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.