RPO30 Gene based PCR for Detection and Differentiation of Lumpy Skin Disease Virus and Sheep Poxvirus Field and Vaccinal Strains

Sherin Reda Rouby

Department of Veterinary Medicine, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.

Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | March 13, 2018; Accepted | March 30, 2018; Published | April 12, 2018

*Correspondence | Sherin Reda Rouby, Department of Veterinary Medicine, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt; Email: sh_alrouby@yahoo.com, shereen.rouby@vet.bsu.edu.eg

Citation | Rouby, S.R. 2018. RPO30 gene based PCR for detection and differentiation of lumpy skin disease virus and sheep poxvirus field and vaccinal strains. Veterinary Sciences: Research and Reviews, 4(1): 1-8.

DOI | http://dx.doi.org/10.17582/journal.vsrr/2018.4.1.1.8

Keyword | Capripoxviruses, LSDV, PCR, RP030 gene, SPPV

Introduction

Lumpy skin disease virus (LSDV), sheep pox (SPPV) and goat pox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae family are causative agents of Lumpy skin disease (LSD), sheep pox and goat pox respectively (Buller et al., 2005; Diallo and Viljoen, 2007), which are important endemic diseases in Egypt. They are now expanding their territory. LSDV was first recognized in 1929 originating in sub-Saharan Africa. The endemic geographic range of LSDV was restricted to the continent of Africa (Woods, 1988), in the past decade, new incursion of LSDV have been reported in the Middle Eastern, European and west Asian regions (Wainwright et al., 2013; Tageldin et al., 2014; Al-Salihi and Hassan, 2015; Ripani and Pacholek, 2015; OIE, 2016). There are obvious differences between the geographical distribution of SPPV and LSDV. Sheep pox and goat pox are found in parts of Africa (north of the equator), Asia, the Middle East, and most of the Indian subcontinent (Asagba and Nawathe, 1981; Mondal et al., 2004; Bhanuprakash et al., 2005).

Pox lesions are considered the most characteristic features of Capripoxvirus infection. LSD is characterized by fever, nodules on the skin, mucous membranes and internal organs, enlargement of lymph nodes, edema of the skin, and sometimes death (Coetzer, 2004; Babiuk et al., 2008). The disease is associated with significant production losses as a result of decreased weight gain, reduced milk yield, damaged to hides and wool. LSDV is an occasionally fatal disease of cattle with a morbidity averaging 10% and usually ranges from 3% to 85% (Thomas and Mare, 1945) and mortality (1–3%) in affected herds but may sometimes reach 40% (Coetzer, 2004; Rouby, 2010) and over 75% as reported by Diesel (1949). In contrast sheep pox is considered a direct cause of death at any stage of the disease (Yeruham et al., 2007). The overall mortality is usually lower than 10% but case fatality rates can be reach 100% in some young animals (Rao and Bandyopadhyay, 2000; Bhanuprakash et al., 2006). In newly imported and highly susceptible flocks high morbidity and mortality rates approaching 100% can be occur and range from 1% to 75% in indigenous breeds. Sheep pox is characterized by fever, widespread pox lesions throughout the skin and mucous membranes, rhinitis, conjunctivitis, and respiratory distress. Production losses are similar to LSD with decreased weight gain and damage to hide and also due to increased abortion rates, and increased susceptibility to pneumonia and fly strike (Babiuk et al., 2008).

Since capripoxviruses cause heavy economic losses among their hosts, it is crucially important to develop fast and specific diagnostic tests. Capripoxviruses are double-stranded DNA viruses with genomes approximately 150 kbp in size (Tulman et al., 2002). Capripoxvirus isolates are extremely conserved with genome identities of at least 96% between LSDV, SPPV, and GTPV (Tulman et al., 2002). Using routine laboratory tests Capripoxviruses cannot be distinguished (Kitching, 1986; Davies and Otema, 1981). Polymerase chain reaction (PCR) offers a rapid and sensitive diagnostic technique for capripoxvirus genome detection (Ireland and Binepal, 1998; Heine et al., 1999). Several PCR assays are currently available for differentiation between LSDV and SPPV, however, they target only one viral species (Stram et al., 2008; Orlova et al., 2006) or prerequisite sequence analyses (Hosamani et al., 2004; Cao et al., 1995). RPO30 gene based PCR designated by (Lamien et al., 2011a) provides a simple and quick differentiation between LSDV and SPPV in one step and is cost effective PCR. The present study elucidates the use of RPO30 gene based PCR for identification of LSDV from different clinical samples and differentiation between LSDV and SPPV recovered from naturally infected animals in Egypt along with the most commonly used vaccine strains in Egypt.

Material and Methods

Sample collection

Between May 2015 and August 2016, a total of 16 different clinical samples were collected from clinically LSD diseased animals during different outbreaks in Beni-suef governorate, Egypt together with three scab samples collected from sheep pox suspected field cases. Data of samples collection are illustrated in Table 1.

Virus strains and vaccines

Tissue culture-adapted cattle LSDV/Ismailyia88 strain was kindly supplied from the Pox Vaccine Production and Research Department, Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, Egypt. This strain was used as a positive control. Sheep poxvirus vaccine was obtained from VSVRI, prepared in Vero cell line and has a titre of 104.5 TCID50/ml. It was supplied in lyophilized ampoules; each of them contained 100 doses. Sheep pox vaccine (RM 65 strain): Sheep poxvirus vaccine (RM65 strain) produced by Jordan Bio- Industries Center (JOVAC) in Jordan prepared in lamb kidney cells and has a titer of 103.5 TCID50/ml was used in this study.

DNA extraction

Skin nodules, scab samples and ticks were triturated and homogenized with 50% phosphate buffered saline and made into 10% suspension. Extraction of viral DNA was performed using a DNA Mini Kit (Thermo, Germany) according to manufacturer’s instructions. DNA from cattle buffy coat, lymph aspirate and tissue culture-adapted cattle LSDV/Ismailyia88 strain were directly extracted using a DNA Mini Kit (Thermo, Germany) according to manufacturer’s instructions. Lyophilized Sheep pox vaccines were

Table 1: Data of samples collection

Table 2: Primer set for PCR

PCR run was performed according to Lamien et al. (2011a) using primer set targeting RP030 gene (Table 2). PCR was carried out in a 25 μL reaction volume in a 200 μL capacity PCR tube containing 12.5 μL PCR Master Mix (Jena bioscience Gmbh, Germany), 1 μL of each primer (20 pmol/μL), 4 μL of extracted DNA and 6.5 μL of nuclease free water nuclease, and free water was used in negative control. The amplification was performed in LabnetR Multigen Gradient thermal cycler, (Catalog TC9600-G- 230V), Labnet international, Inc. Edison, NJ, USA) after initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 95 °C for 45 sec, annealing at 55 °C for 45 sec and extension at 72 °C for 45 sec and final extension at 72 °C for 7min. 72°C and final extension for seven minutes at 72°C. The PCR amplicons were analyzed by running 15 μl of the PCR products in 2% agarose gel stained with ethidium bromide (0.5μg/mL) in comparison with DNA ladder (50 bp and 100bp), (Biomatik R Code No. M7123 and M7508, Biomatik Corporation Ontario, Canada). Under UV illumination using gel documentation and analysis system the gel was photographed.

Results

Between May 2015 and august 2016 a total of 16 different clinical samples were collected from clinically diseased cows with typical clinical pictures of lumpy skin disease where skin nodules were shown scattered in all body (Figure 1) accompanied with edema in fore limbs (Figure 2) and enlargement of superficial lymph nodes (Figure 3). Regarding sheep, samples were collected from three flocks that were shown pox like signs where the infected sheep suffered from pyrexia accompanied with cutaneous papules especially in wool-less areas of skin.

Analysis of the PCR products by agarose gel electrophoresis revealed the positive amplification of the RPO30 gene with correct size for LSDV (172bp) while field skin isolate of SPPV, tissue culture adapted SPPV vaccinal strains were shorter (151 bp) and easily distinguishable, relative to the LSDV amplicons (172 bp) (Figure 4). All DNA extracted from different LSD clinical samples yield a clear band in the gel after amplification of the target gene except that recovered from lymph aspirate. Skin nodules and scabs gave more obvious bands than that from buffy coat and ticks (Figure 4).

In the current study, different clinical samples (n: 16) (Table 1) from naturally infected cattle that exhibited numerous skin lesions and enlargement of superficial lymph nodes with edema in one or more limbs were collected. All these signs account for LSD and came in accordance with that previously reported by (Coetzer, 2004). Samples (n: 3) were also collected from field cases among sheep flocks suffered from pyrexia, oculonasal discharges and characteristic skin pox lesions suggesting sheep pox as recorded by (Yune and Abdela, 2017; Babiuk et al., 2008).

Polymerase chain reaction assay based on 30 kDa RNA polymerase subunit gene- was performed according to Lamien et al. (2011a) on DNA extracted from clinical samples and from tissue culture-adapted cattle LSDV/Ismailyia88 strain as a positive control as well as Sheep poxvirus vaccine (Romanian strain) and Sheep pox vaccine (RM 65 strain). Within RPO30 gene of SPPV there are a 21-nucleotide deletion in the 5’ end compared to that of GTPV and LSDV. Primers employed in the current study bind and amplify the partial RPO30 gene containing the 21-nucleotide deletion. As a result SPPV yields the product size (151 bp) which is lesser than the LSDV product size (172 bp). The same PCR was also used by Yan et al., (2012) for CaPV species identification.

DNA extracts of collected clinical samples yield clear bands in the gel after amplification of the target gene, however; skin nodules and scabs gave more obvious bands than that from buffy coat which reflect their high virus concentration. The results came in accordance with Carn and Kitching (1995) who stated that virus concentration in the nodules is higher than that present in blood during viremia. Therefore skin nodules are considered as better sample for LSDV detection. Scabs also are the best sample material as they are easy to collect without the use of local anaesthesia and withstand long transport in different temperatures. Collected ticks were identified as Rhipicephalus annulatus ticks (nymph and adult) according to (Walker et al., 2003). Recently the role of ticks in the transmission of LSDV was confirmed by Tuppurainen et al. (2011); Tuppurainen and Oura (2012); Tuppurainen et al. (2013); Lubinga et al. (2015); Rouby et al. (2017) suggesting them as excellent sample for LSDV detection. In the current study R. annulatus tick (nymph and adult) yield a clear band in the gel (Figure 4). Unfortunately lymph aspirate failed to give a positive result; it may be related to absence of the virus in the sample.

Owing to cross-protection within the Capripoxvirus genus, SPPV vaccines have been broadly used for cattle against LSDV (Kitching, 2003). Sheep poxvirus vaccines were used in the current study as in Egypt both vaccines are used for cattle to control the spread of LSDV.

Conclusions

RPO30 gene-based PCR is a cost effective, accurate, and simple for detection and differentiation of SPPV and LSDV in a single PCR. The 21-nucleotide deletion in the 5’ end in the RPO30 gene of SPPV makes it a reliable signature for SPPV. Buffy coat and ticks are considered good samples for LSDV detection with skin nodules and scabs are preferred.

Conflict of interest statement

The author declares that she has no conflict of interest.

Acknowledgements

This study was supported by a grant awarded by the Unit of Funding Researches and Projects, Beni-Suef University, Egypt.

References

• • Al-Salihi, K.A, Hassan, I.Q. 2015. Lumpy skin disease in Iraq: study of the disease emergence. Transboundary and Emerging Diseases 62(5):457- 62. https://doi.org/10.1111/tbed.12386
• • Asagba, M.O., and Nawathe, D.R. 1981. Evidence of sheep pox in Nigeria. Tropical Animal Health and Production 13, 61. https://doi.org/10.1007/BF02237892
• • Babiuk, S., Bowden, T.R., Boyle, D.B., Wallace, D,B., Kitching, R.P. 2008. Capripoxviruses: an emerging worldwide threat to sheep, goats and cattle. Transboundary and Emerging Diseases 55:263-72. https://doi.org/10.1111/j.1865-1682.2008.01043.x
• • Bhanuprakash, V., Indrani, B. K., Hosamani, M., and Singh, R. K. 2006. The current status of sheep pox disease. Comparative Immunology, Microbiology & Infectious Diseases 29, 27–60. https://doi.org/10.1016/j.cimid.2005.12.001
• • Bhanuprakash, V., Moorthy, A. R., Krishnappa, G.,. Srinivasa Gowda, R. N, and Indrani, B. K. 2005. An epidemiological study of sheep pox infection in Karnataka State, India. Revue Scientifique Et Technique 24, 909-920. https://doi.org/10.20506/rst.24.3.1621
• • Buller, R.M., Arif, B.M.,. Black, D. N, Dumbell, K.R., Esposito, J.J., Lefkowitz, E.J., McFadden, G., Moss, B., Mercer, A.A.,. Moyer, R.W, Skinner, M.A., and Tripathy, D.N. 2005. Family Poxviridae. In: Fauquet, C. M., M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (eds), Virus Taxonomy: Classification and Nomenclature of Viruses. Eighth Report of the International Committee on Taxonomy of Viruses, pp. 117–133. Elsevier Academic Press, San Diego.
• • Cao, J.X., Gershon, P.D., Black, D.N. 1995. Sequence analysis of HINDIII Q2 fragment of capripoxvirus reveals a putative gene encoding a G-protein-coupled chemokine receptor homolog. Virology 209, 207-212. https://doi.org/10.1006/viro.1995.1244
• • Carn, V.M., Kitching, R.P. 1995. The clinical response of cattle experimentally infected with lumpy skin disease (Neethling) virus. Archives of Virology. 140(3), 503-513.
• • Chand, P., Kitching, R.P., Black, D.N. 1994. Western blot analysis of virus specific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep. Epidemiology and Infection 113, 377-385. https://doi.org/10.1017/S0950268800051803
• • Coetzer, J.A.W. 2004. Lumpy skin disease. In: Coetzer, J.A.W. and R.C. Tustin (eds), Infectious Diseases of Livestock, 2nd edn, pp. 1268–1276. University Press Southern Africa, Oxford.
• • Davies, F. G. Otema, C. 1981. Relationships of capripox viruses found in Kenya with two Middle Eastern strains and some orthopox viruses. Research in Veterinary Science 31, 235-255.
• • Davies, F. G. 1976. Characteristics of a virus causing a pox disease in sheep and goats in Kenya, with observation on the epidemiology and control. The Journal of Hygiene 76, 163-171. https://doi.org/10.1017/S0022172400055066
• • Diallo, A., Viljoen, G. J. 2007. Genus Capripoxvirus. In: Mercer, A. A., A. Schmidt, and O. Weber (eds), Poxviruses, pp. 167–181. Birkhäuser, Basel, Switzerland.
• • Diesel, A. M. 1949. The epizootiology of lumpy skin disease in South Africa. Proc 14th Int Vet Congr. Lond., 2, 492-500.
• • Gershon, P.D., Black, D.N. 1989.The nucleotide sequence around the capripoxvirus thymidine kinase gene reveals a gene shared specifically with leporipoxvirus. Journal of General Virology 70, 525-533. https://doi.org/10.1099/0022-1317-70-3-525
• • Heine, H.G., Stevens, M.P., Foord, A.J., Boyle, D.B. 1999. A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32, the homologue of the vaccinia virus H3L gene. Journal of Immunological Methods. 227, 187-196. https://doi. org/10.1016/S0022-1759(99)00072-1 https://doi.org/10.1016/S0022-1759(99)00072-1
• • Hosamani, M., Mondal, B., Tembhurne, P.A., Bandyopadhyay, S.K., Singh, R.K., Rasool, T.J. 2004. Differentiation of sheep pox and goat poxviruses by sequence analysis and PCR-RFLP of P32 gene. Virus Genes 29, 73-80. https://doi.org/10.1023/B:VIRU.0000032790.16751.13
• • Ireland, D.C., Binepal, Y.S. 1998. Improved detection of capripoxvirus in biopsy samples by PCR. Journal of Virological Methods 74,1-7. https://doi.org/10.1016/S0166-0934(98)00035-4
• • Kitching, R.P. 1986. Passive protection of sheep against capripoxvirus. Research in Veterinary Science 41, 247–250.
• • Kitching, R.P. 2003. Vaccines for lumpy skin disease, sheep pox and goat pox. Developments in biologicals 114, 161-167.
• • Lamien, C.E., Le Goff, C., Silber, R., Wallace, D.B., Gulyaz, V., Tuppurainen, E., Madani, H., Caufour, P., Adam, T., Harrak, M. El Luckins, A.G., Albina, E., Diallo, A. 2011a. Use of the Capripoxvirus homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30) gene as a novel diagnostic and genotyping target: Development of a classical PCR method to differentiate goat poxvirus from sheep poxvirus. Veterinary Microbiolology 149 (1-2), 30-39. https://doi.org/10.1016/j.vetmic.2010.09.038
• • Lamien, C.E., Lelenta, M., Goger, W., Silber, R., Tuppurainen, E., Matijevic, M., Luckins, A.G., Diallo, A. 2011b. Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses. Journal of Virological Methods 171 (1), 134-140. https://doi.org/10.1016/j.jviromet.2010.10.014
• • Lubinga, J.C.1., Tuppurainen, E.S., Mahlare, R., Coetzer, J.A.,Stoltsz, W.H., Venter, E.H. 2015. Evidence of transstadialand mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks. Transboundary and Emerging Diseases 62:174-82.
• • Mondal, B., Hosamani, M., Dutta, T.K., Senthilkumar, V.S., Rathore, R., and Singh, R.K. 2004. An outbreak of sheep pox on a sheep breeding farm in Jammu, India. Revue Scientifique Et Technique 23, 943–949.
• • OIE (World Organisation for Animal Health) 2016. Lumpy skin disease. OIE Terr. Anim. Heal. Code, , 1–4. Available at: www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_lsd.pdf (accessed on 27 August 2016).
• • Orlova, E.S., Shcherbakova, A.V., Diev, V.I., Zakharov, V.M. 2006. Differentiation of capripoxvirus species and strains by polymerase chain reaction. Journal of Molecular Biology, 40, 158-164.
• • Rao, T.V.S., Bandyopadhyay, S.K. 2000. A comprehensive review of goat pox and sheep pox and their diagnosis. Animal Health Research Reviews 1, 127-136. https://doi.org/10.1017/S1466252300000116
• • Ripani, A., Pacholek, X. 2015. Lumpy Skin Disease: Emerging disease in the Middle East-Threat to EuroMed countries. 10th JPC REMESA, Heraklion, Greece, 16-17 March, 2015, 1-24.
• • Rouby, S.R. 2010. Studies on lumpy skin disease in cattle. Thesis, Master of veterinary medicine, Beni-Suef University.
• • Rouby, S.R., Hussein, K., Aboelhadid, S.M., E-Sherif, A.M. 2017. Role of rhipicephalus annulatus tick in transmission of lumpy skin disease virus in naturally infected cattle in Egypt. Advances in Animal and Veterinary Sciences 5(4), 185-191. DO I | http://dx.doi.org/10.17582/journal.aavs/2017/5.4.185.191
• • Stram, Y., Kuznetzova, L., Friedgut, O., Gelman, B., Yadin, H., Rubinstein-Guini, M. 2008. The use of lumpy skin disease virus genome termini for detection and phylogenetic analysis. Journal of Virological Methods 151, 225-229. https://doi.org/10.1016/j.jviromet.2008.05.003
• • Tageldin, M.H., Wallace, D.B., Gerdes, G.H., Putterill, J.F., Greyling, R.R., Phosiwa, M.N., et al. 2014. Lumpy skin disease of cattle: an emerging problem in the Sultanate of Oman. Tropical Animal Health and Production 46(1), 241-6. https://doi.org/10.1007/s11250-013-0483-3
• • Thomas, A.D., Mare, C.V.E., 1945, Knopvelsiekte. Journal of the South African Veterinary Association 16, 36-43.
• • Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Sur, J.H., Sandybaev, N.T., Kerembekova, U.Z., Zaitsev, V.L., Kutish, G.F. Rock, D.L. 2002. The genomes of sheeppox and goatpox viruses. Journal of Virology 76 (12), 6054–6061. doi:10.1128/jvi.76.12.6054-6061.2002.
• • Tuppurainen, E. S, Pearson, C. R., Bachanek-Bankowska, K., Knowles, N. J., Amareen, S., Frost, L., Henstock, M. R., Lamien, C. E., Diallo, A., Mertens P. P. 2014. Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus Antiviral Research 109(100), 1-6. https://doi.org/10.1016/j.antiviral.2014.06.009
• • Tuppurainen, E., Oura, C. 2012. Review: lumpy skin disease:an emerging threat to Europe, the Middle East and Asia. Transboundary and Emerging Diseases 59: 40-48. https://doi.org/10.1111/j.1865 1682.2011.01242.x
• • Tuppurainen, E.S., Stoltsz, W.H., Troskie, M., Wallace, D.B.,Oura, C.A., Mellor, P.S., Coetzer J.A., Vente,r E.H. 2011. A potential role for ixodid (hard) tick vectors in the transmission of lumpy skin disease virus in cattle. Transboundary and Emerging Diseases 58: 93-104. https://doi.org/10.1111/j.1865-1682.2010.01184.x
• • Tuppurainen, E.S.M., Lubinga, J.C., Stoltsz, W.H., Troskie, M., Carpenterm, S.T., Coetzer J.A.W., Venter, E.H., Oura,C.A.L. 2013. Mechanical transmission of lumpy skin disease virus by Rhipicephalus appendiculatus male ticks. Epidemiology and Infection 141, 425-430.
• • Wainwright, S., El Idrissi, A., Mattioli, R., Tibbo, M., Njeumi, F., Raizman, E. 2013. Emergence of lumpy skin disease in the Eastern Mediterranean Basin countries. Empres Watch 29, 1-6.
• • Walker, A.R., Bouattour, A., Camicas, J.L., Estrada-Peña, A., Horak, I.G., Latif, A.A., Pegram, R.G., Preston, P.M. 2003. Ticks of domestic animals in Africa: a guide to identification of species. Bioscience. Reports, Edinburgh.
• • Woods, J. A. 1988. Lumpy skin disease--a review. Tropical Animal Health and Production 20, 11-17 https://doi.org/10.1007/BF02239636
• • Yan, X. M., Chu, Y. F., Wu, G. H., Zhao, Z. X., Li, J., Zhu, H.X., Zhang, Q. 2012. An outbreak of sheep pox associated with goat poxvirus in Gansu province of China. Veterinary Microbiology, 156 (3), 425-428. https://doi.org/10.1016/j.vetmic.2011.11.015
• • Yeruham, I., Yadin, H., Van Ham, M., Bumbarov, V., Soham, A., and Perl, S. 2007. Economic and epidemiological aspects of an outbreak of sheeppox in a dairy sheep flock. Veterinary Record. 160, 236-237. https://doi.org/10.1136/vr.160.7.236
• • Yune, N., Abdela, N. 2017. Epidemiology and Economic Importance of Sheep and Goat Pox: A Review on Past and Current Aspects. Journal of Veterinary Science and Technology 8, 430. doi: 10.4262/2157 7579.1000430