Abstract:
Lumpy skin disease (LSD) is an emerging pox viral disease affecting cattle population worldwide. In India, the first outbreak of LSD is reported during August 2019 in Odisha state, which then followed by outbreaks in crossbred and indigenous cattle population of other states. Present investigation designed to study the prevalence, pathomorphological changes and molecular detection of LSD virus in naturally infected cattle. The overall morbidity of LSD was 4.48% among 30 dairy farms. Skin nodular biopsy, whole blood and serum samples (n= 66) were collected for the diagnosis of LSD by histopathology, PCR and sequencing. The envelope protein gene (P32), Fusion protein (F) and DNA dependent RNA polymerase 30 kDa subunit (RPO30) genes were targeted for PCR testing. Out of 66, 46 cattle showed generalized skin nodules and papules of various sizes (0.5 - 6.5cm) on the skin particularly at neck, face, nose, tail, perineum and udder. Microscopic examination of the skin nodule biopsy tissue revealed presence of diffuse granulomatous inflammation, hyperkeratosis, focal to diffuse vasculitis and lymphangitis, vacuolar degeneration, spongiosis and acanthosis. The inflammatory cells typically comprised of macrophages, lymphocytes, neutrophils and eosinophils along with diffuse necrosis in dermis in chronic cases. The eosinophilic intracytoplasmic viral inclusions in keratinocytes and epithelial cells were detected in few cases. Gel-PCR assay detected P32 gene in 83%, F gene in 72% and RPO30 gene in 77% of skin biopsy samples. Three blood samples were also found positive for P32 gene by PCR. Whereas TaqMan™ probe Real Time PCR targeting EEV glycoprotein gene (LSDV126) detected LSDV in 94% of biopsy samples and three blood samples which indicated its higher sensitive for the diagnosis of LSDV. Phylogenetic analysis of RPO30 gene sequence showed that the isolates from this study were grouped in same cluster with LSDV isolates of Bangladesh, Kenya and other Indian isolates detected during 2019-20.
Keywords: Lumpy Skin Disease Virus (LSDV), pathomorphology, transboundary, poxvirus, TaqMan™ probe Real-Time PCR, eosinophilic intracytoplasmic viral inclusions
Introduction:
Lumpy skin disease (LSD) is an emerging viral disease affecting bovine population world including India. It is spreading rapidly in various parts of world and causing severe economic losses in dairy farming due to reduced milk yield, abortion and hide damage. In recent years, it is believed to be spreading from African region and is an emerging threat to cattle populations in Europe and Asia (Sanz-Bernardo et al., 2020). The incubation period of LSD under field conditions is two to four weeks while, in the experimental infection, it is between 4 to 14 days (Prozesky and Barnard, 1982; Carn and Kitching, 1995). LSD is an arthropod-borne disease and is indirectly transmitted mechanically by biting insects (Aedes aegypti ) and Culicoides, and hard Ixodid ticks (Chihota et al., 2001; Lubinga et al., 2013; Hussein, et al., 2017). Direct transmission also occurs via contact with infected animals. LSD infection in bovines is clinically characterized by high fever (104oF), lacrimation, nasal discharge, and hypersalivation, anorexia, leg edema, enlarged superficial lymph nodes, diffuse papules and raised subcutaneous nodules of 0.5-7 cm all over the body specifically on the neck, head, scrotum, perineum, udder and oral mucosa (Tageldin et al., 2014; OIE, 2018; Sudhakar et al., 2019; Sanz-Bernardo et al., 2020). Microscopic lesions of the LSD include granulomatous inflammation in the dermis and hypodermis with necrotizing and fibrinoid vasculitis, lymphangitis and infarction. In acute to subacute LSD infection presence of intracytoplasmic eosinophilic inclusions in skin histiocytes, keratinocytes, sebaceous gland cells and dermal epithelial cells can be seen (Prozesky, & Barnard, 1982; Al-Salihi and Hassan, 2015; Abdallah, et al., 2018; Sanz-Bernardo et al., 2020).
The LSD virus (LSDV) is highly host-specific, belongs to genus Capripox of the family Capripoxviridae and is 96% antigenically related to goat pox (GTPV) and sheep pox (SPPV) viruses (Tulman et al., 2002; OIE, 2019). The genome of LSDV is a double-stranded DNA with 151 kbp size containing 156 putative genes. The genes regulating host range, virulence and immune evasions are situated at the terminal parts of the genome (Tulman et al., 2001). The serological assays are not useful to distinguish different strains of Capri pox viruses (CaPVs), therefore, molecular studies are useful to distinguish LSDV, SPPV and GTPV (Tulman et al., 2002). Sequencing the host-specific G-protein-coupled chemokine receptor (GPCR), or RNA polymerase (RPO30) genes, species-specific molecular assays have been developed and validated for differentiation of CaPVs and also for phylogenetic grouping of field strains of LSDV (Lamien et al., 2011; Kumar et al., 2021).
Natural infection of LSDV in bovines is diagnosed based on typical clinical symptoms of lumpy skin nodules followed by laboratory detection of virus by PCR, virus isolation, electron microscopy and serological assays. In Asian continent, China and Bangladesh have reported the first LSD outbreaks during 2019 (OIE, 2019) due to uncontrolled movement of cattle and complete lack of vaccination program against LSD. In India, the first outbreak of LSD was reported in Odisha state on the East coast of India during August 2019 affecting 182 of 2,539 cattle with morbidity rate of 7.1% and no mortality (Sudhakar et al., 2020). Presently, in India, there are limited epidemiological and diagnostic studies of LSD. LSD in India is affecting mainly dairy cattle (both crossbreed and indigenous) thereby causing significant economic losses to small and medium farmers. There is no vaccination and any serological assays available in India. Many states in India have reported LSDV occurrence which included Maharashtra, Gujarat, MP, Goa, Karnataka, Kerala, Andhra Pradesh, Odisha, West Bengal and North-Eastern states. Based on phylogenetic analysis, the strains circulating in India were reported to be genetically close to South African NI2490/KSGP-like strains (Sudhakar et al. 2020) and Kenyan LSDV strains (Kumar et al., 2021). Various workers demonstrated gel-based PCR and real-time PCR assays for the detection of LSDV in bovines and differentiation between wild-type LSDV and vaccine virus strains (Lamien, et al., 2011; Vidanovic et al., 2016; Pestova et al., 2018). Sudhakar et al (2020) used Taqman probe-based real-time PCR for rapid diagnosis of LSD outbreak in bovines by targeting the EEV glycoprotein gene (LSDV126) which is specific for field strains of LSDV developed by earlier workers (Pestova et al., 2018). More studies are required to understand the pathology, pathogenesis and to develop suitable in-house diagnostics and vaccine against this disease. In this paper, we have evaluated morbidity, significant pathological changes and the detection of viral genome in bio-samples by employing gel-PCR, teal-time probe PCR assay and sequencing.
Materials and methods
2.1 Ethical clearance: All samples were collected as per standard procedures given by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), New Delhi, India without causing any stress to animals. Skin biopsy specimens were collected under local anesthesia (2% Lignocaine at the site of skin nodule) and blood samples were drawn from jugular vein of the animal. The due consent was obtained from farm owner before collection of the specimens. The laboratory work was carried out at ICAR-Central Coastal Agricultural Research Institute, Goa, India.