The study protocol was reviewed and approved by the Institutional Ethical Committee of Kalasin University, Thailand (approval no. KSU-AE-029; approved on 05 August 2024). All procedures involving animals were conducted in accordance with institutional guidelines for animal care and use and complied with the national regulations for animal welfare in Thailand.

Blood samples were collected from Thai native chickens and fighting cocks using standard veterinary procedures designed to minimize pain, stress, and discomfort. No experimental infection, surgical intervention, or harmful manipulation of animals was performed during the study. All sampling procedures were carried out by trained personnel under appropriate handling conditions to ensure animal safety.

Permission for sample collection was obtained from farm owners before sampling, and informed verbal consent was secured for the use of samples for research purposes. The study involved only routine diagnostic sampling, and therefore, no animals were sacrificed specifically for this research.

All laboratory procedures were performed in accordance with biosafety guidelines for handling biological samples, and appropriate measures were taken to prevent contamination and ensure safe disposal of biological waste.

The study was conducted in compliance with internationally accepted principles for ethical use of animals in research and followed the recommendations for humane treatment of animals in veterinary and biomedical investigations.

A cross-sectional study was conducted from January to April 2025, a period marking the transition from the cool dry season to the hot season, in Kalasin Province, Thailand (Figure 1). Kalasin occupies an area of 6,947 km² and is geographically located at 16.635540°N, 103.772418°E. The study area is situated at an altitude of approximately 147 m above mean sea level, with an average temperature of 26.8°C and annual rainfall of 1,407 mm. Blood samples were collected from all 18 districts of the province, namely Kham Muang, Tha Khantho, Sam Chai, Somdet, Na Khu, Nong Kung Si, Sahatsakhan, Khao Wong, Huai Phung, Huai Mek, Na Mon, Kuchinarai, Muang Kalasin, Don Chan, Yang Talat, Rong Kham, Kamalasai, and Kong Chai. The identification procedures were performed at the Laboratory of the Department of Veterinary Technology, Faculty of Agricultural Technology, Kalasin University.

Samples from 181 Thai native chickens and fighting cocks were collected from all 18 districts. The sample size was determined using the following equation:

Sample size = (1.96² pq) / L²

Here, n = sample size, p = expected prevalence, q = 1 − p, and L = limit of error on the prevalence (0.05). Since the actual prevalence in the Thai native chicken population was unknown but expected to be high based on regional data, the calculation was performed using an assumed prevalence of 90% (p = 0.9, q = 0.1). Based on this calculation (Z = 1.96 for 95% confidence interval), the minimum required sample size was 139 chickens [19, 20].

Farms were selected using convenience sampling based on the availability of animals and the willingness of owners to participate. A total of 181 blood samples were collected from Thai native chickens (n = 112) and fighting cocks (n = 69) within the study area described above. Blood samples (approximately 0.1–0.5 mL) were collected from the wing vein into sterile tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant. Samples were placed in an ice box during transport to the Laboratory of the Department of Veterinary Technology, Faculty of Agricultural Technology, Kalasin University, and stored at −20°C before DNA extraction.

Genomic DNA was extracted from 200 µL of each blood sample using GF-1 Blood DNA Extraction Kit (Vivantis Technologies, Selangor, Malaysia) according to the manufacturer’s instructions. Genomic DNA was eluted in 50 µL of elution buffer. The concentration and purity of the extracted DNA were measured using a spectropho-tometer (NanoDrop, Thermo Scientific, Waltham, MA, USA). Only DNA samples with an A260/A280 ratio between 1.8 and 2.0 were used for further polymerase chain reaction analysis. Each extracted DNA sample was stored at −20°C until molecular identification of Plasmodium spp. was performed.

A fragment of 377 bp of the mitochondrial COXIII gene of Plasmodium spp. was amplified using two primers, PMF (5′-CCTCACGAGTCGATCAGG-3′) and PMR (5′-GGAAACCGGCGCTAC-3′) [21]. Although this primer set has been reported to detect other haemosporidians, the present study specifically focused on Plasmodium spp.

The PCR was performed in a final volume of 25 µL containing 1.5 mM MgSO₄, 0.2 mM dNTPs, 1× PCR buffer, 1 U of Taq polymerase (Vivantis Technologies), 0.2 µL of each primer (10 µM), and 2 µL of template DNA (10–50 ng). The amplification protocol consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 59°C for 90 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. All reactions included one negative control (ddH₂O) for each batch of samples. Amplification products (5 µL) were separated on 2% agarose gel stained with ViSafe nucleic acid stain (Vivantis Technologies) and visualized using Gel Doc™ XR+ imaging system with Image Lab™ software (Bio-Rad, Hercules, CA, USA) under ultraviolet light to confirm positive amplification (Figure 2).

To ensure the reliability of the PCR assay, strict quality control measures were applied. Genomic DNA extracted from a chicken previously confirmed to be infected with P. juxtanucleare by sequencing was used as the positive control, whereas nuclease-free water was used as the negative control in every run. DNA extraction, master mix preparation, and gel electrophoresis were performed in separate areas to prevent cross-contamination. Filtered pipette tips were used throughout the procedure. Samples showing unclear bands were tested again in duplicate to confirm the results.

Thirty-two PCR amplicons showing clear bands of the target gene (377 bp) were selected for nucleotide sequencing (Macrogen, Seoul, South Korea). Chromatograms were checked visually, and sequences were edited and aligned using BioEdit 7.2 [22] and MEGA X software [23]. Sequence identity was verified using the BLAST tool of the National Center for Biotechnology Information. All sequences were deposited in the GenBank database under accession numbers PQ783679–PQ783710.

Before phylogenetic analysis, all sequences were examined in BioEdit software, and low-quality nucleotide regions at the 5′ and 3′ ends were removed. Alignment was performed using the ClustalW algorithm, and gaps or missing data were treated using the complete deletion option. The final dataset contained a uniform sequence length of 377 bp for all isolates. The best-fit nucleotide substitution model was selected based on the lowest Bayesian Information Criterion value using MEGA-XII. Phylogenetic trees were constructed using the Maximum Likelihood method, and branch support was evaluated with 1,000 bootstrap replications. Branch lengths were expressed as the number of substitutions per site.

Statistical analysis was performed using SPSS version 25.0 (IBM Corp., NY, USA). The prevalence of Plasmodium spp. infection was expressed as a percentage. The Chi-square test (χ²) was used to compare infection rates between the two chicken management systems (Thai native chickens vs. fighting cocks) and among different districts. Differences were considered statistically significant when p < 0.05.

Molecular detection using PCR targeting the non-coding region of mtDNA revealed that 114 out of 181 samples were positive for Plasmodium spp., representing an overall prevalence of 62.98%. Regarding chicken management systems, the prevalence in free-range Thai native chickens was 67.85% (76/112), which was higher than in intensively managed fighting cocks (55.07%, 38/69). However, statistical analysis showed no significant difference between these two groups (χ² = 2.470, p = 0.116) (Table 1).

In contrast, the spatial distribution of infection among the 18 districts in Kalasin Province showed statistically significant variation (χ² = 40.416, p = 0.001) (Table 2). The highest prevalence was observed in Kham Muang District (100%), followed by Huai Mek (90%) and Nong Kung Si (85.71%). Conversely, the lowest prevalence rates were found in Na Mon and Kamalasai (20%). Detailed prevalence data for all districts are presented in Table 2.

Sequencing analysis of the 32 PCR products obtained from Kalasin Province revealed two distinct haplotypes of Plasmodium spp. circulating in the study area. BLASTn analysis indicated that the obtained sequences shared 99–100% nucleotide identity with reference Plasmodium sequences, predominantly P. juxtanucleare. The phylogenetic tree, constructed using the Maximum Likelihood method based on the mitochondrial COXIII gene, separated the analyzed sequences into three main clades (Clades A, B, and C). Notably, all isolates from Kalasin Province clustered within Clade A, grouping together with avian Plasmodium lineages, with Eimeria tenella (OP800498) used as the outgroup (Figure 3).

Haplotype I was the predominant lineage, identified in 29 samples. As detailed in Table 3, this haplotype exhibited a broad host range, being detected across all examined Thai native chicken breeds (Chee, Leung Hang Kaw, Dang, and local chickens) as well as in fighting cocks. Phylogenetically, Haplotype I formed a tight cluster with P. juxtanucleare (MG598392) from Brazil and P. elongatum (KY653802) from Colombia, supported by a strong bootstrap value of 94%. These results confirm that Haplotype I represents the pathogenic P. juxtanucleare lineage circulating in the study area.

In contrast, Haplotype II was detected in only three samples (local chickens and fighting cocks) (Table 3). Interestingly, this haplotype formed a distinct sub-branch within Clade A. Although still belonging to the broader avian Plasmodium group, it showed closer phylogenetic affinity to P. relictum (NC012426) and P. matutinum (MW168985) (bootstrap value = 93%) rather than clustering directly with the main P. juxtanucleare lineage. Clades B and C consisted of other reference Plasmodium species (e.g., P. gallinaceum, P. vivax) and did not contain any sequences from the present study.

In the present study, we examined the molecular prevalence and genetic diversity of Plasmodium spp. in Thai native chickens and fighting cocks in Kalasin Province. The overall prevalence detected by PCR was 62.98% (114/181), indicating a hyper-endemic status in this area. This finding is consistent with high infection rates reported across mainland Southeast Asia, including neighboring provinces in Thailand and Myanmar (86.8%–100%) [24, 25], backyard chickens (64.91%) [16], fighting cocks (88.76%) [13], chickens (85%) [26], and Burmese red junglefowls (70.59%) [9]. However, the prevalence observed in the present study is higher than that reported in insular regions such as Indonesia (30%) [27], wild raptors (9%) [28], and Thai native fowls (50%) [10].

Recently, P. knowlesi and P. juxtanucleare were detected in the salivary glands of Anopheles latens in southern Thailand [16]. In northern Thailand, a report described two cases in free-ranging wild birds and caged birds, showing that wild birds had a higher infection rate, possibly because of greater exposure to insect vectors [14]. In addition, P. juxtanucleare and P. gallinaceum were detected in biting midges (Diptera: Ceratopogonidae) in Thailand [3]. Variation in the prevalence of malaria infection among poultry species in Thailand may be due to differences in host susceptibility, presence of competent vectors, variation in vector species or strains, and differences in exposure to vectors [13]. Some studies have attempted to identify transmission-blocking activities against P. gallinaceum, showing that intramuscular injection of low doses of artesunate could block gametocyte production and transmission to the mosquito vector Aedes aegypti [29]. However, many farms in Thailand lack adequate vector control measures, do not prevent mosquito exposure, and may allow reservoir hosts to retain parasites in the blood for prolonged periods before clearance [13]. Rather than reflecting methodological differences alone, these regional variations likely indicate the influence of macro-ecological factors, such as the abundance of dipteran vectors in the Mekong region, on disease transmission intensity [30]. Consequently, because of their continuous exposure to insect vectors in open environments, Thai native chickens may serve as sentinel hosts for monitoring the circulation of haemosporidian parasites in tropical agroecosystems, reflecting real-time transmission risk at the poultry–vector–environment interface.

A critical aspect of this investigation was the direct molecular comparison between poultry types reared under different management systems. Unlike studies focusing on a single host type, this approach allows evaluation of the influence of husbandry practices on disease exposure. Although the difference was not statistically significant (p > 0.05), a higher prevalence was observed in free-range Thai native chickens (67.85%) compared with intensively managed fighting cocks (55.07%).

This trend may be explained by differences in vector exposure related to management practices. Fighting cocks are high-value animals and are usually raised with strict protective measures. They are often kept in cages covered with mosquito nets or cloth during the night, which reduces exposure to Culicoides and Anopheles vectors during peak feeding periods. In contrast, free-range native chickens frequently roost outdoors or in open shelters, increasing the likelihood of repeated vector bites. These findings support the hypothesis that physical barriers and management practices strongly influence infection dynamics, potentially more than intrinsic host immunity [13, 31].

Environmental heterogeneity also played a major role in disease distribution, as evidenced by statistically significant variation in prevalence across the 18 districts (p < 0.05), ranging from 20% to 100%. High prevalence areas such as Kham Muang (100%) are located near large freshwater reservoirs and irrigation systems associated with the Lam Pao Dam. This observation is consistent with previous studies showing that dam construction and irrigation systems can increase malaria risk by creating permanent breeding habitats for mosquito vectors such as Anopheles spp. [32].

These water resources, together with stagnant water, rice fields, and dense vegetation, provide suitable breeding conditions for Culicoides biting midges and Anopheles mosquitoes, which are the primary vectors of P. juxtanucleare and P. gallinaceum in Thailand [3, 33]. Although sampling in the present study was conducted during the dry season (January–April), the high prevalence observed in districts such as Kham Muang suggests that permanent water bodies associated with irrigation systems enable vector populations to persist year-round [29].

P. juxtanucleare is generally considered less pathogenic than P. gallinaceum and typically produces chronic infections rather than acute mortality [34]. Consistent with this, despite the high infection rate (62.98%), most PCR-positive chickens in the present study were asymptomatic or showed only mild clinical signs. This finding differs from previous reports describing morbidity in laying hens in southern Thailand [16] and clinical anemia in northern Thailand [14].

The absence of severe clinical signs may indicate endemic stability or long-term adaptation between native chicken breeds and circulating P. juxtanucleare strains. In such situations, native chickens may act as tolerant reservoir hosts, maintaining parasite transmission without developing severe disease, whereas naïve or exotic breeds may show higher mortality. These findings highlight the importance of PCR-based surveillance, because clinical observation alone cannot detect asymptomatic carriers and may underestimate the true prevalence of infection in endemic regions [7].

This study provides the first molecular characterization of P. juxtanucleare in Kalasin Province. Phylogenetic analysis based on the mitochondrial COXIII gene placed all 32 sequenced isolates within Clade A. Haplotype I (29 isolates) was the dominant lineage and clustered closely with P. juxtanucleare isolates from Brazil [35] and P. elongatum from Colombia [31], with strong bootstrap support (94%). The presence of only two haplotypes despite the high prevalence suggests stable endemic transmission rather than insufficient sampling.

The high nucleotide similarity between Thai isolates and South American strains also indicates strong evolutionary conservation of the mitochondrial COXIII gene. As shown in Table 3, Haplotype I was detected in all examined chicken breeds, suggesting that it represents a generalist lineage capable of infecting multiple hosts. Haplotype II (three isolates) formed a separate branch within Clade A and showed closer similarity to P. relictum (bootstrap support 93%). This limited distribution may indicate a local strain variant or an ancestral lineage, although further sampling is required. These findings highlight the complexity of parasite evolution and suggest possible lineage diversification within the P. juxtanucleare population in Thailand.

Several limitations should be considered when interpreting the results. First, the cross-sectional design provides only a single time-point assessment and does not allow evaluation of seasonal variation or incidence. Second, sampling was limited to the dry season (January–April), and prevalence may be higher during the rainy season when vector populations increase. Third, the study relied on a single-marker PCR assay based on the mitochondrial COXIII gene. Although sensitive for detection, this approach provides lower phylogenetic resolution than multi-locus analysis (e.g., combined with cyt b). Fourth, microscopic examination of blood smears was not performed, preventing estimation of parasitemia levels. Finally, entomological surveys and clinical performance data were not included. Future studies integrating vector ecology, host health, and economic impact within a One Health framework are recommended.