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.

Background and Aim: Trypanosoma evansi infection (Surra) remains a major constraint to equine health and productivity in Thailand. The only available trypanocidal drug, diminazene aceturate (DA), has limited efficacy, poor blood–brain barrier penetration, and toxicity in horses. This study aimed to investigate the in vitro inhibitory effects of commonly used equine antibiotics, gentamicin (GMC), ceftiofur (CTF), and trimethoprim–sulfamethoxazole (TS), against T. evansi (Thai strain isolated from dairy cattle number 953; TEDC 953) to identify potential therapeutic alternatives or adjuncts for equine trypanosomosis. Materials and Methods: An in vitro growth inhibition assay was conducted using the T. evansi TEDC 953 strain cultivated in Hirumi's Modified Iscove's medium 9 (HMI-9 medium) containing 20% horse serum under controlled conditions (37°C, 5% CO2, 75% humidity). Serial dilutions of DA, GMC, CTF, and TS were tested in duplicate across three independent experiments. Parasite viability was assessed after 48 h by microscopic examination, and the half-maximal effective concentration (EC50) was determined using nonlinear regression analysis in GraphPad Prism 5. Results: Among the three antibiotics, GMC and CTF significantly inhibited T. evansi growth in vitro, whereas TS showed no inhibitory effect. The EC50 values were 1.25 × 10⁻5 ± 3.90 × 10⁻6 mg/mL for DA, 0.22 ± 0.08 mg/mL for GMC, and 0.08 ± 0.05 mg/mL for CTF. Parasite viability assays confirmed that GMC (5 mg/mL) and CTF (0.2 mg/mL) completely eliminated T. evansi after 48 h of exposure. These findings provide the first in vitro evidence of the trypanocidal potential of GMC and CTF against the Thai strain of T. evansi. Conclusion: GMC and CTF exhibited substantial inhibitory activity against T. evansi under in vitro conditions, supporting their potential use as repurposed or adjunct antibiotics for trypanocidal therapy in horses. This preliminary evidence underscores the need for in vivo validation, pharmacokinetic profiling, and mechanistic studies to explore synergistic effects with conventional trypanocides such as DA. Keywords: ceftiofur, diminazene aceturate, equine trypanosomosis, gentamicin, in vitro inhibition, Trypanosoma evansi.