Date of Graduation

5-2026

Document Type

Thesis

Degree Name

Bachelor of Science in Agricultural, Food and Life Sciences

Degree Level

Undergraduate

Department

Animal Science

Advisor/Mentor

Danielle Graham

Committee Member

Shawna Weimer

Second Committee Member

Ben Parsons

Abstract

Histomonas meleagridis is a protozoal parasite able to exist in a flagellated or amoeboid form that infects gallinaceous birds, particularly turkeys, and causes significant mortality. There is currently no effective treatment for this disease. The purpose of this study was to assess the anti-histomonal activity of commercially available protease inhibitors, leupeptin, bestatin, and antipain, using an established in vitro model. Three protease inhibitors, leupeptin, bestatin, and antipain, were evaluated across five assays. Treatment groups included: 1) non-treated control, 2) leupeptin 100 µM, 3) bestatin hydrochloride 100 µM, and 4) antipain dihydrochloride 100 µM. Histomonads were enumerated using a hemocytometer after 48 hours of incubation at 40°C in 1.7mL sterile snap cap microcentrifuge tubes (n=4/treatment) in all assays. Replicate tubes were used for viability tests by transferring the culture into 25cm2 tissue culture flasks (2 flasks/treatment) and were incubated at 40°C in assays 2, 3, and 5. Colony forming units (CFU) were evaluated (Log10 CFU/mL) by drop plating onto CHROMagarÔ Orientation in assays 4 and 5. Agar plates were incubated aerobically at 37°C. Data were analyzed using ANOVA, and means were separated using Tukey HSD or t-test where appropriate (P < 0.05). No significant differences were observed in assays 1 and 2 between treatment groups and the non-treated control. In assay 3, both bestatin and leupeptin significantly reduced H. meleagridis cells/mL compared to the non-treated control. In assays 4 and 5, antipain significantly reduced H. meleagridis cells/mL compared to the non-treated control. Bestatin also reduced E. coli and K. pneumoniae in assay 4, whereas no significant differences in bacterial recovery were observed following antipain treatment in assay 5. All cultures remained viable following treatment, indicating that while reductions in parasite concentration were observed, complete elimination was not achieved. Overall, these results demonstrate that leupeptin, bestatin, and antipain may possess some anti-histomonal activity in vitro, with antipain showing the most consistent effect.

These findings support the potential of cysteine protease inhibitors as candidates for further investigation and highlight the need for additional research to evaluate their efficacy in vivo.

Keywords

Turkey; Histomonas meleagridis; Assay; Protease; Inhibitors; Parasite

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