Proteomic analysis of proteins released by Tritrichomonas foetus: Identification of potential targets for the development of new diagnostic methods.

Bovine trichomonosis (BT), a disease of the bovine urogenital tract, is caused by the protozoan Tritrichomonas foetus (Tf). Tf causes endometritis, infertility, and premature death of the embryo, which generates considerable economic losses. The proteins released can mediate fundamental interactions between the pathogen and the host, triggering factors associated with the symptomatology, immune evasion and pathogenesis characteristic of the species. However, little is known about the profile of the proteins released by Tf. In order to contribute to their knowledge, we performed an isolation protocol and a proteomic profiling of the supernatant (SN) content of six Tf isolates. A total of 662 proteins present in the SN of Tf were detected, out of which 121 were shared by the six isolates, while the remaining 541 were found in at least one of the isolates studied. The comparative analyses using the databases of Tf strain genome K revealed 32.9% of uncharacterized proteins. The bioinformatic analyses showed that the main molecular functions predicted were binding (47.9%) and catalytic activity (38.2%). Additionally, we performed immunodetection assays to evidence the antigenic potential of SN proteins. Interestingly, we observed great ability to detect SN proteins from all six isolates using serum from immunized mice and infected bulls. A complementary mass spectrometry assay allowed us to determine that the proteins that showed the strongest signal intensity in the immunoassays were Grp78 (A0A1J4IZS3) and Ap65 (A0A1J4JSR1). This work represents the first proteomic characterization of Tf SN proteins and their antigenic potential, which might be interesting for the future design of new diagnosis and treatment methods for BT.

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

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 (IC50), by flow cytometry, and also to analyze minimum lethal concentration (MLC) by means of recovery tests post-treatment in vitro. IC50 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.

In Vitro Susceptibility to Metronidazole of Tritrichomonas foetus Bovine Isolates from Argentina.

BACKGROUND: Tritrichomonas foetus is the etiologic agent of the sexually transmitted disease Bovine Trichomonosis (BT). In Argentina, BT is endemic and represents a relevant health problem that causes reproductive inefficiency in cattle and large economic losses. Metronidazole is the drug of choice in the treatment of BT. Treatment has been associated with a temporary resolution of the clinical signs but is not able to control the disease. In recent years, the apparition of in vivo and in vitro aerobic and anaerobic resistance leading to ineffective treatments has been reported. AIMS: Thus, the aim of the present study was to explore the susceptibility of six different isolates of T. foetus under aerobic (AC) and anaerobic (ANC) conditions. RESULTS AND DISCUSSION: Six isolates of T. foetus were obtained from samples of preputial smegma of bovine origin. Values of minimum lethal concentration and minimum inhibitory concentration were higher than those observed in other works and represent current data in Argentina and provide information to establish new treatment protocols.