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.

Infectious laryngotracheitis of chickens is one of the most dangerous viral respiratory diseases of chickens, which causes significant economic losses to poultry farms. A key component in this disease control is timely rapid serological diagnosis. To date, the basic method of serological diagnosis and monitoring is enzyme-linked immunosorbent assay (ELISA). The main components of ELISA test systems are purified and concentrated infectious laryngotracheitis virus antigens. Our research aimed to develop a technology for the production of purified and concentrated antigens of infectious laryngotracheitis virus, as well as to test the suitability of epizootic isolates for the production of antigens for ELISA. Based on the results of research, an improved scheme for obtaining purified infectious laryngotracheitis virus antigens using epizootic isolates has been developed. The scheme consists of accumulation of virus raw material, its inactivation, verification of inactivation completeness, concentration of infectious laryngotracheitis virus by PEG-6000 precipitation followed by ultracentrifugation at 14,000 rpm through a 30% sucrose pad. Samples of purified concentrated infectious laryngotracheitis virus antigens from isolates “В 59-11”, “Б 2-10”, “ЧП 96-10”, and “A 4-12” with protein content 1,520–3,720 μg/cm3 have been obtained. The ratio of protein concentration before and after purification ranged from 4.17 to 7.24. ELISA found that all these antigens were suitable for use as antigens. When testing for specificity, it was found that all antigens did not react with heterologous sera to other poultry viral diseases, but reacted only with homologous sera positive for infectious laryngotracheitis, which proves their specificity

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