Abstract
Foot-and-mouth disease (FMD) is a highly contagious transboundary animal disease causing major economic losses worldwide. This study aimed to establish baseline serological data for FMD in cattle from an FMD-free region of Indonesia using a non-structural protein (NSP) antibody lateral flow assay and to evaluate diagnostic interpretation in a low-prevalence setting. A total of 218 serum samples were collected in East Nusa Tenggara Province. Six samples (2.75%) tested positive, 211 (96.79%) were negative, and one was invalid. The apparent prevalence was 2.75% (95% CI: 1.01–5.90%). Adjusted true prevalence using the Rogan–Gladen estimator was 0%, indicating no detectable infection within the study population rather than definitive absence of infection. Considering assay specificity (96.46%), the observed positives are consistent with expected false-positive results. No clinical signs were observed. These findings suggest no evidence of active FMD circulation within the limits of the study and highlight the importance of cautious interpretation of serological data in low-prevalence settings.
Keywords: cattle, foot-and-mouth disease, Indonesia, non-structural protein antibody, surveillance
INTRODUCTION
Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals and is recognized as one of the most economically devastating livestock diseases worldwide [7, 8, 13, 14]. The disease has major implications for livestock productivity, food security, and international trade.
The recent re-emergence of FMD in Indonesia has posed significant challenges to livestock production systems and disease control programs [4, 15, 17]. Despite widespread outbreaks in several regions, East Nusa Tenggara Province remains one of the few areas maintaining FMD-free status.
Serological assays targeting non-structural proteins (NSP) are widely used in surveillance programs because they allow differentiation between infected and vaccinated animals [1, 6, 18]. However, interpretation of results becomes challenging in low-prevalence settings, where even highly specific tests may yield false positives, reducing positive predictive value [5, 9, 16].
Therefore, this study aimed to establish baseline serological data for FMD in cattle from an FMD-free region and evaluate diagnostic interpretation in a low-prevalence epidemiological context.
MATERIALS AND METHODS
Study design and sampling
A cross-sectional study was conducted from April to December 2024 in Kupang City and Regency, East Nusa Tenggara Province, Indonesia (Fig. 1). A total of 218 blood samples were collected from cattle using an opportunistic sampling approach without restriction on age, sex, or clinical condition.
Fig. 1.
Sampling location in Kupang City and Regency, Timor Island-East Nusa Tenggara Province.
Although probabilistic sampling is generally preferred, opportunistic sampling is widely accepted in surveillance studies where rapid baseline data are required. This approach is appropriate in FMD-free regions where the objective is early detection rather than precise prevalence estimation. Inclusion of animals from both slaughterhouse and village settings increased population heterogeneity and improved detection probability.
Serological testing
Serum samples were tested for antibodies against FMD virus NSP (3ABC protein) using a lateral flow immunochromatographic assay (Hangzhou Evegen Biotech Co., Ltd., Hangzhou, China). This method is widely used for rapid field diagnostics due to its simplicity and practicality [14, 19]. The assay sensitivity and specificity were reported as 96.00% and 96.46%, respectively.
Statistical analysis
Apparent prevalence (AP) was calculated as the proportion of positive samples. The 95% confidence interval (CI) was calculated using the exact binomial method. True prevalence (TP) was estimated using the Rogan–Gladen estimator:
| TP=(AP + Sp −1)/ (Se + Sp −1) |
Negative TP values were interpreted as zero.
Ethical approval
Ethical approval was obtained from the Faculty of Veterinary Medicine, Nusa Cendana University (No. 042/KEH/SKet/IX/2024).
Additional note
Confirmatory molecular testing was not performed, as no clinical suspicion of FMD was observed during sampling.
RESULTS
Out of 218 samples, 6 (2.75%) were positive, 211 (96.79%) negative, and 1 (0.46%) invalid (Table 1). The apparent prevalence was 2.75% (95% CI: 1.01–5.90%). Using test sensitivity (0.96) and specificity (0.9646), the estimated true prevalence was 0%, indicating no detectable infection within the study population rather than definitive absence of infection.
Table 1. Test results of serum sample collection by sandwich lateral flow immunochromatographic test method.
| Sampling location | Test results | Total | ||
|---|---|---|---|---|
| Positive | Negative | Invalid | ||
| Oesao Village | 0 | 23 | 1 | 24 samples |
| Pariti Village | 0 | 24 | 0 | 24 samples |
| ReknamoVillage | 0 | 24 | 0 | 24 samples |
| Batubao Village | 0 | 24 | 0 | 24 samples |
| Class 1 Tenau Kupang Quarantine | 0 | 70 | 0 | 70 samples |
| Bimoku slaughterhouse | 6 | 46 | 0 | 52 samples |
| Total | 6 | 211 | 1 | 218 samples |
DISCUSSION
This study provides baseline serological evidence supporting the absence of active FMD virus circulation in East Nusa Tenggara within the limitations of the study design and sampling framework. The observed seropositivity (2.75%) is consistent with the expected false-positive rate given the assay specificity.
In low-prevalence settings, even highly specific tests produce false positives, resulting in low positive predictive value [5, 9, 16]. The estimated true prevalence of zero further supports the absence of infection.
The absence of clinical signs in all sampled animals strengthens this conclusion. NSP antibody assays detect previous exposure rather than active infection and must be interpreted cautiously [12, 20].
The use of lateral flow assays reflects a practical approach for field surveillance in resource-limited and geographically dispersed regions. However, integration with confirmatory diagnostics such as RT-PCR is recommended[11].
Although no evidence of virus circulation was detected, the risk of FMD introduction remains due to animal movement and trade networks [2, 3, 10, 13]. Therefore, continuous surveillance and biosecurity measures are essential.
Overall, this study highlights the importance of epidemiological context in interpreting diagnostic results and provides valuable baseline data to support FMD-free status maintenance.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Acknowledgments
The authors acknowledge the support of the State Agricultural Polytechnic of Kupang and all individuals involved in sample collection.
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