Current situation of Lumpy Skin Disease Virus in some areas in El-Wady El-Gedid Governorate During 2020

Current situation of Lumpy Skin Disease Virus in some areas

 in El-Wady El-Gedid Governorate During 2020

Samia S. Abed EL Naby*, Hala, K. Abd Elmegeed **, Omnia, M. Kattab⃰* and

Hala, A. Salem

*El-Dakhla, El-Wady El-Gedid Provincial Laboratory, Animal Health Research Institute, ARC, Egypt.

**Dep. of Virology, Animal Health Research Institute, ARC , Egypt.

Abstract:

Lumpy skin disease virus (LSDV) was detected in skin biopsies which were collected from diseased animals surfing from LSD. in Eldakhla El-Wady El-Gedid Governorate in 2020.The virus was identified by electron microscopic (EM) examination and confirmed by molecular characterization through sequence and phylogenetic analysis. A phylogenetic analysis was performed using partial sequencing of the ORF103 gene and comparing with reference LSD viruses’ isolates obtained from Gene Bank. The results of the sequence analysis were similar among themselves (99.4-100% identity), it was shown 100% nucleotide genetic similarity with Egyptian isolates of LSDV(CPD/Menofiya 1/2018 MK 342935) and LSD isolate (El Wadi El-Gedid 2018 MN792930)and identities were (99.8% ) with LSD (Serbia/Bujanovac/2016/KY702007).and LSD (Russia/Dagestan/2015MH893760.) where identities were (99.4%) with LSD (Kenya/MN072619) and LSD (KZ- Kostanay MT992618) .and they were  found different from  the other LSD viruses around the world. Further investigation, ELISA test was applied and the specific antibodies of LSDV were detected in serum samples of diseased animals (93.33%) and some of contact animals (10% ).

INTRODUCTION

Lumpy skin disease (LSD) is an infectious viral disease of cattle of all ages and breeds (Tageldin et al. 2014). It is a vector-borne disease transmitted mainly by different biting and biting blood-feeding arthropods (Magori-Cohen et al.2012). It is caused by lumpy skin disease virus (LSDV) in the family Poxviridae, sub-family Chordopoxvirinae, genus Capri poxvirus (Buller et al. 2005). LSDV is enveloped double-stranded DNA virus, closely related antigenic ally to sheep and goat pox virus (Bhanuprakish et al. 2006). LSD. causes considerable economic losses in the livestock industry. The economic losses of this disease are due to reduced milk production, emaciation, poor growth, abortion, temporary or permanent infertility, permanent damage to hides. and pneumonia especially in young animals and respiratory tract lesions and mortality up to 20%. (OIE. 2010, OIE. 2018, Abera et al. 2015) it results in serious restrictions to international trade .Morbidity and mortality vary greatly depending on the activity of insects, susceptibility and the immune status of cattle. Morbidity ranging from 2% to 85% and even higher has been recorded. However, mortality is low (1 – 5%) but can be as high as 40% in some cases (Davies 1991, Tuppurainen and Oura 2012).

   LSD signs range from in apparent to severe disease. There is no current evidence of variation in virulence regarding the different LSDV(Kumar et al. 2011). The incubation period in the field is believed to be 2 to 5 weeks, and lesions first appear at the inoculation site in 4 to 20 days. Fever is the initial sign that is followed by the development of nodules within 48 hour son the skin particularly on the head, neck, limbs, udder, genitalia and perineum. and mucous membranes of the mouth and alimentary tract and in the trachea and lungs and enlargement of superficial lymph nodes (Brenner et al. 2006, Elhaige et al. 2017, Tuppurainen and Oura 2012). Edema of one or more Limbs and other ventral parts of the body, such as the dewlap, brisket, scrotum and vulva, may be edematous, causing the animal to be reluctant to move (Coetzer et al. 2005).

The first record of LSD in Zambia in 1929. the disease is still spread to different parts of Africa until 1983 (Davies 1991). LSD was limited to countries in sub-Saharan Africa; from 1984 to 1988, the disease has extended to the surrounding states. it is considered as an endemic disease in the African continent. (Tuppurainen et al. 2011). However, the disease is moved outside Africa to Madagascar and the Middle East. Recently, the disease is reported in LSD free countries (Jordan, Syria, Lebanon, Iraq,) and Iran and Turkey inOctober 2013 (Sherrilyn et al. 2013, Calistri et al. 2019). with serious economic loss to the livestock industry. Since 2012 it has spread rapidly through the Middle East, South-east Europe, and West Asian regions (Tageldin et al. 2014). In 2015 and 2016 the disease spread to south-east Europe, the Balkans and Caucasus, Russia and Kazakhstan (Alkhamis & Vander Waal. 2016 and OIE. 2017). and since 2016, LSDV has been rapidly spreading into six Balkan countries (Bulgaria, FYR Macedonia, Serbia, Kosovo, Montenegro, and Albania) (Spryginet al. 2019 and Biswas et al. 2020) and in parts of Asia (Azerbaijan, Armenia, Georgia, Kazakhstan and Russia (Toplak et al. 2017), causing serious challenges to the implementation of successful control measures .in Egypt there was an outbreak, for the first time between 1988 and1989, (El-Nahas, et al. 2011, OIE. 2017) and was reported again in 2006, 2011, 2014 ,2017and 2018 (Brenneret al.2006 and Elhaig et al.2017, Abdallah et al. 2018, Hala et al. 2021). In 2017, outbreaks of LSDV in Egyptre-introduced of LSDV to Egypt through imported cattle from Ethiopia or other endemic countries and unrestricted animals, movement across country borders is a major and constant threat for LSD (Hussein et al. 2017).

Diagnosis of LSD is depending upon the basis of the characteristic clinical signs. A confirmed diagnosis is based on laboratory investigations including biopsy material or crust through routine diagnostic techniques either identification of the virus by using the transmission electron microscope (TEM), (OlE. 2010, Gari et al. 2008, Elkenway and EL-Tholoth 2011) Immunoperoxidase (IP) staining, Or detection of its specific antibody using an antigen trapping enzyme-linked immunosorbent assay (ELISA) (Tuppurainen, et al. 2005) and a polymerase chain reaction (PCR) test (Balinsky et al. 2008, Bowden et al. 2009 and OIE 2011) that is the superior test in detecting LSD virus where it detects viral nucleic acid in skin lesions.

This study aimed to investigate the LSDV infection in cattle in El-Wady El-Gedid governorate in 2020based on electron microscopic examination and polymerase chain reaction (PCR) assay and to analyze their phylogenetic against reference genome sequences. ELISA test was applied to detect the specific antibodies of LSDV seroconversion  in serum samples of diseased on vaccinated animals showing clinical symptom which suspect LSD and apparently healthy (contact) animals.

MATERIAL and METHODS.

1.Samples:

Symptoms of lumpy skin disease appears on cattle in different localities of Eldakhla El-Wady El-Gedid Governorate from August , 2020 to early 2021.

1.1 fifteen biopsies of Skin nodules were collected from diseased animals of native, Frisian and mixed (Frisian with native) breeds for detection of LSDV by using of electron microscopic (EM) examination and Polymerase chain reaction (PCR). Samples were collected under aseptic conditions, transported to the laboratory, and stored at −70°C until use

1.2 A total of 80 Serum Samples were collected for ELISA test from cattle of different ages and sex (30 non vaccinated ,natural infected animals which showed clinical signs, paired serum samples were collected, one in initial stages of disease and the other after3 weeks and 20 serum samples were collected from (10 ) apparently healthy (contact) animals in the same time of diseased animals to determine the presence of the specific antibodies of LSDV

2.Electron microscope examination:

Transmission electron microscopy (TEM): Negative staining of skin biopsy for TEM was done according to (OIE. 2004, Tuppurainen, et al. 2005)

3.Polymerase chain reaction (PCR)

3.1-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, 180 μl of ATL buffer was added to 25 mg of the sample and 20 μl QIAGEN protease. For homogenization of samples, tubes were placed into the adaptor sets, which are fixed into the clamps of the Qiagen tissue Lyser. Disruption was performed in 2 minutes high-speed (30 Hz) shaking step. Then samples were incubated at 56˚C. After lysis,  200 µl of the lysate was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 72^OC 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 the kit (Zhuet al. 2013).

3.2-Oligonucleotide Primer. Primers used were supplied from Mutation (Germany) are listed in table (1)

3.3-PCR amplification. 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 thermal cycler