
==== Front
Vet Q
Vet Q
The Veterinary Quarterly
0165-2176
1875-5941
Taylor & Francis

39233648
10.1080/01652176.2024.2398211
2398211
Version of Record
Research Article
Research Article
Pathological, immunological and molecular epidemiological analysis of lumpy skin disease virus in Indian cattle during a high-mortality epidemic
G. B. Manjunathareddy et al.
Veterinary Quarterly
https://orcid.org/0000-0001-8578-7823
Manjunathareddy Gundallhalli Bayyappa a
https://orcid.org/0000-0003-0557-7194
Saminathan Mani b
Sanjeevakumar Lalasangi c
Rao Sugana c
Dinesh Murali b
Dhama Kuldeep b
Singh Karam Pal b
Tripathi Bhupendra Nath d
a ICAR-National Institute of Veterinary Epidemiology and Disease Informatics, Bengaluru, Karnataka, India
b Centre for Animal Disease Research and Diagnosis, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India
c Veterinary College, Karnataka Veterinary, Animal & Fisheries Sciences University, Bengaluru, Karnataka, India
d Vice-Chancellor, Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST), Jammu, Union Territory of Jammu and Kashmir, India
Supplemental data for this article can be accessed online at https://doi.org/10.1080/01652176.2024.2398211.

CONTACT Gundallhalli Bayyappa Manjunathareddy gbmpatho@gmail.com ICAR-National Institute of Veterinary Epidemiology and Disease Informatics, Bengaluru, Karnataka, India
5 9 2024
2024
5 9 2024
44 1 122
14 5 2024
29 7 2024
5 8 2024
KnowledgeWorks Global Ltd.4 9 2024
published online in a building issue4 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

Lumpy skin disease (LSD) is an economically significant, emerging viral disease of Cattle and Buffaloes. This study aimed to investigate the causes of high mortality in a recent LSD epidemic in India. We examined 1618 animals across seventy outbreaks and conducted post-mortem on 48 cattle out of 513 clinically suspected LSD cases. The morbidity, mortality and case fatality rates recorded were 31.70%, 2.97 and 9.37% respectively. Disease stages were categorized as early (20.81%), mid (42.02%), and late (37.17%) and the distribution of skin lesions was classified as mild (34.14%), moderate (39.39%), and severe (26.47%). Post-mortem findings revealed systemic infection with necrotic and ulcerative nodules on multiple internal organs. Histologically, necrotizing vasculitis and mononuclear cell infiltration with intracytoplasmic inclusions were observed in various organs. The highest viral load was found in skin nodules/scabs, trachea, tongue, and lymph nodes. The viral load was significantly higher in mid- and late-stages of skin nodules and internal organs; whereas, blood from early-stage showed high viral load. The expression of Th1-type and Th2-type cytokines varied significantly across different stages of the disease. The downregulation of the apoptotic intrinsic and upregulation of the extrinsic pathway genes, suggesting that the latter plays a role in LSDV infection. Genetic analysis revealed that the LSD virus (LSDV) isolates were derived from a Kenyan ancestral strain with unique nucleotide changes in RPO30 and P32 gene. In conclusion, the high mortality in the recent Indian LSD epidemic can be attributed to a newly identified, highly virulent strain of LSDV causing systemic infection.

Keywords

Lumpy skin disease
cattle
clinical signs
staging
gross pathology
histopathology
Immunohistochemistry
apoptosis
ICAR-NIVEDI ANSCNIVEDISIL202100900123 National Livestock Mission (NLM), DAHD R-300121112022-DADF This work was supported by research grants from the ICAR-NIVEDI (Project code: ANSCNIVEDISIL202100900123, dated 01.09.2021) and National Livestock Mission (NLM), DAHD, India (Grant No. R-300121112022-DADF, dated 27.02.2023).
==== Body
pmc1. Introduction

Lumpy skin disease (LSD) is an economically significant, emerging viral disease affecting cattle and buffaloes, characterised by the development of skin nodules (Möller et al. 2019). It is caused by the lumpy skin disease virus (LSDV) that belongs to the genus Capripoxvirus within the family Poxviridae (Tuppurainen et al. 2017). The disease was first detected in Zambia in 1929 and has become endemic in various African, European and Middle Eastern countries. Recently, since 2019, the Asian continent has been severely impacted by the disease, with several outbreaks reported, including India (Manjunatha Reddy et al. 2023). The disease is predominantly transmitted via biting and blood-feeding arthropods, such as flies, mosquitoes, and ticks (Tuppurainen et al. 2011). Although direct animal contact plays a minimal role in viral transmission, the virus has been detected in milk, saliva, blood, nasal and lacrimal secretions, which can serve as indirect transmission routes via common feeding and watering troughs.

The incubation period of the disease ranges from 7 to 35 days in a naturally transmitted infection and clinically, manifested with a range of symptoms including pyrexia, firm, flat-topped, well-circumscribed papules and nodules of various sizes across the body, lymphadenopathy, oedema of limbs, abdomen, and brisket region. Usually, after 2-3 weeks, the nodules become necrotic and develop into characteristic ‘Sit-fast’, sequestrum of necrotic material (OIE 2024). They will further ulcerate, allowing the virus to find its way into the body cavities and enter bodily fluids like saliva, nasal, ocular, and genital secretions. As a result, the virus exists in skin lesions, blood, scabs, oral, nasal, and ocular fluids, semen, and occasionally on animal skin (Reddy et al. 2024). In certain conditions, the animal may sometimes develop pneumonia due to the formation of lesions on the mouth and the respiratory tract which may sometimes cause the death of the animal. Severe skin, gastrointestinal, and respiratory tract lesions, along with limb oedema, can cause prolonged anorexia and reduced mobility, leading to significant deterioration in body condition and potentially death (Kumar et al. 2021). Additional complications may include reduced milk production, mastitis, orchitis, emaciation, keratitis, and abortion (Möller et al. 2019).

Morbidity rates vary from 2% to 45%, influenced by the host’s immune status and exposure level, while mortality typically remains under 5% but can rise to 15% in severe outbreaks (Babiuk et al. 2008; Tuppurainen et al. 2017). Diagnosing LSD is generally based on its distinctive clinical signs, although differential diagnosis can be challenging when only minor skin lesions or transient fever are present, which may be mistaken for other diseases (OIE 2024).

The pathogenesis of LSD, particularly the immunopathological changes in skin nodules and the host’s immune response to natural LSDV infections, remains poorly understood. As of now, no research has investigated on LSDV-induced pathology, virus distribution, or viral load in the skin and internal organs during the recent LSD epidemics in Indian cattle that resulted in high-mortality. It is well known that, the effective anti-viral defences involve both innate and adaptive immune responses, including apoptosis (Birge and Ucker 2008).

Given these gaps, the present study elucidated the detailed pathology of LSDV, including clinical signs, novel gross and histopathological findings, specific LSDV antigen distribution, viral loads in different organs, Th1-type and Th2-type inflammatory cytokines, and apoptosis pathways during various stages of LSD in naturally infected cattle resulted in better understanding of the pathogenesis of disease and immune responses, which could aid in developing biomarkers to distinguish between susceptible and resistant animals, developing effective vaccines, and designing appropriate control strategies.

2. Materials and methods

2.1. Study location and design

The study was carried out between January 2022 and December 2023 in the states of Karnataka, Uttar Pradesh and Uttarakhand, India. After the first incursion of LSD in these states, high morbidity and mortality were recorded during this period. The animals from LSD affected areas were selected for this study. A total of 1618 animals were examined for clinical signs of LSD, out of which 513 animals with typical skin nodules were included in the present study. During the study, a total of 48 animals died and were subjected to a detailed post-mortem examination. The data regarding the herd size, number of affected animals, disease stage, lesion location and distribution, as well as the age and sex of the animals were recorded.

2.2. Grading and stage of skin lesions

Lesions severity was graded based on the number and distribution of nodular or scab lesions per 5 cm2 on the head, neck, and body: mild (1-2 lesions), moderate (3-4 lesions), and severe (5 or more lesions) (Badr et al. 2022). Early-, mid- and late-stages of the disease were based on disease duration as described by El-Neweshy et al. (2013).

2.3. Sample collection

Skin biopsy samples (4 mm thick) were collected under local anaesthesia from various body regions, preserved in 10% neutral buffered formalin (NBF) for histopathology, and viral transport media for molecular studies. Blood was drawn from the left jugular vein of both 513 affected and 330 healthy animals and stored at 4 °C for transport to the laboratory. Post-mortem examinations were performed on 48 animals that died from LSD, collected tissue samples from skin nodules/scabs, lymph nodes, spleen, trachea, lungs, tongue, liver, gallbladder, kidneys, urinary bladder, rumen, reticulum, intestine, and heart in 10% NBF for histopathology and also collected in a sterile container for LSDV confirmation, viral load quantification, cytokine and apoptotic gene expression analysis. All Institutional and National guidelines were followed during animal handling and sample collection. The study was approved by the Institute Animal Ethics Committee (No. NIVEDI/IAEC/2022/06).

2.4. Haematology and Serum biochemistry

Haematological parameters were analysed for early- (94 cases), mid- (195 cases) and late- (171 cases) stages of LSD using an Auto Haematology Analyser (Mindray BC-2800VET, China) following manufacturer instructions. Analysed parameters included total leukocyte count (TLC), total erythrocyte count (TEC), haemoglobin (Hb), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and total platelet count (TPC). Serum biochemistry tests for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), total protein, creatinine, gamma-glutamyl transferase (GGT), albumin, globulin, and cholesterol were performed using an automatic biochemical analyser (Erba Diagnostics, Mannheim GmbH, Germany).

2.5. Histopathology and Immunohistochemistry (IHC)

Tissue samples from 48 LSDV-positive dead animals were embedded in paraffin, sectioned at 4 µm, and stained with haematoxylin and eosin for microscopic examination. Duplicate sections underwent indirect immunoperoxidase staining to demonstrate LSDV antigen presence (Saminathan et al. 2021). Briefly, the tissue sections were mounted on 3-aminopropyltriethoxy-silane (APES) coated slides, deparaffinized using xylene and rehydrated using descending grades of ethanol. The endogenous peroxidase was quenched, followed by heat-induced epitope retrieval (HIER) with 10 mM citrate buffer (pH 6.0). The non-specific sites were blocked by incubating the sections with 1% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min. Then, the sections were incubated with the monoclonal primary antibody (ORF117/A27Lgene) raised against field strain of LSDV in rabbits for 1 h. The monoclonal antibody used in this study was tested by whole LSDV based iELISA and showed sensitivity of 97% and specificity of 95%. Then, the sections were incubated with HRP conjugated rabbit anti-bovine IgG secondary antibody (Cat No. A10-102P, Bethyl Laboratories, Inc., USA) for 30 min. Then stained with 3,3′-diaminobenzidine (DAB) tetrahydrochloride for 10 min and the reaction was terminated. Then the slides were counter-stained with Harris haematoxylin and followed by mounting. Validation of IHC was done by preparing positive control slides from known LSDV positive samples. The tissue sections from apparently healthy animals were treated identical to infected slides with LSDV primary antibody at similar concentration and served as negative control.

2.6. Molecular detection of LSDV

DNA was extracted from 513 skin nodular biopsy samples and other internal organ tissues from 48 dead animals using a commercial kit (Qiagen, Germany) and quantified using the NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, USA). The PCR was carried out in a 25 µl reaction volume consisting of forward primer (5′-TCCGAGCTCTTTCCTGATTTTTCTTACTAT-3′) and reverse primer (5′-TATGGTACCTAAATTATATACGTAAATAAC-3′) specific for P32 gene of LSDV using 2X DreamTaq Green PCR Master Mix (ThermoFisher Scientific, California, USA) with annealing temperature of 50 °C for 30 s and products were verified by agarose gel electrophoresis.

2.7. Viral load quantification by qPCR

SYBR Green-based quantitative PCR (qPCR) was performed in triplicate to quantify the LSDV across various samples at different stages of the disease (495 skin nodular biopsy samples and other internal organ tissues from 48 dead animals). The primers targeting the EEV glycoprotein gene specific for LSDV (FP-5′-TAGAAAATGGATGTACCACAAATACAG-3′; RP-5′-TTGTTACAACTCAAATCGTTAGGT G-3′) were used (Pestova et al. 2018). The standard curve method was applied for absolute quantification of LSDV, using threshold cycle (Ct) values from early, mid, and late disease stages. The viral copy number in each sample was calculated using the formula: Copy  number=10 [Ct−bM]

where Ct is the cycle threshold, b is the y-intercept, and M is the slope of the standard curve.

2.8. Cytokine and apoptotic gene expression

Total RNA was isolated from the peripheral blood mononuclear cells (PBMCs) of early- (94 cases), mid- (195 cases) and late- (171 cases) stages of LSD and 495 skin nodules/scabs (103 early-, 208 mid-, and 184 late-stages) using TRIzol™ reagent (Invitrogen™, ThermoFisher Scientific, CA, USA) following the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Nanotechnologies, USA). Equal concentrations of RNA were converted to complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, CA, USA) in a Mastercycler Personal thermocycler (Eppendorf, Germany).

The mRNA expression levels of cytokines, including Th1-type (IFN-γ, IL-2, TNF-α, IL-1β, IL-8) and Th2-type (IL-4, IL-10, IL-6), as well as genes involved in intrinsic (Bcl-2, BAX, BAK, caspase 9) and extrinsic (FAS, FAS LG, caspase 8, caspase 3) apoptosis pathways, were quantified using quantitative real-time PCR (qRT-PCR). The qRT-PCR was conducted with the QuantiFast® SYBR® Green PCR Kit (Qiagen, USA) on a CFX96™ Real-Time System (BIO-RAD, USA), using GAPDH as an internal reference gene. The reactions were performed in 12.5 μl volumes using specific primers (Supplementary Tables 1 and 2 for cytokines and apoptotic genes, respectively) in clear 0.2 ml PCR strips with flat optical caps (Genetix Biotech Asia Pvt. Ltd.).

Table 1. Quantification of LSD viral load from various tissues during different stages using real-time PCR assay.

Samples	Early-stage	Mid-stage	Late-stage	
Skin nodule/scab biopsy from live animal	6848.75 ± 334.54a p	27388.75 ± 747.40a q	34073.50 ± 2320.54a q	
Blood	1832.00 ± 127.06a p	964.50 ± 46.42bc pq	419.50 ± 40.98ba p	
Lungs	31121.50 ± 1550.92b p	45935.00 ± 4239.82bd q	52161.50 ± 2383.03bd q	
Superficial lymph nodes	10960.75 ± 1186.78a	12671.00 ± 911.74be	15039.00 ± 625.69bcd	
Skin nodule/scab from carcasses	95419.25 ± 2397.58bc p	101372.25 ± 1966.04bf p	131372.00 ± 4114.54bc q	
Tongue	38754.50 ± 1845.68b p	42180.00 ± 1240.06bg pq	52047.00 ± 1265.94be q	
Trachea	27543.50 ± 1908.53bd p	34113.50 ± 1930.94ag pq	42573.75 ± 1696.67acde q	
Note: Results are presented in the table with mean value and standard error of the mean (SEM). Virus load in different samples was given as copies (103) per 5 μL of DNA sample. The superscripts (a, b, c, d, e, f, g) within a column indicate significant difference between the type of samples in a stage of infection and superscripts (p, q) within a row indicate significant difference between stages of infection within a sample. Two-way ANOVA with Tukey’s post-hoc test was used. p < 0.001 statistically significant compared between samples and stage of disease.

Gene expression was measured by quantification of the cDNA with respect to the cDNA from uninfected cattle as calibrator. Normalization was done against the endogenous control GAPDH. The relative expression levels were calculated using the 2-ΔΔCt method, where ΔCt represents the difference in cycle threshold (Ct) values between the target gene and GAPDH in the same sample, and ΔΔCt is the difference between ΔCt of the test sample and that of the calibrator (Livak and Schmittgen 2001).

2.9. Sequencing and phylogenetic analysis

The LSD virus was isolated from skin and lung tissue samples using the African green monkey kidney (Vero) cell line in minimum essential medium (MEM). The LSDV isolates (n = 9) were subjected to amplification of the complete coding sequence of RPO30 and P32 genes (Manjunatha Reddy et al. 2023). Further, the PCR products were cloned into the pGEMT-Easy vector (Promega, Wisconsin, USA) followed by colony PCR confirmation. The plasmid DNA was extracted using a GeneJET Plasmid Miniprep kit (Cat No. K0502, Thermo Fisher Scientific). The products were sequenced by the sanger-sequencing method (Eurofins Genomics India Private Limited, Bengaluru, India) and nucleotide sequences were submitted to the GenBank database.

For phylogenetic analysis, these sequences were compared with published sequences from India and other countries, retrieved from the GenBank database. Multiple sequence alignment was performed using DNASTAR Lasergene software version 6.0 (DNASTAR, Inc., Madison, USA). The complete RPO30 18 gene sequences of additional CaPVs 75 (LSDVs, GTPVs, and SPPVs), retrieved from GenBank, were included for comparative analyses. The Bayesian phylogenetic inference was performed with BEAST 2.5. The Markov Chain Monte Carlo method was run with BEAST, for 10,000,000 generations with a sample taken each 10,00 generations. TreeAnnotator was used to generate the Maximum Clade Credibility (MCC) after discarding the 3% burn-in. The tree was visualized with Figure tree 1.4 (Tamura et al. 2021).

3. Results

3.1. Detection of LSDV in clinical samples

A total of 70 outbreaks of LSD were attended during the study period. Out of 1618 animals examined, 513 animals revealed skin lesions and of this biopsy samples, 495 were found positive for LSDV by either conventional PCR or real-time PCR with a percent positivity of 96.49%. The morbidity, mortality and case fatality rates recorded were 31.70%, 2.97 and 9.37% respectively.

3.2. Risk factors for LSD in cattle

The highest occurrence of LSD was observed in the cattle aged above 5 years (37.42%) followed by the age group of 2-5 years (36.76%) and the least in the age group of below 2 years (25.82%). The disease was recorded more in females (70.53%) compared to males (29.47%). The highest occurrence of LSD was observed in native breeds (56.29%) followed by Holstein-Friesian (28.15%) and Jersey cross-bred (15.56%) cattle.

3.3. Grading and staging of LSD skin lesions

Out of 495 LSDV positive cases, the LSD skin lesions were graded into moderate grade skin lesions in 195 animals (39.39%) followed by mild in 169 animals (34.14%) and severe skin lesions in 131 animals (26.47%) based on the number of skin nodular/scab lesions per 5 cm2 area around neck (2 locations) and body of the animal (3 locations). Out of 495 LSDV positive cases, the early-stage skin lesions were 20.81% (103/495), mid-stage skin lesions were 42.02% (208/495), and late-stage skin lesions were 37.17% (184/495).

3.4. Clinical sings

The affected animals showed skin nodular/scab lesions at various stages of development with variable size and number.

3.4.1. Early-stage

The skin nodules during early-stage are very small in size, firm, circular, slightly elevated from the surface and clearly demarcated from adjacent normal skin and ruffled hairs on the lesion. The skin nodular lesions were randomly distributed on head, neck, limbs, lateral aspect of the body, tail, limbs, udder, and external genitalia. In mild-grade cases, the number of skin nodules were less than 2 per 5 cm2 area (Figure 1A). In moderate-grade cases, skin nodules were less than 4 per 5 cm2 (Figure 1B). whereas, in severe-grade cases, skin nodules were more than 5 per 5 cm2 (Figure 1C).

Figure 1. Skin nodule/scab lesions in early- (A–C), mid- (D–F), and late- (G–I) stages of LSD. (A) Number of skin nodules are less than 2 per 5 cm2 and firm in consistency, circular, slightly elevated from the surface, and ruffled hairs on the lesion. (B) Number of skin nodules are less than 4 per 5 cm2, very small in size, and distributed on neck and lateral aspect of the body. (C) Number of skin nodules are more than 5 per 5 cm2, round and well circumscribed areas of nodules with erect hair, and distributed throughout the body. (D) Skin nodules are elevated, circumscribed, round, and firm or hard in consistency. (E) Enlarged flat topped skin nodules are warm to touch, painful, and pit on pressure. (F) Enlarged skin nodules of more than 5.0 cm size, coalescing, round, firm or hard, and distributed throughout the body. G. Skin nodules showed scab and scar formation. (H) Skin nodules showed necrotic core become sloughing and forming a characteristic ‘sit-fasts’ lesions. (I) Skin nodules were surrounded by hard indurated borders due to fibrosis and different stages of healing.

The clinical signs in early-stages of LSD were pyrexia (up to 105.4 °F), loss of appetite, enlarged superficial lymph nodes like prescapular (Figure 2A) and prefemoral (Figure 2B), lachrymation, watery and serous nasal discharge, salivation (Figure 2C), conjunctivitis, oedema mainly involving limbs and brisket region, reduced milk yield in dairy animals up to 50%, and decreased feed and water intake. In some animals, skin lesions were soft and pit on pressure, and circumscribed ulcerative lesions were noticed on the muzzle and lips (Figure 2C).

Figure 2. Clinical signs in early- (A–C), mid- (D–F), and late- (G–I) stages of LSD. (A) Prescapular lymph node showed enlargement. (B). Prefemoral lymph node showed enlargement. (C) Watery nasal discharge, excessive salivation and circumscribed ulcerative lesions on muzzle and lips (arrow). (D) Necrotic and ulcerated skin scabs become sloughing off and forming a characteristic ‘sit-fasts’ lesion (arrow). (E) Oedema of dependent parts of the body like brisket (arrow). (F) Animal showed corneal opacity and blindness. (G) Moist, circumscribed nodular lesions with Central ulcerated area covered with fibrinonecrotic material in mouth and dental pad (arrow). (H) Necrotic nodular scab lesions on udder (Inset). (I) Open and ulcerated wounds were infested with maggots (Inset).

3.4.2. Mid-stage

The skin nodules during mid-stage are elevated, round, firm/hard, warm to touch, and painful with pit on pressure. In mild-grade cases, skin nodules are elevated, circumscribed, round, and firm or hard in consistency (Figure 1D). In moderate-grade cases, enlarged flat-topped skin nodules are warm to touch, painful, and pit on pressure (Figure 1E). The number of skin lesions ranged between 3 to 4 per 5 cm2 of the affected area. In severe-grade cases, enlarged skin nodules of more than 5.0 cm in size, round, firm or hard, and distributed throughout the body (Figure 1F). In some animals, skin nodules showed pouch out with fresh open clear-cut bordered ulcerated wounds due to sloughing off of the scabs (Figure 2D) with purulent discharge.

The clinical signs in mid-stages were similar but of a high degree with extensive oedema of dependent body parts (Figure 2E) and corneal opacity (Figure 2F) being noticed in some cases. The nodular lesions in the mouth and nasal cavity were moist and circumscribed, and the central ulcerated area was covered with fibrino-necrotic material. A few cases involving pregnant animals showed abortions and skin lesions in aborted foetuses.

3.4.3. Late-stage

The skin nodules during the late stage of the disease showed scab and scar formation (Figure 1G), sloughing of the necrotic core and forming characteristic ‘sit-fast’ lesions (Figure 1H), fibrosis, and hard indurated borders with different stages of healing (Figure 1I) were the prominent lesions in recovering animals. In severe-grade cases, the number of skin nodules/scabs was more than 5 per 5 cm2 of affected areas, which was taken randomly at 5 locations.

The clinical signs in late-stages of LSD are loss of appetite, mucoid to purulent nasal discharge, and open mouth breathing. Moist, circumscribed nodular lesions, and a central ulcerated area covered with fibrino-necrotic material in the mouth and dental pad were noticed (Figure 2G). Some dairy animals showed nodular scab lesions with pit on pressure and necrosis on the udder and teats (Figure 2H). The late-stage animals also showed weak body condition, emaciation, and recumbency with sit fasts and pus formation due to secondary bacterial infection. In rare cases, maggot-infested wounds were also noticed (Figure 2I). In some animals, corneal opacity and blindness were noticed.

3.5. Blood and serum profile

The haematological values showed a significant decrease in the TEC, HB, HCT, and TLC among LSD-affected cattle in comparison to the respective haematological parameters of normal, healthy cattle during early-stage of the disease; whereas, there was a significant increase in mean corpuscular volume (p < 0.05) in all the stages of the disease. Leukopenia with lymphopenia was observed during early-stage of disease. Decreased TPC was recorded in both mid- and late-stages of LSD. Increased TLC with some cases showed either neutrophilia or lymphocytosis or monocytosis during mid- and late-stages of LSD.

There was a significant increase in the mean value of AST and globulin, and a significant decrease in GGT, albumin, total protein, and creatinine values were observed in all three stages of LSD-affected animals compared to control animals. No significant change was observed in the mean values of BUN, ALT and cholesterol between affected and healthy animals.

3.6. Gross pathological lesions

A total of 48 post-mortem examinations were performed and the gross lesions were recorded for the cattle carcasses of different ages and breeds from LSD outbreaks in various geographical regions.

3.6.1. Early-stage

The skin nodules varied in size and extended throughout the thickness of the skin from the epidermis to the deep subcutis. Deeper areas of the skin where the nodules were present, showed congestion, oedema, and haemorrhages with necrotic areas (Figure 3A). Varying degree of serous atrophy of subcutaneous fat along with enlarged, oedematous lymph nodes with multifocal haemorrhages were recorded (Figure 3B). Most of the cases revealed systemic infection characterized by multiple circumscribed nodular lesions with central paler, slightly depressed, necrotic and ulcerative lesions of varied sizes covered by fibrino-necrotic material, surrounded by a line of hyperaemia on the upper digestive and respiratory tract. Spleen showed enlargement with haemorrhages in serosa (Figure 3C). Lungs showed congestion, hepatisation, and prominent interlobular septa (Figure 3D). Trachea showed frothy exudate with haemorrhagic ulcers on mucosa (Figure 3E). Heart showed necro-haemorrhagic epicarditis (Figure 3F). Serosal surface of rumen (Figure 3G) and mesentery (Figure 3H) showed haemorrhages.

Figure 3. Postmortem lesions in early-stage of LSD. (A) Subcutaneous tissue showed congestion, haemorrhages, oedema, necrosis and serous atrophy of fat with yellow gelatinous appearance. (B) Enlarged, congested and haemorrhagic prescapular lymph node. (C) Enlarged spleen with haemorrhages in serosa. (D) Lungs showed congestion, hepatisation and prominent interlobular septa. (E) Fresh haemorrhagic ulcers on tracheal mucosa with frothy exudate (Inset). (F) Necrohaemorrhagic epicarditis. (G) Serosal surface of rumen showed haemorrhages. (H) Mesentery showed haemorrhages.

3.6.2. Mid-stage

Animals that died during mid-stage showed necrosis and haemorrhages with sero-sanguinous fluid in the subcutaneous tissue of the skin (Figure 4A), in addition to early lesions. Lungs also showed multiple circumscribed nodular lesions (Figure 4B). Multiple circumscribed nodular lesions in digestive and respiratory tract (Figure 4C). Further, these organs contained multiple elevated either circumscribed or irregular erythematous or hyperaemic necro-haemorrhagic lesions on serosal surfaces (Figure 4D). Small intestinal (Figure 4E) and abomasal (Figure 4F) mucosal surfaces also showed congestion and diffuse haemorrhagic ulcerative lesions in mucosa. The liver showed necrotic foci and circumscribed nodular lesions (Figure 4G). The gallbladder was markedly distended with serosal haemorrhages and ulcerative lesions in the mucosa (Figure 4H). Kidneys showed multifocal necrotic lesions on the surface (Figure 4I). Mucosa of the urinary bladder showed severe haemorrhages (Figure 4J).

Figure 4. Postmortem lesions in mid-stage of LSD. (A) Subcutaneous tissue of skin showed congestion, haemorrhages, necrosis with sero-sanguinous edematous fluid. (B) Lungs showed congestion, haemorrhages, focal pneumonic lesions, and multiple circumscribed nodular lesions (arrow). (C) Tracheal mucosa showed necrotic circumscribed nodular lesions. (D) Multiple circumscribed elevated hyperaemic necro-haemorrhagic lesions on serosal surface of rumen (Inset). (E) Small intestine showed congestion and diffuse haemorrhagic ulcerative lesions in mucosa. (F) Congestion and haemorrhages on mucosal surface of abomasum. (G) Liver showed necrotic foci (yellow arrow) and circumscribed nodular lesions (black arrow). (H) Markedly distended gallbladder with serosal haemorrhages and ulcerative lesions in mucosa (Inset). (I) Multifocal necrotic lesions on the surface of kidney (arrow). (J) Mucosa of urinary bladder showed severe haemorrhages.

3.6.3. Late-stage

In late-stage, external sit-fast lesions with maggot wounds were commonly noticed, in addition to early- and mid-stage lesions. Muzzle and lower lip showed necrotic scab lesions with healing (Figure 5A). Dorsal surface of the tongue showed elevated necrotic scab lesions (Figure 5B). Subcutaneous tissue of skin and muscle showed severe congestion, haemorrhages, oedema, and necrosis (Figure 5C). Septicaemic lesions were noticed in internal organs. Lungs showed multifocal areas of consolidation with pneumonic lesions (Figure 5D). Trachea showed congested mucosa and frothy exudate in the lumen with necrotic circumscribed elevated nodular lesions (Figure 5E). Serosal surface of the rumen (Figure 5F), reticulum (Figure 5G) and abomasum (Figure 5H) showed diffuse, multiple circumscribed, elevated hyperaemic, and necro-haemorrhagic ulcerative lesions. Aborted foetuses showed severe congestion of cutaneous blood vessels and necrotic lesions in the skin (Figure 5I).

Figure 5. Postmortem lesions in late-stage of LSD. (A) Muzzle and lower lip showed necrotic scab lesions with healing. (B) Dorsal surface of tongue showed elevated necrotic scab lesions (arrow). (C) Subcutaneous tissue and muscle showed severe congestion, haemorrhages, oedema, and necrosis. (D) Lungs showed circumscribed nodular scab lesions (Inset) and marbling appearance due to prominent interlobular septa, severe congestion, haemorrhages, and pneumonic lesions. (E) Upper tracheal mucosa showed necrotic circumscribed elevated nodular lesions (arrow). (F) Multiple circumscribed elevated hyperaemic necro-haemorrhagic ulcerative lesions on serosal surface of rumen. (G) Diffuse haemorrhages with necrotic ulcerative lesions in serosal surface of reticulum (arrow). (H) Circumscribed ulcerative lesions in the mucosal surface of abomasum (arrow). (I) Aborted foetus showed severe congestion of cutaneous blood vessels and necrotic lesions in skin (Inset).

3.7. Histopathological lesions

A total of 495 skin nodule/scab samples including 103 early-, 208 mid-, and 184 late-stage skin nodules were subjected to histopathological evaluation. The histopathological lesions described in the present study were first time reported from natural cases of LSDV-infected cattle.

3.7.1. Early-stage skin lesions

In the early-stage, acanthosis with hyperplastic epidermal layer, vacuolar degeneration in stratum spinosum (Figure 6A), hyperkeratosis, and parakeratosis were noticed. The epidermis showed severe hydrophic degeneration with intraepithelial microvesicle formation of varying sizes filled with eosinophilic fluid (Figure 6B,E) and necrosis of epithelial cells (Figure 6C,F). The epidermis revealed swollen epidermal cells and spongiosis (inter-cellular oedema) with increased intercellular interdigitating spaces. Epidermis showed marked congestion of blood vessels (Figure 6D). The dermis showed mild to severe perivasculitis with mild to marked infiltration of inflammatory cells especially mononuclear cells (Figure 6C,F) and necrosis of muscle fibres (Figure 6G–1). Mild hyperplasia of sebaceous glands was noticed.

Figure 6. Mild (A,D,G), moderate (B,E,H), and severe (C,F,I) grading of histopathological lesions during early-stage of LSD. (A) Skin nodule/scab biopsy showed mild to moderate hydrophic degeneration in epidermis and inflammatory cells infiltration in dermis. H&E x200. (B) Epidermis showed severe hydrophic degeneration with intraepithelial microvesicle formation of varying sizes filled with eosinophilic fluid (asterisk). H&E x200. (C) Epidermis showed necrosis of epithelial cells (arrowhead) with hyperkeratosis (asterisk) and inflammatory cells infiltration in dermis. H&E x100. (D) Epidermis showed mild vacuolar degeneration and marked congestion of blood vessels (arrowhead). H&E x400. (E) Severe hydrophic degeneration with intraepithelial microvesicle formation (asterisk) and necrosis of epithelial cells. H&E x400. (F) Necrosis of epithelial cells with nuclear debris. H&E x400. (G) Dermis showed mild perivasculitis with infiltration of mononuclear cells. H&E x100. (H) Severe perivasculitis with marked inflammatory cells infiltration and necrosis of muscle fibres. H&E x100. I. Severe perivasculitis with marked infiltration of inflammatory cells and necrosis of muscle fibres. H&E x200.

3.7.2. Mid-stage skin lesions

In this stage, lesions were severe compared to the early-stage. Detaching necrotic scab lesions with acanthosis, hyperkeratosis and necrosis of epidermal cells were noticed (Figure 7A,B). Hyperplastic epidermal layer showed oval or round or elongated eosinophilic intracytoplasmic inclusion bodies, which were varied in size (Figure 7F). In epidermal cells more apoptotic changes (Figure 7D) were noticed. In many cases, epithelial cells of skin and hair follicles showed enlarged round to oval nuclei with margination of chromatin giving vacuolated appearance at the centre, which resembled sheeppox cells or stellate cells, also known as cellules claveleuse (Figure 7E). In severe cases, marked infiltration of mononuclear cells especially dendritic cells, lymphocytes and macrophages were noticed in dermis (Figure 7C). Dermis and hypodermis showed mild to severe vasculitis and perivasculitis at multiple places, severe necrosis of muscle fibres, haemorrhages, vacuolization of endothelial cells, and infiltration of varied number of inflammatory cells especially macrophages around the blood vessels were recorded (Figure 7G–1).

Figure 7. Mild (A,D,G), moderate (B,E,H), and severe (C,F,I) grading of histopathological lesions in mid-stage of LSD. (A) Skin nodule/scab biopsy showed necrotic scab lesion (arrowhead) with acanthosis, hyperkeratosis, and inflammatory cells infiltration in dermis. H&E x100. (B) Epidermis showed detaching necrotic scab lesion (arrowhead) with inflammatory cells infiltration in dermis. H&E x100. (C) Marked infiltration of mononuclear cells especially dendritic cells (inset), lymphocytes and macrophages in subepidermis and dermis. H&E, x100. (D) Epidermis showed mild vacuolar degeneration, haemorrhages, necrosis and apoptosis of epidermal cells (arrowhead), and eosinophilic intracytoplasmic inclusion bodies (Inset). H&E, x200. (E) Cellules claveleuse showed vacuolar degeneration with round to oval nuclei showing margination of chromatin giving vacuolated appearance at the Centre (arrowhead). H&E x400. (F) Necrosis with eosinophilic intracytoplasmic inclusion bodies of varying size (arrowhead). H&E x200. (G) Dermis showed necrosis of muscle fibres, haemorrhages (asterisk) and infiltration of inflammatory cells. H&E x100. (H) Severe perivasculitis with marked inflammatory cells infiltration. H&E x200. (I) Severe necrosis of muscle fibres, congestion (asterisk) and infiltration of inflammatory cells. H&E x200.

3.7.3. Late-stage lesions

In late-stage, lesions were characterized by formation of scabs and fibrotic skin nodules. Histopathologically, scabs appeared as homogenously coagulated eosinophilic hyalinised amorphous masses with necrotic cellular debris, discontinued epidermal layer on both side, and infiltration of inflammatory cells (Figure 8A–C). Epidermis showed necrotic and apoptotic epidermal cells with eosinophilic intracytoplasmic inclusions bodies of varying size in the upper layers of the scab (Figure 8D,E). Complete necrosis of epidermal and dermal layers with infiltration of polymorphonuclear and mononuclear cells was noticed (Figure 8F). Necrotizing vasculitis of dermal blood vessels, particularly the deep dermal plexus and perivasculitis with infiltration of macrophages were noticed (Figure 8I). Regenerative changes were prominent with connective tissue proliferation in dermal region extending up to subcutis with or without hyperaemic blood vessels (Figure 8G,H).

Figure 8. Mild (A,D,G), moderate (B,E,H), and severe (C,F,I) grading of histopathological lesions in late-stage of LSD. (A) Skin nodule/scab biopsy showed homogenous eosinophilic necrotic scab lesion (asterisk) with nuclear debris, and infiltration of inflammatory cells. H&E x100. (B) Necrotic scab lesion (asterisk) with nuclear debris and infiltration of inflammatory cells. H&E x100. (C) Severe necrosis (asterisk) with nuclear debris and infiltration of inflammatory cells in epidermis and dermis. H&E x100. (D) Epidermis showed vacuolar degeneration, necrosis and apoptotic epidermal cells with eosinophilic intracytoplasmic inclusion bodies (Inset). H&E x200. (E) Homogenous eosinophilic necrotic areas with eosinophilic intracytoplasmic inclusion bodies of varying size (Inset). H&E x200. (F) Complete necrosis of epidermal and dermal layers with infiltration of polymorphonuclear and moinonuclear cells. H&E x200. (G) Dermis showed moderate proliferation of fibroblast and connective tissue. H&E x200. (H) Marked proliferation of fibroblast and connective tissue. H&E x200. (I) Necrotizing vasculitis with perivascular infiltration of inflammatory cells. H&E x200.

3.8. Other organs histopathological findings

Lymph nodes: The early-stage showed severe congestion, haemorrhages, subscapular oedema, and occasional lymphoid depletion in the follicles of prescapular and prefemoral lymph nodes (Figure 9A). During mid-stage, necrosis of lymphoid follicles, congestion, and haemorrhages were noticed in the cortex of lymph nodes (Figure 9B). Whereas, in late-stage, multifocal areas of necrosis, large number of macrophages and few neutrophils infiltration were observed in the medulla (Figure 9C).

Figure 9. Histopathological lesions in different internal organs during early- (A,D,G), mid- (B,E,H), and late- (C,F,I) stages of LSD. (A) Prescapular lymph node showed severe congestion, haemorrhages and lymphoid depletion. H&E x100. (B) Necrosis of lymphoid follicles, congestion and haemorrhages in cortex. H&E x100. (C) Severe necrosis of lymphoid follicles. H&E x200. (D) Submucosa of trachea showed severe congestion, haemorrhages, oedema and infiltration of inflammatory cells. H&E x100. (E) Submucosa showed congestion and infiltration of inflammatory cells, and adventitia showed necrotizing vasculitis with infiltration of inflammatory cells. H&E x100. (F) Mucosa showed necrosis of epithelial cells with infiltration of inflammatory cells. H&E x100. (G) Lungs showed severe congestion, haemorrhages and oedematous fluid in alveoli and bronchioles. H&E x100. (H) Periarteritis with infiltration of mononuclear cells and eosinophilic intracytoplasmic inclusion bodies (inset). H&E x400. (I) Bronchopneumonia with accumulation of neutrophils in the alveolar lumen and congestion of interalveolar capillaries. H&E x200.

Spleen: Spleen revealed mild lymphoid depletion in white pulp during early-stage. During mid-stage, necrosis in white pulp areas, severe congestion, and presence of haemosiderin pigments in the red pulp were noticed. In late stage, the central arterioles revealed thickening with infiltration of inflammatory cells, and multifocal areas of necrosis.

Trachea: During early-stage, submucosa of trachea showed severe congestion, haemorrhages, oedema, and infiltration of inflammatory cells (Figure 9D). During mid-stage, congestion and necrotizing vasculitis (periarteritis) with infiltration of mononuclear cells especially macrophages were recorded (Figure 9E). During late-stage, tracheal mucosa showed necrosis and ulceration of epithelial cells covered with fibrinous material along with bacterial colonies and infiltration of inflammatory cells (Figure 9F).

Lungs: During the early stage, the lungs showed severe congestion, haemorrhages, and oedematous fluid in alveoli and bronchioles (Figure 9G). During mid-stage, necrotizing vasculitis with infiltration of varying numbers of mononuclear inflammatory cells especially alveolar macrophages with eosinophilic intracytoplasmic inclusion bodies (Figure 9H). The alveoli and bronchioles were filled with fibrino-purulent necrotic material, bacterial colonies, infiltration of neutrophils, and congestion of interalveolar capillaries were observed during mid- and late-stages (Figure 9I).

Tongue: During early-stage, filiform and fungiform papillae were severely affected and showed swelling, vacuolar degeneration, congestion, necrosis, and loss of squamous cells (Figure 10A). During mid-stage, mucosal squamous cells thickening of mucosal epithelium (acanthosis) and hyperkeratinization with mucosal ulceration with loss of surface epithelium and papillae (Figure 10B). During late-stage, mucosal epithelium showed eosinophilic intracytoplasmic inclusion bodies. Muscular layer showed congestion, severe vasculitis with necrotic lesions, and infiltration of mononuclear cells (Figure 10C).

Figure 10. Histopathological lesions in different internal organs during early- (A,D,G), mid- (B,E,H), and late- (C,F,I) stages of LSD. (A) Tongue epithelial cells showed vacuolar degenerative changes with congestion. H&E x200. (B) Necrosis and ulceration with loss of epithelial layer in tongue. H&E x100. (C) Skeletal muscle of tongue showed congestion, necrosis and infiltration of mononuclear cells with eosinophilic intracytoplasmic inclusion bodies in epithelium (inset). H&E x200. (D) Kidneys showed congestion and degeneration of tubular cells. H&E x200. (E) Renal tubules contained eosinophilic proteinaceous cast and and degeneration of tubular cells. H&E x200. (F) Infiltration of mononuclear cells in kidneys. H&E x200. (G) Liver showed sinusoidal congestion and cholestasis in bile ducts. H&E x200. (H) Liver showed necrosis of hepatocytes and cholestasis. H&E x200. (I) Liver showed severe fatty degenerative changes. H&E x200.

Kidneys: Kidneys showed congestion and degeneration of tubular epithelial cells during early-stage (Figure 10D). Renal tubules contained eosinophilic proteinaceous cast and degeneration of tubular epithelial cells during mid-stage (Figure 10E). Vacuolar degeneration and coagulative necrosis of tubular epithelial cells, vasculitis with thickening of blood vessel wall, and infiltration of mononuclear cells were noticed during late-stage (Figure 10F).

Urinary bladder: Urinary bladder showed vacuolar degeneration of transitional epithelium during early-stage. During mid-stage, necrosis and infiltration of mononuclear cells especially macrophages. During late-stage, thickening of serosal layer with vasculitis and perivascular infiltration of macrophages were noticed.

Liver: Liver showed sinusoidal congestion and stagnation of bile in the lumen of bile ducts during early-stage (Figure 10G). During mid-stage, multifocal areas of necrosis, infiltration of mononuclear cells, cholestasis, mild biliary hyperplasia, and connective tissue proliferation in the periportal areas were noticed (Figure 10H). Severe fatty degenerative changes, necrosis, and infiltration of mononuclear cells were noticed during late-stage (Figure 10I).

Gallbladder: Gallbladder revealed loss of mucosal epithelium, necrosis, congestion, haemorrhage, vasculitis, and perivascular infiltration of macrophages in the submucosa.

Heart: Heart showed epicardial thickening with connective tissue proliferation, vasculitis, perivasculitis, and macrophage infiltration.

Rumen and reticulum: Rumen and reticulum revealed vacuolar degeneration of squamous epithelial cells, loss of lining mucosal epithelium, severe congestion of blood vessels, and areas of haemorrhages in the lamina propria and submucosa during early-stage. During mid-stage, prominently many blood vessels revealed vasculitis with perivascular infiltration of mononuclear cells especially macrophages, in addition to early-stage lesions. During late-stage, multifocal areas of marked infiltration of macrophages were noticed in between the connective tissues.

3.9. LSDV distribution in tissues

Skin: The skin nodular/scab lesions revealed brownish and granular intracytoplasmic immunolocalization of LSDV antigen. In both early- and mid-stages, positive immunoreactivity was observed in the epithelial cells of epidermis, which varied in the intensity among the cells (Figure 11A–C). Strong positive immunoreaction was observed in the infiltrated mononuclear cells, especially macrophages in the subepidermis, dermis, hypodermis, and perivascular areas. Sebaceous glandular epithelial cells showed granular positive immunoreactivity for LSDV antigen (Figure 11E). During late-stage of LSD, granular brownish immunopositive reaction was scattered in clumps in the scabs, cicatrized fibrotic nodules, and necrosed skin scab (Figure 11D).

Figure 11. Immunohistochemical localization of LSDV antigen in the skin and internal organs. (A) Skin nodule/scab showed positive immunoreactivity in epidermal cells and inflammatory cells in dermis. IP-DAB-MH x200. (B) Epidermis of skin nodule/scab showed positive immunoreactivity in epithelial cells. IP-DAB-MH x 400. (C) Skin nodule/scab showed positive immunoreactivity in acanthotic epidermal cells. IP-DAB-MH x 200. (D) Necrosed skin scab showed granular brown colour positive immunoreactivity during late-stage of LSD. IP-DAB-MH x 200. (E) Skin nodule/scab showed positive immunoreactivity for LSDV antigen in sebaceous glandular epithelial cells during mid-stage of LSD. IP-DAB-MH x 400. (F) Submucosa of tongue showed positive immunoreactivity in macrophages infiltrated around the blood vessels during mid-stage of LSD. IP-DAB-MH x400. (G) Spleen showed positive immunoreactivity in the macrophages around perivasculitis. IP-DAB-MH x 200. (H) Lungs showed positive immunoreactivity in the infiltrated alveolar macrophages. IP-DAB-MH x 200. (I) Lymph node showed positive immunoreactivity in medullary macrophages. IP-DAB-MH x 200.

Internal organs: In lungs, intense intracytoplasmic immunopositive reaction was observed in the infiltrated alveolar macrophages (Figure 11H); whereas, mild immunoreaction was observed in the bronchiolar epithelial cells and pneumocytes. Similarly, in lymph nodes, macrophages present in the medulla and perivascular areas showed positive immunostaining for LSDV antigen (Figure 11I). In spleen, splenocytes and macrophages present in the white pulp, subscapsular sinuses, and perivascular areas showed positive immunostaining for LSDV antigen (Figure 11G). In other organs, infiltrated macrophages revealed positive immunostaining. In tongue, positive cytoplasmic immunoreactivity was observed in the cytoplasm of squamous epithelial cells of the mucosal layer, filiform and fungiform papillae, and infiltrated macrophages (Figure 11F). The inclusion bodies in the cytoplasm of affected epithelial cells were intensely stained.

3.10. LSD viral load during different stages of LSD

The present study quantified the number of DNA copies of LSDV during different stages like early, mid and late stages of LSD nodular lesions and in different organs (Figure 12, Table 1). There was a significant difference (p < 0.001) in the copy number of LSDV between early and late-stage lesions; whereas, no significant difference between early and mid-stages, and mid and late-stages of LSD nodular lesions. Among the different post-mortem samples, the highest number of LSDV copies were found in skin nodules/scabs followed by the trachea, tongue, and superficial lymph nodes compared to other organs including blood (Figure 12 and Table 1).

Figure 12. Quantification of LSD viral load from various tissues during different stages using real-time PCR assay: Results are presented in the bar diagram with mean value and standard error of the mean (SEM). Virus load in different samples was given as copies (103) per 5 μL of DNA sample. Two-way ANOVA with Tukey’s post-hoc test was used. P < 0.001 statistically significant compared between samples and stage of disease.

3.11. Cytokine gene expression in PBMC and skin

The kinetics and magnitude of Th1-type (IFN-γ, IL-2, TNF-α, IL-1β and IL-8) and Th2-type (IL-4, IL-10 and IL-6) cytokines were significantly different between the early, mid and late-stages in LSDV-infected cattle (Figure 13A,B). The expression levels of IFN-γ showed significantly (p < 0.05) lower levels in PBMCs and skin nodules in mid and late-stages when compared to early-stage. The IL-2 and IL-1β expression levels were significantly (p < 0.05) higher in PBMCs and skin nodules in late-stage when compared to early-stage of infection. The TNF-α and IL-8 mRNA gene expression levels were significantly (p < 0.05) higher in PBMCs and skin nodules in mid and late-stages when compared to early-stage of infection. These significantly higher pro-inflammatory cytokines were correlated with severe clinical signs and nodular skin lesions, increased viral load, gross and histopathological lesions with infiltration of inflammatory cells and necrosis in tissues. The Th2-type cytokines namely, IL-4 and IL-6 mRNA gene expression levels were significantly (p < 0.05) higher in PBMCs and skin nodules in mid- and late-stages when compared to early-stage of infection. Increased expression of pleiotropic cytokine IL-6 was correlated with increased viral load and vascular lesions like necrotizing vasculitis with haemorrhages and oedema, because IL-6 is mainly produced by damaged endothelial cells and smooth muscle cells in the tunica media of blood vessels. The IL-10 mRNA gene expression levels showed decreased and non-significant changes in PBMCs and skin nodules in early-, mid- and late-stages of LSDV infection. Decreased expression of anti-inflammatory cytokine IL-10 was correlated with increased expression of pro-inflammatory cytokines and severe histopathological lesions.

Figure 13. Quantification of Th1-type (A) and Th2-type (B) cytokines, intrinsic (C) and extrinsic (D) pathways of apoptosis genes expressions in PBMCs and skin nodule/scab by quantitative real-time PCR during early-, mid-, and late-stages of LSD. Two-way ANOVA with bonferroni post-test was used. Results are presented as bar diagram with mean ± SEM at each stage. Error bars indicate SEM. *p < 0.05 and **p < 0.01 statistically significant compared with early-stage of LSD.

3.12. Apoptotic gene expression in PBMC and skin

The mRNA expression levels intrinsic (Bcl-2, BAX, BAK, and caspase 9) and extrinsic (FAS, FAS LG, caspase 8, and caspase 3) pathways of apoptosis were significantly different between the early-, mid- and late-stages in LSDV-infected cattle (Figure 13C,D). The intrinsic pathway genes of apoptosis namely, Bcl-2, BAX, BAK, and caspase 9 mRNA expression levels were significantly downregulated in PBMCs and skin nodules in mid- and late-stages as compared to early-stage of LSDV infection. The extrinsic pathway genes (FAS, FAS LG, caspase 8, and caspase 3) expression levels were significantly upregulated in PBMCs and skin nodules during mid- and late-stages of infection when compared to early-stage of LSDV infection. Increased expression of apoptotic genes were correlated with increased viral load, histopathological lesions with apoptosis of cells in tissues, and leukopenia.

3.13. Phylogenetic analysis of RPO30 and P32 genes of LSDV

In the present study, 33 complete RPO30 (OM362828 to OM362845 and OP903443- OP903456) and 9 P32 (OP903431 to OP903433, OP903439 to OP903441 and OR47296969- OR 47296971) gene sequences were submitted to the GenBank database and the accession numbers were obtained. The phylogenetic analysis revealed separate clustering of LSDV with that of vaccines strains of LSDV, SPV, and GPV. All isolates in the present study were closely related to LSDV form Kenya (Figure 14). The field LSDV isolates from cattle in the current study formed distinct sub-group with in the field LSDV with respect to both the gene sequences. The isolates from first-wave (2020-2021) grouped away from the isolates of the second-wave (2021-2022). There was only one nucleotide change at the position of 54 (A to G) in RPO30 gene among the second-wave isolates. But this nucleotide change did not lead to change in the protein sequence. However, the P32 gene (OR472969) showed the highest number of variations in the nucleotides at the positions 8 (A to G), 30 (A to C), 70 (A to C), 98 (A to C), 124 (A to C), 112 (A to C) and 226 (A to C), which led to change in amino acid changes at 3 (D to G), 24 (K to Q), 33 (K to T), 42 (K to Q) and 76 (K to Q) positions.

Figure 14. Bayesian Evolutionary phylogenetic analysis of full-length RPO30 gene of LSDV from LSD outbreaks in cattle. The Bayesian time based maximum clade credibility (MCC) tree was constructed using MEGA11. The Bayesian phylogenetic inference was performed with BEAST 2.5. The markov chain monte carlo method was run with BEAST, for 10,000,000 generations with a sample taken each 10,00 generations. Tree annotator was used to generate the maximum clade credibility (MCC) after discarding the 3% burn-in. The tree was visualized with figure tree 1.4. The LSDV isolates from cattle were clustered with LSDV field strains circulating in India and closely related to Kenya LSDV isolates. The green color indicates indicate the newly obtained isolates in this study.

Multiple sequence alignment of protein selected sequences shown variation at gap 6-12 position was observed in SPPV where as in LSDV and GPV gap was not observed but at 6 position isoleucine (I) and LSDV (I) present, GTPV and LSDV gap was shown the NSYSDN 7 – 12 positions. 51 position GTPV (T), LSDV and SPPV (I), 92 position GTPV and LSDV (E) whereas at SPPV (D), 96 position GTPV and LSDV (D) whereas at SPPV (E), 98 GTPV and SPPV (S) whereas at LSDV (P), 102 position GTPV and SPPV (N) whereas at LSDV (D), 135 position LSDV and SPPV (N) whereas at GTPV (D), 141 position GTPV and SPPV (Y) whereas at LSDV (H), 160 position GTPV and LSDV (K) and SPPV (E) aminoacid variations was observed. Sheep pox RNA Polymerase (Acc. no- MK607151) were shown Percent Identity Matrix - created by Clustal 2.1 values are SHPV 100.00, GTPV and LSDV (Acc.no- MK607149)- 95.36, LSDV (Acc.no- OP903447)- 94.85 and GTPV with LSDV (Supplementary Figure 1).

4. Discussion

Lumpy skin disease is a vector transmitted, economically important and emerging viral disease of cattle and buffaloes. LSD was for a long time restricted to sub-Saharan Africa during 1929 to 1986 and exotic to India (OIE 2017). However, over the past decades, LSD has been slowly infiltrated into new territories first into the Middle East in 1988, Turkey during 2013 to 2015, since 2015 into most of the Balkan countries, Caucasus and the Russian Federation, and recently into Southern Asia in 2019 (OIE 2017; Mercier et al. 2018). In India, first outbreak of LSD was reported in Odisha state in the year 2019 (Sudhakar et al. 2020). The disease later spread to all the states of India within a span of 2 to 3 years and caused high morbidity and significant mortality. In the present study, case fatality rate was 9.70%, which was similar to variable case fatality rates that have been reported during earlier LSD outbreaks from different regions of world (Tuppurainen et al. 2017; Sudhakar et al. 2020; Mathewos et al. 2022). The case fatality rate of LSD is less than 10%; however, during recent natural outbreaks in India up to 15% was reported. Geletu et al. (2024) reported the case fatality rate of 16.44% in West Hararghe Zone, Eastern Ethiopia. However, the case fatality rate of 3.03 − 5.26 was reported in cattle of south-west part of Bangladesh (Biswas et al. 2020). The variation in case fatality, morbidity and mortality rates have been attributed to several biological and climatic factors such as breed of cattle, immunological status of the affected population, insect vectors involved in the transmission, and the virus isolate (Mathewos et al. 2022).

In the present study, the LSDV strains responsible for the cattle outbreak in India were distinct from the first outbreak reported from Odisha, India. But, genetically related to the field strains of LSDV originated from Kenyan LSDV strains circulating in India and other Asian countries except China, in which Russian strains are causing the LSDV outbreaks (Sudhakar et al. 2020). All the field isolates from study were falling under cluster 1.2. The RPO30 gene is used originally to differentiate between the three capripoxviruses, but any nucleotide sequence variation is highly indicative of a true difference between vaccine strains, field isolates and differentiating the Cluster 1.1 and 1.2 (Mazloum et al. 2023). In the recent LSD outbreak from Sikkim, India, which notably occurred in Yaks, an atypical host, also revealed similar clinical pattern and the LSD virus was akin to the Kenyan LSDV predominantly found in the Indian subcontinent with high case fatality rate (Manjunatha Reddy et al. 2023). The whole genome sequencing revealed high variations in LSDV field strains at nucleotides might be responsible for severe form of disease especially with high mortality in Belagavi region of Karnataka and Gujarat states, which had reported highest number of cases and deaths during the year 2022 (Yadav et al. 2024). While variations in clinical manifestations are observed in atypical or spill-over host, with generally milder symptoms reported, it is important to note that no genetic variations have been identified in the LSDV responsible for these spill-over events (Manjunatha Reddy et al. 2023).

Variable lesions concerning the number, size, shape, and distribution pattern of skin nodules and their morphology depending on the stage, duration, and severity of the disease were contrary to the observations of earlier workers (Sudhakar et al. 2020; Rouby et al. 2021; Parvin et al. 2022). It has been reported that the development of lesions was due to epitheliotropism of the LSD virus and extensive vasculitis. However, in the current study, vasculitis with perivascular infiltration of inflammatory cells was one of the most prominent histopathological lesions observed in all the organs. Vasculitis leads to thrombosis and disrupts the normal blood supply to the tissues causing degeneration and ultimately death of the cell.

The nodular lesions were mild in 34.14% of cases, moderate in 39.39% of cases, and severe in 26.47% of cases, however, earlier studies have reported that the severity of LSD skin lesions varied among animals (Hasib et al. 2021) in both natural infections and in experimentally challenged animals (Dietze et al. 2018; Shumilova et al. 2023). Further, as per the previous reports, where the LSD lesions were mostly reported only on the skin and lungs of the affected animals (Kononov et al. 2019; Sanz-Bernardo et al. 2020; Shumilova et al. 2023), we observed that the infection was mostly systemic with necrotic nodular lesions on various organs including the tongue, larynx, trachea, lungs, rumen, reticulum, abomasum, liver, and gallbladder. These lesions in various internal organs could be attributed to the dissemination of the LSD virus following leukocyte-associated viraemia mainly through circulation, which could have been because of the increased viral load in blood. Immunohistochemically, macrophages were shown to contain LSD viral antigen and reported to play a role in the spread of LSDV through leukocyte-associated viraemia to different organs of the body.

In this study, many of the histopathological lesions described are reported first time in natural cases of LSD during different stages of infection, i.e. early-, mid-, and late-stages, where the LSDV-induced lesions were observed in the skin nodule/scab, lymph nodes, trachea, lungs, tongue, heart, rumen, reticulum, liver, gallbladder, kidneys, and urinary bladder. There were scarce reports on microscopic descriptions of the LSDV-induced lesions in various organs (Dietze et al. 2018).

The mucous membranes of digestive and respiratory tracts were often affected with multiple discrete ulcerative and necrotic nodular lesions causing difficulty in swallowing, severe dyspnoea, and asphyxia. Further, the involvement of internal organs like lymph nodes, lungs, heart, liver, kidneys, uterus, udder, and testes during LSDV infection might have contributed to high mortality (Kononov et al. 2019). The post-mortem examination of the deceased animals also revealed secondary bacterial infections. Hence, use of antibiotics during LSD infection is necessary to combat the secondary bacterial infections. The secondary infections might have occurred due to the LSDV-induced immunosuppression, which was evident by lymphoid depletion in lymphoid organs and the lesions of fibrino-purulent necrotic lesions on the mucous membranes of the trachea and bronchopneumonia, might have been another factor for mortality and also for the late recovery of clinical cases (Parvin et al. 2022).

The main histopathological lesions in the skin during the mild grade of early-stage were vasculitis with infiltration of inflammatory cells and rarely eosinophilic intracytoplasmic inclusions in the epithelial cells of the prickle cell layer, indicating that infection begins with the affection of vascular endothelium (vasculitis) rather than inflammation of epithelial cells. Further, in some of the moderate-grade early and mid-stage cases, there was ballooning degeneration, microvesicle formation and necrosis in the epidermal layer which could be attributed to the tropism of the LSD virus towards keratinocytes (Vasković et al. 2019; Mathewos et al. 2022; Parvin et al. 2022).

The blood profile of the LSD-affected animals revealed a reduction in TEC which was probably attributed to the slight haemolysis due to vasculitis, a consistent finding in LSD lesions (Parvin et al. 2022). As reported earlier by Abutarbush (2015) and Rouby et al. (2021), there was leukocytosis accompanied by either neutrophilia or lymphocytosis or monocytosis during mid- and late-stages of the disease. Leukopenia is usually seen during the early stage of the acute infection, after which, the production of neutrophils is intensified leading to leukocytosis due to secondary bacterial pyogenic infections Reddy GBM, Mounica P S, Sudeep N, Vikram R, Garam GB,. Increased AST enzyme is an indication of liver and muscle injury in LSD-affected animals. Hypoalbuminemia with decreased total protein was in agreement with Abutarbush (2015), who opined that it could be due to loss of appetite, decreased protein synthesis, and higher catabolic rate with progressive emaciation in the LSD-affected animals. However, on the contrary, increased albumin and total protein were reported by Abutarbush (2015) and Rouby et al. (2021), who stated that these changes were due to dehydration in the LSD-affected animals. Further, the elevated globulin levels may be due to increased γ globulins consequent upon LSD infection in the affected cattle, which was concurrent with the observation of earlier reports (Rouby et al. 2021).

Among the inflammatory cells, macrophages and dendritic cells among the mononuclear cells during early- and mid-stages, and neutrophils during late-stage were majorly concentrated around the affected blood vessels in the sub-epidermis, dermis, and hypodermis (Vasković et al. 2019; Rouby et al. 2021). The infiltration of inflammatory cells could be primarily due to virus-induced chemotaxis and/or due to secondary bacterial or fungal infection that is correlated with the increased expression of IFN-γ and IL-8, which is a macrophage activator and neutrophil chemotactic factor, respectively.

There are no reports available on the probable tropism of LSDV to the cells of monocyte-macrophage series and immunolocalization of LSDV in internal organs. The present study for the first time demonstrated the localization of LSDV antigen in different internal organs during the early, mid, and late stages of LSD, which is going to be a unique contribution for understanding the pathology and pathogenesis of LSDV involving multi-systems. The cytoplasmic immunostaining of LSDV antigen was demonstrated more in the epithelial cells of the epidermis and hair follicles of the skin, and macrophages infiltrated into the sub-epidermis, dermis, hypodermis and perivascularly. These findings were in concurrence with the observations of earlier workers (Sanz-Bernardo et al. 2020; Rouby et al. 2021) and indicated the active replication of LSDV in these cells because the LSD virus is epitheliotropic.

In the dead animals, the highest copies of LSDV were recorded in the skin followed by the trachea, tongue and superficial lymph nodes, which could be attributed to the active replication of LSDV in the constituent epithelial cells of these organs and infiltrated macrophages surrounding the vasculitis, as shown by immunohistochemistry and systemic spread of LSDV mainly through circulation by monocytic leukocytes during viraemia. Similar observations were reported by Tuppurainen et al. (2005), where high concentration was observed in skin and lymph nodes, moderate in the lungs and rumen, and lesser concentration in the liver and kidneys. Only two reports in the literature are available on the distribution of LSDV antigen in lymph nodes and the absence of immunostaining was reported in the lungs, liver, spleen and heart (El-Neweshy et al. 2013). The observations of the present study indicated that the trachea, tongue and superficial lymph nodes apart from skin lesions are suitable tissue samples from dead animals for the diagnosis of LSDV infection.

The progressive increase in the LSDV DNA copies from early-stage to late-stage of the skin lesion development indicates that, as the disease progresses, the viral load increases with its active virus replication in the infected tissues. However, no significant difference was observed between the mid- and late-stages of skin lesions, indicating the persistence of the virus in the late-stage of skin lesions like scabs and scars in higher concentration for a longer period. These observations were amply supported by immunohistochemistry, which detected higher concentrations of LSDV antigen during mid- and late-stages of skin lesions compared to early-stage lesions. This might be attributed to high and persistent titres of LSDV in the lesions for 30-92 days following the onset of acute disease (Tuppurainen et al. 2005; Babiuk et al. 2008), which was similar to that of sheeppox and goatpox viruses, and indicated that skin scab lesions are most suitable samples for diagnostic purposes.

Information on the role of pro- and anti-inflammatory cytokines in the pathogenesis of LSDV-affected animals is scarce. This study, for the first time, evaluated the role of pro- and anti-inflammatory cytokines and apoptosis-mediated immune responses in LSDV naturally infected cattle. The Th1-type cytokines (IFN-γ, TNF-α, IL-1β, and IL-8) contribute to cell-mediated immune responses and host defence against intracellular viruses (Kak et al. 2018). IFN-γ is the main Th1 cytokine, which enhances the host’s defence against viral infection and activates pathways that can directly inhibit viruses (Shtrichman and Samuel 2001). In the current study, IFN-γ levels were significantly upregulated in PBMCs and skin nodules in the early stages of LSDV infection which is in agreement with the report of Badr et al. (2022), where it was stated that there is an increase in the IFN-γ expression in cutaneous lesions of naturally occurring LSD cases in cattle. El-Neweshy et al. (2013) reported that LSDV causes immune-mediated vasculopathy, but not a direct endothelial damage-mediated vasculopathy, because endothelial cells were negative for LSDV antigen. IFN-γ activates immune cells especially macrophages resulting in the production of IL-6, IL-1β and cytotoxic mediators, which are responsible for the pro-inflammatory action leading to the pathogenesis of LSDV including granulomatous vasculitis (42). It was reported that IFN-γ is a double-edged sword and therefore, it is possible that increased levels of IFN-γ might have caused exaggerated clinical signs and pathological lesions during early-stage of LSD (Kak et al. 2018).

Tumour necrosis factor-alpha (TNF-α) is a pleiotropic multi-functional cytokine, which acts on various cell types and is involved in the pathogenesis of various diseases. In this study, increased TNF-α mRNA gene expression levels during the mid and late stages of LSDV infection could potentially be involved in the pathogenesis of LSD. El-Mandrawy and Alam (2018) and Kamr et al. (2022) reported that LSDV-infected cattle showed increased concentrations of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 during viraemic states. IL-2 is a pro-inflammatory cytokine, that regulates the growth and differentiation of T-lymphocytes, and enhances the NK cell activity and antibody production (Wang et al. 2018). In the current study, increased IL-2 and IL-1β mRNA gene expression levels during the late-stage of LSDV infection indicated the involvement of potent inflammatory responses in the pathogenesis of LSD. Hence, uses of anti-inflammatory drugs especially non-steroidal anti-inflammatory drugs (NSAIDs) are highly recommended to use all stages of LSD. Offerman et al. (2015) reported similar results in a mouse model and Kamr et al. (2022) reported increased concentrations of pro-inflammatory cytokine IL-1β in the cows infected with LSD during the viraemic state. In this study, IL-8 or CXCL8, a pro-inflammatory cytokine and the main neutrophil chemotactic factor responsible for recruitment, activation, and accumulation of neutrophils was found to have increased expression correlated with secondary bacterial infections and infiltration of neutrophils.

The Th2-type cytokines (IL-4, IL-10, and IL-6) activate and maintain the humoral or antibody-mediated immune responses (Raphael et al. 2015; Li et al. 2018). IL-4 is the main Th2-type multifunctional cytokine, a key regulator of humoral and adaptive immune responses, stimulates the proliferation and differentiation of B-cells into antibody-producing plasma cells, alternative macrophage activation pathways, and inflammation (Raphael et al. 2015; Li et al. 2018). In this study, IL-4 expression levels were significantly upregulated during mid- and late stages of LSDV infection, which indicated its involvement in the disease pathogenesis during mid-stage and tissue repair processes during the late-stage. Earlier studies reported that LSDV-infected animals showed increased concentrations of IL-4 (El-Mandrawy and Alam 2018; Ahmad et al. 2023).

IL-6 is a pleiotropic pro-inflammatory cytokine, a primary mediator of cytokine storms, and stimulates both the innate and adaptive immune responses during viral infection (Velazquez-Salinas et al. 2019; Vitenberga-Verza et al. 2022). In the present study, during the mid and late stages of LSDV infection in the cattle, there was significant upregulation of IL-6 mRNA levels, indicating cytokine storm involvement and mounting of effective immune responses against LSDV. Kamr et al. (2022) reported the increased concentrations of IL-6 in cows infected with LSD during viraemic states.

The IL-10 is a potent anti-inflammatory cytokine with immunomodulatory properties that downregulates the expression of Th1 cytokines and host immune responses during viral infections thereby limiting pathogen clearance and worsening the disease pathology (Couper et al. 2008). In this study, IL-10 mRNA levels showed non-significant changes during the early, mid, and late stages of LSDV infection, which was correlated with increased expression of pro-inflammatory cytokines, and increased tissue damage as evident from the severe clinical signs and pathological lesions. These findings were corroborated by the findings of Ahmad et al. (2023), who reported non-significant differences in IL-10 expression in LSD-affected animals.

Apoptosis is one of the important self-defence mechanisms of the innate immune response against viral infection where the virus-infected host cells may impair virus propagation. Interestingly, poxviruses inhibit apoptosis-mediated host defence mechanisms using anti-apoptotic proteins encoded by poxviral genes such as M11L, F1L, CrmA protein, SPI-1, and SPI-2 (Nichols et al. 2017). In this study, the genes of the intrinsic pathway of apoptosis namely, Bcl-2, BAX, BAK, and caspase 9 mRNA expression levels were downregulated in PBMCs and skin nodules during mid and late stages of LSDV infection, which indicated that the intrinsic pathway had no role in apoptosis. Offerman et al. (2015) reported that LSDV did not up-regulate the caspase 1 gene expression; whereas, other poxviruses like modified vaccinia Ankara virus, Fowlpox virus and Canarypox virus upregulated the caspase 1 expression. LSDV significantly up-regulated the caspase 4 (commonly referred to as caspase 11 in mouse) gene expression. Caspase 1 and caspase 4-dependent programmed cell death are known as pyroptosis, which is an inflammatory process mediated, but apoptosis is a non-inflammatory mediated process (Bergsbaken et al. 2009).

Apoptosis may be initiated through a caspase cascade, which is activated by receptor-ligand interactions, such as FAS and TNF. In the present study, genes of the extrinsic pathway of apoptosis namely, FAS, FAS LG, caspase 8, and caspase 3 were significantly upregulated in PBMCs and skin nodules on mid and late stages of infection, which indicated the involvement of extrinsic pathway of apoptosis during LSDV infection. Sonowal et al. (Sonowal et al. 2021) reported that sheeppox virus follows the FAS-mediated extrinsic signalling pathway for apoptosis by upregulating the expression of the FAS gene during immediate-early infection. Further, Offerman et al. (2015) reported that LSDV up-regulated the Fas-associated death domain (FADD) protein gene expression in the mouse model. Royo et al. (2014) reported from their in vitro studies that attenuated and non-attenuated poxviruses downregulated the anti-apoptotic genes namely, Bcl-2 and upregulated the apoptosis inducers such as Fas ligand or caspase genes.

The present study was conducted in few states of India with specific areas and timeframe, which limits generalizability. Future studies need with more number states with large sample size and timeframe. Further, conditions like environmental factors on LSD transmission and management practices were not included in this study, and these warrants further investigation. The results of present study provide valuable insights that result in the better understanding of pathogenesis of LSD. The results highlight the need for continued surveillance and monitoring of the disease to avoid further spread. Further, these results can be exploited to design the suitable preventive and effective control measures against LSDV results in mitigation of the significant economic losses associated with the disease. In addition, new strategic policies should be developed to effectively control and eradicate the LSD. Public awareness programs are necessary to improve the vector control measures and knowledge about the disease.

5. Conclusions

The results of this study confirmed that there was a systemic inflammatory response with increased concentrations of pro-inflammatory cytokines, which was correlated with severe clinical disease and pathology during different stages of LSDV infection in cattle. There was a significant difference in LSDV antigen, inflammatory cells and the copy number of LSDV between the early and late stages of the disease. During the apoptosis-mediated pathogenesis of LSDV in natural settings, the downregulation of intrinsic pathway genes and upregulation of extrinsic pathway genes were recorded. These findings form a significant basis for better understanding the molecular mechanisms that are responsible for the differential clinical manifestations in the LSDV-affected cattle population. Further studies on genomics and experimental pathogenesis on the Indian variant of LSDV are warranted. The results highlight the need for continued surveillance and monitoring of the disease to avoid further spread. Further, these results can be exploited to design the suitable preventive and effective control measures against LSDV results in mitigation of the significant economic losses associated with the disease.

Supplementary Material

Supplemental Material

Acknowledgments

We thank Director, ICAR-NIVEDI, Bengaluru and Director, ICAR-IVRI, Bareilly for providing all the facilities to carry out this research work. The authors are also grateful to the field veterinary doctors and animal owners for their tremendous help during the clinical sample collection and outbreak investigations.

Compliance with ethical standards

The authors confirm the ethical policies of the journal have been followed.

Compliance with ethics requirements

All Institutional and National Guidelines for the care and use of animals (fisheries) were followed. The study was approved by the Institute Animal Ethics Committee (No. NIVEDI/IAEC/2022/06).

Disclosure statement

There was no conflict of interest among the authors.
==== Refs
References

Abutarbush SM. 2015. Hematological and serum biochemical findings in clinical cases of cattle naturally infected with lumpy skin disease. J Infect Dev Ctries. 9 (3 ):283–288. doi: 10.3855/jidc.5038.25771466
Ahmad SF, Patra MK, Mahendran K, Paul BR, Khanna S, Singh AK, De UK, Agrawal RK, Gaur GK, Dutt T. 2023. Hematological and serum biochemical parameters and profiling of cytokine genes in lumpy skin disease in Vrindavani cattle. 3Biotech. 13 :66.
Babiuk S, Bowden TR, Parkyn G, Dalman B, Manning L, Neufeld J, Embury-Hyatt C, Copps J, Boyle DB. 2008. Quantification of lumpy skin disease virus following experimental infection in cattle. Transbound Emerg Dis. 55 (7 ):299–307. doi: 10.1111/j.1865-1682.2008.01024.x.18503511
Badr Y, Noreldin AE, Elewa YHA, Ahmed MS, Inoshima Y, Baker NM, Aamer WN, Abas OM, Nayel M, Rahman MM, Elgendy E, Saleh AG, El-Neweshy MS. 2022. Cellular infiltration, cytokines, and histopathology of skin lesions associated with different clinical forms and stages of naturally occurring lumpy skin disease in cattle. Comp Immunol Microbiol Infect Dis. 90-91:101894. doi: 10.1016/j.cimid.2022.101894.
Bergsbaken T, Fink SL, Cookson BT. 2009. Pyroptosis: host cell death and inflammation. Nat Rev Microbiol. 7 (2 ):99–109. doi: 10.1038/nrmicro2070.19148178
Birge RB, Ucker DS. 2008. Innate apoptotic immunity: the calming touch of death. Cell Death Differ. 15 (7 ):1096–1102. doi: 10.1038/cdd.2008.58.18451871
Biswas D, Saha SS, Biswas S, Sayeed MA. 2020. Outbreak of lumpy skin disease of cattle in south-west part of Bangladesh and its clinical management. VSRR. 6 (2 ):100–108. doi: 10.17582/journal.vsrr/2020.6.100.108.
Couper KN, Blount DG, Riley EM. 2008. IL-10: the master regulator of immunity to infection. J Immunol. 180 (9 ):5771–5777. doi: 10.4049/jimmunol.180.9.5771.18424693
Dietze K, Moritz T, Alexandrov T, Krstevski K, Schlottau K, Milovanovic M, Hoffmann D, Hoffmann B. 2018. Suitability of group-level oral fluid sampling in ruminant populations for lumpy skin disease virus detection. Vet Microbiol. 221 :44–48. doi: 10.1016/j.vetmic.2018.05.022.29981707
El-Mandrawy SA, Alam RT. 2018. Haematological, biochemical and oxidative stress studies of lumpy skin disease virus infection in cattle. J Appl Anim Res. 46 (1 ):1073–1077. doi: 10.1080/09712119.2018.1461629.
El-Neweshy MS, El-Shemey TM, Youssef SA. 2013. Pathologic and immunohistochemical findings of natural lumpy skin disease in Egyptian cattle. Pak Vet J. 33 (1 ):60–64.
Geletu US, Musa AA, Usmael MA, Keno MS. 2024. Molecular detection and isolation of lumpy skin disease virus during an outbreak in West Hararghe Zone, Eastern Ethiopia. Vet Med Int. 29 :9487970.
Hasib FMY, Islam MS, Das T, Rana EA, Uddin MH, Bayzid M, Nath C, Hossain MA, Masuduzzaman M, Das S, et al. 2021. Lumpy skin disease outbreak in cattle population of Chattogram, Bangladesh. Vet Med Sci. 7 (5 ):1616–1624. doi: 10.1002/vms3.524.33993641
Kak G, Raza M, Tiwari BK. 2018. Interferon-gamma (IFN-γ): exploring its implications in infectious diseases. Biomol Concepts. 9 (1 ):64–79. doi: 10.1515/bmc-2018-0007.29856726
Kamr A, Hassan H, Toribio R, Anis A, Nayel M, Arbaga A. 2022. Oxidative stress, biochemical, and histopathological changes associated with acute lumpy skin disease in cattle. Vet World. 15 (8 ):1916–1923. doi: 10.14202/vetworld.2022.1916-1923.36313851
Kononov A, Prutnikov P, Shumilova I, Kononova S, Nesterov A, Byadovskaya O, Pestova Y, Diev V, Sprygin A. 2019. Determination of lumpy skin disease virus in bovine meat and offal products following experimental infection. Transbound Emerg Dis. 66 (3 ):1332–1340. doi: 10.1111/tbed.13158.30811855
Kumar N, Chander Y, Kumar R, Khandelwal N, Riyesh T, Chaudhary K, Shanmugasundaram K, Kumar S, Kumar A, Gupta MK, et al. 2021. Isolation and characterization of lumpy skin disease virus from cattle in India. PLoS One. 16 (1 ):e0241022. doi: 10.1371/journal.pone.0241022.33428633
Li W, Chen H, Deng H, Kuang Z, Long M, Chen D, Liao X, Li M, Rock DL, Luo S, et al. 2018. Orf virus encoded protein ORFV119 induces cell apoptosis through the extrinsic and intrinsic pathways. Front Microbiol. 9 :1056. doi: 10.3389/fmicb.2018.01056.29896166
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25 (4 ):402–408. doi: 10.1006/meth.2001.1262.11846609
Manjunatha Reddy GB, Pabbineedi SM, Nagaraj S, Bijalwan S, Tadakod S, Bhutia Z, Palmu D, Rai S, Bhutia PD, Bhutia PT, et al. 2023. Lumpy Skin Disease (LSD) in Yak (Bos grunniens): an Evidence of Species Spillover from Cattle in India. Microorganisms. 11 (12 ):2823. doi: 10.3390/microorganisms11122823.38137967
Mathewos M, Dulo F, Tanga Z, Sombo M. 2022. Clinicopathological and molecular studies on cattle naturally infected with lumpy skin diseases in selected districts of Wolaita Zone, Southern Ethiopia. BMC Vet Res. 18 (1 ):297. doi: 10.1186/s12917-022-03403-4.35922813
Mazloum A, Van Schalkwyk A, Babiuk S, Venter E, Wallace DB, Sprygin A. 2023. Lumpy skin disease: history, current understanding and research gaps in the context of recent geographic expansion. Front Microbiol. 14 :1266759. doi: 10.3389/fmicb.2023.1266759.38029115
Mercier A, Arsevska E, Bournez L, Bronner A, Calavas D, Cauchard J, Falala S, Caufour P, Tisseuil C, Lefrançois T, et al. 2018. Spread rate of lumpy skin disease in the Balkans, 2015-2016. Transbound Emerg Dis. 65 (1 ):240–243. doi: 10.1111/tbed.12624.28239954
Möller J, Moritz T, Schlottau K, Krstevski K, Hoffmann D, Beer M, Hoffmann B. 2019. Experimental lumpy skin disease virus infection of cattle: comparison of a field strain and a vaccine strain. Arch Virol. 164 (12 ):2931–2941. doi: 10.1007/s00705-019-04411-w.31538254
Nichols DB, De Martini W, Cottrell J. 2017. Poxviruses utilize multiple strategies to inhibit apoptosis. Viruses. 9 (8 ):215. doi: 10.3390/v9080215.28786952
Offerman K, Deffur A, Carulei O, Wilkinson R, Douglass N, Williamson AL. 2015. Six host-range restricted poxviruses from three genera induce distinct gene expression profiles in an in vivo mouse model. BMC Genomics. 16 (1 ):510. doi: 10.1186/s12864-015-1659-1.26153454
OIE. 2017. Infection with lumpy skin disease virus. In OIE terrestrial animal health code (2017), Chapter 11.9. Article 11.9.1. Paris: OIE. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_lsd.pdf.
OIE. 2024. Lumpy skin disease. Chapter 3.4.12. OIE Terrest Man. 13:1–13.
Parvin R, Chowdhury EH, Islam MT, Begum JA, Nooruzzaman M, Globig A, Dietze K, Hoffmann B, Tuppurainen E. 2022. Clinical epidemiology, pathology, and molecular investigation of lumpy skin disease outbreaks in Bangladesh during 2020-2021 indicate the re-emergence of an old African Strain. Viruses. 14 (11 ):2529. doi: 10.3390/v14112529.36423138
Pestova YE, Artyukhova EE, Kostrova EE, Shumoliva IN, Kononov AV, Prygin AV. 2018. Real time PCR for the detection of field isolates of lumpy skin disease virus in clinical samples from cattle. S-h. Biol. 53 (2 ):422–429. doi: 10.15389/agrobiology.2018.2.422eng.
Raphael I, Nalawade S, Eagar TN, Forsthuber TG. 2015. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 74 (1 ):5–17. doi: 10.1016/j.cyto.2014.09.011.25458968
Reddy GBM, Mounica PS, Sudeep N, Vikram R, Garam GB, Lalzampuia H, Ragulraj S, Pal S, Khate K, Bijalwan S, Girish PS, Gulati BR. 2024. First evidence of lumpy skin disease in mithun (Bos frontalis) in India. Arch Virol. 169(3):65. doi: 10.1007/s00705-024-05996-7.
Rouby SR, Shehata O, Abdel-Moneim AS, Hussein KH, Mahmoud MM. 2021. Lumpy skin disease in calves: the association between clinical signs and biochemical alterations. Adv Anim Vet Sci. 9 :1863–1868.
Royo S, Sainz B, Jr, Hernández-Jiménez E, Reyburn H, López-Collazo E, Guerra S. 2014. Differential induction of apoptosis, interferon signaling, and phagocytosis in macrophages infected with a panel of attenuated and nonattenuated poxviruses. J Virol. 88 (10 ):5511–5523. doi: 10.1128/JVI.00468-14.24599993
Saminathan M, Singh KP, Maity M, Vineetha S, Manjunathareddy GB, Dhama K, Malik YS, Ramakrishnan MA, Misri J, Gupta VK. 2021. Pathological and immunological characterization of bluetongue virus serotype 1 infection in type I interferons blocked immunocompetent adult mice. J Adv Res. 31 :137–153. doi: 10.1016/j.jare.2021.01.007.34194838
Sanz-Bernardo B, Haga IR, Wijesiriwardana N, Hawes PC, Simpson J, Morrison LR, MacIntyre N, Brocchi E, Atkinson J, Haegeman A, et al. 2020. Lumpy skin disease is characterized by severe multifocal dermatitis with necrotizing fibrinoid vasculitis following experimental infection. Vet Pathol. 57 (3 ):388–396. doi: 10.1177/0300985820913268.32314676
Shtrichman R, Samuel CE. 2001. The role of gamma interferon in antimicrobial immunity. Curr Opin Microbiol. 4 (3 ):251–259. doi: 10.1016/s1369-5274(00)00199-5.11378475
Shumilova I, Sprygin A, Mazloum A, Pronin V, Byadovskaya O, Babiuk S, Donnik I, Chvala I. 2023. Comparison of gross pathology between classical and recombinant lumpy skin disease viruses. Viruses. 15 (9 ):1883. doi: 10.3390/v15091883.37766289
Sonowal J, Lal Patel C, Kumar Gandham R, Sajjanar B, Ishaq Nabi Khan R, Ranjan Praharaj M, Akram Malla W, Kumar D, Dev K, Barkathullah N, et al. 2021. Genome-wide expression analysis reveal host genes involved in immediate-early infections of different sheeppox virus strains. Gene. 801 :145850. doi: 10.1016/j.gene.2021.145850.34274484
Sudhakar SB, Mishra N, Kalaiyarasu S, Jhade SK, Hemadri D, Sood R, Bal GC, Nayak MK, Pradhan SK, Singh VP. 2020. Lumpy skin disease (LSD) outbreaks in cattle in Odisha state, India in August 2019: Epidemiological features and molecular studies. Transbound Emerg Dis. 67 (6 ):2408–2422. 2020. doi: 10.1111/tbed.13579.32304275
Tamura K, Stecher G, Kumar S. 2021. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 38 (7 ):3022–3027. doi: 10.1093/molbev/msab120.33892491
Tuppurainen ES, Stoltsz WH, Troskie M, Wallace DB, Oura CA, Mellor PS, Coetzer JA, Venter EH. 2011. A potential role for ixodid (hard) tick vectors in the transmission of lumpy skin disease virus in cattle. Transbound Emerg Dis. 58 (2 ):93–104. doi: 10.1111/j.1865-1682.2010.01184.x.21114790
Tuppurainen ES, Venter EH, Coetzer JA. 2005. The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques. Onderstepoort J Vet Res. 72 (2 ):153–164. doi: 10.4102/ojvr.v72i2.213.16137133
Tuppurainen ESM, Venter EH, Shisler JL, Gari G, Mekonnen GA, Juleff N, Lyons NA, De Clercq K, Upton C, Bowden TR, et al. 2017. Review: capripoxvirus diseases: current status and opportunities for control. Transbound Emerg Dis. 64 (3 ):729–745. doi: 10.1111/tbed.12444.26564428
Vasković N, Debeljak Z, Vidanović D, Šekler M, Matović K, Aničić M, Marinković D. 2019. Morphological characteristics of skin lesions in cattle naturally infected with lumpy skin disease virus in Serbia. Acta Veterinaria. 69 (4 ):369–378. doi: 10.2478/acve-2019-0031.
Velazquez-Salinas L, Verdugo-Rodriguez A, Rodriguez LL, Borca MV. 2019. The role of Interleukin 6 during viral infections. Front Microbiol. 10 :1057. doi: 10.3389/fmicb.2019.01057.31134045
Vitenberga-Verza Z, Pilmane M, Šerstņova K, Melderis I, Gontar Ł, Kochański M, Drutowska A, Maróti G, Prieto-Simón B. 2022. Identification of inflammatory and regulatory cytokines IL-1α-, IL-4-, IL-6-, IL-12-, IL-13-, IL-17A-, TNF-α-, and IFN-γ-producing cells in the milk of dairy cows with subclinical and clinical mastitis. Pathogens. 11 (3 ):372. doi: 10.3390/pathogens11030372.35335696
Wang T, Hu Y, Wangkahart E, Liu F, Wang A, Zahran E, Maisey KR, Liu M, Xu Q, Imarai M, et al. 2018. Interleukin (IL)-2 is a key regulator of T helper 1 and T helper 2 cytokine expression in fish: functional characterization of two divergent IL2 paralogs in Salmonids. Front Immunol. 9 :1683. doi: 10.3389/fimmu.2018.01683.30093902
Yadav P, Kumar A, Nath SS, Devasurmutt Y, Shashidhar G, Joshi M, Puvar A, Sharma S, Raval J, Pandit R, et al. 2024. Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks. BMC Genomics. 25 (1 ):196. doi: 10.1186/s12864-024-10061-3.38373902
