Molecular Characterization of lumpy Skin Disease Virus in Cattle (2017)

Molecular Characterization of lumpy Skin Disease Virus in Cattle (2017)

Hala, A. Salem,  Ebtsam, A. Abouelyazeed ,  Ali, H.M., Khattab, O. M and Saad, A. Moussa

 Virology Dept., Animal Health Research Institute, ARC, Egypt

INTRODUCTION

Lumpy skin disease (LSD) is viral disease of cattle that is caused by lumpy skin disease virus, LSD is caused by an enveloped double-stranded DNA virus called LSD virus (LSDV), which together with sheep poxvirus (SPV) and goat poxvirus (GPV) constitutes the genus Capripoxvirus of the Chordopoxvirinae subfamily of the Poxviridae family   (Buller et al., 2005). It causes abortion, infertility, and damage to cattle hide. LSD is considered a notifiable disease, and in affected countries, it results in serious restrictions to international trade (Davies, 1991a, b, c); Tuppurainen and Oura 2012). Soon after the fever, few to several variable-sized cutaneous nodules appear on different regions of the body. The entire body of the animal can be covered with nodules, and lesions may be seen in the mouth and nose as well as the mucous membranes of the eye in affected animals (Haig 1957; Coetzer 2004; Babiuk et al. 2008). Bulls may become permanently or temporarily infertile and the virus can be excreted in the semen for prolonged periods (Irons et al., 2005, Şevik M, Doğan 2017).

Lumpy skin disease is considered as an economically important disease of cattle; serious economic losses can follow outbreaks that have a high morbidity and can produce a chronic debility in infected cattle (OIE, 2018).

The economic losses due to this disease is due to reduced milk production, in appetite and weight loss, poor growth, abortion, infertility, skin damage and pneumonia especially in animals with mouth and respiratory tract lesions (OIE, 2018). Morbidity, mortality, and case fatality rates are influenced by many factors including the immune status of the affected cattle and the abundance of the vectors (Thomas and Mare 1945; Tuppurainen and Oura 2012).. Experimental seminal transmission of LSDV in cattle has been reported recently (Annandale et al. 2010 and 2014). Although not commonly seen, the disease can be transmitted by direct contact (cutaneous lesions, saliva, respiratory secretions, milk, and semen) and using of contaminated needles (Davies 1991a; Hunter and Wallace 2001). Most cases are believed to result from transmission by an arthropod vector (Chihota et al., 2001). Lumpy skin disease (LSD) affects primarily cattle and occasionally buffalo (Sharawi and El-Rahim 2011, El-Tholoth and El-Kenawy 2016). The first diagnosis of LSD in Egyptian cattle was in the summer of 1989, followed by outbreaks in 2006, 2011, 2014, and 2017 (Tuppurainen and Oura 2012) and (Abdallah et al., 2018). In 2017, outbreaks of fLSDV in Egypt re-introduced of LSDV through imported cattle from Ethiopia or other endemic countries and unrestricted animals’ movement across country borders is a major and constant threat for LSD (Şevik and Doğan 2017, Hussein et al., (2017). LSD is currently endemic in almost all African countries except a few northern countries (Libya, Tunisia, Algeria and Morocco) and in the Middle East (Tuppurainen and Oura, 2012; Stram et al., 2008; Yeruham et al., 1995). The disease has recently emerged in Israel, Jordan, Lebanon, Turkey, Iraq, Iran and Azerbaijan (Tuppurainen et al., 2014). With the exception of a few northern and southern African countries, all three capripox diseases co-exist in Africa. As a use of a homologous vaccine is recommended for all CaPVs, the molecular characterization of the causative agent will lead to better control of the spread of disease and will allow the use of the most appropriate vaccine

The objective of this study was to observe the clinical signs of the cattle infected with LSD , detect ORF103 gene of capripox virus  from selected sample by PCR and Phylogeny was done using sequence of the partial ORF103 gene and by homology comparison with other capripox virus .

Material and Methods

Samples:

During 2017, a total of 42 samples (skin nodules) from cattle suspected to have LSD were submitted to the animal health research Institute (7 Menofya, 19 ElQalyubia, 5 Kafr El-shikh, 5 El-Behera, 3 Asuit and 3 Domiat). Skin nodules were aseptically collected from infected cattle with typical clinical signs of lumpy skin disease. Each sample was prepared for virological examination in 50 % glycerin buffer saline, 10% suspension in phosphate buffer saline (PBS) containing 100 IU/ml pencillin, 100 ug/ml streptomycin and 50 units mycostatin, then stored at  -80°C till used. the mixture was incubated at 25 C 1 hr then frozen and thawed 3 times at –20˚C and centrifuged at 3000rpm /10 min., the supernatant fluid was used as inoculums for virus isolation in embryonated chicken egg (ECE). 

Virus Isolation:

SPF twelve days old embryonated chicken eggs (ECE) were inoculated with the prepared samples via chorioallantoic membrane (CAM) route which was used for virus isolation. Supernatant fluids of nodules injected on chorioallantoic membrane (CAM). Samples were isolated in 12 days old in specific pathogen free (SPF) embryonated chicken egg (Koum Oshiem SPF, Fayoum, Egypt) and harvesting of CAMs.

DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56°C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in kit. Extraction step for viral DNA directly from processed CAMs was done using a commercial kit (QIAamp. viral DNA Mini Kit).

Oligonucleotide Primers and thermocycling.

Primers used were supplied from Metabion (Germany) are listed in table (1). Primers were utilized in a 25 µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermocycler.

Polymerase chain reaction PCR:

Agarose gel electrophoreses Electrophoresis grade agarose (1.5 g) was prepared in 100 ml TBE buffer in a sterile flask, it was heated in microwave to dissolve all granules with agitation and allowed to cool at 70˚C, then 0.5μg/ml ethedium bromide was added and mixed thoroughly. The warm agarose was poured directly in gel casting apparatus with desired comb in apposition and left at room temperature for polymerization. The comb was then removed, and the electrophoresis tank was filled with TBE buffer. Twenty μl of each PCR product samples, negative control and positive control were loaded to the gel. The power supply was 1-5 volts/cm of the tank length. For gel analysis, 15 µl of the products was loaded in each gel slot. Gelpilot 100 bp DNA Ladder  (Qiagen, Germany, GmbH) was used to determine the fragment sizes. The run was stopped after about 30 min. and the gel was transferred to UV cabinet. The gel was photographed by a gel documentation system and the data was analyzed through computer software (Sambrook et al., 1989).