This study was reviewed and approved by the Research Ethics Committee of the National Research and Innovation Agency (BRIN), Indonesia, under Animal Ethical Approval No. 071/KE.02/SK/04/2023. All procedures involving animals were conducted in accordance with institutional guidelines for the ethical use and care of animals in research, as well as internationally accepted standards for animal welfare and biosafety.

Prior to field activities, verbal informed consent was obtained from all participating farmers after explaining the objectives of the study, the sampling procedures, and the intended use of the collected data. Participation was entirely voluntary, and confidentiality of farm-related information was maintained throughout the study.

Blood collection was performed aseptically by trained personnel using standard veterinary procedures to minimize pain, stress, and handling time. Approximately 1.5 mL of blood was collected from the wing vein of each bird using sterile syringes and needles. Birds were gently restrained during sampling, and all efforts were made to ensure humane handling and reduce distress. No experimental infection, invasive surgical procedure, or euthanasia was performed as part of this study.

Necropsy and histopathological examinations were conducted only on naturally diseased or deceased birds identified during field investigations. Tissue collection and laboratory procedures were carried out following established biosafety and biosecurity protocols to prevent environmental contamination and pathogen dissemination.

The study complied with national regulations governing animal experimentation and field epidemiological investigations in Indonesia and adhered to One Health principles by integrating animal health, environmental considerations, and public health perspectives during surveillance activities in the transboundary region of West Kalimantan.

The study was conducted between 5 and 28 May 2023 in West Kalimantan Province, Indonesia, a transboundary region bordering Sarawak, Malaysia. Sampling was carried out across three districts, namely Sambas, Bengkayang, and Sanggau, selected due to their proximity to the international border and their potential role in cross-border poultry movement and shared ecological interfaces. Figure 1 illustrates the geographical distribution of the sampling locations.

A cross-sectional study design was employed using purposive sampling. Flocks were selected based on proximity to border areas, free-range management systems, flock size (>20 birds), and farmers’ willingness to participate, targeting populations at higher risk of exposure to transboundary diseases.

A total of 245 waterfowl blood samples were initially collected from 26 smallholder flocks between 5 and 28 May 2023. Approximately 1.5 mL of blood was collected aseptically from the wing vein of each bird. Of these, 211 serum samples (52 ducks and 159 Muscovy ducks) with sufficient volume for all assays were included in the final analysis. Due to extensive management practices and field constraints, the number of birds sampled per flock varied. Consequently, analyses were conducted at the individual bird level without formal adjustment for intra-flock correlation. The study population consisted of apparently healthy ducks and Muscovy ducks raised under free-range systems. Vaccination history was assessed through structured farmer interviews, and all flocks were reported as unvaccinated at the time of sampling.

Clinical observations and gross pathological examinations were conducted during field sampling to assess the presence of disease in the waterfowl population. During sample collection, all waterfowl were clinically examined for signs of DVH, AI, and ND. Observations focused on identifying typical clinical signs of these diseases, including neurological symptoms, respiratory distress, diarrhea, weakness, and other abnormalities. Birds exhibiting morbidity or mortality underwent further pathological assessment.

Necropsy and histopathological examinations were conducted on a subset of clinically affected birds from selected flocks (notably Farms A and B) where mortality or suggestive lesions were observed. Gross pathological examinations were performed to assess lesions, with particular attention to the liver, air sacs, lungs, intestines, and immune organs such as the bursa of Fabricius.

Tissue samples from affected birds were collected from Farms A and B, located in Sambas and Sanggau, respectively, for histopathological analysis. Samples were fixed in 10% neutral buffered formalin, processed using standard paraffin embedding procedures, and stained with hematoxylin and eosin. Histopathological examination focused on detecting lesions consistent with viral infections, including inflammation, necrosis, hemorrhage, and cellular infiltration in relevant organs.

Cloacal swab samples from Farm A that showed diarrhea were collected for bacteriological examination using standard culture methods and the Analytical Profile Index 20E system (bioMérieux, Marcy-L’Etoile, France) to detect Salmonella spp.

Enzyme-linked immunosorbent assay for DVH: A commercial Duck Viral Hepatitis antibody ELISA kit (Catalog No. SL0018Du; SunLong Biotech Co., Ltd., Hangzhou, China) was used to qualitatively assess antibodies against DVH in duck and Muscovy duck serum. The ELISA was performed according to the manufacturer’s instructions.

The microplate wells were pre-coated with DVH-specific antigen to form a solid-phase antigen. Test samples were added to the wells, allowing binding between the antigen and the antibodies present in the samples. Subsequently, horseradish peroxidase-conjugated antigen was added to facilitate antigen–antibody complex formation. After incubation, wells were washed to remove unbound reagents. Tetramethylbenzidine substrate was then added, producing a color change from blue to yellow upon addition of the stop solution. Optical density was measured at a wavelength of 450 nm using a spectrophotometer. Results were interpreted qualitatively based on the kit cutoff value. According to the manufacturer’s documentation, diagnostic sensitivity and specificity were 95% and 98%, respectively.

Serological testing for AI and ND: Serological testing was conducted using hemagglutination (HA) and hemagglutination inhibition (HI) methods, following the guidelines of the World Organization for Animal Health [16, 17].

A V-shaped microplate and micro-technique were used to conduct the HA test. The AI antigen (H5N1 subtype clade 2.3.2) and ND antigen (La Sota strain) were obtained from Pusat Veteriner Farma (Pusvetma, Surabaya, Indonesia). Each well was filled with 25 µL of phosphate-buffered saline. An additional 25 µL of antigen was added to the first well and serially diluted twofold across subsequent wells (1/2 to 1/1024). The 12th well served as the red blood cell control. After dilution, phosphate-buffered saline and 1% chicken red blood cells were added, and the mixture was incubated at 26°C (room temperature) for 30 min. The antigen titer was determined, and a 4 HA unit antigen was prepared for the HI assay.

The HI assay was performed using a similar micro-technique. A standard anti-H5N1 antibody (Pusvetma) was used as the positive control. Test samples were serially diluted as in the HA test. In each well, 4 HA units of antigen were added, followed by incubation. Subsequently, 1% chicken red blood cells were added, and the mixture was incubated further. The inhibition of hemagglutination indicated the presence of antibodies against AI or ND, confirming viral exposure [16, 17].

Seropositivity was defined as an HI titer ≥ 1:16 (4 log₂), in accordance with World Organization for Animal Health recommendations. Geometric mean titers were calculated for seropositive samples where applicable.

Data were recorded using Microsoft Excel 365 (Microsoft Corporation, Redmond, WA, USA). Herd-level and animal-level prevalence were calculated using the following formula [18]:

Prevalence (%) = (Number of birds that tested positive / Total number of birds examined) × 100

The Chi-square (χ²) test was used to examine differences between species, location, and age groups. The strength of association between potential risk factors and seroprevalence was estimated using odds ratios with 95% confidence intervals [19], calculated using univariate logistic regression with the lowest-risk category as the reference group (Sambas for district, duck for species, and birds aged < 3 months for age).

For variables with sparse data, descriptive comparisons were prioritized to avoid unstable estimates. Statistical significance was set at p < 0.05. Descriptive statistics were used to summarize qualitative and quantitative variables. Due to uneven sampling across flocks, adjustment for intra-flock correlation was not performed, and analyses were conducted at the individual level.

No clinical signs suggestive of DVH were observed in Farm B. However, clinical signs, gross lesions, and histopathological findings were detected in both Muscovy ducks from Farm A and ducks from Farm B in Sambas and Sanggau districts (Table 1).

In Farm A, 10 Muscovy ducks showed weakness, lethargy, and white diarrhea, with four deaths recorded. In contrast, birds in Farm B were emaciated, exhibited feather loss, and showed a high prevalence of ectoparasites (> 90%), accompanied by fever and respiratory distress. Gross examination revealed hemorrhagic intestines in Farm A, whereas Farm B showed cloudy air sacs, hyperemic lungs and intestines, and enlargement of the bursa of Fabricius.

Histological findings in Farm A included hemorrhagic pneumonia, lymphoid depletion of the bursa of Fabricius, and intestinal hemorrhage. In Farm B, lesions included pulmonary and intestinal hemorrhage, hepatic necrosis, cerebral and cerebellar necrosis, and bursal necrosis. These lesions are consistent with systemic viral infections commonly associated with ND or AI rather than DVH.

Representative lesions are illustrated in Figure 2. The lesion patterns were compatible with viral etiologies, with findings in Farm B suggestive of ND virus infection. In contrast, hemorrhagic changes in Farm A, accompanied by the detection of Salmonella spp., may indicate secondary bacterial involvement.

A total of 211 waterfowl, comprising ducks and Muscovy ducks, were tested for the presence of antibodies against DVH, AI, and ND in the border areas of West Kalimantan, Indonesia.

Table 2 shows that none of the ducks or Muscovy ducks in the three study districts had detectable DVH antibodies at any age. No seropositive cases of DVH antibodies were detected in the sampled waterfowl population (Table 2). All 211 samples tested negative, resulting in an overall seroprevalence of 0.0%. As no positive cases were identified, inferential statistical analysis was not applicable.

Serological evidence of ND exposure was detected in 28 out of 211 samples, yielding an overall seroprevalence of 13.27% (Table 3). Seroprevalence of ND varied slightly across districts, ranging from 11.29% in Bengkayang to 14.29% in Sanggau. Using Sambas as the reference district, no significant differences were observed among districts (p = 0.860). Muscovy ducks showed a slightly lower seroprevalence (12.58%) compared to ducks (15.38%); however, this difference was not statistically significant (p = 0.780).

Using birds aged < 3 months as the reference group, those aged 3–6 months (OR = 1.33, 95% CI: 0.78–2.27) and > 6 months (OR = 2.16, 95% CI: 1.29–3.62) showed higher odds of ND seropositivity. Although the overall age effect was not statistically significant (p = 0.281), birds aged > 6 months had numerically higher odds of seropositivity than the reference group.

AI antibodies were detected in 12 of 211 samples, yielding an overall seroprevalence of 5.69% (Table 4). Seropositivity differed significantly across districts (p = 0.014), with the highest proportion observed in Sanggau (12.50%), whereas no positive cases were detected in Bengkayang. Regarding species, Muscovy ducks exhibited a higher AI seroprevalence (6.92%) compared to ducks (1.92%); however, this difference was not statistically significant (p = 0.310).

Age-related differences were not statistically significant (p = 0.061), although descriptively higher seroprevalence was observed in birds aged < 3 months (9.43%) compared with older age groups. Given the relatively small number of AI-seropositive cases (n = 12), inferential comparisons should be interpreted with caution.

This study investigated the seroprevalence of DVH, AI, and ND in waterfowl (ducks and Muscovy ducks) inhabiting the border region between West Kalimantan, Indonesia, and Malaysia. The sero-epidemiology of infectious diseases in waterfowl, such as DVH, AI, and ND, is critical for understanding disease dynamics and potential threats posed by these pathogens. The findings provide valuable epidemiological insights into the presence and distribution of these economically significant avian diseases in a transboundary context.

The clinicopathological findings, together with bacteriological examination of cloacal swab samples, indicated the presence of Salmonella spp. in Farm A. Histopathological examination of the liver revealed multifocal-to-diffuse hepatic necrosis accompanied by degenerative changes and hemorrhages within the hepatic parenchyma. Similar hepatic lesions have been reported in ducks with Salmonella infections, where systemic bacterial dissemination leads to hepatic necrosis and tissue damage [20]. The occurrence of such bacterial infections may also reflect suboptimal farm management and inadequate biosecurity practices that facilitate the introduction and spread of pathogenic bacteria in duck production systems.

Non-suppurative proventriculitis and interstitial hemorrhagic pneumonia observed in Farm B were consistent with NDV infection. Viral antigen localization in bronchiolar and alveolar epithelial cells has been demonstrated using immunohistochemical techniques [21]. Highly pathogenic AI is considered an important differential diagnosis of NDV infection due to similarities in clinicopathological findings and tissue tropism. Hemorrhages observed in visceral organs, including the lungs, liver, proventriculus, and intestines, are consistent with previous findings in Peking ducks experimentally infected with highly pathogenic AI in Vietnam [22]. The major differential diagnoses for ND include highly pathogenic AI, infectious bronchitis, infectious laryngotracheitis, and the diphtheritic form of fowl pox. Accurate differentiation among these diseases is essential for establishing a tentative diagnosis [23].

The absence of detectable antibodies to DVH in waterfowl across all age groups and districts in West Kalimantan suggests limited natural exposure to DHAV or the absence of vaccination programs in the study area. Vaccination status was determined through farmer interviews and may be subject to recall bias, which should be considered when interpreting serological findings. In younger birds, maternally derived antibodies may also influence serological interpretation, although their impact under field conditions remains uncertain.

The lack of serological evidence aligns with limited documentation of DHAV circulation in Indonesia, particularly in Borneo. Although DHAV is widely reported in parts of East and Southeast Asia [2], data from the Indonesia–Malaysia border region remain scarce. Regional variations in management practices, biosecurity levels, and poultry movement patterns may contribute to differences in disease occurrence, especially considering the proximity to Sarawak.

Previous studies have demonstrated that DHAV primarily affects ducklings under three weeks of age, often causing acute disease and high mortality rates [24]. The absence of antibodies across age groups highlights the vulnerability of naïve populations, which may face severe outbreaks if the virus is introduced. This finding emphasizes the importance of strengthening biosecurity measures to prevent potential DHAV incursions.

Duck diseases are often multifactorial, and co-infections are common. Liu et al. [25] reported co-infection of DHAV with Muscovy duck parvovirus, AI virus, Escherichia coli, Salmonella, and other pathogens. The absence of DVH detection in this study should be interpreted with caution, as it does not exclude low-level or undetected viral circulation. Detection may also be influenced by sample size, sampling timing, and diagnostic sensitivity [26, 2, 24].

The overall seroprevalence of ND in waterfowl (13.27%, 95% CI: 9.34–18.51) indicates low-level exposure or sporadic circulation of NDV in West Kalimantan. Such circulation suggests that waterfowl may contribute to the epidemiological cycle of the virus, potentially facilitating spillover into domestic poultry [27].

The absence of statistically significant differences in seroprevalence among districts (p > 0.05) indicates relatively uniform exposure risk, possibly due to similar farming practices, bird movement patterns, and environmental conditions. Ducks exhibited a slightly higher seroprevalence (15.38%) compared to Muscovy ducks (12.58%), although this difference was not statistically significant.

In univariate logistic regression, Muscovy ducks had an odds ratio of 1.26 (95% CI: 0.71–2.25), indicating comparable exposure between species. These findings are consistent with reports describing natural NDV infection across multiple waterfowl species. Experimental evidence has demonstrated that Muscovy ducks infected with velogenic NDV (genotype VII) can act as carriers and transmit the virus to in-contact chickens [28].

Age-based analysis showed increasing ND seroprevalence with age, ranging from 10.38% in birds < 3 months to 20.00% in birds > 6 months. Although not statistically significant (p > 0.05), this pattern likely reflects cumulative exposure over time. Similar trends have been reported in endemic regions, where sustained infection pressure results in continuous exposure [29]. Longitudinal studies have also shown persistent circulation of lentogenic avian paramyxovirus-1 strains in waterfowl populations [30]. Furthermore, waterfowl can shed NDV without clinical signs, contributing to silent maintenance of the virus in mixed-species systems [31].

The overall AI seroprevalence (5.69%) indicates limited but detectable exposure in the study population. In contrast to ND, significant differences were observed among districts (p < 0.05), with the highest seropositivity in Sanggau (12.50%).

This spatial heterogeneity suggests localized transmission dynamics influenced by ecological factors, poultry trade, and interactions between domestic and wild birds. The higher seroprevalence in Sanggau may be associated with increased interaction between backyard poultry and wetland environments. Similar observations have been reported in Indonesia, where proximity to wetlands facilitates the introduction of viruses via migratory birds [15].

Comparable ecological interfaces have been described in Bangladesh, where wetlands and backyard duck systems support virus circulation at the domestic–wild interface [32]. Although Muscovy ducks exhibited a higher seroprevalence (6.92%) than ducks (1.92%), the difference was not statistically significant and should be interpreted with caution.

Field evidence suggests that Muscovy ducks can be exposed to AI viruses and seroconvert without clinical disease. Studies in Indonesia have reported the detection of non-H5 AI viruses in clinically healthy Muscovy ducks and seropositivity in birds near farms [33, 34]. These findings suggest that Muscovy ducks may contribute to the silent circulation of the AI virus. Waterfowl are recognized as natural reservoirs of influenza A viruses and play a key role in virus maintenance and transmission [35].

Age-stratified analysis showed the highest AI seroprevalence in birds aged < 3 months (9.43%), although the difference was not statistically significant (p = 0.061). This trend is consistent with previous studies indicating higher susceptibility and viral shedding in younger birds [14, 15].

The decline in seroprevalence with age may reflect survival bias or prior exposure leading to immunity. Experimental studies have demonstrated greater susceptibility and viral shedding in juvenile ducks, supporting their role as key amplifiers in transmission dynamics [36].

From a One Health perspective, the detection of AI antibodies in apparently healthy waterfowl indicates subclinical or past infection events that may pose risks to poultry and human health. Low-pathogenic AI is naturally maintained in waterfowl populations, and viral exchange between wild and domestic birds can lead to the emergence of highly pathogenic strains [37].

The proximity of West Kalimantan to Malaysia increases the risk of cross-border virus introduction, particularly given the active poultry trade and migratory bird pathways. Continued surveillance is essential to safeguard poultry production and mitigate zoonotic risk, as certain AI subtypes, including H5 and H9, have demonstrated zoonotic potential [38].

This study has several limitations. First, purposive sampling may introduce selection bias and limit generalizability. Second, clustering at the flock level was not accounted for in the statistical analyses, which may have underestimated the variance. Third, serological testing cannot distinguish between vaccination and natural exposure, and maternally derived antibodies may influence results.

Additionally, cross-reactivity in HI assays may affect antibody interpretation. Sampling was conducted during a single period (May 2023), limiting seasonal inference. The relatively small number of AI-positive cases reduces statistical power and may affect the stability of estimates.

Future studies should incorporate longitudinal designs, larger sample sizes, and molecular approaches to better understand transmission dynamics and the ecological role of waterfowl in maintaining viral pathogens in transboundary regions.