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CLINICAL, PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF SHEEPPOX VIRUS IN SAUDI ARABIA.

Byline: M. Hamouda, F. Al-Hizab and I. El-Sabagh

ABSTRACT

The present study was aimed to asses clinical, histopathology, PCR and phylogenetic analysis of sheep pox virus (SPPV) in indigenous sheep. A sheep farm at Al Hassa District was struck by the sheep pox virus. The clinical signs were a sudden onset of fever, which peaks at 40-42degC, with discharges from the nose and eyes, and excessive salivation. The animal loses its appetite and is reluctant to move. Mortality was mostly seen in lambs. At necropsy, the skin lesions were erythematous macules and papules, measured 2 to 4 mm in diameter which may become larger and nodular. These lesions were frequently seen in face, back and tail. Multiple firm white nodules disseminated throughout the lobes of the lungs, particularly in lambs. Specific histopathologic features like epidermal hyperplasia with ballooning degeneration and the presence of intracytoplasmic eosinophilic inclusion bodies associated with hyperplasia of pneumocyte type II were seen in the affected animals.

The collected samples were screened for the presence of SPPV DNAs using KS-1.5/KS-1.6 and InS-1.1/Ins-1.1/based multiplex PCR. The p32 gene of selected two positive samples was sequenced and aligned with different SPPV, GTPV and LSDV available in GenBank. The phylogenetic analysis revealed that sheep pox virus strain; SPPV/Al-Hassa/2014/Saudi Arabia (accession number, KP342531) clustered on SPPV clad with SPPVs from India and China.

Keywords: Sheep pox, Histopathology,PCR, Saudi Arabia.

INTRODUCTION

Sheeppox virus (SPPV) is a member of Capripox viruses (CaPVs) which belongs to the Poxviridae family (Fauquet et al., 2005). Various strains of the virus cause disease only in sheep (SPPV), others only in goats (GTPV), and some in both sheep and goats (Bhanuprakash et al., 2010). Sheeppox virus (SPPV) has been recorded in Africa, the Middle East and Asia (Bhanuprakash et al., 2006; Babuik et al., 2008). In Saudi Arabia, Sheep pox is a serious problem and has been reported from different regions of the country (Abu-Elzein et al., 2003; Abdulkarem et al., 2014). Generally, sheep pox isa disease of considerable economic importance for indigenous sheep farming. Heavy economic losses in sheep pox outbreaks are due to mortality, abortions and loss of market value of the affected animals (Senthilkumar and Thirunavukkarasu, 2010; Rinku et al., 2013). A risk factors of the disease, including age, sex, breed and physiological status of animal (Yashpal et al., 1997; Senthilkumar et al., 2006; Selvaraju, 2014).

All age groups can be affected, however, mortality may be up to 50% in a fully susceptible flock and as high as 100% in young animals (Bhanuprakash et al., 2005). Disease occurrence is also affected by ecosystem, physiography, soil types, rainfall and relative humidity and temperature (Bhanuprakash et al., 2006). Sheeppox is characterized by fever, ocular and nasal discharges, papular dermatitis, and nodular lesions in a variety of organs, including the lungs, trachea, and the abomasums (Diallo and Viljoen, 2007). Histopathology was manifested by hyperkeratosis and dermatitis in skin with cytoplasmic inclusion bodies and the lung lesions are characterized by proliferation of type II pneumocyte with focal areas of neutrophils and lymphocytes (Gulbahar et al., 2006; Beytut, 2010).

Diagnosis of sheep pox is usually based on highly characteristic clinical signs (Ozmen et al., 2009), virus isolation, virus neutralization test, ELISA test (Tian et al., 2010) and PCR assays (Balinsky et al., 2008; Manjunatha et al., 2015). However, conventional serological assays could not distinguish SPPV, GTPV and LSDV due to the close antigenic and virulence relationship (Balinsky et al., 2008). Characterization of these viruses needs molecular techniques targeting CaPVs specific genes like P32, GPCR and RPO30 genes (Zhou et al., 2012; Yan et al., 2012).The present article planned to study clinical signs, pathological description and the molecular identification of SPPV with PCR.Phylogenetic analysis based on sequencing ofthe PCR products was also performed using genome sequence data from capripoxvirus isolates from all over the world.

MATERIALS AND METHODS

Animals: A sheep farm at Al Hassa District was struck by the sheep pox virus during winter 2014.The farm has not been vaccinated and there is also history of new entrance of animals.The clinical signs were a sudden onset of fever, which peaks at 40-42degC, with discharges from the nose and eyes, depression and excessive salivation.The animal loses its appetite and is reluctant to move.Mortality rate and mortality rate was up to 60% and 90% respectively.

Necropsy and samples: Necropsy examination was carried out on dead animals. Tissue samples from skin lesions of different parts as well as from the lungs were fixed in 10% neutral buffered formalin and processed routinely. Paraffin wax-embedded sections were stained with haematoxylin and eosin (HE). Similar tissues werealso collected, transferred to the Central Biotechnology Laboratory at the College of Veterinary Medicine and Animal Resources, King Faisal University, Saudi Arabia, and stored at -80oC until used.

Molecular detection of sheep pox virus: According to manufacturer`s instruction, DNA was extracted from skin and lung samples as well as commercial live attenuated sheep pox virus as positive control using DNeasy Blood and Tissue Kit (QIAGEN, USA). DNAs were subjected to multiplex PCR assay to diagnose sheep pox virus infection using KS-1.5 5/-GTGTGACTTTCCTGCCGAAT-3/, KS-1.6 5/-TCTATTTTATTTCGTATATC-3/, InS-1.1 5/-AGAAACGAGGTCTCGAAGCA-3/ and InS-1.1/5/-GGAGGTTGCTGGAAATGTGT-3/ primers (Mangana-Vougiouka et al., 2000). Thermal cycling parameters were initial denaturation at 95oC for 5 minutes then 35 cycles of 94oC for 30 second, 43oC for 30 second and 72 oC for 30 second and a final extension step at 72 oC for 10 minutes. PCR products were electrophoresed in 1.5% agarose gel stained with ethidium bromide and documented using ultraviolet gel documentation system (BIORAD).

Sequencing and phylogenetic tree analysis: P32 gene (envelope protein) was amplified using B68 5/-CTAAAATTAGAGAGCTATACTTCTT-3/and B695/-CGATTTCCATAAACTAAAGTG-3/ primers (Heine et al., 1999). The thermocycling conditions as in duplex PCR except the annealing temperature was 48 oC. 390bp PCR specific band was sequenced in an automated ABI 3730 DNA sequencer (Applied Biosystems, USA). The obtained sequence was analyzed using online BLAST server and compared with capripoxviruses sequences available in GenBank (Table 1). A phylogenetic tree was constructed using MEGA version 5.2 software.

GenBank accession number: The obtained P32 gene sequence of detecting SPPV during 2014 was submitted to the GenBank database with the accession number (KP342531), Sheeppox virus strain SPPV/Al-Hassa/2014/KSA.

RESULTS

Necropsy findings: All of the necropsied sheep had numerous skin lesions included erythematous macules and papules, measured 2 to 4 mm in diameter, which may become larger and nodular with typical demarcation. When the necrotic centers of the nodules were removed, the lesions appeared ulcerated. Pustular changes were also observed.The lesions were found all over the body, but were most prominent on the face, back and tail (Fig. 1, a, b, c). The lungs contained areas of congestion, oedema and consolidation, and often had multiple firm white nodules(2-15 mm diameter) disseminated throughout the lobes (Fig. 1d). These nodules were red and surrounded by a zone of hyperemia.

Microscopic findings: The epidermal layers showed thickened, hyperplastic epithelium with hydropic degeneration, particularly in stratum spinosum. Intracytoplasmic eosinophilic inclusion bodies were visible (Fig. 2a). The dermal layer revealed inflammatory exudate characterized by fibrin and cellular debris and massive number of neutrophils (Fig. 2b). Other mononuclear cells consisting of lymphocytes, plasma cells, and some macrophages were observed (Fig. 2c). The blood vessels were congested and showed perivascular cuffs with lymphocytes and macrophages. In the lung, epithelial hyperplasia with squqmous metaplasia were noticed in the terminal bronchioles. In some areas of lung, alveolar walls were thickened due to hyperplasia of type II pneumocytes (Fig. 2d). In most cases alveoli were filled with inflammatory exudate consisting of fibrin, macrophages and lymphocyte

Molecular detection and sequencing of SPPV: Suspicious papule and nodule samples were positive in duplex and P32 PCR test. Amplicons of approximately 149 and 289 bp and 390 bpwere obtained in both PCR assays, respectively (Fig.3). Partial open reading frame (ORF) of P32 gene was sequenced and analyzed using the online BLAST server. The result of BLAST analysis showed that the Sheeppox virus strain SPPV/Al-Hassa/2014/KSA was very close relationships with other Capripoxviruses isolated worldwide with nucleotide sequence identity from 95 to 100%. The nucleotide sequence identities were 99 ~ 100%; 95 ~ 98% and 96 ~ 98% with SPPV, GTPV and LSDV, respectively.

Phylogenetic analysis: Phylogenetic tree was performed using MEGA program version 5.2 (Fig.4). Analysis of the phylogenetic tree showed that the strain (SPPV/Al-Hassa/2014/KSA) was clustered with (SPPV/Al-Hassa/2013/Saudi Arabia) and closely related to SPPV strains identified in China and India.

Table 1. Capripoxviruses used in the phylogenetic tree construction.

Virus name###Country###GenBank Accession

SPPV/Al-Hassa/2013/Saudi Arabia###Saudi Arabia###KF204447

SPPV/Rumanian Fanar/India###India###AY368684

SPPV/Rainpet/India###India###DQ431989

SPPV/Makhdoom-2007/India###India###EU314721

SPPV/Pune-08/India###India###FJ882029

SPPV/Shanxi/China###China###HM770955

SPPV/Maha/sheep/22/India###India###KF468761

SPPV/GanSuGT/11/2012/China###China###KF661974

SPPV/GanSuHN/12/2012/China###China###KF661977

SPPV/Jilin/China###China###KF991005

SPPV/Kurdistan/Iraq###Iraq###KF992798

SPPV/Lx/Gs/China###China###KJ026555

SPPV/Anyang/Henan/China###China###KJ026553

LSDV/2/Slemani/Kurdistan/2014/Iraq###Iraq###KM047051

LSDV/9/Slemani/Kurdistan2014/Iraq###Iraq###KM047058

LSDV/Slemani/Kurdistan/2013/Iraq###Iraq###KF996498

GPPV/Yemen (Sanaa/1983)/Yemen###Yemen###EU625262

GPPV/Maha/goat/19/India###India###KF468762

GPPV/JK/sheep/27/India###India###KF468758

GPPV/ChongQ/2009/China###China###HM572329

GPPV/Viet Nam (NinhThuan/2005) / Viet Nam###Viet Nam###EU625263

GPPV/JK/goat/27/India###India###KF468757

LSDV/Neethling vaccine LW 1959/South Africa###South Africa###AF409138

LSDV/Neethling###South Africa###AF336131

LSDV/Neethling 2490###Kenya###AF325528

LSDV/NeethlingWarmbaths NW-LW###South Africa###AF409137

GPPV/G20-LKV###Kazakhstan###AY077836

GPPV/Pellor###Kazakhstan###AY077835

SPPV/NISKHI###Kazakhstan###AY077834

SPPV/ strain A###Kazakhstan###AY077833

SPPV/TU-V02127###Turkey###AY077832

SPPV/Nigeria 1###Nigeria###AF124517

SPPV/RM/65/Iran###Iran###FJ917518

DISCUSSION

Capripoxviruses have the potential to become an emerging and bioterrorism agents because of worldwide climate change, an alteration in trade and animal products (Babiuk et al., 2008). So far, the current situation of sheep pox infection among sheep and goats in Saudi Arabia still needs more investigation about the varieties of strains, the economic losses and the definitive diagnosis.These attributes increase the prospect of successfully implementing country control program. During winter, 2014 an outbreak of the disease strucka sheep farm which is not previously vaccinated and also has a history of new entrance of animlas. Previous studies stated that varying degree of disease might be due to the influence of host, agent, environmental conditions, the increase of illegal animal movement through trade and husbandry practices (Eroksuz et al., 2008).

Clinical signs of illness were fever and respiratory signs, Mortality rate reach to 60 %, while mortality rate was up to 90% and mostly confined in lambs (Bhanuprakash et al., (2005) and Sharma et al., (2013).There is a consistent specific pox skin lesion in all infected animals and the lesions were frequently in the form of macules and papules, and obviously seen in unwooled areas in the head, abdomen and tail (Babiuk et al., 2008 and Bhanuprakash et al., 2010).At necropsy, Lung was the only organ affected and there was multiple nodules distributed throughout lung lobes, and this findingwas considered a consistent necropsy findings in all dead lambs (Chani, 2011). Histopathological examination of skin lesion in all infected sheep showed that the epidermal and dermal changes associated with hyperplasia of pneumocyte type IIand the presence of intracytoplasmic inclusions were considered a characteristic lesion of the disease in sheep (Singh et al., (2007); Zangana and Abdullah, (2013)).

Furthermore, the molecular diagnosis of disease which based on alignment and phylogenetic analysis of P32 gene showed that that there is a close relationship among Saudi Arabia SPPVs detected during 2013 and 2014 as they are clustered together in a separate sub-cluster (Abdulkarem et al., 2014).Additionally, the Saudi Arabian SPPVs detected during 2013 and 2014 were closely related to Indian and Chinese SPPVs.The occurrence of this outbreak together with the previous outbreak in 2013 highlights the fact that sheep pox is an emerging pathogen in this District and an affect susceptible host during stress weather. Therefore, it is important that the state animal husbandry along with other stakeholders keep strict vigil on the development of disease among animals in susceptible areas.

Also, quarantine of new animals before introduction to the herd and immediate implementation of mass vaccination, slaughter and proper disposal of affected animals is very important to prevent any further losses of disease.

Acknowledgements: The authors are thankful to the Deanship of Scientific Research, King Faisal University, Saudi Arabia for support this work with a financial grant number: 150048

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Publication:Journal of Animal and Plant Sciences
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Date:Feb 28, 2017
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