Immuno-pathological studies of lumpy skin disease outbreaks in cattle

1 Introduction

Lumpy skin disease is a highly contagious viral disease caused by lumpy skin disease virus, a member of the Capripoxvirus genus within the Poxviridae family (1). Although historically confined to Africa, the disease has rapidly expanded into the Middle East and Asia and has emerged as one of the most significant transboundary animal diseases affecting cattle (2), mithun (3), and yak (4). The disease, which affects cattle, buffalo, and wild ruminants, is characterized by the development of cutaneous skin nodules that progress to necrotic lesions, predisposing affected animals to secondary bacterial infections such as myiasis (5). The disease results in substantial economic losses through reduced milk yield, weight loss, infertility, abortion, hide damage, treatment costs, restrictions on animal movement and trade, and, in severe cases, death (6). The LSD outbreak in India resulted in over 155,000 cattle deaths and caused an estimated economic loss of 2217.26 million USD, highlighting the significant threat posed by the disease to the livestock sector (7).

Transmission of LSDV occurs through both direct and indirect routes. Direct transmission occurs through contact with infected body fluids, while indirect transmission is via biting vectors like flies and ticks (2). Following infection, the virus replicates in susceptible host cells, leading to the development of clinical signs including fever, nasal discharge, enlargement of lymph nodes, characteristic skin nodules, pneumonia, and mastitis. Infected animals shed the virus through various body fluids such as blood, serum, milk, semen, and oral, nasal, and lacrimal discharges, contributing to disease transmission (8).

The disease severity varies considerably among affected animals and is influenced by factors such as age, breed, immune status, and environmental conditions (9). Based on the clinical signs, LSD infection is categorized into early, mid, and late stages (6, 10). Diagnosis of LSD is based on characteristic clinical signs, pathological lesions, and various laboratory techniques such as PCR (11), ELISA (12), and virus isolation (13). Although clinical diagnosis is often straightforward in advanced cases, laboratory confirmation remains essential because several dermatological and poxviral infections can produce similar lesions. Among the available diagnostic techniques, PCR provides rapid and specific detection, whereas histopathology and immunohistochemistry facilitate understanding of tissue alterations and viral localization.

The genetic makeup of individual animals plays a crucial role in determining their immune response to the same strain of the LSD virus, as well as the distribution of disease transmitting vectors within the environment influence the epidemiology and severity of the disease (14, 15). Understanding the genetic factors underlying host responses to LSDV is essential to uncover the mechanisms behind the immune response to LSD (16). In particular, Investigating the regulation of cytokines involved in inflammation can provide valuable biomarkers. These biomarkers could help distinguish between animals that are susceptible to infection and those that are more tolerant or resilient to the disease. Although recovered animals develop immunity, factors such as virus strain, host immunity, and vector prevalence influence disease severity (6, 17). Despite the growing impact of LSD worldwide, there is a paucity of information on the understanding of the genetic and immune responses to LSD in Cattle, including cytokine involvement, to improve diagnostic and treatment strategies.

Despite the increasing prevalence of LSD in India, information integrating clinical observations, pathological lesions, molecular diagnosis, immunohistochemical localization and cytokine responses under natural field conditions remains limited. Therefore, the present study was designed and undertaken to diagnose LSD by various techniques and also to study the hematological alterations and the expression of pro-inflammatory and anti-inflammatory cytokine genes in LSD-affected cattle in Pondicherry and adjacent areas of Tamil Nadu, India.

2 Methodology

2.1 Study design and sample collection

The study population is confined to Puducherry and contiguous areas of Tamil Nadu, India. The study was conducted on 21 cattle (adults, n = 4; calves, n = 17) clinically suspected of lumpy skin disease. The healthy cattle (n = 6) were included as controls for the study. Among the affected group, the animals were re-classified into early (0 to 7 days, n = 9), mid (7 to 14 days, n = 6), and late (14 to 21 days and more, n = 6) stages of the LSD based on the days from which the animal has been suffering from the clinically shown LSD signs after the incubation period.

During the LSD outbreak, field investigations were carried out, and clinical samples were collected aseptically from clinically affected and healthy cattle in accordance with the guidelines set by the World Organisation for Animal Health (WOAH) for laboratory investigations. Additionally, a structured questionnaire was employed to gather essential information regarding the sampled animals, including age, sex, breed, and demographic details during sample collection. All animals were subjected to detailed clinical examinations; clinical observations included fever, body temperature, respiratory signs, edema, presence and distribution of skin nodules, ulceration, lesion progression with pyrexia, and decreased production performance.

Nasal swabs were collected by carefully inserting a sterile, flexible, fine-shafted swab into the nasopharyngeal cavity and gently rotating it to absorb secretions. The swabs were subsequently placed into sterile collection tubes containing 1 mL of viral transport medium (RPMI-1940). Whole blood was drawn via jugular vein puncture and collected in ethylene diamine tetra acetic acid (K₂EDTA, Labtech-coated) vacutainers. Skin nodules or scabs from the lesions were collected in RNA later using the punch biopsy needle. To ensure sample integrity, all specimens were transported in insulated shipping containers with dry-ice packs, maintaining a strict cold chain. Samples were analyzed at the BSL-2+ virology laboratory, Indian Council of Agricultural Research-National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), under the approval of the Institute Animal Ethics Committee (No. NIVEDI/IAEC/2022/06), Bengaluru. All samples were subsequently stored at -20 °C except for blood samples, which were preserved at 4 °C.

2.2 Hematological analysis

Whole blood samples collected aseptically from each cattle into EDTA-coated vacutainers were transported to the laboratory for hematological evaluation. Hematological parameters including hemoglobin concentration (Hb; g/dL), packed cell volume (PCV; %), total erythrocyte count (TEC; ×10⁶/mm³), total leukocyte count (TLC; ×10³/mm³), absolute leukocyte count (all expressed as ×10³/µL), mean corpuscular volume (MCV; fL), mean corpuscular hemoglobin (MCH; pg), mean corpuscular hemoglobin concentration (MCHC; g/dL), and platelet count (×10⁵/µL) were analyzed using an automated Veterinary Hematology Analyzers (Mindray, 5-part differential hematology analyzer, China) according to the manufacturer’s instructions.

Data were represented as mean ± standard deviation (SD). Statistical analyses were conducted using the Statistical Package for the Social Sciences (IBM^® SPSS^® version 27). Differences among the control, clinically affected early, mid, and late-stage LSD groups were analyzed using one-way analysis of variance (ANOVA). Following the observed significant differences, post hoc multiple comparison analysis was performed to determine differences between groups. A value of p < 0.05 was considered as statistically significant.

2.3 Molecular detection of LSDV

The collected samples were thawed to room temperature before processing. Nasal swabs were vortexed thoroughly and aliquoted for extraction. Approximately 30 mg of skin nodules or scab samples were finely chopped using a sterile Bard Parker blade, and a tissue homogenate was prepared by triturating the sample with 1 mL of chilled 1X phosphate-buffered saline (PBS, pH 7.2) using a mortar and pestle. The total genomic DNA was extracted from the processed clinical samples using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. DNA concentration and purity were assessed using a Nanodrop spectrophotometer (NABI, Microdigital Co., Ltd., South Korea). The extracted DNA was stored at -20 °C until further use. All biological materials were disposed of in accordance with the standard operating procedures (SOPs) established by the Institute Biosafety Committee (IBSC).

Diagnostic screening for the presence of lumpy skin disease virus (LSDV) was conducted on clinical affected samples, including nasal swabs (n = 21), and skin tissue (n = 21) from the same cattle. DNA was amplified for the Capripoxvirus-specific partial P32 gene using respective forward (F: 5’-TTTCCTGATTTTTCTTACTAT-3’) and reverse (R: 5’-AAATTATATACGTAAATAAC-3’) primers. Amplification of the expected 192 bp DNA fragment was considered positive for LSDV infection (6).

Conventional polymerase chain reaction (PCR) assay was performed using a Bio-Rad thermal cycler (Hercules, CA, USA) with a total reaction volume of 25 μL. The reaction mixture comprised 12.5 μL of PCR master mix (DreamTaq Green PCR Master Mix 2X, Thermo Scientific, Waltham, MA, USA), 1 μL each of forward and reverse primers (10 pmol/μL), 8.5 μL of nuclease-free water, and 50 ng of template DNA. PCR amplification was performed with an initial denaturation step at 94 °C for 5 minutes, followed by 30 amplification cycles consisting of denaturation at 94 °C for 1 minute, annealing at 50 °C for 30 s, and extension at 72 °C for 1 minute. A final extension step was carried out at 72 °C for 5 minutes. Following amplification, PCR products were analyzed via agarose gel electrophoresis on a 1.5% agarose gel prepared in 1X Tris-acetate-EDTA (TAE) buffer, stained with ethidium bromide (10 mg/ml, HiMedia Laboratories Pvt. Ltd.), and visualized under a UV transilluminator using a Gel Documentation System (Vilber Bio-Print, Vilber Lourmat, France).

2.4 Histopathological examination

The skin tissues from suspected LSD-affected and apparently from healthy cattle specimens were fixed in 10% Neutral Buffered Formalin (NBF), stored at room temperature and processed by the routine paraffin-embedding technique. 3-5µm thick tissue sections were prepared with a rotary microtome (Leica Biosystems). After deparaffinization, tissue sections were stained with Hematoxylin and Eosin, were further examined microscopically for histopathological investigations associated with LSDV infection (18).

2.5 Immunohistochemistry

Immunohistochemistry (IHC) was performed to confirm LSDV infection to determine the LSD viral antigen distribution in the skin biopsy tissues. Tissue sections were mounted on poly-L-lysine pre-coated slides and allowed to dry overnight at room temperature. The sections were subsequently deparaffinized, rehydrated and subjected to antigen retrieval using pressure-cooking method in citrate buffer at 37 °C for 5 minutes.

Endogenous peroxidase activity was blocked by covering the whole tissue sections with 3% hydrogen peroxide (H₂O₂) in methanol and incubated at room temperature for 10 minutes, followed by three washes with 0.01 M PBS. Non-specific antibody was blocked using 1.5% rabbit serum for one hour at room temperature.

The primary antibody was added directly to completely cover the tissue sections and incubated at room temperature for one and a half hour. Rabbit anti-bovine IgG-horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilution) was added and incubated at room temperature for one hour. Immunoreactivity was visualized using 3,3’-diaminobenzidine tetrahydrochloride (DAB) as the chromogen and incubated at room temperature for 2–4 minutes, or until the desired color (light brown) was developed. Between each step, the sections were washed with 0.01 M PBS.

Nuclear staining was performed using Harris Hematoxylin for 1 minute 30 seconds, followed by washing in distilled water, dehydrated in graded alcohol, clearing in xylene, and mounted with DPX (Dystrene Polystyrene Xylene). Positive immunostaining for LSDV antigen was visualized as granular golden-brown deposits within the affected tissues. Immunohistochemically stained sections were captured using an Optika B5 microscope (Optika, Italy) and documented with an Optika digital camera.

2.6 Indirect enzyme-linked immunosorbent assay

An in-house indirect ELISA was performed to detect antibodies against lumpy skin disease virus (LSDV) in serum samples. Recombinant LSDV antigen developed by ICAR-NIVEDI was coated onto ELISA plates using carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. The plates were subsequently washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and blocked with 1% bovine gelatin prepared in PBST for 1 hour at 37 °C.

After blocking, the plates were washed three times with PBST, and serum samples along with known positive and negative control sera, diluted at 1:150 in PBST, were added and incubated for 1 hour at 37 °C. After incubation, plates were washed and rabbit anti-bovine IgG horseradish peroxidase (HRP) conjugate (Bethyl Laboratories), diluted 1:7500 in blocking buffer was added to each well and incubated for an additional 1 hour at 37 °C.

Color development was achieved using ortho-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich) as the substrate, and optical density (OD) was measured at 492 nm using a Tecan Infinite F50 ELISA plate reader. The percentage positivity (PPV) was obtained for each serum sample by calculating the ratio of the difference in the OD value of the test serum and negative serum control to the difference in the OD value of the positive serum control and negative serum control. Serum samples exhibiting a PPV greater than 35% were considered positive for LSDV antibodies.

2.7 Cytokine expression analysis

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood (4 ml) samples using Histopaque (HiSep™ LSM 1077, HiMedia, LS001) according to the manufacturer’s instructions. The isolated PBMCs were stained with Trypan Blue, and viable cells were counted using a hemocytometer.

For cytokine expression analysis, samples from cattle representing the early (n = 2), mid (n = 2), and late (n = 2) stages of LSD infection and control (n = 2) were selected. Total RNA was isolated from both the PBMCs and skin tissues samples using the Hybrid-R™ RNA Isolation Kit (HB3500, v.2.1; GeneAll Biotechnology Co., Ltd., Korea) according to the manufacturer’s protocol. The quality and integrity of the RNA was assessed prior to downstream applications. RNA was then reverse transcribed into cDNA using a commercial cDNA synthesis kit.

The relative expression of five cytokine genes representing Th1 and Th2 immune responses was evaluated by quantitative real-time PCR (qPCR). The selected Th1-associated cytokines included interleukin-2 (IL-2), interferon-gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas interleukin-4 (IL-4) and interleukin-10 (IL-10) represented Th2-associated cytokines. Gene-specific primers used for amplification were adopted from 17, and their sequences, annealing temperatures, and expected amplicon sizes are presented in Table 1.

Table 1. Shows primer sequences used for quantitative real-time PCR analysis of cytokine gene expression in peripheral blood mononuclear cells (PBMCs) and skin tissues of cattle (17).

IL-2, Interleukin-2; IL-4, Interleukin-4; IL-10, Interleukin-10; IFN-γ – Interferon-gamma; GM-CSF – Granulocyte Macrophage, Colony Stimulating Factor; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Real-time PCR was performed in a 20 µL reaction volume containing 2 µL of cDNA template, 10 µL of PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific), 1 µL each of forward and reverse primers, and nuclease-free water to make up the final volume. Amplification was carried out using a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific), and fluorescence data were analyzed using QuantStudio™ Design and Analysis Software v1.4.3.

The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference gene for normalization. Relative gene expression levels were calculated using the comparative Ct (2^−ΔΔCt) method and expressed as fold changes relative to the healthy control group (19).

3 Results

3.1 Epidemiological observations

The present study was carried out during the period October 2023 to March 2024 at Puducherry and the adjoining states. Clinically affected cattle for the study were selected from both field cases and veterinary dispensaries. From all the animals, signalment (breed, age, and sex), clinical signs, and stage of the disease were recorded in a proforma. The data are presented in Table 2. Among the 21 clinically affected cattle, crossbred Jersey constituted the majority of cases. Females accounted for 90.5% of affected animals, where males represented 9.5%. A higher proportion of cases were observed in cattle below 1 year of age compared with adults.

Table 2. Shows the demographic and epidemiological data of cattle affected with LSD.

CBJ*, Jersey Cross-bred cattle; CBHF^#, Holstein-Friesian Cross-bred cattle.

3.2 Clinical findings

Affected cattle exhibited a wide range of clinical symptoms that varied according to the stage of the disease progression. Nodules varied considerably in size and distribution, were round in shape, soft to hard in consistency. In the early stage, clinical findings included mild fever, nasal and lacrimal discharge, brisket and limb edema, reduced milk production, and the presence of mild to moderate cutaneous nodules distributed over the neck, lateral abdomen region, and perineum (Figures 1A–F). These nodules were initially firm, well-circumscribed, and confined to the skin and subcutaneous tissues. During the mid stage of the disease, cattle exhibited higher rectal temperatures, persistent nasal discharge and respiratory distress. Cutaneous nodules were more numerous and distributed over multiple body regions (Figures 2A–D). In the late stage, affected cattle showed respiratory distress and characterized by severe cutaneous nodular lesions. Many nodules underwent necrosis and rupture, resulting in ulcerative wounds, sit-fast lesions, and subsequent scab formation (Figures 3A–D). These lesions were also associated with delayed healing and secondary bacterial infections. Lesions were predominantly distributed over the head, neck, lateral abdomen, limbs, hindquarters, udder and perineal regions. Least frequent observation recorded include brisket and limb edema, decreased milk production, and the presence of severe ulcerative skin lesions associated with secondary complication of skin wounds.

3.3 Hematological alterations

The hematological data of LSD affected cattle and healthy controls are presented in Table 3. Significant alterations were observed in erythrocytic and leukocytic indices during the course of infection. Among erythrocytic parameters, TEC showed a significant increase in LSD affected cattle compared with controls (p = 0.049). In contrast, MCV and MCH were significantly reduced in affected animals, particularly in the late stage of disease compared with controls (p = 0.036 and p = 0.013, respectively). No significant differences were observed in PCV, Hb, or MCHC among the study groups (p > 0.05).

Table 3. Compares hematological data of control and LSD affected cattle at different stages of infection.

Different superscripts within the same row indicate significant differences between groups (p* < 0.05) as determined by one-way ANOVA followed by post hoc comparison. P value indicates significance at a 5% level.

Marked alterations were observed in leukocyte profiles. TLC increased significantly with disease progression and was highest in late stage (13.17 ± 2.21 ×10³/mm³), which differed from both control and early stage affected cattle (p = 0.001). Similarly, absolute lymphocyte counts were significantly elevated in infected cattle, with the highest values recorded in late-stage cases (7.22 ± 1.40 ×10³/mm³) compared with controls (p < 0.001). Absolute monocyte counts also increased progressively with disease severity and were significantly higher in late-stage animals than in early-stage, mid-stage and control groups (p < 0.001). Although absolute neutrophil counts tended to be higher in infected cattle than in controls, the differences were not statistically significant (p = 0.222). Significant increases were observed in absolute eosinophil counts (p = 0.043) and absolute basophil counts (p = 0.007) in LSD-affected cattle compared with healthy animals. However, platelet counts did not differ significantly among the groups (p = 0.738). Overall, the hematological profile of LSDV-infected cattle was characterized by leukocytosis, lymphocytosis, monocytosis, eosinophilia, and basophilia, accompanied by reduced MCV and MCH values, indicating systemic inflammatory and immune responses associated with disease progression.

3.4 Diagnosis of LSDV by PCR

PCR targeting partial P32 gene detected LSDV DNA in both nasal swab and skin tissue samples. Out of twenty-one nasal swab samples examined, total 11 samples that is 6 from early stage, 3 from mid stage and 2 from late stage (52.4%) were positive for LSDV. Positivity rate rates were highest in early-stage animals and gradually decreased with disease progression. Among skin tissue samples collected from LSD-affected cattle, 12 samples that is 6 from early stage, 3 each from mid and late stage (57.1%) were positive for LSDV. Positive amplification was observed across different stages of disease, indicating persistence of viral DNA within cutaneous lesions. PCR targeting the partial P32 gene amplified the expected 192 bp product in 11 of 21 nasal swabs and 12 of 21 skin biopsies (Table 4, Figure 4).

Table 4. Presents a comparison of the diagnostic assays used for the detection of LSDV infection in the study.

3.5 Histopathological findings

Histopathology was carried out for LSD-affected animals (n=21). In the epidermis, the epithelium is intact and there were no significant histopathological findings except hyperplasia of the epithelium present in the epidermis. In the dermis, necrosis and severe infiltration of histiocytic cells. There was severe infiltration of histiocytic cells around the perivascular space in the dermis and also in between the muscular bundles present in the deep dermis. These histopathological changes were associated with vasculitis and myositis in the dermis and deep dermis region. Representative images of histochemical staining of LSD-affected skin tissues are shown in Figures 5A–F.

3.6 Immunohistochemical localization of LSDV antigen

Immunohistochemical staining of skin tissues was performed according to the procedure detailed under materials and methods. Among 12 samples (n=6 from early stages, n=3 each from mid and late stages) subjected to immunohistochemistry, 10 samples (n=6 from early stages and n=2 each from the mid and late stages) showed positive immunoperoxidase staining of LSDV. The positive immunoperoxidase staining was observed by the granular golden-brown color in the skin tissues where the LSDV is localized. Representative images of IHC-stained tissues are shown in Figures 6A–F.

In general, LSDV was detected in the cytoplasm of all the epithelial layers of the epidermis but more in the basal layer of the epidermis, the epithelial matrix lining the hair follicle, the epithelial cells of sweat glands and the sebaceous glands. The localisation of LSDV in the epithelial matrix lining the hair follicle was observed in a higher number of cases, followed by the epithelial cell layers of the epidermis and the epithelial cells of sweat glands and sebaceous glands.

3.7 Serological analysis

Indirect ELISA detected antibodies against LSDV in four of twenty-one serum samples, 2 from early stage and one each from mid and late stages. The results correspond to an overall seropositivity rate of 19%. The relatively low seropositivity observed in the present study may reflect variation in the timing of sample collection, differences in individual immune responses and delayed seroconversion in naturally infected animals.

3.8 Expression profiles of cytokine genes from PBMCs and skin tissues

Cytokine expression analysis revealed marked suppression of both Th1- and Th2-associated cytokines in affected cattle compared with healthy controls. Expression of IL-2 was substantially reduced in infected animals, indicating suppression of cell-mediated immune responses. Similarly, IL-4 expression remained lower than control levels, suggesting impairment of humoral immune regulation. IL-10 expression also demonstrated downregulation in affected cattle. The reduced expression of this regulatory cytokine may contribute to immune dysregulation and persistent inflammatory responses. A pronounced reduction in IFN-γ expression was observed in both PBMCs and skin tissues. As IFN-γ plays a critical role in antiviral immunity, its suppression may facilitate viral persistence within host tissues. GM-CSF exhibited marked downregulation compared with healthy controls. Reduced expression of GM-CSF may adversely affect activation and differentiation of innate immune cells, thereby compromising effective antiviral responses.

Overall, the cytokine expression profile demonstrated broad suppression of both systemic and local immune responses in naturally infected cattle. Relative expression patterns of IL-2, IL-4, IL-10, IFN-γ and GM-CSF in PBMCs and skin tissues are presented in Tables 5, 6 respectively.

Table 5. Shows relative expression (fold change ± SD) of cytokine genes in PBMCs of LSDV-infected cattle and healthy controls.

IL-2, Interleukin-2; IL-4, Interleukin-4; IL-10, Interleukin-10; IFN-γ, Interferon-gamma; GM-CSF, Granulocyte-Macrophage Colony-Stimulating Factor; PBMCs, Peripheral Blood Mononuclear Cells.

Table 6. Shows relative expression (fold change ± SD) of cytokine genes in skin tissues of LSDV-infected cattle and healthy controls.

IL-2, Interleukin-2; IL-4, Interleukin-4; IL-10, Interleukin-10; IFN-γ, Interferon-gamma; GM-CSF, Granulocyte-Macrophage Colony-Stimulating Factor.

4 Discussion

The present study provides a comprehensive evaluation of the clinical manifestations, hematological alterations, pathological and diagnostic findings, and cytokine expression profiles associated with LSDV infection in cattle. The majority of affected animals were crossbred cattle, females, and calves, findings that are consistent with previous reports indicating greater disease severity among young and crossbred animals (1, 20, 21).

The clinical findings during sampling reflected the progressive nature of LSDV infection. Early-stage of disease was characterized by fever, lacrimation, edema, and localized cutaneous nodules, whereas mid-stage animals exhibited more extensive skin lesions and respiratory distress. Late-stage cases showed severe respiratory distress, sit-fast lesions, scab formation, rupture of nodules, and wound healing responses. These observations are in agreement with the classical clinical descriptions of LSD reported by Prozesky and Barnard (22) Coetzer and Tuppurainen (23), and Weiss (24) confirming the multisystemic nature of LSDV infection.

Further, molecular diagnosis of the clinical samples targeting the partial P32 gene detected LSDV DNA in both nasal swabs and skin biopsies. Detection rates were higher in skin tissues than nasal swabs, indicating prolonged persistence of viral DNA within cutaneous lesions. These findings support earlier studies demonstrating that skin biopsies remain one of the most reliable samples for LSD diagnosis throughout the course of infection (25). Among the nasal swabs, 11 of 21 animals tested positive, with the highest detection rate occurring during the early stage of disease. The greater PCR positivity in acute phase of infection indicates active viral replication and shedding in respiratory secretions. Similar findings were reported by (26), who demonstrated the presence of LSDV in nasal secretions during early infection. In contrast, the low seropositivity observed by indirect ELISA may reflect delayed antibody development and variability in humoral responses during LSDV infection, as reported previously by (27, 28).

Analysis of blood samples demonstrated significant hematological alterations, including leukocytosis with increased lymphocyte and monocyte counts, particularly in the late stage of disease. These results indicate persistent immune stimulation and inflammatory responses associated with ongoing viral infection. Davies (29)Kitching (30) Tuppurainen and Oura (31) studies showed similar hematological alterations in infected cattle and are considered indicative of host immune activation during disease progression. The observed reductions in MCV and MCH may reflect alterations in erythrocyte dynamics associated with chronic inflammatory processes.

In this study, histopathological analysis of LSD-affected cattle (n=21) revealed significant findings primarily in the dermis and deep dermis, with notable changes in the epidermis. The epidermis of LSD-affected animals showed no significant histopathological findings except for hyperplasia of the epithelium. This finding aligns with previous studies of Prozesky and Barnard (22), who documented epidermal hyperplasia in LSD cases. The intact epithelium with hyperplasia suggests that while the virus causes significant immune responses and tissue changes, it does not necessarily disrupt the integrity of the epidermal layer in the early to moderate stages of the disease. In the dermis, necrosis and severe infiltration of histiocytic cells were observed. The severe infiltration of histiocytic cells indicates a robust immune response to the viral infection. These findings are consistent with the observations by Coetzer and Tuppurainen (23), who noted marked infiltration of inflammatory cells, including histiocytes, in the dermis of LSD-affected cattle. The perivascular space in the dermis also showed severe infiltration of histiocytic cells. This is indicative of vasculitis, which is a characteristic finding in LSD. Vasculitis can lead to increased vascular permeability, resulting in oedema and further recruitment of inflammatory cells to the site of infection. Manjuanthareddy et al., 2024 reported similar findings of perivascular cuffing and vasculitis in LSD cases, highlighting the role of vascular inflammation in the pathogenesis of the disease. Vasculitis, as documented disrupts normal blood flow and contributes to tissue damage and necrosis. Myositis, resulting from both direct viral effects and secondary inflammatory responses, underscores the severity of LSD in advanced stages. Vasculitis and myositis reflect the systemic nature of LSDV infection and its ability to induce widespread inflammatory responses, leading to significant tissue damage.

The immunohistochemical findings in this study enhance the understanding of the tissue tropism in LSD and are consistent with previous reports on the localization of LSDV in skin tissues. According to Al-Salihi and Hassan (32), LSDV exhibits a marked affinity for the basal layer of the epidermis and the hair follicle epithelium, where viral replication and cytopathic effects are prominent. Similarly, a study by (25) demonstrated that LSDV predominantly infects the epidermal and follicular epithelial cells, which are rich in target receptors facilitating viral entry and replication. The predilection of LSDV for the basal layer of the epidermis and the hair follicle epithelial matrix can be attributed to several factors. The basal layer of the epidermis contains proliferative keratinocytes that may provide a favorable environment for viral replication. Additionally, the hair follicle epithelium is known to harbor stem cells with high metabolic activity, which may support efficient viral replication and persistence. The detection of LSDV in the epithelial cells of sweat glands and sebaceous glands, although less frequent, is significant as it suggests that these structures can also serve as sites of viral replication and persistence. This finding is supported by a study conducted by (33), which reported the presence of LSDV antigen in sweat gland epithelial cells, indicating that these glands may contribute to viral shedding and transmission. The extensive involvement of the hair follicle epithelium and basal layer of the epidermis likely contributes to the characteristic nodular lesions and ulcerations observed in LSD. The viral replication in sweat and sebaceous glands may enhance the inflammation and contribute to the secondary bacterial infections commonly associated with LSD.

A notable finding of this study was the marked downregulation of IL-2, IL-4, IL-10, IFN-γ, and GM-CSF in both PBMCs and skin tissues of affected cattle. Their suppression suggests substantial immune modulation during LSDV infection and may represent a viral strategy to evade host immune system and establish persistence. Reduced IL-2 expression suggests impaired adaptive immunity and diminished antiviral responses. Similar suppression of IL-2 during viral infections has been reported by (6), who described cytokine downregulation as a viral immune-evasion strategy. IL-4 promotes Th2 differentiation and antibody-mediated immunity. IL-4 downregulation may compromise antibody production and impair viral neutralization during viral infections which facilitate immune evasion. Downregulation of IL-4 may also contribute to prolonged inflammation due to loss of its anti-inflammatory functions. Similar mechanisms of cytokine dysregulation of IL-10, INF-gamma, and GM-CSF have been reported in LSDV and other viral infections by (34) and Hamilton (35). The collective downregulation of IL-2, IL-4, IL-10, IFN-γ, and GM-CSF in both blood and skin tissues indicates that LSDV induces systemic immune suppression. Similar cytokine modulation has been reported for LSDV by (26) and for other viral infections by (36). (6) further demonstrated that modulation of host immune responses is an important mechanism through which LSDV enhances survival, persistence, and disease severity. Therefore, the observed cytokine suppression likely represents a critical immune-evasion strategy contributing to progression of infection.

A limitation of the present study is that sampling based on stage of disease was obtained from naturally infected cattle rather than from the same animals following longitudinal disease progression. Under field conditions, longitudinal sampling was not feasible because animals were at different stages of infection, and repeated sampling of the same individuals could not be ensured. Consequently, the stage wise comparisons represent cross-sectional observations rather than temporal changes within individual cattle. Nevertheless, the inclusion of naturally infected cattle at distinct clinical stages provides valuable insights into disease alterations with different phases of LSDV infection under field conditions.

The combined use of PCR, histopathology, immunohistochemistry and iELISA can provide a more comprehensive understanding of the infection status and the immune response in affected cattle. These findings provide valuable insights into the pathological modifications underlying the varying clinical outcomes seen in LSDV-affected cattle. These results can be leveraged to develop effective preventive strategies and control measures, ultimately helping to reduce the substantial economic losses caused by the LSD in the cattle population.

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Footnotes

Edited by: Ganesh Sonawane, Central Sheep and Wool Research Institute (ICAR), India

Reviewed by: Manimuthu Prabhu, Tamil Nadu Veterinary and Animal Sciences University, India

Nitin Thakur, Guru Angad Dev Veterinary and Animal Sciences University, India