Genetic polymorphism in IL10 exon 1 and their association with antibody response following lumpy skin disease vaccination in Hallikar and crossbred Holstein Friesian cattle

Introduction

Lumpy skin disease (LSD) is an economically important transboundary viral disease of cattle caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus within the family Poxviridae. The disease may occur in acute or subacute forms and is characterized by fever, anorexia, and distinctive cutaneous nodules, typically 2-5 cm in diameter, which may also involve the mucous membranes of the nostrils and mouth, as well as the udder, rectum, and genitalia (Moda et al., 2024; Manjunatha Reddy et al., 2025) [1,2] . LSDV is primarily transmitted mechanically by blood-feeding arthropod vectors, including biting flies, mosquitoes, and ticks. The disease has emerged as a major threat to cattle production, causing substantial morbidity, mortality, and production losses. Milk yield losses associated with LSD vary considerably across regions, with estimated losses of approximately 8-90 litres per affected animal (Naidu et al., 2025) [3] . In recent years, LSD outbreaks in India have resulted in severe economic losses, estimated at approximately USD 2.2 billion (Manjunatha Reddy et al., 2023; Tadakod S et al., 2026) [4,5] . Vaccination remains the principal strategy for the prevention and control of LSD, particularly in endemic and highrisk regions (Tuppurainen et al., 2017; Reddy GB et al., 2026) [6,7] .

Despite vaccination, considerable animal-to-animal variation may occur in the magnitude of the antibody response. This variability can be influenced by several factors, including breed, age, sex, nutritional status, physiological condition, management practices, and genetic variation in immune response genes (Meeusen et al., 2007 [8] ; Glass et al., 2012 [9] ; Bayyappa et al., 2025) [10] . Genetic variation in candidate genes involved in pathogen recognition, antigen presentation, and cytokine-mediated immune regulation may therefore contribute to differences in vaccine responsiveness among cattle (Leach et al., 2010 [11] ; Glass et al., 2012 [9] ). Interleukin-10 (IL-10) is a pleiotropic cytokine with potent anti-inflammatory and immunoregulatory activity. It is produced by regulatory T cells, helper T-cell subsets, macrophages, dendritic cells, monocytes, B cells and other leukocytes. IL-10 limits excessive production of proinflammatory cytokines, reduces antigen-presenting-cell activation and contributes to the resolution of inflammation thereby preventing immunopathology while maintaining tissue homeostasis (Saraiva and O'Garra, 2010) [12] . IL-10 regulates the interface between innate activation and adaptive immunity, variation in the IL10 signalling pathway may influence vaccine responsiveness. Changes in coding sequence may alter cytokine structure or stability whereas promoter and regulatory variants may influence transcription. In cattle, polymorphisms in IL10, IL10RA and IL10RB have been investigated in relation to somatic cell score, mastitis and Mycobacterium avium subsp. paratuberculosis infection (Verschoor et al., 2009 [13] ; Verschoor et al., 2010 [14] ). These studies indicate that IL10pathway diversity may contribute to disease-related phenotypes, although effects can depend on the genomic region, population and phenotype examined. The present study selected IL10 as a candidate immune response gene because its anti-inflammatory function could influence the magnitude and duration of antibody production after LSD vaccination. However, information on IL10 sequence variation associated with antibody response to LSD vaccination in indigenous Hallikar and CB-HF cattle is scarce. The objective of the present analysis was therefore to characterize IL10 exon 1 in higher and lower antibody responders of both breeds and to test whether identified nucleotide variants were associated with vaccine-induced humoral response.

Materials and Methods

Experimental animals and pre-vaccination management

A total of 154 cattle, comprising 86 Hallikar and 68 Crossbred Holstein Friesian (CB-HF) cattle were identified from different villages of Bagepalli and Shidlaghatta taluks of Chikkaballapur District, Karnataka. Animals were selected based on dentition, and only cattle possessing a minimum of one pair of permanent incisor teeth were considered for screening. The age of each animal was recorded in years based on the incisor eruption pattern. Before vaccination, all the identified animals were dewormed with fenbendazole bolus 1.5g and were supplemented with a trace mineral bolus to minimize the possible confounding effects of parasitic infestation and mineral deficiency on vaccine-induced immune responses.

Animal selection and vaccination

Before enrolment, all identified animals were screened for pre-existing antibodies against LSDV using indirect enzyme-linked immunosorbent assay (iELISA) and Serum Neutralization Test (SNT). Animals exhibiting an iELISA percentage positivity value of less than 35 PPV and an SNT antibody titre of less than 1:8 were considered seronegative and included in the study. Based on these criteria, 50 Hallikar and 50 CB-HF cattle were enrolled in the study and vaccinated subcutaneously with 1 ml of live attenuated Goatpox vaccine (Uttarkashi strain; Hester Bioscienes Ltd., India) containing not less than 1 X 10³ TCID₅₀ per dose, as per the manufacturer's recommendations and the Government of India guidelines for heterologous vaccination against LSD.

Classification of antibody responder groups

Post-vaccination antibody responses were assessed at 28 days using iELISA and SNT. Since SNT is the reference assay for evaluating functional neutralizing antibodies, SNT titres were used for fold-change calculations and responder classification. Animals were categorized into higher and lower antibody responder groups based on the fold change in log₂-transformed SNT titres by using quartile based distribution. Animals with fold-change values ≤ Q1 (25th percentile) were designated as lower responders, those with values between Q1 and Q3 (25th-75th percentile; including Q2, the median) as average responders, and those with values ≥ Q3 (75th percentile) as higher responders. Only the higher and lower responder groups were selected for sequencing and subsequent SNP association analysis.

DNA isolation and quality assessment

Genomic DNA was isolated from EDTA-anticoagulated whole blood samples using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The concentration and purity of the extracted DNA were assessed using a UV spectrophotometer, and DNA integrity was confirmed by electrophoresis on 1.5% agarose gel. DNA samples of adequate quality and concentration were used for amplification of IL10 exon 1.

Selection of the target region and primer design

IL10 exon 1 was selected as one of the candidate immune response gene regions for molecular characterization. The Bos taurus reference sequence was obtained from the Ensembl Genome Browser (IL10 gene/transcript ID: ENSBTAT00000008190, IL10 exon ID: ENSBTAE00000066213). Primers were designed using NCBI Primer-BLAST and synthesized by Eurofins Genomics India, Bengaluru, the details of the primers were represented in Table 1. PCR thermal cycling conditions PCR amplification of IL10 exon 1 was performed using the following standardized thermal profile, the details of the PCR thermal cycling conditions were mentioned in Table 3. Step 2 to 4 were repeated 35 times 6.

Final extension 72 °C 7 min 7.

The optimum annealing temperature for IL10 exon 1 was determined as 57.8 °C based on the production of a sharp and distinct band of the expected size without visible nonspecific amplification.

Agarose gel electrophoresis

PCR products were evaluated using 1.5 percent agarose gel electrophoresis in 1× TBE buffer. Electrophoresis was performed with a 100 bp DNA ladder at 80V for 60 minutes. Amplified products were visualized under ultraviolet illumination and documented using a gel documentation system.

Purification and Sanger sequencing PCR amplified products selected from the higher and lower responder groups were purified and subjected to separate forward and reverse sequencing. Bidirectional sequencing was performed using the Sanger sequencing method.

Sequence editing and consensus sequence generation

Forward and reverse Sanger sequencing reads were quality checked and low quality bases at the 5′ and 3′ ends were trimmed using CLC Genomics Workbench (version 9.5.2). The reverse reads were reverse-complemented and assembled with the corresponding forward reads to generate consensus sequences.

Sequence alignment and SNP identification

The consensus sequences were aligned with the Bos taurus reference sequence using the CLUSTAL W algorithm implemented in BioEdit (version 7.2.5) to identify SNPs. Chromatograms corresponding to the identified polymorphic positions were manually inspected to confirm single peaks and overlapping heterozygous peaks using Chromas Lite (version 2.6.6).

Statistical analysis

The association between identified IL10 SNPs and antibody response category was evaluated using the two-tailed Fisher's exact test. Contingency tables were constructed by comparing the distribution of reference and variant nucleotides between higher and lower antibody responder groups. The statistical analysis was performed using IBM SPSS Statistics. A P-value below 0.05 was considered statistically significant.

Results

Post vaccination humoral immune response

A marked increase in antibody titres was observed in both breeds at 28 days post-vaccination (dpv). The postvaccination indirect ELISA PPV values, SNT titres, and geometric mean titres (GMT) are presented in Table 4.

Genomic DNA isolation and quality assessment

High quality genomic DNA was successfully isolated from all selected animals. The DNA concentration ranged from 60 to 110 ng/µl, with a mean concentration of approximately 85 ± 12 ng/µl. The A260/A280 ratio ranged from 1.75 to 2.00, indicating good purity. Agarose gel electrophoresis showed intact high-molecular-weight genomic DNA suitable for PCR amplification.

PCR amplification of IL10 exon 1 PCR amplification of IL10 exon 1 was successful in all selected animals. Agarose gel electrophoresis revealed distinct bands of the expected size without non-specific amplification or primer-dimer formation (Figure 1) Consequently, association analysis between IL10 exon 1 polymorphisms and antibody response was not performed.

Association of IL10 exon 1 polymorphisms with antibody response

The distribution of IL10 exon 1 SNP between higher and lower antibody responder groups was evaluated using the two-tailed Fisher's exact test. A single nucleotide polymorphism was identified at nucleotide position 207, where the reference nucleotide 'G' was substituted by A. The identified G→A transition was not significantly associated with antibody response. The identified IL10 exon 1 polymorphism and its association with antibody response in CB-HF cattle are summarized in Table 5.

Discussion

The present study identified marked breed-specific differences in nucleotide diversity within the analysed IL10 exon 1 fragment. Complete conservation in Hallikar cattle contrasted with a single G→A transition at position 207 in CB-HF cattle. IL-10 acts mainly as a negative regulator of inflammation. It suppresses excessive activation of macrophages and antigen-presenting cells and limits the production of inflammatory mediators, thereby reducing collateral tissue injury (Saraiva and O'Garra, 2010 [12] ). During vaccination, this regulation may be beneficial by preventing immunopathology, but excessive IL-10 signalling can also restrict antigen presentation and effectorcell activation. Thus, the relationship between IL-10 and vaccine response is unlikely to be linear and may depend more strongly on expression kinetics, cellular source and receptor signalling than on a single coding-region variant. Previous bovine studies have reported greater diversity in regulatory and receptor components of the IL10 pathway. Verschoor et al. (2009) [13] identified IL10 promoter and IL10 receptor polymorphisms in dairy cattle and reported an association between IL10RA variation and somatic cell score whereas, the IL10 promoter variants themselves were not significantly associated. Verschoor et al. (2010) [14] further identified IL10-pathway variants in cattle evaluated for Mycobacterium avium subsp. paratuberculosis phenotypes. These findings support the possibility that receptor or regulatory variation may exert stronger phenotypic effects than variation in the limited exon 1 fragment studied in the present study. The absence of polymorphism in Hallikar cattle suggests that the IL10 exon 1 region is highly conserved in this indigenous breed. In contrast, a G>A polymorphism at nucleotide position 207 was identified in Crossbred Holstein Friesian cattle, likely reflecting their admixed genetic background. Although the 'A' allele was observed only among higher antibody responders, the association was not statistically significant, indicating that the identified variant was not associated with antibody response following LSD vaccination. The lack of significance may be attributed to the low allele frequency, limited sample size or absence of a functional effect. Therefore, further validation in larger cattle populations, together with functional characterization of the variant, is required to determine its biological significance and potential role in vaccine responsiveness.

Conclusions

IL10 exon 1 was completely conserved in Hallikar cattle whereas a single G→A transition at nucleotide position 207 was identified in CB-HF cattle. Although the variant A allele occurred exclusively in higher antibody responders, its association with LSD vaccine-induced antibody response was not significant (P = 0.1092). These findings indicate limited polymorphism within the analysed IL10 exon 1 region and provide no evidence for a major influence on humoral vaccine responsiveness. Larger population studies incorporating broader genomic, expression and functional analyses are required to establish the potential relevance of IL10 variants for marker-assisted selection.

Acknowledgement

AcknowledgementThe authors gratefully acknowledge the Department of Animal Husbandry and Veterinary Services, Government of Karnataka, for their support and cooperation in facilitating the field investigations. The assistance extended by the Deputy Director, Assistant Directors, Veterinary Officers and field staff during animal selection, vaccination, sample collection and coordination with livestock owners is sincerely appreciated.

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