| Summary: | African horse sickness (AHS) was reported as an outbreak in Thailand in 2020. Hematophagous insects from the genus <i>Culicoides</i> are the suspected vector responsible for AHS transmission. Horses in Hua Hin district, Prachuab Khiri Khan province, Thailand, were affected and died from AHS in 2020. However, the potential <i>Culicoides</i> species and its host preference blood meal in the affected areas are unknown. To investigate the potential vectors of AHS, <i>Culicoides</i> were collected using ultraviolet light traps placed near horse stables. Six horse farms, including five farms with AHS history and one farm without AHS history, were included in this study. Morphological and molecular identification of the <i>Culicoides</i> species was performed. Polymerase chain reaction (PCR) targeting the cytochrome b oxidase I (COXI) gene for confirmation of the <i>Culicoides</i> species, identification of the prepronociceptin (PNOC) gene for host preference blood meal, and bidirectional sequencing were conducted. Consequently, 1008 female <i>Culicoides</i> were collected, consisting of 708 and 300 samples captured at positions A and B at a distance of <2 and >5 m from the horse, respectively. Twelve <i>Culicoides</i> species identified by morphology were noted, including <i>C. oxystoma</i> (71.92%), <i>C. imicola</i> (20.44%), <i>C. actoni</i> (2.28%), <i>C. flavipunctatus</i> (1.98%), <i>C. asiana</i> (0.99%), <i>C. peregrinus</i> (0.60%), <i>C. huffi</i> (0.60%), <i>C. brevitarsis</i> (0.40%), <i>C. innoxius</i> (0.30%), <i>C. histrio</i> (0.30%), <i>C. minimus</i> (0.10%), and <i>C. geminus</i> (0.10%). The PCR detection of the <i>Culicoides</i> COXI gene confirmed <i>Culicoides</i> species in 23 DNA samples. PCR targeting the PNOC gene revealed that the <i>Culicoides</i> collected in this study fed on <i>Equus caballus</i> (86.25%), <i>Canis lupus familiaris</i> (6.25%), <i>Sus scrofa</i> (3.75%), and <i>Homo sapiens</i> (3.75%) for their blood meal. Human blood was identified from two samples of <i>C. oxystoma</i> and a sample of <i>C. imicola</i>. Three dominant species including <i>C. oxystoma</i>, <i>C. imicola,</i> and <i>C. actoni</i> that were reported in the Hua Hin area prefer to feed on horse blood. Moreover, <i>C. oxystoma</i>, <i>C. imicola</i>, and <i>C. bravatarsis</i> also feed on canine blood. This study revealed the species of <i>Culicoides</i> in Hua Hin district, Thailand, after the AHS outbreak.
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