Flow cytometry evaluation of in vitro susceptibility of bovine isolates of Tritrichomonas foetus to metronidazole

Abstract

Bovine Trichomonosis, an endemic sexually transmitted disease in countries with extensive livestock and natural service, represents one of the most common causes of reproductive failure. 5-nitroimidazoles and their derivatives are used for its treatment, mainly metronidazole (Mz). The emergence of resistance mechanisms adopted by the parasites against the drug and failure of the treatments suggest the need to investigate susceptibility and obtain current values. The available information of in vitro susceptibility of these drugs comes from the use of a diversity of methodologies and criteria, especially observation of the mobility of the parasite under the optical microscope to evaluate its viability. These techniques are arduous and time consuming and lead to a subjective assessment dependent on the operator, the methodology used, and the morphology adopted by the protozoan. In this sense, flow cytometry has proven to be a fast and efficient method to evaluate viability in other protozoa. The aim of this study was to evaluate the in vitro susceptibility of six bovine isolates of Tritrichomonas foetus to Mz in aerobic (AC) and anaerobic (ANC) conditions by means of the calculation of the 50% inhibitory concentration (IC 50 ), by flow cytometry, and also to analyze minimum lethal concentration (MLC) by means of recovery tests post-treatment in vitro. IC 50 values ranged from 1.06 to 1.25 μM in ANC and from 1.44 to 3.03 μM in AC, these being the only ones reported at 48 h for these protozoa. With respect to MLC at 48 h, the results were from 3.67 to 7.35 μM in ANC, and from 7.35 to 14.7 μM for AC, where two isolates (Tf0 and Tf2) for AC and one (Tf2) for ANC showed higher values than those described in the literature. Flow cytometry has proven to be an effective, rapid and objective methodology and very useful in susceptibility tests. The data obtained through these tests allow us to describe the susceptibility exhibited by these protozoa, this being valuable information when establishing dosages in Mz treatments.