisera against it reacted strongly with the homologous antigen, but with lower titers when using the reference Australian antigen. The amino acid motifs of the HA cleavage site and receptor-binding site were different from those in the Australian viruses. The new virus, together with an H15 virus from Siberia from 2008, constitutes a new clade of H15 AIV isolates.

Despite the existence of 10 avian paramyxovirus (APMV) serotypes, very little is known about the distribution, host species, and ecological factors affecting virus transmission. To better understand the relationship among these factors, we conducted APMV wild bird surveillance in regions of Ukraine suspected of being intercontinental (north to south and east to west) flyways. Surveillance for APMV was conducted in 6,735 wild birds representing 86 species and 8 different orders during 2006 to 2011 through different seasons. Twenty viruses were isolated and subsequently identified as APMV-1 ( n = 9), APMV-4 ( n = 4), APMV-6 ( n = 3), and APMV-7 ( n = 4). The highest isolation rate occurred during the autumn migration (0.61%), with viruses isolated from mallards, teals, dunlins, and a wigeon. The rate of isolation was lower during winter (December to March) (0.32%), with viruses isolated from ruddy shelducks, mallards, white-fronted geese, and a starling. During spring migration, nesting, and postnesting (April to August) no APMV strains were isolated out of 1,984 samples tested. Sequencing and phylogenetic analysis of four APMV-1 and two APMV-4 viruses showed that one APMV-1 virus belonging to class 1 was epidemiologically linked to viruses from China, three class II APMV-1 viruses were epidemiologically connected with viruses from Nigeria and Luxembourg, and one APMV-4 virus was related to goose viruses from Egypt. In summary, we have identified the wild bird species most likely to be infected with APMV, and our data support possible intercontinental transmission of APMVs by wild birds.

Wild bird surveillance for avian influenza virus (AIV) was conducted from 2001 to 2012 in the Azov - Black Sea region of the Ukraine, considered part of the transcontinental wild bird migration routes from northern Asia and Europe to the Mediterranean, Africa, and southwest Asia. A total of 6281 samples were collected from wild birds representing 27 families and eight orders for virus isolation. From these samples, 69 AIVs belonging to 15 of the 16 known hemagglutinin (HA) subtypes and seven of nine known neuraminidase (NA) subtypes were isolated. No H14, N5, or N9 subtypes were identified. In total, nine H6, eight H1, nine H5, seven H7, six H11, six H4, five H3, five H10, four H8, three H2, three H9, one H12, one H13, one H15, and one H16 HA subtypes were isolated. As for the NA subtypes, twelve N2, nine N6, eight N8, seven N7, six N3, four N4, and one undetermined were isolated. There were 27 HA and NA antigen combinations. All isolates were low pathogenic AIV except for eight highly pathogenic (HP) AIVs that were isolated during the H5N1 HPAI outbreaks of 2006-08. Sequencing and phylogenetic analysis of the HA genes revealed epidemiological connections between the Azov-Black Sea regions and Europe, Russia, Mongolia, and Southeast Asia. H1, H2, H3, H7, H8, H6, H9, and H13 AIV subtypes were closely related to European, Russian, Mongolian, and Georgian AIV isolates. H10, H11, and H12 AIV subtypes were epidemiologically linked to viruses from Europe and Southeast Asia. Serology conducted on serum and egg yolk samples also demonstrated previous exposure of many wild bird species to different AIVs. Our results demonstrate the great genetic diversity of AIVs in wild birds in the Azov-Black Sea region as well as the importance of this region for monitoring and studying the ecology of influenza viruses. This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.