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Tritrichomonas foetus is an extracellular parasite of the reproductive tract in cattle. To investigate the cytopathic effects of T. foetus in deeper parts of the reproductive tract, a bovine primary oviduct epithelial cell system (BOECs) was developed. Reproductive tracts were obtained from cows and the effect of co-incubating T. foetus with BOECs was analyzed by scanning electron, transmission electron and fluorescence microscopy. Viability tests were performed using colorimetric methods, TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling), fluorescein diacetate, propidium iodide, JC-1 and annexin-V. The results demonstrate that: (1) the in vitro oviduct epithelium is useful for interaction experiments with T. foetus; (2) T. foetus adheres to the BOECs as single separate cells, and later on the cells aggregate as large clusters; (3) the posterior region of the cell initiates the process of adhesion and forms filopodia and digitopodia; (4) T. foetus severely damages BOECs leaving imprints in the epithelial cells, wide intercellular spaces, and large lesions in the epithelium; and (5) T. foetus provokes bovine oviduct cell death by apoptosis and secondary necrosis. Our observations indicate the possibility that T. foetus can move through the reproductive tract to the oviduct and that infertility in cows can be mediated by an attack on the oviduct cells by T. foetus.

Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process

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To verify the response of host cells to trichomonad infection, a primary culture of bovine epithelial cells (BOECs) was grown to model host cells, following previously published procedures (Midlej et al. 2009). The JT strain of T. vaginalis (Fig. 1A) and the K strain of T. foetus (Fig. 1B) were used as models for human and bovine parasites, respectively. Parasites were cultivated in trypticaseyeast extract-maltose (TYM) medium (Diamond 1957) Financial support: CNPq, FAPERJ, CAPES, PRONEX, AUSU + Corresponding author: marlenebenchimol@gmail.com Received 28 January 2012 Accepted 18 July 2012 supplemented with 10% foetal bovine serum. Host cell cultures were exposed to a parasite:BOECS cell ratio of 5:1 for 12 h in 37ºC (Fig. 1C, D). Cells were equilibrated in incubation medium for 5 min at 37ºC (5% CO 2 ) prior to parasite addition. The medium contained two parts of complete DMEM (pH 7.2) and one part TYM medium (W/D 2:1) without serum. The incubation medium and temperature were chosen after testing parasite and host cell viabilities in several different conditions. The supernatants of cultures with parasites or without (controls) were collected and cytokine levels were quantified with specific kits for measuring