Microfilarial morphometry and molecular characterization of Setaria spp. from naturally infected cattle in Thailand

Filarioid nematodes cause reduced livestock productivity and economic losses worldwide. In cattle, differentiation of microfilariae based on morphology is challenging due to overlapping morphological characteristics. This study aimed to identify microfilariae in cattle blood and determine the prevalence and risk factors of filarial worm infection in Thailand using two microscopic diagnostic methods and molecular techniques. A cross-sectional study was conducted from June 2023 to June 2024 across 15 provinces in Thailand. Cattle blood samples (n = 419) were collected, and microfilariae were examined using thin blood smears, the acetone-modified Knott's technique, and conventional PCR targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, followed by sequencing and phylogenetic analysis. Parasitologically, microfilariae were detected in 10.28% of samples by the thin smear technique and 24.11% by Knott's technique, with morphometry confirming sheathed microfilariae of the genus Setaria. Molecular screening revealed a 17.9% PCR positivity rate, with sequencing confirming Setaria digitata as the predominant species, alongside Setaria labiatopapillosa. The overall combined prevalence of Setaria spp. infection was 25.77%, with the highest prevalence in the central region (39.33%) and the lowest in the southern region (3.16%). Risk factor analysis revealed that beef cattle (OR = 8.336, P = 0.048) and dairy cattle (OR = 4.400, P = 0.004) had significantly higher infection risks than bullfighting cattle. At the farm level, geographical region (P < 0.001) and the presence of water ponds (OR = 5.885, P = 0.004) were significant risk factors. This study provides the first comprehensive report of Setaria spp. infection in Thai cattle and highlights the importance of combined diagnostic approaches for improved veterinary surveillance and targeted vector control.