Studies on a Tritrichomonas from the Nasal Cavity of Swine

~e growth an~ morphology of a nasal porcine trichomonad (probably Tritrichomonas .suzs) were studied on organisms grown in axenic cultures m C.P.L.M. medium under a variety of environmental conditions including temperature changes (20°C., 25°C., 30°C., 35°C., 40°C.) and :anges in pH (4.1, 4.6, 5.1, 5.6, 6.1, 6.6, 7.1, 7.6, 8.1, H.6, and 9.1). Temperatures of 25°C. , 30°C. , and 35°C. supported active growth with maxi:nal populatio~s of 3,050,000, 5,150,000, and 5,200,000 per ml. , respectively. The optimal pH for growth was found to be within the range of 5.1-7.6, but the organism can be successfully grown between a pH range of 4.6 to 8.6. The morphology of the organism was studied in relation to the environmental temperatures and a change in size was noted. At cooler temperatures the organisms were wider, nucleus was smaller, and the axostyle tip was longer. Both temperature and hydrogen ion concentration influenced the size of the organisms. At both 30°C. and 35°C. the smallest organisms were in the pH range of 6.1-7.1; whereas, the organisms were longer in the pH range of 4.6-5.6 and also in the pH range of 7.6-8.6. At each temperature the organisms were wider in the acid range. Such morphological changes associated with changing environmental conditions must be taken into consideration when taxonomy is based upon morphological structures. In an earlier paper Buttrey ( 1956) described a Tritrichomonas from the nasal cavity of swine. Hibler et al. (1960) confirmed the presence of this organism in the nasal cavity, considering it to be Tritrichomonas suis (Gruby & Delafond, 1843), a species also found in the stomach and caecum of swine. Their conclusions were based primarily on morphological data from stained organisms. The true significance of morphological differences and their taxonomic interpretation is often questioned. To further interpret the porcine trichomonad taxonomy, Buttrey ( 1960) studied the normal pattern of growth of the porcine Tritrichomonas from the nose and later ( 1968) made a similar study on the form from the caecum. To further study the nasal porcine trichomonad and to better understand its taxonomic position in relation to the trichomonads throughout the alimentary tract of swine, it was felt appropriate to reinvestigate certain aspects of its physiology and morphology and correlate the results with those of earlier studies. The experiments using a strain of trichomonad (Buttrey's strain 3/N) from the nose of swine were designed to: (1) determine the growth curves for the organism grown at different temperatures; (2) determine if the populations vary in morphology when grown at different temperatures; and (3) determine the relation of hydrogen ion concentration to growth and size of the organism. 1 Iowa State University, Ames. 423 1 Buttrey and Sankaranarayanan: Studies on a Tritrichomonas from the Nasal Cavity of Swine Published by UNI ScholarWorks, 1969 424 IOWA ACADEMY OF SCIENCE [Vol. 76 MATERIALS AND METHODS The culture strain used in the study, originally isolated from the nose of a pig sacrificed 9 years and 10 months prior to the study, was in its 449th subculture. The axenic population was first obtained by using a U-shaped migration tube and aseptic laboratory procedures were subsequently followed during the extended period of subculturing. The stock culture had been maintained at 20°C. + 1° and had been subcultured every 3 to 7 days. C.P.L.M. medium (Johnson, 1947) with 10% bovine serum was used for growing the organisms throughout the experiments. To each 10 ml. of the final C.P.L.M. medium mixture, 25,000 units of penicillin and 25 mg. of dihydrostreptomycin were added. The organisms for inoculation were prepared from a two-dayold flourishing culture containing 1-2 million active trichomonads per ml. which was diluted to approximately 2,000 organisms per ml. One-half ml. of this dilution ( 1,000 organisms) was inoculated into 91'2 ml. of culture medium, making 100 organisms per ml. of medium. T):ie study consists of two experiments. In the first, the pH value (pH 6.1) was constant but the temperatures varied (20°C., 25°C., 30°C., 35°C., and 40°C.). This experilnent consisted of a series of 12 "identical" subcultures at each temperature. Counts of living cells were made every 12 hours, but data to form the growth curve are recorded at 24-hour intervals except where significant changes occurred between these longer periods. The populations were determined with a brightline improved Neubauer haemacytometer after thoroughly shaking the culture. The second experiment regarding the relationship between pH values and growth involved varying the pH values but keeping the temperature constant. The pH values of 4.1, 4.6, 5.1, 5.6, 6.1, 6.6, 7.1, 7.6, 8.1, 8.6, and 9.1 were used at temperatures of 20°C. , 25°C. , 30°C. , 35°C. , and 40°C. Six "identical" subcultures were used at each pH value and temperature. Morphological studies were based upon stained slides fixed in Schaudinn's and Bouin's and stained with iron-hematoxylin and protein silver (Moskowitz, 1950), respectively. In order to compare the morphological changes during the growth cycle, smears were made from the experimental cultures at 24 hour intervals throughout the growth cycle. All other smears were made at or near the maximal population levels. All measurements are based upon the average of ten to twenty-five organisms. RESULTS AND DrscussroN Growth Curves for the Organism Grown at Different Temperatures. Of the five temperatures (20°C. , 25°C. , 30°C. , 35°C. , 2 Proceedings of the Iowa Academy of Science, Vol. 76 [1969], No. 1, Art. 56 http://scholarworks.uni. edu/pias/vol76/iss1/56