Vector-borne parasitic diseases (VBPDs) are diseases that have a major impact on the health and productivity of livestock. VBPDs, including trypanosomosis, theileriosis, and anaplasmosis, are caused by blood parasites and Rickettsiales infections that can be transmitted by biting insects. The control and elimination of VBPDs is difficult and challenging. This study contributed to better understanding the impact of hematophagous insects on carrying pathogens to livestock by investigating the detection of Trypanosoma evansi, Theileria spp. and Anaplasma spp. in hematophagous insects collected from five livestock farms in Northeastern Thailand using polymerase chain reaction (PCR). The Stomoxys and Tabanus samples were collected using Nzi and Vavoua traps. Morphological and molecular identifications of hematophagous insects were performed. The Stomoxys samples were dissected into head and abdomen parts, while the Tabanus samples were dissected into head, salivary glands, and abdomen parts before DNA extraction individually. The host blood meal in the DNA of each part of the hematophagous insects was identified using PCR targeting the prepronociceptin (PNOC) gene. PCR detection of T. evansi, Theileria spp., and Anaplasma spp. were performed by targeting the ITS2, 18s rRNA, and msp4 genes, respectively. In total, 131 insect samples were collected, with Stomoxys calcitrans being the most common species at 40 (30.53%), followed by Tabanus spp. at 14 (10.69%) and the other Stomoxys spp. at 7 (5.34%), respectively. The hemoparasite detection results identified Theileria spp. in the abdomen (21.43%; 3/14) of three Tabanus samples. Neither Anaplasma spp. nor T. evansi were detected in this study. Additionally, host blood meal DNA was found in the abdomen of Stomoxys calcitrans (4.17%; 1/24) and Stomoxys spp. (20%; 1/5). For the Tabanus spp., host blood meal DNA was detected in the head (7.14%; 1/14), salivary glands (7.14%; 1/14), and abdomen (14.29%; 2/14). These results highlighted that Tabanus spp. might play an important role in Theileria transmission.

Trypanosomosis (Surra), caused by the hemoflagellate protozoan Trypanosoma evansi, has a negative impact on animal health and the livestock economy in many countries, including Thailand. Molecular epidemiological data and integrative control approaches for small-scale farms remain limited. This study determined the prevalence of Trypanozoon infection and its associated risk factors in Thai livestock under an integrative control approach. Molecular tools were used to characterize T. evansi typing. A total of 947 blood samples were collected from cattle and buffalo across 123 small-scale farms in 16 provinces covering four regions of Thailand, with diagnostic results and control recommendations communicated to farmers within 14 days. The ITS2-positive samples were then subjected to further molecular characterization, together with two dogs, seven horses, and an in vitro T. evansi isolate, comprising 40 Trypanozoon-positive samples. These were characterized using PCR targeting the TBR primer, maxicircle NADH5, VSG RoTat 1.2, and minicircle B, followed by ITS2-based phylogenetic analysis. Risk factors were assessed using Chi-square and Fisher’s exact tests, logistic regression, a generalized linear mixed model, and Firth’s penalized logistic regression. The overall prevalence of Trypanozoon infection was 3.17% (30/947), detected exclusively in beef cattle in the Northern region (15.30%; 30/196), specifically in Lampang (46.15%, 12/26), Tak (23.08%, 3/13), and Phrae (17.24%, 15/87) Provinces. No infections were reported in the Northeastern, Central, or Southern regions. Male sex, age under one year, and residence in the Northern region were significant individual-level risk factors, while farm-level lacrimation and the presence of Stomoxys were significantly associated with infection. Molecular characterization identified T. evansi type A and type non-A/B, with no type B or maxicircle-positive samples detected. Phylogenetic analysis grouped the Thai isolates with previously reported isolates from Thai deer and cattle, Iranian camels, and Colombian dogs. On follow-up, no Trypanozoon infection was detected in revisited farms. These findings confirmed that T. evansi, comprising types A and non-A/B, continues to circulate in Thai livestock, particularly in beef cattle in Northern Thailand. Data from this study can be used for surveillance and future development of more effective diagnostic systems.