doi: 10.15389/agrobiology.2018.2.422eng

UDC 636.2:619:57.083.2:577.2

 

REAL TIME PCR FOR THE DETECTION OF FIELD ISOLATES OF
LUMPY SKIN DISEASE VIRUS IN CLINICAL SAMPLES FROM CAT-TLE

Ya.E. Pestova, E.E. Artyukhova, E.E. Kostrova, I.N. Shumoliva, A.V. Kononov, A.V. Sprygin

Federal Center for Animal Health Control, FGBU VNIIZZh, mkr. Yurievets, Vladimir, 600901 Russia, e-mail kononov@arriah.ru, spriginav@mail.ru (✉ corresponding author);

ORCID:
Pestova Ya.E. orcid.org/0000-0002-7974-623X
Shumoliva I.N. orcid.org/0000-0002-4663-3845
Artyukhova E.E. orcid.org/0000-0003-2963-1415
Sprygin A.V. orcid.org/0000-0001-5982-3675
Kostrova E.E. orcid.org/0000-0001-7150-7632

Received July 6, 2017

 

Lumpy skin disease caused by lumpy skin disease virus (LSDV, Capripoxvirus, Poxviridae) is a capripoxviral disease with significant morbidity in cattle, which necessitates the development of reliable diagnostic tools in the context of live vaccine administration. OIE-recommended PCR assays target not only LSDV but also sheep pox virus and goat pox virus. Conventional PCR is prone to carry-over contamination, whereas real-time PCR offers more advantages, including prevention of amplicon carryover contamination post amplification. In this paper we report the development of a PCR real time assay for the detection of field isolates of lumpy skin disease virus in clinical samples from cattle. The specificity was validated against a panel of homologous and heterologous viruses retrieved from the strain depository of FGBI ARRIAH. The PCR assay was shown to be highly specific toward field LSDV. When tested in the presence of vaccine strain DNA and related capripoxviruses, no false-positive results were obtained. Using a series of 10-fold dilutions the assay proved to be highly sensitive with a detection limit of 0.21 lg TCD50/ml. The calculated efficiency of amplification was 98.6 %, with SD ranging from 0.11 to 0.33 over five orders of magnitude. The PCR assay was also validated on samples from experimentally inoculated bulls. The animals received a subcutaneous injection of a field LSDV and were tested for the presence of LSDV DNA in blood and nasal swab in comparison to PCR by D.C. Ireland и Y.S. Binepal (1998) (data not shown). Overall, the presented assay demonstrated high specificity and sensitivity and can be recommended as a diagnostic tool for the detection of field isolated of LSDV.

Keywords: lumpy skin disease, diagnostics, real-time PCR, genome, virus.

 

Full article (Rus)

Full article (Eng)

 

REFERENCES

  1. Tuppurainen E.S., Venter E.H., Shisler J.L., Gari G., Mekonnen G.A., Juleff N., Lyons N.A., De Clercq K., Upton C., Bowden T.R., Babiuk S., Babiuk L.A. Capripoxvirus diseases: current status and opportunities for control. Transbound. Emerg. Dis., 2017, 64: 729-745 CrossRef
  2. Beard P.M. Lumpy skin disease: a direct threat to Europe. Vet. Rec., 2016, 28: 557-558 CrossRef
  3. Sevik M., Dogan M. Epidemiological and molecular studies on lumpy skin disease outbreaks in Turkey during 2014-2015. Transbound. Emerg. Dis., 2016, 64(4): 1268-1279 CrossRef
  4. Abutarbush S.M., Ababneh M.M., Al Zoubi I.G., Al Sheyab O.M., Al Zoubi M.G., Alekish M.O., Al Gharabat R.J. Lumpy skin disease in Jordan: disease emergence, clinical signs, complications and preliminary-associated economic losses. Transbound. Emerg. Dis., 2015, 62(5): 549-554 CrossRef
  5. Mishchenko A.V., Karaulov A.K.  Mishchenko V.A. Veterinariya, 2016, 4: 3-6 (in Russ.).   
  6. Tasioudi K.E., Antoniou S.E., Iliadou P., Sachpatzidis A., Plevraki E., Agianniotaki E.I., Fouki C., Mangana-Vougiouka O., Chondrokouki E., Dile C. Emergence of lumpy skin disease in Greece, 2015. Transbound. Emerg. Dis., 2016, 63(3): 260-265 CrossRef
  7. Mercier A., Arsevska E., Bournez L., Bronner A., Calavas D., Cauchard J., Falala S., Caufour P., Tisseuil C., Lefrançois T., Lancelot R. Spread rate of lumpy skin disease in the Balkans, 2015-2016. Transbound. Emerg. Dis., 2017, 00: 1-5 CrossRef 
  8. Tulman E.R., Afonso C.L., Lu Z., Zsak L., Kutish G.F., Rock D.L. Genome of lumpy skin disease virus. J. Virol., 2001, 75(15): 7122-7130 (doi:  10.1128/JVI.75.15.7122-7130.2001).
  9. Chihota C.M., Rennie L.F., Kitching R.P., Mellor P.S. Mechanical transmission of lumpy skin disease virus by Aedes aegypti (Diptera: Culicidae). Epidemiol. Infect., 2001, 126: 317-321 CrossRef
  10. Irons P.C., Tuppurainen E.S., Venter E.H. Excretion of lumpy skin disease virus in bull semen. Theriogenology, 2005, 63: 1290-1297 CrossRef
  11. Annandale C.H., Holm D.E., Ebersohn K., Venter E.H. Seminal transmission of lumpy skin disease virus in heifers. Transbound. Emerg. Dis., 2013, 61: 443-448 CrossRef
  12. Tuppurainen E.S., Lubinga J.C., Stoltsz W.H., Troskie M., Carpenter S.T., Coetzer J.A., Venter E.H., Oura C.A. Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks. Ticks Tick Borne Dis., 2013, 4: 329-333 CrossRef
  13. Lubinga J.C., Tuppurainen E.S., Mahlare R., Coetzer J.A., Stoltsz W.H., Venter E.H. Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks. Transbound. Emerg. Dis., 2013, 62: 174-182 CrossRef
  14. Makarov B.V., Grubyi V.A., Gruzdev K.N., Sukharev O.I. Spisok MEB i transgranichnye infektsii zhivotnykh: monografiya [OIE List and transboundary animal infections: monograph]. Moscow, 2012: 76-79 (in Russ.).  
  15. Beard P., Lubisi B.A. Manual of diagnostic tests and vaccines for terrestrial animals 2017. Chapter 2.4.13. Lumpy skin disease. Available http://www.oie.int/fileadmin/Home/eng/Health_stand-ards/tahm/2.04.13_LSD.pdf. Accessed December 21, 2017.
  16. Abera Z., Degefu H., Gari G., Ayana Z. Review on epidemiology and economic importance of lumpy skin disease. International Journal of Basic and Applied Virology, 2015, 4(1): 8-21 CrossRef
  17. Mishchenko A.V., Mishchenko V.A., Kononov A.V., Shevkoplyas V.N., Dzhailidi G.A., Dresvyannikova S.G., Chernykh O.Yu. Veterinariya Kubani, 2015, 5: 3-6 (in Russ.).   
  18. Ireland D.C., Binepal Y.S. Improved detection of capripoxvirus in biopsy samples by PCR. J. Virol. Methods, 1998, 74: 1-7 CrossRef
  19. Bowden T.R., Babiuk S.L., Parkyn G.R., Copps J.S., Boyle D.B. Capripoxvirus tissue tropism and shedding: a quantitative study in experimentally infected sheep and goats. Virology, 2008, 371(2): 380-393 CrossRef
  20. Lamien C.E., Lelenta M., Goger W., Silber R., Tuppurainen E., Matijevic M., Luckins A.G., Diallo A. Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses. J. Virol. Methods, 2011, 171: 134-140 CrossRef
  21. Kononova S.V., Shumilova I.N., B'yadovskaya O.P. Metodicheskie rekomendatsii po vydeleniyu virusa nodulyarnogo dermatita krupnogo rogatogo skota v kul'ture kletok: metodicheskii material [Guidelines on the isolation of the nodular dermatitis virus in cattle cell culture]. Moscow, 2017: 5-17 (in Russ.).   
  22. Orlova E.S., Shcherbakov A.V., Diev V.I., Zakharov V.M. Molekulyarnaya biologiya, 2006, 40(1): 158-164 (in Russ.).    
  23. Menasherow S., Rubinstein-Giuni M., Kovtunenko A., Eyngor Y., Fridgut O., Rotenberg D., Khinich Y., Stram Y. Development of  an assay to differentiate between virulent and vaccine  strains of  lumpy skin disease virus (LSDV). J. Virol. Methods, 2014, 199: 95-101 CrossRef
  24. Sevik M., Avci O., Dogan M., Ince Ö.B. Serum biochemistry of lumpy skin disease virus-infected cattle. Biomed. Res. Int., 2016, 2016: 6257984 CrossRef
  25. Balinsky C.A., Delhon G., Smoliga G., Prarat M., French R.A., Geary S.J., Rock D.L., Rodriguez L.L. Rapid preclinical detection of sheep pox virus by a real-time PCR assay. J. Clin. Microbiol., 2008, 46: 438-442 CrossRef

back