Skip to main content
Download PDF
  • Research Note
  • Open access
  • Published:

Development and application of a real-time polymerase chain reaction assay to detect lumpy skin disease virus belonging to the Kenyan sheep and goat pox group

BMC Research Notes volume 16, Article number: 247 (2023) Cite this article

Abstract

Lumpy skin disease (LSD) outbreaks in Southeast and South Asia are attributed to different lineages of LSD virus (LSDV). Variants belonging to the novel recombinant cluster 2.5 circulate in China and Thailand, while a Kenyan sheep and goat pox (KSGP) strain from cluster 1.1 circulates in India, Pakistan, and Bangladesh. The clusters representing these circulating strains are vastly different. However, if their distribution encroaches into each other’s ranges, it will be impossible to differentiate between them due to the lack of suitable molecular tools. Thus, fit-for-purpose molecular tools are in demand to effectively and timeously diagnose and investigate the epidemiology of LSDVs in a region. These could significantly contribute to the phylogenetic delineation of LSDVs and the development of preventive measures against transboundary spillovers. This work aimed to develop a real-time polymerase chain reaction assay targeting open reading frame LW032, capable of specifically detecting KSGP-related isolates and recombinant LSDV strains containing the KSGP backbone. The analytical specificity was proven against the widest possible panel of recombinant vaccine-like LSDV strains known to date. The amplification efficiency was 91.08%, and the assay repeatability had a cycle threshold variation of 0.56–1.1 over five repetitions across three runs. This KSGP-specific assay is reliable and fast and is recommended for use in LSDV epidemiological studies where the accurate detection of KSGP genetic signatures is a priority, particularly in regions where KSGP-like and other lineages are circulating.

Peer Review reports

Introduction

Lumpy skin disease (LSD) is a disease caused by the lumpy skin disease virus (LSDV), which belongs to the genus Capripoxvirus along with sheep pox virus (SPPV) and goat pox virus (GTPV), which are considered emerging pathogens that pose a significant threat to the global livestock industry [1]. All three diseases are listed as notifiable to the World Organization for Animal Health ( [2]. LSDV primarily affects cattle, while SPPV and GTPV affect sheep and goats, respectively [3]. Similar to other viruses in the Poxviridae family, LSDV has a brick-shaped structure containing a linear double-stranded DNA genome of approximately 151 kilo-base pairs (kbp) in length and encoding 156 open reading frames (ORFs). The genome consists of a central coding region flanked by identical 2.4 kbp-inverted terminal repeat regions [4].

LSD has been reported not only in cattle but also in water buffaloes and game animals, particularly antelopes in sub-Saharan Africa [5,6,7]. This poses a serious economic threat not only to the global cattle but also to the wildlife industries. This disease was first reported in Zambia in the 1920s and has been confined to the African continent for the majority of the 20th century [8]. However, in the last decade, the virus spread beyond the borders of Africa and was reported in the Middle East, Turkey, and Azerbaijan, reaching the European Union, the Balkans, Russia, and Kazakhstan in 2015. Subsequently, the disease was reported in China, Vietnam, Thailand, India, Bangladesh, and Nepal in 2019 [9,10,11,12,13].

The molecular characterization of the known LSDV isolates has led to their subdivision into nine clusters. Before 2017, the two main clusters were clusters 1.1 (Neethling) and 1.2 (Kenyan sheep and goat pox (KSGP)-like). Since 2017, molecular epidemiology has changed, with the first reports of novel LSDV recombinant vaccine-like variants responsible for outbreaks in Russia between 2017 and 2021 [14]. Since the description of Saratov/Russia/2017 (cluster 2.1) in 2017, additional full genome sequences of novel recombinants have been described, resulting in subclusters 2.1–2.5 [15,16,17]. The novel lineage first described in China in 2019 (cluster 2.5) is currently the dominant lineage circulating in Southeast Asia [18, 19].

Genetic evidence from the ongoing pandemics in India and Bangladesh revealed that the causative agent belongs to the KSGP-like subgroup within cluster 1.2 [20, 21]. Although the origin of this vaccine strain to the Indian subcontinent and the multiple recombinant strains in Southeast Asia remain elusive, the circulation of these strains raises concerns regarding the diagnostics and epidemiology in the region [22,23,24]. Currently, assays are available to detect LSDV at the species level and even to differentiate between vaccine and field strains. However, no assay has been reported to detect specifically KSGP-like strains [25, 26].

This study aimed to develop a specific real-time polymerase chain reaction (PCR) assay capable of detecting and differentiating KSGP-like genetic signatures. The sensitivity and specificity of the assay were evaluated using the largest available panel of LSDV strains. The assay was capable of selectively detecting not only KSGP but also recombinant strains containing the KSGP backbone.

Methods

Samples and viruses

A panel of 27 unique isolates representing all the identified genome clusters was used to validate the reported real-time PCR assay (Table 1).

Table 1 A panel of strains used for the assay specificity validation
Full size table

DNA extraction

Viral genomic DNA was extracted using the phenol–chloroform extraction protocol as described previously [27].

Primer design

The poly(A) polymerase catalytic subunit encoded by ORF LW032 was selected since it is conserved across the available sequences. No recombination event has yet been observed in this locus. Subsequently, the primers F 5-ACCCATGGTTTTATCCGTCA-3 and 5-TGAAGACATATCTAGCGTTTGTAAAGA-3 were designed to amplify a 610 bp region, while the probe FAM 5-[C-lna]GATGAAG[G-lna]TACAAACTTTTTCAC-3 BHQ-1 selectively annealed to KSGP-like strains. The alignment of the binding region of the probe is shown in Fig. 1.

Fig. 1
figure 1

An alignment of the primers and probe-binding region, containing all the available LSDV strains, representing the seven different clusters

Full size image

PCR protocol

PCR reaction setup, protocol, and statistical analysis where performed as previously described [28, 29]. We focused on the minimal critical parameters needed to ascertain the assay reliability: analytical specificity, analytical sensitivity (limit of detection), reaction efficiency, repeatability.

Analytical specificity

The specific detection of KSGP-like signatures was verified using DNA containing individual strain nucleic acids and a mixture of strain DNA mimicking the co-infection. All strains used for validation had a Ct value not higher than 30 to ensure the specificity. The assay did not produce any false positives when tested against sheep pox virus with a high titer (skin scabs and cell culture with an average Ct value of 13.11–17.12). Cross-reactivity against sheep pox virus is chosen due to the high genetic similarity across the Capripoxvirus genus [4].

Analytical sensitivity

The limit of detection (LOD) of the PCR assay was determined using a serial dilution of different LSDV genomic DNA, starting with the Udmurtiya/2019 virus strain containing a titer of 5.75 lg TCD50/cm3. LOD was defined as at least 95% positive replicates at the terminal dilution of 20 replicates tested [28]. Five tenfold dilutions were initially prepared, followed by three twofold serial dilutions. The reaction efficiency was determined from the slope using the following equation:

E = [10 (slope) − 1] × 100,

where E is the reaction efficiency, and E = 100 corresponds to 100% efficiency. The repeatability and coefficient of variation (CV) were assessed by examining the same five tenfold dilutions (Udmurtiya/2019, with a starting titer of 5.75 lg TCD50/cm3) in five repetitions on three different days. Statistic evaluation was performed using Statistica v.10 (StatSoft, Tulsa, OK, USA).

Results

Before evaluating the analytical characteristics of the assay, the specificity was first examined against a wide collection of naturally occurring recombinant vaccine-like strains and other capripoxviruses (Table 1). This is the most diverse panel of LSDV currently described, providing a unique opportunity to validate diagnostic tools for LSDV research. Since the probe annealing region differs by a few nucleotides between KSGP and non-KSGP strains, the probe contained locked nucleic acid bases to enhance the binding specificity. Therefore, the assay demonstrated specific detection of only target LSDV sequences. The latter is specific to the original KSGP virus DNA or DNA of recombinant strains whose major parental strain is KSGP (Table 1) [30]. No cross-reaction with related LSDV DNA or nontarget SPPV samples was detected (Table 1). Since KSGP-like strains of Indian origin were unavailable for the study, considering the conservative nature of poxvirus genomes [17], the assay is confidently deemed readily specific toward Indian KSGP.

The amplification efficiency over five orders of magnitude was 91.08%, with the variation ranging from 0.54 to 1.18 (Fig. 2).

Fig. 2
figure 2

Linear regression constructed over serial 10-fold dilutions. The equation of the standard curve obtained was y = 3.556x + 18.644, and the coefficient of determination (R2) was 0.9983

Full size image

The repeatability of the assay was assessed across three replicates, calculated by the percentage of total variance obtained from five repetitions of a single sample in one run. A low variation in CV was obtained: the SD and CV ranged from 0.56 to 1.1 and 1.87–3.64%, respectively, over five repetitions across three runs (Table 2). As for LOD, the assay sensitivity was found to be the fifth tenfold dilution with a titer of 0.75 lg TCD50/cm3.

Table 2 Repeatability of the assay in one run (5 repetitions) and across 3 runs (15 repetitions)
Full size table

Discussion

LSD can be successfully controlled by proper diagnostic tools [31]. Recently, a few lineage-specific assays have been reported. Along with the pan LSDV assay by Sprygin [25], the PCR assay as a DIVA (Differentiating Infected from Vaccinated Animals) strategy has also been developed and validated on clinical samples [32,33,34]. The ORF LW008 vaccine assay can detect Neethling vaccine DNA and DNA of some recombinant vaccine-like strains [31, 35], whereas the GPCR vaccine assay targets Neethling DNA, KSGP DNA, and DNA of some recombinant vaccine-like strains, which significantly limits the application of these assays in regions where recombinant vaccine-like LSDV is present [36]. Haegeman A. et al., 2023 published the first manuscript on development and validation of a new DIVA qPCR for differentiation between the Neethling vaccine strain from the LSDV recombinant strains currently circulating in Asia [37]. With the emergence of recombinant vaccine-like strains of LSDV in Southeast Asia coupled with one more pandemic with another lineage within cluster 1.2 (KSGP) in India and Bangladesh, it is crucial to have reliable and thoroughly validated laboratory instruments for timely detection and diagnosis [38]. The protocol developed by Haegeman A. et al., is capable on identifying recombinant isolates that have Neethling genome as a major parental backbone, but doesn’t fit for recombinant isolates with KSGP-vaccine genome as a major parental backbone.

While the risks of mutual spillovers are high in countries located at the interface of affected regions, followed by coinfection of animals, the lack of instruments to differentiate between cluster 2.5 and cluster 1.2 KSGP subcluster would have a profound influence on the understanding and control of LSD in Southeast and South Asia [17]. Interestingly, the Kenyan strain lineage was restricted to some African regions in the past, so its molecular detection was not the priority, and then it fell within the field cluster 1.2 [18]. However, a recent resurgence of recombinant LSDV whose parental strain is KSGP and the concomitant rise of KSGP-related outbreaks in South Asia showed an objective need for such tools [21]. In this study, a specific PCR assay capable of detecting KSGP genetic signatures was developed (Tables 1 and 2). Notably, the assay was proved to be KSGP-specific regardless of whether it is a recombinant strain with the KSGP backbone, such as Kurgan/2018 or Udmurtiya/2019, or the original KSGP strain (Table 2). Unfortunately, KSGP-like strains of Indian origin were unavailable for the study. However, considering the conservative nature of poxvirus genomes [39], this assay will readily differentiate Indian LSDV strains as well. The performance characteristics, such as analytical sensitivity and amplification efficiency, were validated according to recommended Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [28], OIE Validation Guideline [29] and compared well with reported assays.

This KSGP assay opens up new possibilities in molecular diagnostics. In combination with the ORF126 assay that targets isolates from cluster 1.2, the differentiation of field isolates into subclusters is now accessible: one including Dagestan/2015, Serbia, Warmbaths, and Israel [17] and the other including KSGP, Indian LSDV [20], Bangladesh LSDV [21], and recombinants (Kurgan/2018, Samara/2018, and Udmurtiya/2019) (Table 2). As a complementary add-on to this tool, since the Kenyan Lumpivax contains KSGP, this tool can be used for DIVA if this KSGP-based vaccine is administered.

In this work, we developed a novel PCR assay against a panel of unique LSDV strains available in FGBI ARIAH (Vladimir, Russia), that can specifically detect KSGP-like genetic signatures. Not only outbreaks can be investigated, but a DIVA approach (differentiation infected animals from vaccinated animals) can also be implemented with particular regard to the Kenyan vaccine (KSGP strain), the use of which precipitated the emergence of all currently known recombinant stains [17]. This new tool will significantly improve the molecular epidemiological studies tracking the ongoing spread of different LSDV lineages, which is essential for the understanding of LSDV transmission throughout affected regions and in tandem with the assay by Haegeman et al. (2023) more detailed information on the molecular epidemiology of LSDV worldwide will be gained. Overall, this assay holds promise and is recommended for use in countries where the risks of KSGP-related outbreaks can occur or where the KSGP based vaccine is in use. In the future, this assay will be validated and tested on a larger collection of samples to estimate such parameters as the diagnostic sensitivity and diagnostic specificity, where WOAH guidelines 3.2.6 will be considered as reference for research methodology [29].

Limitations

This assay demonstrated good specificity based on the available samples. Samples of LSDV DNA from India and Bangladesh, where the target lineage is circulating also, would complement the specificity testing and guarantee that the proposed assay perfectly fit for purpose.

Data Availability

All data are presented within the manuscript.

Abbreviations

LSD:

Lumpy skin disease

LSDV:

LSD virus

KSGP:

Kenyan sheep and goat pox

SPPV:

sheep pox virus

GTPV:

goat pox virus

kbp:

kilobase pairs

ORF:

open reading frame

LOD:

limit of detection

CV:

coefficient of variation (CV)

TCD50:

50% Tissue Culture Infectious Dose

R2 :

coefficient of determination

DIVA:

Differentiating Infected from Vaccinated Animals

BHQ-1:

Black Hole Quencher-1

Ct:

threshold cycle

FAM:

6-carboxy fluorescein

LNA:

Locked Nucleic Acid

References

  1. Babiuk S, Bowden TR, Boyle DB, Wallace DB, Kitching RP, Capripoxviruses. An emerging worldwide threat to sheep,goats and cattle. Transbound Emerg Dis. 2008;55:263–72.

    Article  PubMed  CAS  Google Scholar 

  2. OIE—Listed, Diseases. Infections and Infestations in Force in 2021. OIE—Terrestrial Animal Health Code, Twenty-Eighth Edition.2019. Available online: https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/2.04.13_LSD.pdf) (accessed on14 November 2022).

  3. Hamdi J, Munyanduki H, Omari Tadlaoui K, El Harrak M. Fassi Fihri, O. Capripoxvirus Infections in Ruminants. Rev Microorganisms. 2021;23:902.

    Article  Google Scholar 

  4. Tulman ER, Afonso CL, Lu Z, Zsak L, Kutish GF, Rock DL. Genome of lumpy skin disease virus. J Virol. 2001;75:7122–30.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Ahmed EM, Eltarabilli MMA, Shahein MA, Fawzy M. Lumpy skin disease outbreaks investigation in egyptian cattle andbuffaloes: serological evidence and molecular characterization of genome termini. Comp Immunol Microbiol Infect Dis. 2021;76:101639.

    Article  PubMed  CAS  Google Scholar 

  6. Hasib FMY, Islam MS, Das T, Rana EA, Uddin MH, Bayzid M, Nath C, Hossain MA, Masuduzzaman M, Das S, et al. Lumpy skin disease outbreak in cattle population of Chattogram. Bangladesh Vet Med Sci. 2021;7:1616–24.

    Article  PubMed  Google Scholar 

  7. Molini U, Boshoff E, Niel A, Phillips J, Khaiseb S, Settypalli T, Dundon W, Cattoli G, Lamien C. Detection of LumpySkin Disease Virus in an asymptomatic Eland (Taurotragus oryx) in Namibia. J Wildl Dis. 2021;57:708–11.

    Article  PubMed  CAS  Google Scholar 

  8. Alkhamis MA, VanderWaal K. Spatial and temporal epidemiology of lumpy skin disease in the Middle East, 2012–2015. Front Vet Sci. 2016;3:19.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Agianniotaki E, Tasioudi K, Chaintoutis S, Iliadou P, Mangana-Vougiouka O, Kirtzalidou A, Alexandropoulos T, Sachpatzidis A, Plevraki E, Dovas C, et al. Lumpy skin disease outbreaks in Greece during 2015–16, implementation ofemergency immunization and genetic differentiation between field isolates and vaccine virus strains. Vet Microbiol. 2017;201:78–84.

    Article  PubMed  Google Scholar 

  10. Khan YR, Ali A, Hussain K, Ijaz M, Rabbani AH, Khan RL, Abbas SN, Aziz MU, Ghaffar A, Sajid HA. A review:Surveillance of lumpy skin disease (LSD) a growing problem in Asia. Microb Pathog. 2021;158:105050.

    Article  PubMed  Google Scholar 

  11. Kumar N, Chander Y, Kumar R, Khandelwal N, Riyesh T, Chaudhary K, Shanmugasundaram K, Kumar S, Ku-mar A, Gupta M et al. Isolation and characterization of lumpy skin disease virus from cattle in India. PLoS ONE 2021,16, e0241022.

  12. Lu G, Xie J, Luo J, Shao R, Jia K, Li S. Lumpy skin disease outbreaks in China, since 3 August 2019. Transbound Emerg Dis. 2020;68:216–9.

    Article  PubMed  Google Scholar 

  13. Sprygin A, Babin Y, Pestova Y, Kononova S, Byadovskaya O, Kononov A. Complete genome sequence of the lumpy skin disease virus recovered from the first outbreak in the Northern Caucasus Region of Russia in 2015. Microbiol Resour Announc. 2019;8:e01733–18.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Sprygin A, Babin Y, Pestova Y, Kononova S, Wallace DB, Van Schalkwyk A, Kononov A. Analysis and insights into recombination signals in lumpy skin disease virus recovered in the field. PLoS ONE. 2018;13:e0207480.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Sprygin A, Van Schalkwyk A, Shumilova I, Nesterov A, Kononova S, Prutnikov P, Byadovskaya O, Kononov A. Full-length genome characterization of a novel recombinant vaccine-like lumpy skin disease virus strain detected during the climaticwinter in Russia, 2019. Arch Virol. 2020;165:2675–7.

    Article  PubMed  CAS  Google Scholar 

  16. Sprygin A, Pestova Y, Bjadovskaya O, Prutnikov P, Zinyakov N, Kononova S, Ruchnova O, Lozovoy D, Chvala I, Kononov A. Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease.PLoS ONE. 2020, 13, e0232584.

  17. Krotova A, Byadovskaya O, Shumilova I, van Schalkwyk A, Sprygin A. An in-depth bioinformatic analysis of thenovel recombinant lumpy skin disease virus strains: from unique patterns to established lineage. BMC Genom. 2022;23:396.

    Article  CAS  Google Scholar 

  18. Krotova A, Mazloum A, Byadovskaya O, Sprygin A. Phylogenetic analysis of lumpy skin disease virus isolates in Russia in2019–2021. Arch Virol. 2022;167:1693–9.

    Article  PubMed  CAS  Google Scholar 

  19. Wang J, Xu Z, Wang Z, Li Q, Liang X, Ye S, Li S. Isolation, identification and phylogenetic analysis of lumpy skin diseasevirus strain of outbreak in Guangdong, China. Transbound Emerg Dis. 2022;69:e2291–301.

    Article  PubMed  CAS  Google Scholar 

  20. Sudhakar SB, Mishra N, Kalaiyarasu S, Jhade SK, Singh VP. Genetic and phylogenetic analysis of lumpy skin disease viruses (LSDV) isolated from the first and subsequent field outbreaks in India during 2019 reveals close proximity with unique signatures of historical kenyan NI-2490/Kenya/KSGP-like field strains. Transbound Emerg Dis. 2022;69(4):e451–62. https://doi.org/10.1111/tbed.14322.

    Article  PubMed  Google Scholar 

  21. Badhy SC, Chowdhury MGA, Settypalli TBK, Cattoli G, Lamien CE, Fakir MAU, Akter S, Osmani MG, Talukdar F, Begum N, Khan IA, Rashid MB, Sadekuzzaman M. Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains. BMC Vet Res. 2021;17(1):61. https://doi.org/10.1186/s12917-021-02751-x.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Wang J, Xu Z, Wang Z, Li Q, Liang X, Ye S, Cheng K, Xu L, Mao J, Wang Z, Meng W, Sun Y, Jia K, Li S. Isolation, identification and phylogenetic analysis of lumpy skin disease virus strain of outbreak in Guangdong, China. Transbound Emerg Dis. 2022;69(5):e2291–301. https://doi.org/10.1111/tbed.14570.

    Article  PubMed  CAS  Google Scholar 

  23. Ma J, Yuan Y, Shao J, Sun M, He W, Chen J, Liu Q. Genomic characterization of lumpy skin disease virus in southern China. Transbound Emerg Dis. 2022;69(5):2788–99. https://doi.org/10.1111/tbed.14432.

    Article  PubMed  CAS  Google Scholar 

  24. Maw MT, Khin MM, Hadrill D, Meki IK, Settypalli TBK, Kyin MM, Myint WW, Thein WZ, Aye O, Palamara E, Win YT, Cattoli G, Lamien CE. First Report of Lumpy skin disease in Myanmar and Molecular Analysis of the Field Virus isolates. Microorganisms. 2022;10(5):897. https://doi.org/10.3390/microorganisms10050897.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Alexander S, Olga B, Svetlana K, Valeriy Z, Yana P, Pavel P, Aleksandr K. A real-time PCR screening assay for the universal detection of lumpy skin disease virus DNA. BMC Res Notes. 2019;12(1):371. https://doi.org/10.1186/s13104-019-4412-z.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Haegeman A, De Vleeschauwer A, De Leeuw I, Vidanović D, Šekler M, Petrović T, Demarez C, Lefebvre D, De Clercq K. Overview of diagnostic tools for Capripox virus infections. Prev Vet Med. 2020;181:104704. https://doi.org/10.1016/j.prevetmed.2019.104704.

    Article  PubMed  Google Scholar 

  27. Sprygin A, Mazloum A, Van Schalkwyk A, Krotova A, Shalina K, Dmitric M, Byadovskaya O, Prokhvatilova L. Chvala, I. The development of a real-time PCR assay for specific detection of the NISKHI Sheep Pox Vaccine Virus strain DNA. Appl Microbiol. 2022;2:956–64.

    Article  Google Scholar 

  28. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–22. https://doi.org/10.1373/clinchem.2008.112797.

    Article  PubMed  CAS  Google Scholar 

  29. OIE—Development and optimization of nucleic acid detection assays., Validation Guideline 3.6.3., Version adopted by the World Assembly of Delegates of the OIE in May 2014 Available online: https://www.woah.org/fileadmin/Home/eng/Health_standards/aahm/current/GUIDELINE_3.6.3_NAD_ASSAYS.pdf (accessed on14 November 2022).

  30. Krotova A, Mazloum A, van Schalkwyk A, Prokhvatilova L, Gubenko O, Byadovskaya O, Chvala I, Sprygin A. The characterization and differentiation of recombinant lumpy skin Disease isolates using a region within ORF134. Appl Microbiol. 2023;3:35–44. https://doi.org/10.3390/applmicrobiol3010003.

    Article  Google Scholar 

  31. Byadovskaya O, Pestova Y, Kononov A, Shumilova I, Kononova S, Nesterov A, Babiuk S, Sprygin A. Performance of the currently available DIVA real-time PCR assays in classical and recombinant lumpy skin disease viruses. Transbound Emerg Dis. 2021;68(6):3020–4. https://doi.org/10.1111/tbed.13942.

    Article  PubMed  CAS  Google Scholar 

  32. Vidanović D. Šekler, Milanko, Petrović, Tamaš, Debeljak, Zoran, Vasković, Nikola, Matović, Kazimir and Hoffmann, Bernd. “Real-Time PCR Assays for the Specific Detection of Field Balkan Strains of Lumpy Skin Disease Virus” Acta Veterinaria, vol.66, no.4, 2016, pp.444–454. https://doi.org/10.1515/acve-2016-0038.

  33. Vidanović D, Tešović B, Šekler M, Debeljak Z, Vasković N, Matović K, Koltsov A, Krstevski K, Petrović T, De Leeuw I, Haegeman A. Validation of TaqMan-Based assays for specific detection and differentiation of wild-type and neethling vaccine strains of LSDV. Microorganisms. 2021;9(6):1234. https://doi.org/10.3390/microorganisms9061234.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Sprygin AV, Pestova YE, Kostrova ES, Kononova SV, Byadovskaya OP, Zhbanova TV, Kononov AV. One-run real time PCR assays for the detection of capripoxviruses, field isolates and vaccine strains of lumpy skin disease virus. Sel’Skokhozyaistvennaya Biologiya. 2019;54(2):347–58.

    Article  Google Scholar 

  35. Sprygin A, Pestova Y, Prutnikov P, Kononov A. Detection of vaccine-like lumpy skin disease virus in cattle and Musca domestica L. flies in an outbreak of lumpy skin disease in Russia in 2017. Transbound Emerg Dis. 2018;65(5):1137–44. https://doi.org/10.1111/tbed.12897.

    Article  PubMed  CAS  Google Scholar 

  36. Agianniotaki EI, Chaintoutis SC, Haegeman A, Tasioudi KE, De Leeuw I, Katsoulos PD, Sachpatzidis A, De Clercq K, Alexandropoulos T, Polizopoulou ZS, Chondrokouki ED, Dovas CI. Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains. J Virol Methods. 2017;249:48–57. https://doi.org/10.1016/j.jviromet.2017.08.011.

    Article  PubMed  CAS  Google Scholar 

  37. Haegeman A, De Leeuw I, Philips W, De Regge N. Development and Validation of a New DIVA Real-Time PCR Allowing to Differentiate Wild-Type Lumpy Skin Disease Virus Strains, Including the Asian Recombinant Strains, from Neethling-Based Vaccine Strains. Viruses 2023, 15, 870. https://doi.org/10.3390/v15040870.

  38. Arjkumpa O, Suwannaboon M, Boonrod M, Punyawan I, Liangchaisiri S, Laobannue P, Lapchareonwong C, Sansri C, Kuatako N, Panyasomboonying P, Uttarak P, Buamithup N, Sansamur C, Punyapornwithaya V. The First Lumpy skin Disease Outbreak in Thailand (2021): epidemiological features and spatio-temporal analysis. Front Veterinary Sci. 2022;8:799065. https://doi.org/10.3389/fvets.2021.799065.

    Article  Google Scholar 

  39. Roy P, Jaisree S, Balakrishnan S, Senthilkumar K, Mahaprabhu R, Mishra A, Maity B, Ghosh TK, Karmakar AP. Molecular epidemiology of goat pox viruses. Transbound Emerg Dis. 2018;65(1):32–6. https://doi.org/10.1111/tbed.12763.

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

This work was supported by the grant no. 075-15-2021-1054 from the Ministry of Education and Science of Russia to implement objectives of the Federal Scientific and Technical Program for the Development of genetic technologies during 2019–2027.

Author information

Authors and Affiliations

  1. Federal Center for Animal Health, Vladimir, Russia

    Alexander Sprygin, Ali Mazloum, Alena Krotova, Olga Bydovskaya, Larisa Prokhvatilova & Ilya Chvala

  2. Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa

    Antoinette Van Schalkwyk

  3. Department of Biotechnology, University of the Western Cape, Bellville, South Africa

    Antoinette Van Schalkwyk

Authors
  1. Alexander Sprygin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ali Mazloum
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Antoinette Van Schalkwyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alena Krotova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Olga Bydovskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Larisa Prokhvatilova
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ilya Chvala
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SA contributed to designing the primers and probe, planning out the experimental evaluation, and contributed to the analysis of data and writing of the manuscript. AM, AK, AvS contributed to the analysis of data and writing of the manuscript. AK contributed to performing the DNA isolation as well as performed the real-time PCR. AS, AM and AvS wrote the original draft and performed editing. OB, LP and IC project administration. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Alexander Sprygin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All methods were performed in accordance with the relevant guidelines and regulations. No work with live animals was conducted in this study. All protocols with DNA extraction procedures and PCR testing were approved before implementation by the Ethics Committee of the Federal Center for Animal Health (Vladimir, Russia).

Consent for publication

Not applicable.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sprygin, A., Mazloum, A., Van Schalkwyk, A. et al. Development and application of a real-time polymerase chain reaction assay to detect lumpy skin disease virus belonging to the Kenyan sheep and goat pox group. BMC Res Notes 16, 247 (2023). https://doi.org/10.1186/s13104-023-06502-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13104-023-06502-z

Keywords

BMC Research Notes

ISSN: 1756-0500

Contact us