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doi: 10.15389/agrobiology.2019.2.369eng

UDC: 619:578:57.083.2:577.2

Acknowledgements:
The work was carried out according to the state task No. 007-01359-17-00 as part of the implementation of the Federal Scientific and Technical Program for the Development of Agriculture for 2017-2025, the subprogram “Creating domestic competitive meat crosses of broiler-type hens”.

 

MULTIPLEX MULTILOCUS REAL TIME PCR FOR ANALYSIS AND CONTROL OF AVIAN LEUKOSIS VIRUS SUBGROUPS A, B, J AND K IN RUSSIA

А.М. Borodin1, 5, Ya.I. Alekseev2, 3, N.V. Konovalova3, Е.V. Terentyeva3, N.Yu. Serova4, D.N. Efimov5, Zh.V. Emanuilova5, S.V. Smolov5, О.А. Ogneva5, V.I. Fisinin6

1Non-profit Partnership Institute of Medico-Biological Research,10, ul. Studenaya, Nizhnii Novgorod, 603000 Russia, e-mail Aborodinm@sinn.ru;
2Institute for Analytical Instrumentation RAS, 31-33, ul. Ivana Chernyh, St. Petersburg, 198095 Russia, e-mail jalex@syntol.ru;
3LLC Syntol, 42, ul. Timiryazevskaya, Moscow, 127550 Russia, e-mail jalex@syntol.ru (✉ corresponding author);
4All-Russian Research Veterinary Institute of Poultry Science — Branch of Federal Scientific CenterAll-Russian Research and Technological Poultry Institute RAS,48, ul. Chernikova, St. Peterburg—Lomonosov, 198412 Russia, e-mail vnivip.lab@gmail.com;
5Breeding and Genetic Center Smena, pos. Bereznyaki, Moscow Province, 141327 Russia, e-mail Smena@tsinet.ru, dmi40172575@yandex.ru;
6Federal Scientific Center All-Russian Research and Technological Poultry Institute RAS

ORCID:
Borodin А.М. orcid.org/0000-0002-1478-1261
Efimov D.N. orcid.org/0000-0002-4152-2476
Alekseev Ya.I. orcid.org/0000-0002-1696-7684
Emanuilova Zh.V. orcid.org/0000-0002-8855-2947
Konovalova N.V. orcid.org/0000-0003-4316-1077
Smolov S.V. orcid.org/0000-0001-6058-3672
Terentyeva Е.V. orcid.org/0000-0003-2777-0948
Ogneva О.А. orcid.org/0000-0002-8698-1975
Serova N.Yu. orcid.org/0000-0003-2121-2048
Fisinin V.I. orcid.org/0000-0003-0081-6336

Received February 2, 2018

 

The avian leukosis virus (ALV) belongs to genus Alpharetrovirus of Retroviridae family and has a diploid genome consisting of a single-stranded RNA. ALV subgroups A, B, C, D, E, J and K are specific for chicken. The classification is based on differences in the viral coat protein structure. ALV, in particular ALV subgroup J, causes huge damage to industrial poultry. The gold standard for detecting ALV is virus isolation in CEFs or DF-1 cell cultures. This method has significant disadvantages, i.e. it takes 7-9 days, requires specialized facilities and equipment. Enzyme-linked immunosorbent assay based on detection of the ALV p27 group-specific antigen is the most widely used, but it also has significant deficiencies, the main of which are false positive results due to the expression of p27 by endogenous viruses and lack of sensitivity. In this study, a test system has been developed to detect exogenous viruses of the most common ALV subgroups A, B, J, and K and to control the spread of ALV. To test the developed system, we use 1200 samples of broiler DNA from a poultry farm of the Moscow Province. Analysis of the samples detected ALV subgroup J in 51 % poultry flock and ALV subgroup K in 8 % poultry flock. No viruses of subgroups A and B were found. We also analyzed 97 DNA samples from chickens from the regions of Russia, i.e. Orenburg, Chelyabinsk, Kemerovo, Tyumen, Kaliningrad, Leningrad, Sverdlovsk, Novgorod regions and Krasnodar Territory. ALV subgroups K were found in samples from the Kaliningrad, Leningrad, Sverdlovsk, and Novgorod regions, ALV subgroups A in samples from the Leningrad region, and ALV subgroups J in the Sverdlovsk and Leningrad regions. ALV subgroup B has not been identified, that is, it may indicate that this subgroup of ALV is not common in Russia at the present time. At the next stage, measures were taken to eradicate the ALV of subgroups J and K found in the broiler-type meat cross lines in one of the farms of the Moscow Province. For this, a multiplex multilocus real-time PCR test system was developed and applied for the simultaneous detection of ALV subgroups J and K. Using the proposed test system, several screening cycles of four broiler-type chicken meat cross lines with an initial total of 9029 chickens were performed. Prior to the start of the program for control and eradication of ALV, the proportion of poultry with neoplasia ranged from 17 to 26 % depending on the line of chickens (the maximum was observed in the line with the ev21 locus). On the 265th day after the start of the program for the control and eradication of ALV subgroups J and K, only three out of 2621 individuals (0.10 %) were diagnosed with a diagnosis of neoplasia, confirmed by positive results of real-time PCR as ALV subgroup J. Total percentage of individuals’ samples containing ALV DNA of subgroups J and K in a sample of 2621 individuals at the age of 265 days were 0.67 % and 0.04 %, respectively.

Keywords: Avian leukosis virus, ALV subgroups A, B, J and K, real time PCR, ALV detection.

 

 

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