Tavassoli et al.: Novel in Vitro Efficiency of Chitosan Biomolecule … Introduction Iantiparasitic drugs has been criticized by n recent years, application of synthetic many researchers and animal health care professionals because of some drawbacks including, the emergence and re-emergence of parasitic diseases, the appearance of drug resistant organisms and toxic side effects (1). As a result, the development of new natural agents for animal parasitic diseases treat- ment has unexpectedly increased to over- come these problems. Among them, plant and animal based naturally produced agents can be used as alternatives or adjuncts to current antiparasitic therapies (2, 3). Avian trichomoniasis is caused by a single celled protozoan T. gallinae which is mainly responsible for infections of mouth pharynx, esophagus, and crop of domestic pigeons, wild columbids and raptons (4). It is likely that T. gallinae is found wherever domestic pigeons and mourning doves are found (5). Chitosan is a linear co-polymer of glucosa- mine (2-desoxy-2-amino-β-D-glucopyranose, glcN) and N-acetylglucosamine (2-desoxy- 2-acetamido-β-D-glucopyranose, glcNAc) in β -1,4-glycosidic linkages (4). It is converted from chitin, which is a natural carbohydrate polymer found in the skeleton of crustaceans, marine zooplankton, insects and cell walls of fungi (6). Chitosan is found in nature, to a lesser extent than chitin, in the cell walls of fungi. In contrast to cellulose (the fiber- forming component of plant cell walls) and chitin (the fiber-forming component of fun- gal cell walls), chitosan possess positive charges at physiological pH values due to their free amino groups (Fig.1) (7). Among abundant naturally occurring polysaccha- rides, which are neutral or acidic, chitosan is distinguished for its cationic nature (pKa ≈ 6.5), which allows it to interact with the many polyanionic polymers such as proteins, Available at: http://ijpa.tums.ac.ir DNA, and membranes (8). Chitosan pos- sesses many desirable properties including, antimicrobial, antifungal and antioxidant ac- tivity and also film forming properties which have been reviewed in great details (9). Moreover, as reported earlier (10, 11), chi- tosan and its derivatives could consider as a novel antiparasitic agent. The efficacy of several nitroimidazoles such as metronidazole, dimetridazole, ronidazole, carnidazole and ornidazole against T. galli- nae has been reported (12). In addition, the susceptibility of this protozoan to four syn- thetic compounds was evaluated in vitro (13). The aim of the present study was to evaluate possible antiprotozoal activities of various concentrations of low molecular weight chi- tosan on trophozoites of T. gallinae in vitro. Materials and Methods Chitosan solution preparation Chitosan solution was prepared by dissolv- ing chitosan with low molecular weight (Sigma, Aldrich, St. Louis, MO, USA) in aqueous solution of 1% (w/v) acetic acid to a final concentration of 20000 μgml-1 with agi- tation overnight at room temperature to achieve complete chitosan dispersion. The pH of solution was then adjusted to 7 with 1 mol equi/L NaOH. Parasite collection and culture In this study which conducted at Laboratory of Parasitology (Faculty of Veterinary Medi- cine, Urmia University, Urmia, Iran,), T. gallinae isolated from an adult pigeon was cultured axenically in vitro in trypticase- yeast extract-maltose (TYM) medium (pH 7.2) supplemented with 10% (v/v) heat- inactivated cold horse serum (14), and pas- saged every two weeks. Stock suspensions 93