Molecular prevalence and phylogenetic characterization of Plasmodium juxtanucleare in Thai native chickens and fighting cocks across Kalasin Province, Northeast Thailand

Background and Aim: Avian malaria caused by Plasmodium spp. is an important vector-borne disease affecting poultry production in tropical and subtropical regions. Thai native chickens (Gallus gallus domesticus) and fighting cocks represent valuable genetic and economic resources in Thailand, yet information on the molecular epidemiology of avian malaria in northeastern Thailand remains limited. Kalasin Province contains diverse ecological environments with freshwater reservoirs, irrigated agricultural systems, and backyard poultry farming, which may facilitate transmission of haemosporidian parasites. Molecular tools targeting mitochondrial genes provide higher sensitivity than microscopic examination and allow accurate phylogenetic characterization. This study aimed to determine the molecular prevalence of Plasmodium spp., compare infection rates between free-range Thai native chickens and intensively managed fighting cocks, and analyze the phylogenetic relationships of circulating parasites using the mitochondrial cytochrome c oxidase subunit III (COXIII) gene. Materials and Methods: A cross-sectional survey was conducted from January to April 2025 using 181 blood samples collected from Thai native chickens (n = 112) and fighting cocks (n = 69) across 18 districts of Kalasin Province, Thailand. Genomic DNA was extracted using a commercial kit, and Plasmodium infection was detected by polymerase chain reaction targeting the mitochondrial COXIII gene. Positive amplicons were sequenced, and phylogenetic relationships were reconstructed using the Maximum Likelihood method. Differences in prevalence between host types and districts were evaluated using the Chi-square test, with p < 0.05 considered significant. Results: The overall molecular prevalence of Plasmodium spp. infection was 62.98% (114/181). Infection was higher in Thai native chickens (67.85%) than in fighting cocks (55.07%), but the difference was not statistically significant (p > 0.05). In contrast, prevalence varied significantly among districts (p < 0.05), ranging from 20% to 100%. Sequencing analysis revealed two haplotypes of Plasmodium. Phylogenetic analysis showed that all isolates clustered within Clade A and shared 99%–100% nucleotide identity with Plasmodium juxtanucleare. Haplotype I was dominant and detected in multiple chicken breeds, whereas Haplotype II formed a minor lineage closely related to P. relictum. Conclusion: This study confirms the hyper-endemic circulation of P. juxtanucleare in Kalasin Province and demonstrates genetic diversity within the local parasite population based on COXIII gene analysis. Similar infection rates in free-range and intensive systems indicate that environmental vector exposure plays a major role in transmission. These findings highlight the importance of molecular surveillance and vector control strategies and provide baseline data for future One Health studies on avian haemosporidian infections in Thailand. Keywords: avian malaria, COXIII gene, molecular prevalence, phylogenetic analysis, Plasmodium juxtanucleare, poultry parasite, Thailand, Thai native chicken.