Lumpy Skin Disease (LSD): Pathomorphological Features and Molecular Detection in Dairy Cattle of West Coastal India

Lumpy skin disease (LSD) is an emerging pox viral disease affecting cattle population worldwide. In India, the rst 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 inammation, hyperkeratosis, focal to diffuse vasculitis and lymphangitis, vacuolar degeneration, spongiosis and acanthosis. The inammatory 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.


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 eld 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 (104 o F), lacrimation, nasal discharge, and hypersalivation, anorexia, leg edema, enlarged super cial lymph nodes, diffuse papules and raised subcutaneous nodules of 0.5-7 cm all over the body speci cally on the neck, head, scrotum, perineum, udder and oral mucosa (Tageldin et  The LSD virus (LSDV) is highly host-speci c, 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-speci c G-proteincoupled chemokine receptor (GPCR), or RNA polymerase (RPO30) genes, species-speci c molecular assays have been developed and validated for differentiation of CaPVs and also for phylogenetic grouping of eld 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 rst LSD outbreaks during 2019 (OIE, 2019) due to uncontrolled movement of cattle and complete lack of vaccination program against LSD. In India, the rst 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

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.

Sample collection and processing:
Total 30 affected dairy farms (backyard -13 and organized -17) rearing both crossbred and indigenous cows were investigated. The size of backyard farms was 2-10 animals with limited oor space and close contact between animals. The organized dairy farms consisted of 10 to 600 animals. Sampling was done from 66 cows showing varying degree of clinical signs of LSDV and 15 healthy/asymptomatic animals which were in close contact with affected animals. Skin biopsy specimens were collected under local anesthesia (2% lignocaine administration at the nodule site). Blood in EDTA and without anticoagulant was collected from all the animals. The samples were transported to laboratory on ice.

Histopathology:
Skin biopsy specimens of size 5-10mm were cut and xed in 10% buffered formalin for 48 hours. The tissues were sequentially dehydrated in ethanol (65-100%) and cleared in three changes in xylene. The para n blocks were prepared and 5µm sections were prepared for staining with hematoxylin and eosin staining method. The histological lesions were documented under 10x and 40x objective in compound microscope (DM3000, Leica, Germany)

Extraction of DNA from Skin biopsies:
DNA extraction from skin biopsies and blood samples was done by using Wizard® Genomic DNA puri cation kit (Promega #A1120, USA) by following manufacturer's instructions. DNA samples were stored at -20 o C for further testing.

PCR ampli cation of LSDV P32 and F gene:
For ampli cation of LSDV genes, OIE recommended primers were employed (Table -1 Table-1. The PCR products were electrophoresed in 1.5% agarose gel and visualized under UV transilluminator. DNA from goat pox vaccine used as positive control in PCR reaction.

PCR and Sequencing of RPO30 gene for phylogenetic analysis:
PCR ampli cation of RPO30 gene was carried out by using the speci c primers (Table 1) and sequenced using Sanger's sequencing method. The sequence was submitted to NCBI GenBank and obtained accession number MW590715. The reference sequences of LSDV from China, Bangladesh, Kenya, Indian states of Odisha and Haryana, GTPV and SPPV virus strains retrieved from GenBank and the phylogenetic analysis of RPO30 gene generated in present investigation. The sequences were aligned and compared using BioEdit and the phylogenetic analysis was carried out using MEGA-X.

Clinical infection and morbidity:
The major clinical ndings observed in affected animals includes fever (upto 105 o F), inappetence and drastic reduction in milk yield with lacrimation, nasal discharge, super cial lymph node enlargement, localized/generalized subcutaneous nodules, leg edema and pustular lesions in feet with lameness. The overall morbidity of LSDV infection in both backyard and organized dairy farms (n=30) was 4.48% (66/1471) with no mortality ( Table 2). The cattle in backyard farms showed higher morbidity (91.6%) and the risk factors were attributed to limited oor space and close contact with infected animals. In organized farms, though the number of susceptible animals was more, however, early detection of clinical signs and timely separation of affected animals by farmers resulted in lower morbidity (3.17%). Backyard farms <10 animals (2 to 10); Organized farms -10 to 600 animals; ND-Non-descript; HF; Holstein Friesian

Pathological observations:
Out of 66 clinical cases, 46 cattle (69.7%) showed generalized skin nodules and papules of various sizes ranged from 0.5 cm to 6.5 cm located all over the body particularly, neck, head, face, nose, tail, perineum and udder (Fig. 1A).
Twenty showed few small patchy nodules at the neck region, lumbar and face region. The number of nodules varied from few to more than a hundred based on the severity of the disease. These subcutaneous nodules were round, circumscribed, rm and raised above the skin and diffusely present on the body (Fig. 1B). In severe cases, these nodules were ruptured, and whitish-grey serous uid was oozing out of lesions. In few cases, nodules at the vulva, perineum and hindquarters were ruptured leading to ulcers and suppurative wounds (Fig. 1C). The prescapular lymph nodes were enlarged in nineteen cases (19/66, 38%). Edema of the ventral body parts of the body (brisket edema) and legs was consistent in some cases. Nasal and ocular discharge, conjunctivitis and salivation were noticed in all the animals. Lameness was observed due to severe edema and pustular lesions on the feet of affected animals (Fig. 1D).

Detection of LSDV nucleic acid:
PCR assay targeting capripoxvirus-speci c P32 gene, detected viral genome in 83% and LSDV-speci c F gene in 72% skin biopsy specimens ( Fig. 2A). Whole blood, nasal swabs and ies were found negative for both the genes. Blood samples collected from close contact animals were negative for both the genes.

Ampli cation and phylogenetic analysis of RPO30 gene:
PCR ampli cation of RPO30 gene detected genome in 77% skin biopsy samples (Table 4). Nucleotide sequencing of RPO30 gene revealed that LSDV isolate LSDV/CCARI-Goa1/India/2021 (Acce.No.MW590715) showed highest similarity (~100%) with Neethling (NI-2490) strain (Acce.No.AF325528) and Kenyan strain (Acce.No.MN072619), however it was distinctly different from GTPV and SPPV isolates of India. The phylogenetic tree generated ve clusters. Cluster I had the LSDV isolates from India, Kenya and Bangladesh. Cluster II contained LSDV from China, cluster III with GTPV isolates and cluster IV with SPV isolates (Fig. 2B). The recovered isolate in this study was similar to LSDV isolates reported from eld outbreaks in Odisha and other parts of India during 2019. 3.5 Detection of LSDV by TaqMan TM probe: The TaqMan™ probe real time PCR assay detected LSDV in 62 skin biopsy samples (94%), 2/66 blood samples and 3/5 ocular swab samples, which proved that this assay could be very sensitive and rapid in the detection of LSDV from eld outbreaks (

Discussion
This study mainly described the overall morbidity, pathomorphological features, molecular detection and phylogenetic analysis of LSD in cattle of organized and backyard dairy cattle in the west coastal region of India. The overall morbidity of LSDV infection in cattle was 4.48% with no mortality. It was observed that the backyard farms were more affected due to lack of adequate oor space to keep the affected animals isolated, lack of timely diagnosis and lack of awareness about the disease, presence of stray cattle around and grazing practice. Our ndings were similar to the outbreak report from Europe, where higher morbidity has been reported in cattle in small farms with fewer animals than in medium and large farms (EFSA, 2017; Sevik & Dogan, 2017).
The pathology investigation in present study revealed that the animals exhibited characteristic lesions of LSD such as fever, cutaneous nodules, lymphadenopathy, pustules on various body parts, lameness, etc., which were described earlier also (Prozesky, & Barnard1982;Abdallah, et al., 2018;Sanz-Bernardo et al., 2020;Sudhakar et al., 2020;Kumar et al., 2021). This study observed morbidity rate of 4.48%, which is in the range of the LSD morbidity rate reported earlier (Sudhakar et al., 2020;Kumar et al., 2021). Higher morbidity rate of 40-75% is also reported by some workers (Babiuk et al., 2008;Tuppurainen&Oura, 2012). The characteristic microscopic lesions of LSD were vasculitis, perivascular in ltration of in ammatory cells around the dermal blood vessels, spongiosis and necrosis (Tageldin et al., 2014;Abdallah et al., 2018;Vaskovic, et al., 2019;Prozesky and Barnard,1982). The characteristic lesions of pox viral infection viz.,vacuolar degeneration of keratinocytes, spongiosis, and intracytoplasmic eosinophilic inclusion bodies were also observed in our study (Body, et  Three major genes namely, capri pox speci c -P32 and LSD speci c fusion protein gene (F) and RPO30 were targeted for PCR ampli cation to detect the LSDV nucleic acid. The P32 gene encodes envelope protein which is homologous to vaccinia virus H3L gene located on the membrane surface of a mature virion (Tulman et al., 2001). Several studies demonstrated that the P32 gene is highly conserved among capripox viruses and therefore it has been used as a

Conclusion
This study was primarily focused on the prevalence of LSDV infection in small and organized farms, its pathological features and molecular detection using gel-based and real-time PCR assays. LSDV causes signi cant economic losses to dairy cattle and there is an urgent need to develop diagnostic assays and homologous vaccine for the control of the disease in the Indian subcontinent.