619:578.828.11:636.2 ОПРЕДЕЛЕНИЕ УРОВНЯ ПРОВИРУСНОЙ НАГРУЗКИ У ЖИВОТНЫХ ПРИ ЛЕЙКОЗЕ КРУПНОГО РОГАТОГО СКОТА
DOI:
https://doi.org/10.52578/2305-9397-2025-2-2-3-12Ключевые слова:
вирус лейкоза, крупный рогатый скот, полимеразная цепная реакция, провирусная нагрузка, диагностикаАннотация
Провирусная нагрузка является важным диагностическим показателем, который позволяет определить уровень инфицированности животных и оценить риск прогрессирования заболевания. Высокая провирусная нагрузка связана с большей вероятностью развития клинических проявлений и передачи вируса внутри стада, что делает данный показатель критически важным для ветеринарной эпизоотологии. В статье представлены материалы и методы исследования провирусной нагрузки вируса лейкоза крупного рогатого скота (BLV). Объектом исследования послужили 54 особи крупного рогатого скота. Отбор проб крови проводился с использованием вакуумных систем в пробирки с ЭДТА. Выделение ДНК осуществлялось коммерческим набором «Diatom DNA Prep 200», а её концентрация определялась флуориметром Qubit 4. Для количественного определения провирусной нагрузки применяли метод ПЦР в реальном времени с набором реагентов «РеалБест-Вет ДНК вируса лейкоза КРС». Праймеры и зонд использовались в соответствии с протоколом Marzena Rola-Łuszczak и соавт. (2004). Стандартная кривая строилась на основе серийных разведений ПЦР-продукта (10³–10⁷ копий/реакция). Анализ данных проводился с использованием программного обеспечения Bio-Rad CFX Maestro Software и Microsoft Excel. Статистическая обработка включала расчет средних значений, стандартного отклонения и коэффициентов вариации. Исследование выявило расхождения между результатами коммерческого набора для выявления провируса лейкоза крупного рогатого скота и результатами, количественной ПЦР, что подчеркивает необходимость стандартизации методов для повышения точности диагностики. В результате был проведен анализ корреляций между уровнем провирусной нагрузки и клиническим течением заболевания, что позволяет дифференцированно подходить к диагностике и контролю BLV-инфекции. Показатели провирусной нагрузки находились в диапазоне от 0 копий/100нг ДНК до 192257,23 копий/100 нг ДНК. Средний уровень провирусной нагрузки составлял 32691 копий/100нг ДНК. Данные исследования имеют большое значение в разработке эффективных программ борьбы с вирусом лейкоза крупного рогатого скота.
Библиографические ссылки
СПИСОК ЛИТЕРАТУРЫ
Aida, Y. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T
cell leukemia virus [Text] / Y. Aida, H. Murakami, M. Takahashi et all. // Front. Microbiol. – 2013. –
V.4. – P. 328. doi: 10.3389/fmicb.2013.00328.
Polat, M., Takeshima S.N., Aida Y. Epidemiology and genetic diversity of bovine leukemia virus
[Text] / M. Polat, S.N. Takeshima, Y. Aida. // Virol J. – 2017. – Vol. 14, Issue 1. – P. 209-1-209-16.
Lefkowitz, E.J. Virus taxonomy: the database of the International Committee on Taxonomy of
Viruses (ICTV) [Text] / E.J. Lefkowitz, D.M. Dempsey, R.C. Hendrickson et al. // Nucleic Acids
Research. – 2018. – Vol. 46. – P. 708-717.
Buehring, G.C. Bovine leukemia virus DNA in human breast tissue [Text] / G.C. Buehring, H.M.
Shen, H.M. Jensen, K.Y. Choi, D.J. Sun, G. Nuovo. // Emerg Infect Dis. – 2014. – Vol. 20. –
P. 772–782. 10.3201/eid2005.131298
Hamada, R. Detection and Molecular Characterization of Bovine Leukemia Virus in Egyptian
Dairy Cattle [Text] / R. Hamada, S. Metwally, M. Polat, et. al. // Front Vet Sci. – 2020. – Vol. 7. –
P. 608-1-608-13.
Nekouei, O. Lifetime effects of infection with bovine leukemia virus on longevity and milk
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Yang, Y. Bovine leukemia virus infection in cattle of China: Association with reduced milk
production and increased somatic cell score [Text] / Y. Yang, W. Fan, Y. Mao, et. al. // J Dairy Sci. –
– Vol. 99, Issue 5. – P. 3688-3697.
Panei, CJ. Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte
subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV
CoCoMo-qPCR [Text] / C.J. Panei, S.N. Takeshima, T. Omori, T. Nunoya, W.C. Davis, H. Ishizaki,
K. Matoba, Y. Aida // BMC Vet Res. – 2013. – V9. – P. 95. doi: 10.1186/1746-6148-9-95.
Juliarena, M.A. Determination of proviral load in bovine leukemia virus-infected cattle with and
without lymphocytosis [Text] / M.A. Juliarena, S.E. Gutierrez, C. Ceriani // Am J Vet Res. – 2007.
Vol.68. – B.1220–1225.
Nakatsuchi, A. BoLA-DRB3 Polymorphism Controls Proviral Load and Infectivity of Bovine
Leukemia Virus (BLV) in Milk [Text] / A. Nakatsuchi, S. Watanuki, L. Borjigin, H. Sato,
L. Bai, R. Matsuura, M. Kuroda, H. Murakami, R. Sato, S. Asaji, A. Ando, Y. Matsumoto,
S. N. Takeshima, Y. Aida [Text] // Pathogens. – 2022. – Vol. 11. – № 2. – P. 210. DOI:
3390/pathogens11020210.
Xinyue. Wu Development of droplet digital PCR for quantification of bovine leukemia virus
proviral load using unpurified genomic DNA [Text] / Xinyue Wu, Kousuke Notsu, Yuichi Matsuura,
Shuya Mitoma, Hala El Daous, Junzo Norimine, Satoshi Sekiguchi. // J Virol Methods. – 2023. –
V. 315. – P.114706. doi: 10.1016/j.jviromet.2023.114706.
Sulav, Sh. Removing bovine leukemia virus-infected animals with high proviral load leads to
lower within-herd prevalence and new case reduction [Text] / Sulav Shrestha, Karin Orsel, Casey
Droscha, Sanjaya Mijar, Frank van der Meer. // J Dairy Sci. – 2024. – V.107(8). – P. 6015-6024.
Lew, A.E. Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease
PCR using a 3′ minor groove binder fluorogenic probe [Text] / A.E. Lew, R.E. Bock,
J.B. Molloy, C.M. Minchin, S.J. Robinson, P. Steer. //J Virol Methods. – 2004. – V.115. – P.167–175.
Martin, D. Comparative study of PCR as a direct assay and ELISA and AGID as indirect assays
for the detection of bovine leukaemia virus [Text] / D. Martin, A. Arjona, I. Soto, N. Barquero, M. Viana,
E. Gomez-Lucia // J Vet Med B Infect Dis Vet Public Health. – 2001. – V. 48. – V.97–106.
Takeshima, S N. Single nucleotide polymorphisms in the bovine MHC region of Japanese
Black cattle are associated with bovine leukemia virus proviral load [Text] / S.N. Takeshima,
S. Sasaki, P. Meripet, Y. Sugimoto, Y. Aida. // Retrovirology. – 2017. – V.14. – P. 24.
Frie, M.C. Bovine leukemia virus: a major silent threat to proper immune responses in cattle
[Text] / M.C. Frie, P.M. Coussens // Vet Immunol Immunopathol. – 2015. – № 163(3-4). –
P. 103-114.
Heenemann, K. Development of a bovine leukemia virus polymerase gene-based real-time
polymerase chain reaction and comparison with an envelope gene-based assay [Text] / K. Heenemann, S.
Lapp, J.P. Teifke, D. Fichtner, T.C. Mettenleiter, T.W. Vahlenkamp. // J Vet Diagn Invest. – 2012. – V.
– P.649–655. doi: 10.1177/1040638712447524
Van Eijk, M. J. T. Extensive polymorphism of the BoLA‐DRB3 gene distinguished by
PCR‐RFLP [Text] / M. J. T. Van Eijk, J. A. Stewart‐Haynes, H. A. Lewin. // Animal genetics. – 1992. –
Т. 23. – №. 6. – P. 483-496.
Bai, L. Development of a new recombinant p24 ELISA system for diagnosis of bovine leukemia
virus in serum and milk [Text] / L. Bai, K. Yokoyama, S. Watanuki, H. Ishizaki,
S.N. Takeshima, Y. Aida. // Arch Virol. – 2019. – V. 164(1). – P. 201-211.
Saushkin, N.Y. Strip-dried blood sampling: applicability for bovine leukemia virus detection
with ELISA and real-time PCR. [Text] / N.Y. Saushkin, J.V. Samsonova, A.P. Osipov, S.E. Kondakov //
J Virol Methods. – 2019. – V. 263. – P.101-104.
Pluta, A., An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell
epitopes on the Gag protein of bovine leukemia virus [Text] / A. Pluta, T. M. Taxis, F. van der Meer, et
al. // Scientific reports. – 2023. – V. 13(1). – P. 22356.
Felmer, R. Association of bovine leukocyte antigen (BoLA) DRB3 alleles with resistance and
susceptibility to bovine leukemia virus infection in Holstein cattle [Text] / R. Felmer, et al. // Veterinary
Immunology and Immunopathology. – 2011. –V. 144(1-2). – P.102-110.
Rola-Łuszczak, M. Development of an improved real time PCR for the detection of bovine
leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results
[Text] / M. Rola-Łuszczak, C. Finnegan, M. Olech, B. Choudhury, J. Kuzmak. // J Virol Methods. –
– V.189. – P.258–264.
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REFERENSES
Aida, Y. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T
cell leukemia virus [Text] / Y. Aida, H. Murakami, M. Takahashi et all. // Front. Microbiol. – 2013. –
V.4. – P. 328. doi: 10.3389/fmicb.2013.00328.
Polat, M., Takeshima S.N., Aida Y. Epidemiology and genetic diversity of bovine leukemia virus
[Text] / M. Polat, S.N. Takeshima, Y. Aida. // Virol J. – 2017. – Vol. 14, Issue 1. –
P. 209-1-209-16.
Lefkowitz, E.J. Virus taxonomy: the database of the International Committee on Taxonomy of
Viruses (ICTV) [Text] / E.J. Lefkowitz, D.M. Dempsey, R.C. Hendrickson et al. // Nucleic Acids
Research. – 2018. – Vol. 46. – P. 708-717.
Buehring, G.C. Bovine leukemia virus DNA in human breast tissue [Text] / G.C. Buehring, H.M.
Shen, H.M. Jensen, K.Y. Choi, D.J. Sun, G. Nuovo. // Emerg Infect Dis. – 2014. – Vol. 20. –
P. 772–782. 10.3201/eid2005.131298
Hamada, R. Detection and Molecular Characterization of Bovine Leukemia Virus in Egyptian
Dairy Cattle [Text] / R. Hamada, S. Metwally, M. Polat, et. al. // Front Vet Sci. – 2020. – Vol. 7. –
P. 608-1-608-13.
Nekouei, O. Lifetime effects of infection with bovine leukemia virus on longevity and milk
production of dairy cows [Text] / O. Nekouei, J. Van Leeuwen, H. Stryhn, et. al. // Prev Vet Med. – 2016. – Vol. 133. – P. 1-9.
Yang, Y. Bovine leukemia virus infection in cattle of China: Association with reduced milk
production and increased somatic cell score [Text] / Y. Yang, W. Fan, Y. Mao, et. al. // J Dairy Sci. –
– Vol. 99, Issue 5. – P. 3688-3697.
Panei, CJ. Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte
subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV
CoCoMo-qPCR [Text] / C.J. Panei, S.N. Takeshima, T. Omori, T. Nunoya, W.C. Davis, H. Ishizaki,
K. Matoba, Y. Aida // BMC Vet Res. – 2013. – V9. – P. 95. doi: 10.1186/1746-6148-9-95.
Juliarena, M.A. Determination of proviral load in bovine leukemia virus-infected cattle with and
without lymphocytosis [Text] / M.A. Juliarena, S.E. Gutierrez, C. Ceriani // Am J Vet Res. – 2007.
Vol.68. – B.1220–1225.
Nakatsuchi, A. BoLA-DRB3 Polymorphism Controls Proviral Load and Infectivity of Bovine
Leukemia Virus (BLV) in Milk [Text] / A. Nakatsuchi, S. Watanuki, L. Borjigin, H. Sato,
L. Bai, R. Matsuura, M. Kuroda, H. Murakami, R. Sato, S. Asaji, A. Ando, Y. Matsumoto,
S. N. Takeshima, Y. Aida [Text] // Pathogens. – 2022. – Vol. 11. – № 2. – P. 210. DOI:
3390/pathogens11020210.
Xinyue. Wu Development of droplet digital PCR for quantification of bovine leukemia virus
proviral load using unpurified genomic DNA [Text] / Xinyue Wu, Kousuke Notsu, Yuichi Matsuura,
Shuya Mitoma, Hala El Daous, Junzo Norimine, Satoshi Sekiguchi. // J Virol Methods. – 2023. –
V. 315. – P.114706. doi: 10.1016/j.jviromet.2023.114706.
Sulav, Sh. Removing bovine leukemia virus-infected animals with high proviral load leads to
lower within-herd prevalence and new case reduction [Text] / Sulav Shrestha, Karin Orsel, Casey
Droscha, Sanjaya Mijar, Frank van der Meer. // J Dairy Sci. – 2024. – V.107(8). – P. 6015-6024.
Lew, A.E. Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease
PCR using a 3′ minor groove binder fluorogenic probe [Text] / A.E. Lew, R.E. Bock,
J.B. Molloy, C.M. Minchin, S.J. Robinson, P. Steer. //J Virol Methods. – 2004. – V.115. – P.167–175.
Martin, D. Comparative study of PCR as a direct assay and ELISA and AGID as indirect assays
for the detection of bovine leukaemia virus [Text] / D. Martin, A. Arjona, I. Soto, N. Barquero, M. Viana,
E. Gomez-Lucia // J Vet Med B Infect Dis Vet Public Health. – 2001. – V. 48. – V.97–106.
Takeshima, S N. Single nucleotide polymorphisms in the bovine MHC region of Japanese
Black cattle are associated with bovine leukemia virus proviral load [Text] / S.N. Takeshima,
S. Sasaki, P. Meripet, Y. Sugimoto, Y. Aida. // Retrovirology. – 2017. – V.14. – P. 24.
Frie, M.C. Bovine leukemia virus: a major silent threat to proper immune responses in cattle
[Text] / M.C. Frie, P.M. Coussens // Vet Immunol Immunopathol. – 2015. – № 163(3-4). –
P. 103-114.
Heenemann, K. Development of a bovine leukemia virus polymerase gene-based real-time
polymerase chain reaction and comparison with an envelope gene-based assay [Text] / K. Heenemann, S.
Lapp, J.P. Teifke, D. Fichtner, T.C. Mettenleiter, T.W. Vahlenkamp. // J Vet Diagn Invest. – 2012. – V.
– P.649–655. doi: 10.1177/1040638712447524
Van Eijk, M. J. T. Extensive polymorphism of the BoLA‐DRB3 gene distinguished by
PCR‐RFLP [Text] / M. J. T. Van Eijk, J. A. Stewart‐Haynes, H. A. Lewin. // Animal genetics. – 1992. –
T. 23. – №. 6. – P. 483-496.
Bai, L. Development of a new recombinant p24 ELISA system for diagnosis of bovine leukemia
virus in serum and milk [Text] / L. Bai, K. Yokoyama, S. Watanuki, H. Ishizaki,
S.N. Takeshima, Y. Aida. // Arch Virol. – 2019. – V. 164(1). – P. 201-211.
Saushkin, N.Y. Strip-dried blood sampling: applicability for bovine leukemia virus detection
with ELISA and real-time PCR. [Text] / N.Y. Saushkin, J.V. Samsonova, A.P. Osipov, S.E. Kondakov //
J Virol Methods. – 2019. – V. 263. – P.101-104.
Pluta, A., An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell
epitopes on the Gag protein of bovine leukemia virus [Text] / A. Pluta, T. M. Taxis, F. van der Meer, et
al. // Scientific reports. – 2023. – V. 13(1). – P. 22356.
Felmer, R. Association of bovine leukocyte antigen (BoLA) DRB3 alleles with resistance and
susceptibility to bovine leukemia virus infection in Holstein cattle [Text] / R. Felmer, et al. // Veterinary
Immunology and Immunopathology. – 2011. –V. 144(1-2). – P.102-110.
Rola-Łuszczak, M. Development of an improved real time PCR for the detection of bovine
leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results
[Text] / M. Rola-Łuszczak, C. Finnegan, M. Olech, B. Choudhury, J. Kuzmak. // J Virol Methods. –
– V.189. – P.258–264.
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