Genetic polymorphisms in TLR4 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, a member of the genus Capripoxvirus under the family Poxviridae. It is an acute, subacute, viral disease of cattle which is genetically similar to the goat and sheep pox virus. The disease is characterized by fever, anorexia, and distinctive nodules of 2-5cm on the skin and mucous membranes of nostrils, mouth, udder, rectum, and genitals (Moda et al., 2024, Manjunatha Reddy et al., 2025) [1,2] . The virus is primarily transmitted mechanically by blood-feeding arthropod vectors, including biting flies, mosquitoes, and ticks. LSD has emerged as a major threat which leads to significant reductions in the productivity and deaths of the animals. The impact of LSD on milk yield differs by region, producing per-animal losses of roughly 8-90 litres (Naidu et al., 2025) [3] . In recent years, LSD outbreaks in India have caused severe economic damage with 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 preventing and controlling the disease, particularly in endemic and high-risk regions (Tuppurainen et al., 2017, Reddy GB et al., 2026) [6,7] . Although vaccination can produce protective immunity, considerable animal to animal variation is often observed in the magnitude of the antibody response. Such variation may arise from the combined effects of breed, age, sex, nutritional status, physiological condition, management, and genetic variation in immune response genes (Meeusen et al., 2007; Glass et al., 2012; Bayyappa et al., 2025) [8,9,10] . Candidate genes involved in antigen recognition, antigen presentation and cytokine regulation may contribute to differences in vaccine responsiveness. Genetic differences in genes involved in pathogen recognition, antigen presentation and cytokine signalling may therefore contribute to variation in vaccine responsiveness among cattle (Leach et al., 2010; Glass et al., 2012) [11,9] . Toll-like receptors are germline encoded pattern-recognition receptors that constitute an important component of innate immunity. These receptors recognize conserved pathogenassociated molecular patterns and endogenous damageassociated molecular patterns and initiate intracellular signalling pathways that regulate inflammatory and antimicrobial responses (Akira, 2001;Akira et al., 2006 ] ; Kawai and Akira, 2010) [12,13,14] . Among the functional Toll-like receptors identified in cattle, Toll-like receptor 4 (TLR4) plays a central role in recognizing bacterial lipopolysaccharide and several endogenous danger signals (Miller et al., 2005; Kawai and Akira, 2010) [15,14] . The bovine TLR4 gene has been mapped to chromosome 8 and is expressed in several immune-cell populations, including macrophages, dendritic cells and neutrophils (McGuire et al., 2006) [16] . Activation of TLR4 initiates MyD88-dependent and TRIF-dependent signalling pathways. Through its effects on dendritic-cell maturation, macrophage activation, antigen presentation and cytokine production, TLR4 provides an important functional link between innate and adaptive immunity (Akira et al., 2006; Vlasova and Saif, 2021) [13,17] . Enhanced activation of antigen-presenting cells promotes T-lymphocyte stimulation, B-cell differentiation and antibody production indicating that TLR4-mediated signalling may influence the magnitude and quality of vaccine induced immunity (Glass et al., 2012 [9] ; Vlasova and Saif, 2021 [17] ) [9,17] . Polymorphisms in the bovine TLR4 gene have been associated with variation in mastitis resistance, paratuberculosis, inflammatory responses, reproductive traits and vaccine responsiveness (Wang et al., 2007; Glass et al., 2012; Girish et al., 2016; Gopi et al., 2020; Chen et al., 2022) [18,9,19,20] . Despite the recognized role of TLR4 in innate immune activation, information regarding its genetic variation in relation to antibody response following LSD vaccination is limited. Hallikar cattle represent an indigenous Bos indicus breed adapted to tropical environmental conditions whereas Crossbred Holstein Friesian cattle possess both exotic and indigenous genetic contributions. These contrasting genetic backgrounds may contribute to breed wise differences in immune-gene polymorphisms and vaccine responses. Therefore, the present study was designed to identify single nucleotide polymorphisms (SNPs) in exon 1 of the TLR4 gene in Hallikar and Crossbred Holstein Friesian cattle and to evaluate their association with variation in vaccine induced antibody responses following LSD vaccination (Bayyappa MR et al., 2025) [23] .

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 (25 th percentile) were designated as lower responders, those with values between Q1 and Q3 (25 th -75 th percentile; including Q2, the median) as average responders, and those with values ≥ Q3 (75 th 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 percent agarose gel. DNA samples of adequate quality and concentration were used for amplification of TLR4 exon 1.

Selection of the target region and primer design

TLR4 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 (TLR4 gene/transcript ID: ENSBTAT00000008190, TLR4 exon ID: ENSBTAE00000066213). Primers were designed using PerlPrimer and synthesized by Eurofins Genomics India, Bengaluru, the details of the primers are represented in Table 1.

PCR thermal cycling conditions

PCR amplification of TLR4 exon 1 was performed using the standardized thermal profile, the details of the PCR thermal cycling conditions are mentioned in Table 3. Step 2 to 4 were repeated 35 times 6.

Final extension 72 °C 7 min 7.

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 80 V 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 bidirectional sequencing 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 TLR4 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.

Classification of antibody responder groups

The fold change in log₂-transformed SNT titres was calculated separately for Hallikar and Crossbred Holstein Friesian (CB-HF) cattle and classified using a quartile based distribution. In Hallikar cattle, the first quartile (Q1), median (Q2) and third quartile (Q3) values were 4.03, 4.04 and 5.03, respectively. Accordingly, animals with foldchange values ≤ 4.03 were classified as lower responders, those with values > 4.03 and < 5.03 were classified as average responders, and animals with fold-change values ≥ 5.03 were classified as higher responders. In CB-HF cattle, the Q1, Q2 and Q3 values were 3.04, 4.03 and 4.04, respectively. Animals with fold-change values ≤ 3.04 were categorized as lower responders, those with values > 3.04 and < 4.04 were categorized as average responders, and animals with fold-change values ≥ 4.04 were categorized as higher responders. Based on this classification, 12 higher and 12 lower antibody responder animals from each breed were selected for bidirectional Sanger sequencing and subsequent SNP association analysis.

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 85 ± 12 ng/µl. The A260/A280 nm 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 TLR4 exon 1

PCR amplification of TLR4 exon 1 was successful in all selected animals. Agarose gel (1.5%) electrophoresis revealed distinct bands of the expected size without nonspecific amplification or primer-dimer formation (Figure 1).

Association of TLR4 exon 1 polymorphisms with antibody response

The distribution of TLR4 exon 1 SNPs between higher and lower antibody responder groups was evaluated using the two-tailed Fisher's exact test. Among the three polymorphic positions identified, only the A>C substitution at nucleotide position 534 in Hallikar cattle showed a significant (P = 0.0084) association with antibody response following vaccination. In contrast, the G>A substitution at position 413 in Hallikar cattle and the G>C substitution at position 245 in CB-HF cattle were not significantly associated with antibody responder status. The identified TLR4 exon 1 polymorphisms and their association with antibody response in Hallikar and Crossbred Holstein Friesian (CB-HF) cattle are summarized in Table 5.

Discussion

In the present study, sequence analysis of TLR4 [18,19,21] , but the polymorphic positions identified in the present study differed from those reported earlier. The differences may be attributed to variation in cattle breeds, population structure, genomic regions analysed and evolutionary selection pressures. At nucleotide position 413 in Hallikar cattle, G>A transition was identified. Although the variant A nucleotide occurred more frequently among lower antibody responders than higher responders, the association was not statistically significant. Similarly, in CB-HF cattle, G>C transversion was identified at nucleotide position 245, where the C nucleotide occurred more frequently among lower responders, the difference was also not statistically significant. These findings suggest that the identified polymorphisms may not have a measurable influence on vaccine-induced antibody response in the studied population. Comparable observations have been reported by Girish et al. (2016) [19] who identified several TLR4 polymorphisms in the entire gene in indigenous and crossbred cattle but did not observe significant associations between all variants and mastitis resistance. Likewise, Sonwane et al. (2023) [22] reported TLR4 polymorphisms in exon 3 in HF × Gir crossbred cattle without detecting significant association with somatic cell count among 40 subclinical mastitis affected and 20 healthy cows. In contrast, nucleotide position 534 in Hallikar cattle showed a significant association (P = 0.0084) with antibody response. The variant 'C' nucleotide was more frequently observed among higher antibody responders whereas the reference 'A' nucleotide predominated among lower responders. This observation suggests that the polymorphism at position 534, or another functional variant in linkage disequilibrium with it, may influence vaccine induced humoral immunity. Although the exact biological effect of this nucleotide substitution could not be determined in the present study, the observed association indicates that this locus may serve as a potential genetic marker for variation in antibody response following LSD vaccination.

The present findings are consistent with previous studies demonstrating the involvement of TLR4 polymorphisms in bovine immune responses. Glass et al. (2012) [9] reported that genetic variation in TLR4 influenced IgG1 and IgG2 responses following vaccination against bovine respiratory syncytial virus and foot-and-mouth disease virus, indicating that innate immune receptor polymorphisms contribute to variation in adaptive immune responses. Similarly, Chen et al. (2022) [21] demonstrated that the missense mutation rs8193069 within the TLR4 gene was significantly associated (p < 0.05) with mastitis resistance and somatic cell count in Holstein cattle. Functional analysis suggested that the mutation altered the receptor structure and downstream signalling, thereby influencing inflammatory responses. Wang et al. (2007) [18] also reported associations between TLR4 polymorphisms at nucleotide 4525 in intron 1, and a C→T substitution at nucleotide 1397 in exon 3 and mastitis resistance traits in dairy cattle, supporting the importance of TLR4 gene in disease resistance. However, the present investigation provides the first evidence to our knowledge, of an association between a TLR4 exon 1 polymorphism and antibody response following LSD vaccination in Hallikar cattle. Future studies integrating genome wide association analyses, transcriptomics, functional assays and protein modelling would help determine the biological significance of this polymorphism and evaluate its potential application in marker-assisted selection for improved vaccine responsiveness and disease resistance.

Conclusions

The present study identified two polymorphic nucleotide positions (413 and 534) in Hallikar cattle and one polymorphic position (245) in CB-HF cattle. Among these, the A>C substitution at nucleotide position 534 in Hallikar cattle showed a significant association (P = 0.0084) with the vaccine-induced antibody response. These findings suggest that the TLR4 polymorphism at position 534 may represent a potential candidate marker for variation in vaccine responsiveness in Hallikar cattle. However, validation in larger and genetically diverse cattle populations, together with functional characterization of the identified variants is required before their application in marker assisted selection programmes aimed at improving vaccine responsiveness and disease resistance.

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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