Open Veterinary Journal, (2026), Vol. 16(4): 2271-2280

Research Article

10.5455/OVJ.2026.v16.i4.27

Immune and biochemical evaluation of a live attenuated lumpy skin disease vaccine in cattle

Pummarin Tippramuan^1,2, Thanapol Nongbua¹, Worapol Aengwanich^1,3, Satitpong Promsatit⁴ and Piyarat Srinontong^1,2,3*

¹Faculty of Veterinary Sciences, Mahasarakham University, Mahasarakham, Thailand

²Bioveterinary Research Unit, Faculty of Veterinary Sciences, Mahasarakham University, Mahasarakham, Thailand

³Stress and Oxidative Stress in Animal Research Unit, Faculty of Veterinary Sciences, Mahasarakham University, Mahasarakham, Thailand

⁴Department of Livestock Development, Amnat Charoen Provincial Livestock Office, Amnat Charoen, Thailand

*Corresponding Author: Piyarat Srinontong. Faculty of Veterinary Sciences, Mahasarakham University, Mahasarakham, Thailand.
Email: piyarat [at] msu.ac.th

Submitted: 14/11/2025 Revised: 28/02/2026 Accepted: 14/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Vaccination remains one of the most effective strategies to control infectious diseases in cattle, and early immune response evaluation is essential for understanding initial vaccine-induced immune activation.

Aim: This study aimed to evaluate hematological and biochemical parameters, cytokine expression, including Interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), and antibody titers in cattle following administration of a live attenuated Neethling lumpy skin disease virus (LSDV) vaccine.

Methods: Cattle were divided into two groups: i) 14 healthy, unvaccinated cattle (control group) and ii) 21 cattle vaccinated subcutaneously with the Neethling LSDV vaccine. Hematological and biochemical profiles, cytokine gene expression, and LSDV-specific antibody titers were measured 14 days after vaccination.

Results: Vaccinated cattle showed significantly higher lymphocyte counts and gamma-glutamyl transferase levels than unvaccinated controls (p < 0.05). IFN-γ gene expression was upregulated 1.9-fold in vaccinated cattle (p < 0.05). LSDV-specific antibody titers were significantly higher in vaccinated cattle; however, enzyme-linked immunosorbent assay sample-to-positive (S/P) values remained below the diagnostic cutoff value at 14 days post-vaccination.

Conclusion: Our results indicate that 14 days after vaccination, cattle exhibit increased lymphocyte activity and IFN-γ expression, consistent with an early cellular immune response, although elevated gamma-glutamyl transferase levels may reflect mild physiological stress associated with early vaccine response.

Keywords: Cattle, Immunity, Lumpy skin disease, Vaccine.

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Introduction

Lumpy skin disease (LSD) is an emerging infectious disease of cattle that has caused major outbreaks in several countries (Roche et al., 2020; Arjkumpa et al., 2022). LSD causes large economic losses in the livestock industry due to morbidity, immunosuppression, and reproductive disorders. Lumpy skin disease virus (LSDV) is a double-stranded DNA virus of the genus Capripoxvirus in the family Poxviridae. LSDV is mechanically transmitted by blood-feeding arthropods, such as mosquitoes (Aedes spp. and Culex spp.), biting flies (Stomoxys calcitrans), and ticks (Roche et al., 2020). The clinical manifestations are skin nodules, anorexia, fever, enlarged lymph nodes, nasal discharge, reduced milk production, and abortion (Namazi and Khodakaram Tafti, 2021). LSD-associated hematological alterations commonly include leukocytosis, neutrophilia, and lymphocytosis. Alterations in serum gamma-glutamyl transferase (GGT) concentrations have been reported in cattle with LSD and may reflect physiological or metabolic responses associated with infection or stress rather than direct indicators of immune protection (Abutarbush, 2015).

Elimination and prevention of LSD mainly depend on controlling the spread of the disease, as well as vaccination, biosecurity, control of insect vectors, quarantine, and active surveillance (Roche et al., 2020). Currently, vaccination is a key and indispensable component of LSDV control programs in endemic areas and in protection zones adjacent to affected regions, whereas comprehensive disease management in newly affected or disease-free areas relies on vector control, biosecurity measures, surveillance, movement restrictions, and culling strategies (Tuppurainen et al., 2021). Live attenuated vaccines, such as the Neethling LSD vaccine, have been widely used for LSD control in several countries (Haegeman et al., 2021), including Israel, the European Union and the Balkans (Abutarbush, 2015). The vaccine elicits protective immunity approximately 2–3 weeks following vaccination and reaches its peak 30 days after vaccination. Although humoral immunity has gradually declined, cellular immunity still protects vaccinated animals (Tuppurainen et al., 2021). Live attenuated vaccines also elicit effective cellular immune responses that produced high level of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) and enhanced lymphocyte proliferation (Norian et al., 2017; Varshovi et al., 2017).

Despite the widespread use of vaccination-based disease control measures, knowledge regarding the efficacy of vaccines and early immune responses of cattle to live attenuated Neethling LSDV vaccination under field conditions in endemic areas remains limited. Early immune and physiological responses following vaccination have been poorly characterized, particularly through integrated evaluation of hematological, biochemical, cellular, and humoral immune parameters. This represents an important knowledge gap, as early immune activation during emergency vaccination campaigns may influence vaccine efficacy, animal welfare, and stress-related physiological responses. Therefore, this study aimed to evaluate short-term immune responses in cattle vaccinated with a live attenuated Neethling LSDV vaccine by assessing hematological and biochemical parameters, cytokine expression (IFN-γ and tumor necrosis factor-alpha (TNF-α), and antibody titers under field conditions in Thailand. The results of this study will be useful for understanding vaccine-induced immunity in cattle in endemic areas and may inform future LSD control and prevention strategies.

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Materials and Methods

Animal management

The study was conducted on a farm in Mahasarakham Province, northeastern Thailand, between June and July 2021. Thirty-five mixed-bred Brahman × Thai native cattle aged 2–5 years were included. Body condition score (BCS) was assessed before starting the experiment using a 5-point scale (1=thin to 5=fat) (Bell et al., 2018). Cattle (body weight 200–260 kg) were allocated based on BCS ranging from 2.5 to 3.5. All cattle were dewormed. They were vaccinated with the foot-and-mouth disease (FMD) vaccine every 4 months, following the routine vaccination program on the farm, and were checked for health before and throughout the experimental period. All cattle were housed in free-stall barns. Feed and water were supplied ad libitum.

Experimental design

A total of 35 clinically healthy cattle were enrolled in this study. Before the experiment, all animals underwent clinical examination by a veterinarian and showed no clinical signs suggestive of LSD at the time of examination. Farm records indicated no history of LSD vaccination and no outbreaks during the study period. The cattle were divided into two groups: a control group consisting of 14 unvaccinated animals and a vaccinated group comprising 21 cattle that received a single subcutaneous dose of a commercial live attenuated vaccine (Lumpyvac®; Vetal Animal Health Product S.A., Adiyaman, Turkey) according to the manufacturer’s instructions. The vaccine contained 10^3.5 TCID₅₀ of the LSDV Neethling virus strain per 2 ml dose. Blood samples were collected 14 days after vaccination to measure hematological, biochemical, and immunological parameters.

Collection of blood samples

Blood samples were aseptically collected from all cattle using jugular venipuncture. Whole blood samples (20 ml/cattle) were placed into tubes containing ethylenediaminetetraacetic acid (EDTA) and plain blood collection tubes. EDTA-anticoagulated blood was used for hematology analysis and isolation of peripheral blood mononuclear cells (PBMCs) for RT-qPCR. Plasma was separated from EDTA-anticoagulated blood by centrifugation and stored for biochemistry analysis. Blood samples without anticoagulants were used to separate serum and were kept for antibody assessment. Samples were delivered on ice to the laboratory of the Faculty of Veterinary Sciences, Mahasarakham University, Mahasarakham, Thailand, for subsequent analysis.

Hematological and biochemical analysis

Hematological parameters were measured using the IDEXX Procyte DX Hematology Analyzer (IDEXX Laboratories, Inc., Maine, USA). Biochemical parameters [aspartate aminotransferase (AST), GGT, and total protein] were measured using the IDEXX Catalyte One Analyzer (IDEXX Laboratories, Inc., Maine, USA).

LSDV-specific antibody determination by enzyme-linked immunosorbent assay

A commercial enzyme-linked immunosorbent assay (ELISA) kit ID Screen® Capripox Double Antigen Multi-species (IDvet, Grabels, France) Lot: I97 was used to detect LSDV-specific antibodies in serum, following the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader (TECAN, Männedorf, Switzerland). Optical density (OD) values of samples were determined as sample-to-positive percentage (S/P%) according to the following formula: [(OD_sample-OD_NC)/(OD_PC-OD_NC)] × 100, where OD_sample, OD_PC, and OD_NC were the mean OD of the cattle serum sample, positive control, and negative control, respectively. A sample with an S/P% value ≥ 30% was considered positive, and an S/P% < 30% was considered negative for antibodies against LSDV.

Isolation of peripheral blood mononuclear cells

Whole blood was diluted 1:1 with phosphate-buffered saline (PBS), carefully layered over Ficoll-Paque Plus (Cytiva^TM, MA, USA), and centrifuged at 800 × g for 35 minutes at room temperature without brake as previously described (Chalalai et al., 2025). The mononuclear cell layer located at the interface between PBS and Ficoll-Paque Plus was transferred to a new tube. Contaminating erythrocytes were lysed using red blood cells (RBCs) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The PBMCs were washed with PBS at 1,400 × g for 10 minutes and stored immediately at −20°C in NucleoProtect RNA (Macherey-Nagel, Düren, Germany) for further processing.

RNA isolation and reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA of PBMCs was isolated using the Nucleospin RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) was used to measure RNA concentrations and quality (A260/A280 ratio). Reverse transcription was performed using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). Quantitative polymerase chain reaction (PCR) was performed using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, MA, USA). Reactions were performed in a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, CA, USA). The program consisted of 1 cycle at 95^°C for 10 minutes, 40 cycles at 95^°C for 15 seconds, and 60^°C for 60 seconds. Melt curve analysis was conducted by gradually increasing the temperature from 65°C to 95°C. The fold change in gene expression was determined using the 2^−∆∆Ct method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The primer sequences are listed in Table 1.

Table 1. Real-time PCR primers.

Statistical analysis

Hematological and biochemical parameters, LSDV-specific antibody titers, and cytokine gene expression (IFN-γ and TNF-α) were compared between the unvaccinated and vaccinated groups using an independent t-test. Data are presented as mean ± standard deviation (SD). Differences between groups were considered significant at p < 0.05.

Ethical approval

The study protocol and experimental procedures were approved by the Institutional Animal Care and Use Committee of Mahasarakham University (IACUC-MSU-33/2022).

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Results

Hematological parameters

Routine hematological profiles were assessed to investigate the association between vaccination and hematological alterations. Vaccination selectively increased lymphocyte counts without affecting other hematological parameters. Lymphocyte counts were significantly higher in vaccinated cattle compared with the unvaccinated group (p < 0.05). In contrast, no significant differences were observed in total white blood cell, neutrophil, monocyte, or eosinophil counts between vaccinated and unvaccinated cattle (p > 0.05) (Fig. 1). Similarly, red blood cell count, packed cell volume, hemoglobin concentration, platelet count, and erythrocyte indices [mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC)] did not differ significantly between groups (p > 0.05) (Fig. 2).

Fig. 1. Differential leukocyte counts in unvaccinated and vaccinated cattle at 14 days post-vaccination. Total white blood cell (WBC), neutrophil (NEU), lymphocyte (LYM), eosinophil (EOS), and monocyte (MON) counts were determined in unvaccinated and vaccinated cattle 14 days post-vaccination. Data are expressed as mean ± standard deviation (SD). * Indicates a significant difference between groups (p < 0.05).

Fig. 2. Hematological parameters in unvaccinated and vaccinated cattle at 14 days post-vaccination. Red blood cell count (RBCs), packed cell volume (PCV), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) were measured in unvaccinated and vaccinated cattle 14 days after administration of the live attenuated Neethling LSDV vaccine. Data are presented as mean ± SD.

Biochemical parameters

Plasma biochemical analyses were performed in both vaccinated and unvaccinated cattle to determine the impact of vaccination on biochemical parameters. The GGT level was significantly higher in vaccinated cattle compared with the unvaccinated group (p < 0.05) (Fig. 3A). However, the total protein and AST levels did not differ significantly between groups (p > 0.05) (Fig. 3B and C).

Fig. 3. Biochemical parameters of unvaccinated and vaccinated cattle at 14 days post-vaccination. (A) Plasma gamma-glutamyl transferase (GGT), (B) total protein concentrations, and (C) aspartate aminotransferase (AST) are presented as mean ± SD. * Indicates a statistically significant difference between groups (p < 0.05).

Expression of inflammatory cytokine genes in PBMCs

To assess early cellular immune responses after immunization, RT-qPCR was used to measure cytokine gene expression profiles in PBMCs. IFN-γ gene expression was significantly increased in vaccinated cattle, with a 1.9-fold increase compared with unvaccinated controls (p < 0.05) (Fig. 4A). In contrast, TNF-α gene expression did not differ significantly between groups (p > 0.05) (Fig. 4B).

Fig. 4. Relative gene expression of cytokines, IFN-γ (A), and TNF-α (B) of PBMC between the unvaccinated and vaccinated groups. * Indicate statistically significant differences (p < 0.05). IFN-γ=interferon-gamma; TNF-α=tumor necrotic factor-alpha; GAPDH=glyceraldehyde-3-phosphate dehydrogenase.

Antibody titers of LSDV

To assess humoral immune responses following vaccination with the live attenuated Neethling LSDV vaccine, LSDV-specific antibody titers were measured by ELISA at 14 days post-vaccination. Vaccinated cattle exhibited significantly higher ELISA S/P% values compared with unvaccinated controls (p < 0.05). However, the mean S/P% values in both groups remained below the diagnostic cutoff value (S/P% ≥ 30%) (Table 2).

Table 2. LSDV antibody titer in cattle on day 14 post-vaccination.

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Discussion

This study evaluated hematological, biochemical, and immunological responses to LSDV vaccination to characterize early immune activation in cattle. This was needed as there have been very limited reports or data on the monitoring and evaluation of live LSDV Neethling vaccinated cattle using these parameters.

White blood cell (WBC) count is an important pathophysiological indicator related to the nonspecific immune response in animals (Koch et al., 2025). Although total WBC counts increased slightly in the vaccinated group, this was mainly due to an increase in lymphocytes compared with the unvaccinated group. This observation is consistent with Shawky et al. (2016), who reported higher lymphocyte counts in cattle vaccinated against FMD. Similarly, Monir et al. (2020) found increased lymphocyte counts in vaccinated tilapia. The observed lymphocytosis may reflect the activation of lymphocyte populations involved in early adaptive immune responses (Barkakati et al., 2015; Reddy and Sivajothi, 2020). However, PCV, hemoglobin, RBC count, platelet count, and erythrocyte indices did not differ significantly between groups. These findings align with previous studies (Shawky et al., 2016; Reddy and Sivajothi, 2020), suggesting that the live LSDV vaccine does not significantly affect RBC profiles.

Liver-associated biochemical parameters were included as part of routine physiological monitoring to detect potential metabolic or stress-related responses following vaccination, rather than to assess vaccine efficacy or hepatic pathology (Shawky et al., 2016; Monir et al., 2020; Reddy and Sivajothi, 2020). Accordingly, total protein concentration and hepatic enzyme activities, including AST and GGT, were evaluated, as these enzymes are commonly used indicators of hepatocellular integrity and cholestatic or metabolic responses, respectively (Aba et al., 2020). Plasma GGT levels were significantly higher in vaccinated cattle compared with unvaccinated controls, whereas AST activity and total protein concentration remained unchanged. The absence of AST elevation suggests that the increase in GGT was unlikely to be associated with hepatocellular damage. In cattle, GGT has been reported to respond to metabolic adaptation, oxidative stress, and physiological stress (Tufarelli et al., 2023). Live attenuated Neethling LSDV vaccines have been widely used in endemic regions and evaluated under field conditions, with safety assessments primarily focusing on clinical outcomes, productivity, and mortality, and without evidence of vaccine-associated hepatotoxic effects (Morgenstern and Klement, 2020; Tuppurainen et al., 2021). Therefore, although live vaccines contain stabilizers or excipients, the observed increase in GGT in the present study is unlikely to reflect direct liver injury and may instead represent a transient metabolic or stress-related response during early immune activation (Hudson et al., 2020; Efe et al., 2022). The unchanged AST activity observed in this study is consistent with previous reports showing no significant alteration in AST levels following vaccination, including studies on FMD vaccination in cattle and buffaloes (Shawky et al., 2016; Reddy and Sivajothi, 2020). Similarly, total protein concentrations did not differ between vaccinated and unvaccinated groups, supporting the absence of overt hepatic dysfunction, as also reported in earlier studies (Kudair and Al-Hussary, 2010; Shawky et al., 2016; Reddy and Sivajothi, 2020). Further investigation of serum protein fractions, including albumin and globulin, is recommended to clarify these findings.

The relevance of cellular immune responses after vaccination has been studied (Gari et al., 2015; Varshovi et al., 2017). Elevated cytokine levels after vaccination serve as indicators of immune activation and may correlate with vaccine-induced protection. Generally, higher IFN-γ expression correlates with protection against diseases, as vaccination enhances Th1 and cytotoxic T-cell responses (Sadarangani et al., 2021; Kurteva et al., 2022). Th1 cytokines such as IFN-γ and TNF-α contribute to antiviral activity (Pandey and Karupiah, 2022). However, information on cytokine responses after single-dose emergency vaccination against LSDV remains limited. In the present study, IFN-γ gene expression in PBMCs increased significantly after vaccination, whereas TNF-α expression did not differ between groups. These findings are consistent with Norian et al. (2017), who reported elevated IFN-γ levels in dairy cattle vaccinated with a live goat pox virus, and with Khafagy et al. (2016), who observed similar responses in animals immunized with a live Romanian sheep pox vaccine. These results indicate that the Neethling LSDV vaccine stimulates cellular immune responses in cattle through upregulation of IFN-γ expression. Further investigation of Th2-associated cytokines would provide a more comprehensive understanding of the balance between cellular and humoral immunity following vaccination.

Although LSDV-specific antibody titers were significantly increased in vaccinated cattle at 14 days post-vaccination, the observed ELISA S/P% values remained below the diagnostic positivity threshold. This indicates that humoral immunity was still in an early developmental phase and had not yet reached levels associated with protective antibody responses (Milovanović et al., 2019; Samojlović et al., 2024). These findings are consistent with previous reports demonstrating that antibody responses to live attenuated Neethling LSDV vaccines typically peak at later time points, generally between 21 and 28 days post-vaccination (Hamdi et al., 2020). Accordingly, the early increase in antibody levels observed in this study likely reflects initial activation of the humoral immune response rather than established seroconversion. A limitation of the present study is that cattle were examined at a single time point; thus, long-term antibody kinetics could not be described. Future studies should address the application of long-term vaccination monitoring programs. Notably, antibody responses have been detected as early as one week after vaccination with live sheep pox or goat pox virus strains (Norian et al., 2017) and up to 28 days in some studies (Hamdi et al., 2020; Haegeman et al., 2021). Such variability may reflect differences in vaccine strains, host factors, or antibody detection methods (Milovanović et al., 2019; Fay et al., 2022).

Importantly, low antibody levels at this early stage do not imply a lack of protection, as cell-mediated immunity plays a critical role in protection against LSDV infection (Bamouh et al., 2021; Fay et al., 2022). All unvaccinated cattle remained seronegative (S/P% < 30%), indicating no detectable humoral response at the time of sampling. However, baseline serological testing was not performed before vaccination; therefore, the complete absence of prior subclinical exposure cannot be definitively confirmed. This should be considered when interpreting the antibody findings.

IgG2 production was closely related to the Th1 cytokine expression profile, particularly IFN-γ, indicating an association between cellular and humoral immunity (Wilson-Welder et al., 2021; Lesta et al., 2025). In the present study, LSDV-specific antibodies increased following vaccination, corresponding with elevated IFN-γ gene expression in PBMCs of vaccinated cattle. However, the use of a commercial ELISA kit is limited, as it cannot identify the antibody isotypes (IgG1, IgG2, or IgM). Consequently, the relative contribution of specific immunoglobulin subclasses to the observed humoral response could not be determined. Nevertheless, the concurrent increase in antibody levels and IFN-γ expression supports the interpretation that vaccination induced immunological activation of PBMCs, contributing to the differences observed between vaccinated and unvaccinated groups.

Despite the clear evidence of early immune activation following vaccination, several limitations of this study should be considered. Immune responses were evaluated at a single time point (14 days post-vaccination), which does not allow assessment of peak antibody responses or the durability of vaccine-induced immunity. In addition, the relatively small sample size and the use of cattle from a single farm may limit the generalizability of the findings. Future studies incorporating multiple sampling time points, larger populations, and cattle from different management systems are warranted to better characterize the magnitude and duration of immune responses induced by the live attenuated Neethling LSDV vaccine under field conditions.

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Conclusion

Early immune responses to a live attenuated Neethling LSDV vaccine were evaluated 14 days after vaccination. Increased lymphocyte counts, elevated GGT levels, upregulated IFN-γ gene expression, and higher antibody titers collectively indicate the initiation of both cellular and humoral immune responses. However, these early immune responses may not yet represent fully developed protective immunity and could reflect transient physiological or immunometabolic adaptation during the initial post-vaccination period. Further studies are required to evaluate the durability of immune responses and assess the duration of protection under field conditions.

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Acknowledgments

This research project was financially supported by Mahasarakham University. The authors are grateful to the Faculty of Veterinary Sciences, Mahasarakham University, for providing research facilities.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding

This research project was financially supported by Mahasarakham University.

Authors’ contributions

Pummarin Tippramuan: Conceptualization, data curation, investigation, methodology, validation, and writing of the original draft. Thanapol Nongbua: Methodology and formal analysis. Worapol Aengwanich: Resources, original draft writing, and validation. Satitpong Promsatit: Methodology and formal analysis. Piyarat Srinontong: Conceptualization, funding acquisition, investigation, formal analysis, supervision, validation, original draft writing, review, and editing.

Data availability

Data supporting the findings of this study are available upon request from the corresponding author.

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