Seroprevalence and Risk Factors for Bovine Herpesvirus 1 Infection Among Dairy Cattle in Western Thailand

1. Introduction

Bovine herpesvirus 1 (BoHV-1), a member of the genus Varicellovirus in the amily Herpesviridae, is the causative agent of infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis (IPV) in cattle. IBR is a highly contagious viral disease that causes substantial economic losses worldwide, particularly in dairy production systems [1]. BoHV-1 establishes lifelong latency in infected cattle, with periodic viral reactivation and shedding. Following genital infection, the virus primarily establishes latency in the sacral ganglia, whereas respiratory infection is typically associated with latency in the trigeminal ganglion [2]. Infectious bovine rhinotracheitis (IBR) can range from subclinical infection to mild or severe clinical disease. Early clinical signs include fever, lethargy, and profuse nasal discharge, which is initially serous but may subsequently become mucopurulent. The nasal mucosa becomes hyperemic, and intranasal lesions may develop. These lesions can progress from localized necrosis and purulent inflammation to extensive, shallow hemorrhagic ulcerations covered by a diphtheritic membrane. Dyspnea, salivation, and a deep bronchial cough may also occur. Conjunctivitis is another common clinical manifestation of IBR in cattle. Viral infection damages the respiratory mucosal epithelium, predisposing affected cattle to secondary bacterial infections, including those caused by Mannheimia haemolytica and Pasteurella multocida. These secondary infections may contribute to the development of the bovine respiratory disease complex (BRDC) [1,3].

BoHV-1 infection occurs worldwide in both beef and dairy cattle populations, although its prevalence varies across geographical regions. In Ireland, a prevalence of 21.43% was reported among cattle younger than 30 months [4]. In Colombia and Brazil, reported prevalence estimates were 44.9% and 43.1%, respectively [5,6]. In Iran, the reported prevalence was 50% [7], whereas a higher prevalence of 72% was reported in Ethiopia [8]. In Thailand, data on the individual animal-level seroprevalence of BoHV-1 remain limited. A previous study reported a farm-level seroprevalence of 67% in northern and northeastern Thailand [9].

The epidemiology of bovine herpesvirus 1 (BoHV-1) infection in dairy cattle is influenced by a combination of herd characteristics, animal movements, and farm management practices that affect pathogen introduction and transmission [10]. The establishment of lifelong latent infection in recovered cattle represents a major challenge for disease control, as latently infected animals may reactivate viral replication and shed infectious virus under certain conditions. Consequently, the introduction of infected cattle, particularly animals with an unknown infection status, may facilitate the introduction and subsequent circulation of BoHV-1 within susceptible herds [11]. Herd size, cattle purchasing practices, replacement animal management, and contact with cattle from other herds have therefore been considered important factors in the epidemiology of BoHV-1. In addition, differences in biosecurity implementation, herd health management, and the intensity of animal movement may contribute to variation in infection risk between dairy farms [4,10]. Farm-level management systems may also influence the likelihood of BoHV-1 exposure. Practices involving the isolation of newly introduced cattle, control of animal movements, routine health monitoring, and veterinary supervision may reduce opportunities for pathogen introduction and transmission [10]. Conversely, inadequate biosecurity and frequent introduction of cattle from herds with unknown health status may increase the potential for infection. Vaccination history should also be considered when interpreting BoHV-1 serological results, because conventional serological tests may not distinguish antibodies induced by natural infection from those induced by vaccination [12].

This study aimed to assess the epidemiological prevalence of BoHV-1 infection in dairy farms across western Thailand and identify associated risk factors of infection.

2. Materials and Methods

2.1. Sample Size Calculation and Sample Collection

According to the Department of Livestock Development [13], the dairy cattle population in western Thailand was 117,373. The required sample size was calculated using ProMESA software (version 2.3.0.2), assuming a simple random sampling design. An expected prevalence of 67%, based on a previous study by Kampa et al. [9], was used for the calculation. The sample size was determined using a 95% confidence level and an acceptable relative error of 0.1, according to the following formula:

n = p×(1-p) ×z² /e²

where p stands for assumed prevalence of the event in the population under study, z is the critical value obtained from a standard normal distribution, e is the maximum acceptable absolute error and n stands for sample size. The minimal sample size was 256 samples.

A total of 290 blood samples were collected from the coccygeal vessels of randomly selected dairy cattle on 22 farms between June and August 2025. To minimize the potential influence of maternally derived antibodies on serological results, only cattle older than 6 months of age were included in the study [14]. The study was conducted on selected dairy farms in three provinces in western Thailand: Kanchanaburi, Nakhon Pathom and Ratchburi (Figure 1).

2.2. Data Collection

Data collected through farmer interviews were analyzed to identify potential risk factors associated with bovine herpesvirus 1 (BoHV-1) seropositivity. Variables were evaluated at both the individual-animal and farm levels. Individual-level variables included cattle production status (heifer or cow), age category, breed, and history of respiratory problems. Farm-level variables included farm size, introduction of new animals, province, housing type (tie-stall or free-stall), and farm certification status (certified or non-certified).

2.3. Serological Laboratory Analysis

Blood samples were centrifuged at 3,000 × g for 10 min, and the resulting serum was collected and stored at −20 °C until analysis. Serum samples were tested for antibodies against bovine herpesvirus 1 (BoHV-1) using a commercial enzyme-linked immunosorbent assay (ELISA; IDEXX IBR Pool Ab Test, IDEXX Laboratories, Inc., USA), according to the manufacturer's instructions. The test had a reported sensitivity of 90.70% and specificity of 99.90%.

2.4. Statistical Analysis

The seroprevalence of BoHV-1 was estimated using ProMESA software (version 2.3.0.2). Apparent prevalence (Pa) was adjusted for the diagnostic test's sensitivity (Se) and specificity (Sp) to estimate true prevalence (TP) using the following formula.

TP = (P_a + Sp – 1)/ (Se + Sp – 1)

A farm was classified as seropositive if at least one animal from that farm tested positive. Univariate binary logistic regression analyses were performed to evaluate the associations between the investigated variables and BoHV-1 seropositivity at both the individual-animal and farm levels. Variables with p values ≤ 0.20 in the univariate analyses were considered for inclusion in the multivariable logistic regression models. Associations were expressed as odds ratios (OR) with 95% confidence intervals (CI) for both univariate and multivariable analyses.

3. Results

A total of 290 serum samples from dairy cattle were included in this study. Of these, 53 samples tested positive for antibodies against BoHV-1. The overall apparent prevalence of BoHV-1 infection in dairy cattle in western Thailand was 18.28% (95% CI: 13.83-22.72%). After adjustment for the diagnostic sensitivity and specificity of the test, the estimated true prevalence was 20.07%. At the farm level, 15 of the 22 farms tested positive, representing the farm prevalence of BoHV-1 infection of 68.18% (95% CI: 48.77-87.59%).

The results of the univariate analysis of the associations between BoHV-1 serostatus and potential risk factors at the individual-animal level are presented in Table 1, while the corresponding results at the farm level are presented in Table 2. In the univariate analysis, cattle status, breed, and age were not significantly associated with BoHV-1 seropositivity. Heifers showed lower odds of seropositivity than cows, although the association did not reach statistical significance. Similarly, mixed-breed cattle had slightly higher odds of seropositivity than Holstein Friesian cattle. Age was also not significantly associated with seropositivity, with wide confidence intervals for both the 2–4-year and >4-year age groups. In contrast, a significant association was observed between respiratory problem history and BoHV-1 seropositivity. Cattle without a history of respiratory problems had significantly higher odds of being seropositive than those with a history of respiratory problems.

At the farm level (Table 2), none of the evaluated factors showed a statistically significant association with BoHV-1 seropositivity. Seropositivity varied among provinces, ranging from 57.14% in Kanchanaburi to 77.78% in Ratchaburi, but the differences were not statistically significant. Medium-sized farms showed a higher seropositivity than small and large farms, although no significant association with farm size was observed. Farms that introduced new animals had higher seropositivity than farms without animal introduction, but this difference was not statistically significant. Farm certification showed a borderline association with BoHV-1 seropositivity, with certified farms tending to have lower seropositivity than non-certified farms and substantially lower odds of seropositivity. Farm type was also not significantly associated with seropositivity, although tie-stall farms showed lower odds than free-stall farms.

Firth logistic regression was performed to evaluate the independent associations between individual-level variables and BoHV-1 seropositivity (Table 3). The overall model was statistically significant (likelihood ratio test = 17.49, df = 4, p = 0.0015). Cattle status and age range were not significantly associated with BoHV-1 seropositivity after adjustment. Compared with cows, heifers had lower adjusted odds of seropositivity. Compared with cattle younger than 2 years, neither the 2–4-year group nor the >4-year group showed a significant association. In contrast, respiratory problem history remained significantly associated with BoHV-1 seropositivity. Cattle with a history of respiratory problems had approximately threefold higher adjusted odds of seropositivity than those without such a history (adjusted OR = 2.937, 95% CI: 1.584–5.458; p = 0.001)

4. Discussion

The present study revealed an estimated true individual-level seroprevalence of 20.07% and a farm-level seroprevalence of 68.18% for BoHV-1 among dairy cattle in western Thailand. The individual-level seroprevalence was comparable to that reported in Ireland (21.43%) [4] but lower than the estimates reported in Colombia (44.9%) [5], Brazil (43.1%) [6], and Iran (50.0%) [7]. Differences in seroprevalence among these studies may reflect variations in cattle management practices, stocking density, biosecurity measures, animal movement controls, and vaccination strategies across regions. However, direct comparisons should be interpreted cautiously because differences in study populations, sampling designs, diagnostic methods, and approaches to prevalence estimation may also contribute to the observed variation. At the farm-level, the prevalence observed in this study was 68.18% which was like the study in Iran (65.0%) [7] but lower than that reported in Brazil (89.5%) [6]. The high farm prevalence reported in this study indicates that BoHV-1 infection is widely distributed among dairy farms in western Thailand. Historically, a 2004 study in Thailand detected BoHV-1 antibodies in 67% of bulk tank milk samples [9]. Although bulk tank milk testing reflected farm-level exposure rather than individual animal serostatus, these findings confirm long-standing endemicity in Thai dairy populations. In the present study, the relatively moderate individual-level seroprevalence contrasted with the substantially higher farm-level seroprevalence, indicating that BoHV-1 exposure was widespread among dairy farms in the study area. This pattern may reflect the presence of seropositive animals across multiple farms, although the cross-sectional study design did not establish when or how the virus was introduced or transmitted within individual farms.

In the present study, cattle with a history of respiratory disease demonstrated approximately three times higher odds of being seropositive for BoHV-1 compared to those without a prior history of respiratory illness. This strong association is biologically plausible given the pivotal role of BoHV-1 as a primary viral pathogen in the BRDC. Infection with BoHV-1 causes IBR, an acute disease of the upper respiratory tract characterized by fever, mucopurulent nasal discharge, conjunctivitis, tracheal lesions, and severe respiratory distress, leading to significant economic losses and reduced farm productivity [11]. Beyond primary pathogenesis, BoHV-1 contributes to respiratory disease through profound immunosuppression. Primary viral replication causes mucosal damage and necrosis within the respiratory epithelium, impairing mucociliary clearance and compromising host innate defense mechanisms [15]. Additionally, BoHV-1 targets leukocytes and alters macrophage function, creating a opportunistic window for secondary bacterial invaders—such as Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis—which exacerbate severe fibrinous pleuropneumonia [15]. Furthermore, the lifelong viral persistence characteristic of alphaherpesviruses must be considered. Following primary infection, BoHV-1 establishes latency in the trigeminal ganglia. Stress factors—such as transportation, overcrowding, co-infections, or nutritional changes—can trigger viral reactivation and shedding, potentially causing recurrent clinical signs or secondary transmission events within the farm [3].

The statistical analysis revealed no significant associations between BoHV-1 seropositivity and either cattle production status (heifer or cow) or age category. These findings suggested that, within the study population, neither variable was demonstrably associated with differences in the odds of BoHV-1 seropositivity. One possible explanation is that exposure to BoHV-1 may occur across different age groups and production categories within affected farms, resulting in relatively similar seropositivity patterns among these groups [16]. Because BoHV-1 establishes lifelong latency following primary infection, seropositive animals may remain antibody-positive over time, potentially contributing to the accumulation of seropositive animals across age categories [11]. Consequently, differences in age or production status may not necessarily translate into detectable differences in seroprevalence, particularly when exposure has occurred throughout the farm. Furthermore, the absence of a significant association with age does not necessarily indicate that age has no influence on the epidemiology of BoHV-1. The likelihood of exposure may be influenced by farm management practices, animal contact patterns, introduction of replacement animals, and previous exposure to the virus. These factors may affect cattle across different age groups and potentially obscure age-related differences in seropositivity [10]. Similarly, the lack of a significant association with production status may reflect overlapping exposure histories between heifers and cows or similarities in their management and housing conditions. However, these explanations remained hypothetical because the present study did not directly assess the timing of infection, cumulative exposure, or age-specific transmission patterns.

The wide confidence intervals observed for some age categories indicate limited precision in the estimated associations, potentially reflecting small sample sizes within certain groups and the resulting uncertainty in the odds ratio estimates. The distribution of seropositive and seronegative animals across categories may also have influenced the statistical power to detect differences. Therefore, the non-significant findings should be interpreted cautiously and should not be taken as evidence that age and production status are epidemiologically irrelevant [17]. Further studies involving larger sample sizes, more balanced age-group distributions, and longitudinal monitoring would help clarify whether these factors influence the risk of BoHV-1 exposure in Thai dairy cattle.

The lower likelihood of BoHV-1 seropositivity among dairy farms certified under Good Agricultural Practices (GAP) suggests that adherence to standardized farm management practices may contribute to reducing the risk of BoHV-1 exposure. GAP certification provides a framework for promoting appropriate animal health management, farm hygiene, biosecurity, and systematic herd management [10]. These practices may help reduce opportunities for pathogen introduction and transmission, particularly through the movement of cattle between herds and the introduction of animals with unknown infection status. This is especially relevant to BoHV-1 epidemiology because infected cattle can maintain lifelong latent infections and may subsequently reactivate the virus, potentially serving as sources of infection for susceptible animals [2]. Accordingly, the association observed in this study may reflect the combined benefits of management practices that limit the introduction and spread of infectious agents. Nevertheless, GAP certification should be interpreted as a potential indicator of farm management and biosecurity rather than a direct protective mechanism against BoHV-1 infection. Because the present study was observational, the association does not establish causality, and residual confounding by herd characteristics, cattle purchasing practices, vaccination history, or other management factors cannot be excluded. Further studies incorporating detailed assessments of GAP compliance and longitudinal monitoring of BoHV-1 seroconversion are warranted to identify the specific management components associated with reduced infection risk.

The multivariable Firth logistic regression model was statistically significant, demonstrating strong overall goodness-of-fit for identifying independent factors associated with BoHV-1 seropositivity. The Firth’s method was specifically chosen to mitigate potential bias arising from small sample sizes or sparse data within sub-categories, thereby providing robust adjusted odds ratio estimates [18]. A history of respiratory problems emerged as the strongest independent predictor of BoHV-1 exposure. This finding aligns with the central role of BoHV-1 as a primary pathogen in the BRDC, where upper respiratory mucosal damage facilitates localized inflammation and secondary respiratory infections [3]. Furthermore, the capacity of BoHV-1 to establish life-long latency in the trigeminal ganglion, periodic reactivation triggered by production stress, parturition, or transport, explains the sustained antibody responses and persistent circulation observed in animals with a history of respiratory disease [2]. Alternatively, as noted with the wide confidence intervals, reduced statistical power within specific age categories may have obscured subtle age-related variations. Consequently, these findings highlight clinical respiratory history as a far more reliable indicator of BoHV-1 exposure than age or production class alone.

5. Conclusions

BoHV-1 infection remains endemic within dairy cattle in western Thailand, characterized by a moderate individual true seroprevalence of 20.07% alongside a high farm-level prevalence of 68.18%. This pattern indicated extensive circulation of the virus among dairy farms, even while within-farm prevalence remains limited. Multivariable Firth logistic regression identified a history of respiratory problems as the primary independent risk factor for seropositivity, underscoring the virus's central role in the Bovine Respiratory Disease complex and its propensity for lifelong latency. Conversely, age and production status were not significantly associated with seropositivity, likely reflecting shared housing, common management practices, and equalized exposure risks across age classes within farms. Overall, these findings emphasize the crucial role of clinical respiratory monitoring, early case isolation, and strict farm-level biosecurity measures over demographic targeting for the effective control and surveillance of BoHV-1 infection.