INFECTION AND IMMUNITY, Nov. 1990, p. 3627-3632 0019-9567/90/113627-06$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 58, No. 11

Cytotoxic and Hemolytic Effects of Tritrichomonas foetus Mammalian Cellst


DONALD E. BURGESS,* KENNETH F. KNOBLOCK, TOM DAUGHERTY, AND NANCY P. ROBERTSON Veterinary Molecular Biology Laboratory, Montana State University, Bozeman, Montana 59717 Received 30 April 1990/Accepted 10 August 1990

Geographically distinct lines of Tritrichomonas foetus were assayed for their ability to cause cytotoxicity in nucleated mammalian cells and lysis of bovine erythrocytes. T. foetus was highly cytotoxic toward a human cervical cell line (HeLa) and early bovine lymphosarcoma (BL-3) but displayed low levels of cytotoxicity against African green monkey kidney (Vero) cells. In addition to variation in the extent of cytotoxicity toward different targets, differences in the levels of cytotoxicity in the same nucleated target occurred with different parasite lines. Whole T. foetus, unfractionated whole-cell extracts, and parasite-conditioned medium (RPMI 1640 without serum) all caused lysis of bovine erythrocytes. Lytic activity in the conditioned medium was substantially reduced by repeated freezing and thawing or heating to 90°C for 30 min. Damage of mammalian target cells by live T. foetus could be reduced by the presence of protease inhibitors; however, such inhibitors did not diminish the lytic effects of conditioned medium. These results suggested that proteolytic enzymes were necessary for the lytic mechanism of the live parasites but were not required once lytic factors were released into the parasite-conditioned medium. They further suggested that the lytic molecules were either proteins or had proteinaceous components.

Microorganisms have a variety of cytopathic effects on mammalian cells, including lysis via toxins (6, 12, 23, 35), production of pore-forming proteins (38, 39), and loss of adhesion of host cells to their substrate (3, 4, 25). Parasitic protozoa can damage host tissues and cells (2, 22) via contact-dependent cytotoxicity (2-4, 22, 32), production of enzymes which attack tissue components such as collagen (17, 28), and production of soluble lytic factors which mediate host cell membrane damage (38, 39). Trichomonads, which are considered parasites of the surfaces of mammalian tissues, can be cytotoxic (2, 4, 25). The human pathogen Trichomonas vaginalis has been shown to mediate cytotoxicity against adherent human cell lines, as evidenced by the parasite's ability to cause adherent target cells to take up vital dyes and detach from their substrate (25). In addition, target cells labeled with radioactive DNA precursors were shown to release labeled DNA when incubated with T. vaginalis (4), indicating that T. vaginalis damaged the membrane of these cells. Tritrichomonas foetus has been reported to attach to bovine primary vaginal epithelial cells (13), damage cultured cells and inhibit their division (16, 22), produce enzymes capable of attacking host tissues (14, 26, 29), cause inflammation of the uterus (30), and invade bovine placental tissue (33). By using monoclonal antibodies specific for surface epitopes of T. foetus (9, 10) we recently confirmed this bovine tissue invasion (11). These results suggested that T. foetus could directly damage bovine tissue and was likely to cause considerable cell destruction, although they revealed little about the mechanism of host tissue damage. Our goal in this study was to investigate the capacity of T. foetus to damage mammalian targets in an in vitro culture system and to begin to study the mechanism of host cell destruction. In this report we present the initial description

of this system and data that show that T. foetus can mediate substantial cytotoxicity toward adherent and nonadherent mammalian cell lines and can lyse bovine erythrocytes (BRBC). We also show that different lines of T. foetus cause different degrees of cytotoxicity in the same target cells and that a single, individual parasite line displays different degrees of cytotoxicity against distinct target cells. In addition, we present evidence that the mechanism of this cytotoxicity involves parasite-derived, proteinaceous factors that are apparently not proteases but require parasite protease activities. MATERIALS AND METHODS Growth of the parasite. The following lines of T. foetus were used in these studies: MT 85-330 and MT 84-685 from Montana; clone MT 84-685.2, derived from MT 84-685; CAVMC 84 from California; and UT MS-1 from Utah, kindly provided by Mark Hall, University of Nevada, Reno. All parasites were grown and passaged every 2 days in the peptone-yeast extract-maltose medium of Diamond (15) without agar and containing 10% newborn bovine serum (HyClone, Logan, Utah) and 20 ,ug of gentamicin sulfate (GIBCO Laboratories, Grand Island, N.Y.) per ml as previously described (9, 10). Cloning of T. foetus. Cloning of T. foetus lines MT 84-685 and CAVMC 84 was performed exactly as described previously for T. vaginalis (21) except that it was done at pH 7.2. Cytotoxicity assays. The dye assay to measure the cytotoxicity of T. foetus toward adherent mammalian cells was done as described for T. vaginalis by Alderete and Pearlman (4). This assay uses crystal violet to stain targets remaining after exposure to trichomonads, thus assessing damage to anchorage-dependent cells (4). Briefly, African green monkey kidney (Vero) cells, Madin-Darby bovine kidney cells (MDBK), or human cervical cells (HeLa) were plated into 96-well, flat-bottom plates (no. 25860; Corning Science Products, Corning, N.Y.) at 2 x 104 to 4 x 104 cells per well 24 h prior to addition of T. foetus at 5 x 104 to 1 x 105 parasites per well. At 12 to 24 h later, the monolayers were washed twice

Corresponding author. t Contribution no. J-2510 from the Montana Agricultural Exper-


iment Station.





with warm Dulbecco phosphate-buffered saline (pH 7.2), fixed, stained, and processed as described before (4), and the A590 was read with an enzyme immunoassay plate reader (Bio-Tek Instruments, Burlington, Vt.). Cytotoxicity in the dye assay was determined as 1 - EIC, where E is the mean A590 of the experimental plate and C is the mean A590 of the control plate (4). To assess cytotoxicity mediated by T. foetus toward adherent and nonadherent target cells, cells were labeled with 300 ,uCi of 51Cr (sodium chromate, 200 to 900 Ci/g; Dupont-NEN Products, Wilmington, Del.) per flask for 1 h at 37°C. After labeling, cells were removed, washed, and plated into 96-well plates 2 to 4 h prior to addition of T. foetus. Plates were incubated further at 37°C and centrifuged after incubation (400 x g, 10 min, 4°C). One-half of the supernatant volume was removed, and the radioactivity of these samples was determined by liquid scintillation spectrometry. Quadruplicate or triplicate wells were used for each experimental condition and included control wells containing host cells without parasites for spontaneous release and host cells lysed by detergent to determine maximum release. Cytotoxicity as determined by the 51Cr release assay was expressed as percent specific release = [(experimental cpm - control cpm)/(maximum cpm - control cpm)] x 100, where cpm is the mean counts per minute (31). Hemolytic activity of T. foetus. To determine the hemolytic activity of T. foetus and its products, BRBC were asceptically collected from venous blood in Alsever solution, washed in assay buffer (0.1 M KH2PO4 [pH 6], 0.15 M NaCl), and adjusted to 108 BRBC per ml. Then 1 ml of the BRBC suspension was labeled with 51Cr as described for nucleated cell targets (above) and washed in assay buffer, and 106 labeled BRBC in 100 ,ul of buffer were added to each well of a 96-well plate. T. foetus, extracts, or parasiteconditioned medium (100 pI) was then added to BRBC targets at effector (parasite)-to-target (BRBC) (E/T) ratios of 1:1 to 1:40. The plates were incubated for 12 h at 37°C and centrifuged (400 x g, 10 min, 10°C); 100 pul of supernatant was removed from each well, and the released radioactivity was determined by liquid scintillation counting. Controls included BRBC with buffer (spontaneous release) and BRBC plus 100 ,ul of 1% Triton X-100 in water (maximum release). Results were expressed as percent specific release as described above for the cytotoxicity experiments with nucleated targets. In some cytotoxicity assays, protease inhibitors (see below) were added to targets at the same time that parasites were added, and the cytotoxicity assay was completed as described above. Conditioned medium. To examine the ability of T. foetus to secrete lytic factors into the medium, T. foetus was washed twice in RPMI 1640 (GIBCO Biologicals, Grand Island, N.Y.) without serum (200 x g, 10 min, 20°C), suspended to a concentration of 5 x 107 parasites per ml, and incubated at 37°C for 12 h. At the end of incubation, parasite viability was determined by phase-contrast microscopy; the parasite suspension was pelleted (400 x g, 10 min, 20°C), and the supernatants were removed, concentrated by ultrafiltration (Amicon), and stored at -20°C until used. Protease activity and inhibition. To demonstrate protease activity in whole T. foetus, a substrate gel system similar to that employed by Henssen and Dowdle (20) was used. Whole-parasite preparations were washed in Dulbecco phosphate-buffered saline, disrupted in sample buffer (0.0625 M Tris hydrochloride [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol), and electrophoresed




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FIG. 1. Cytotoxicity of four lines of T. foetus (MT 84-685, MT 84-685.2, CAVMC 84, and UT MS-1) against three adherent target cell lines, MDBK (hatched bars), HeLa (open bars), and Vero (solid bars). Parasites and target cells were incubated at an E/T of 10:1 for 20 h at 37°C in 95% air-5% C02, and cytotoxicity was evaluated by the dye assay (4).

on 12.5% sodium dodecyl sulfate-polyacrylamide substrate gels containing 0.1% collagen gelatin (Sigma Chemical Co., St. Louis, Mo.) as previously described (20, 37). After electrophoresis, gels were soaked in three changes of 2.5% Triton X-100 for 1 h and incubated for an additional 18 h at 37°C in 0.1 M phosphate buffer (pH 6) without inhibitors or with 0.1 mM leupeptin or 1 mM phenylmethylsulfonyl fluoride (PMSF). The ability of dithiothreitol (DTT) to restore proteinase activity in the presence of inhibitors was tested by addition of 5 mM DTT to the gel and inhibitor mixture. After incubation, all gels were fixed in 50% trichloroacetic acid, stained with Coomassie blue solution, and destained (37). Protease activity in parasite-conditioned RPMI was assayed by the method of Aschom and Jacobson exactly as described before (5) with agarose slabs and 5'-iodoacetamidofluorescein (Molecular Probes, Junction City, Oreg.) linked to bovine serum albumin. In addition to PMSF and leupeptin, EDTA (disodium salt) and iodoacetamide were used as protease inhibitors with conditioned RPMI. RESULTS Cytotoxic effects of T. foetus on host cells. Initially the cytopathic effect of several lines of T. foetus on three lines of adherent, nucleated mammalian cells was examined by the dye assay, which assesses monolayer destruction by trichomonads (4). Four lines of T. foetus exhibited marked cytotoxicity against HeLa cells, while only two of the four lines (MT 84-685 and clone MT 84-685.2) were cytotoxic against MDBK cells (Fig. 1). No significant cytotoxicity was seen against the Vero cell line with any of the T. foetus lines tested. Examination of monolayers by phase-contrast microscopy just prior to processing for the dye assay confirmed that numerous T. foetus adhered tightly to cells, while other parasites were free-swimming and highly motile (data not shown). Kinetics studies revealed that T. foetus displayed cytotoxicity against HeLa cells as early as 16 h after T. foetus inoculation and increased until maximum levels were reached at 20 h. Other kinetics analyses showed that by 20 h after inoculation of T. foetus, cytotoxicity was consistently demonstrable, and subsequent dye assays were done at 20 h after inoculation (data not shown). We reasoned that if T. foetus could cause cytotoxicity in

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Or.Two.t FIG. 4. Lysis of 51Cr-labeled BRBC by whole T. foetus. T. foetus (1 X 106, 1 X 1O5, 5 104 and 2.5 x 104 per well) and 51Cr-labeled Ef


FIG. 2. Comparison of cytotoxicity of four lines of T. foetus (MT 85-330, MT 84-685, CAVMC 84, and UT MS-1) against HeLa targets as assessed by the dye assay (hatched bars) or 51Cr release assay (open bars). Parasites and targets were incubated as described in the legend to Fig. 1.

adherent target cells that was detectable in the dye assay as X ss of monolayer adherence, the membranes of these tar-'cis could probably be damaged as well. To test for such effects, we developed a 51Cr release assay in which target cells were labeled prior to exposure to parasites. Kinetics studies with the 51Cr release assay revealed that T. foetus caused cytotoxicity as early as 12 h after inoculation of parasites onto labeled HeLa cells and that this effect was pronounced by 20 h of incubation (Fig. 2). The relative levels of cytotoxicity measured for the four lines of T. foetus by the dye assay and by the 51Cr release assay against HeLa cells were comparable (Fig. 2), indicating that for HeLa targets loss of adhesion and cell membrane damage were concomitant events.

Experiments with nonadherent targets indicated that T. foetus could be cytotoxic toward these cells as well. When T. foetus was incubated with BL-3 targets, a bovine lymphosarcoma, cytotoxicity was detected as early as 4 h at an E/T (parasite-target cell) ratio of 10:1 and at 6 h at an E/T ratio of 2.5:1 in the 51Cr release assay (Fig. 3). Further kinetics experiments verified these results and indicated that cytotoxicity was consistently detectable at 6 h of incubation (data not shown). To examine the mechanism of T. foetus cytotoxicity toward mammalian targets, extracts of T. foetus were as-



BRBC (1 x 106 per well) were incubated in a final volume of 200 ,ul of RPMI 1640 for 12 h at the indicated E/T ratio.

sayed for their ability to mediate cytotoxicity against mammalian target cells. Cytotoxicity against HeLa targets and BL-3 cells was consistently demonstrated with frozenthawed lysates of T. foetus, although at lower levels (approximately 17% specific release) than with live parasites, indicating that soluble products of T. foetus are sufficient to cause some target cell damage (data not shown). Hemolytic activity. Intact T. foetus and parasite extracts (data not shown) were hemolytic, as shown by their lytic activity on BRBC (Fig. 4). Parasite-conditioned RPMI, concentrated 10 times by ultrafiltration, also contained lytic factors capable of mediating BRBC lysis (Fig. 5), suggesting active release of the lytic factors by T. foetus. Various physical treatments of the lytic supernatants revealed that lysis could be ablated by treatment at 90°C for 30 min or three cycles of freezing and thawing (liquid N2 [37°C]) but that lytic activity was resistant to 37°C for 20 min. These data suggested that a soluble, proteinaceous factor(s) was released by T. foetus which mediated hemolysis. Association of protease activity with cytotoxicity. Since proteases have been associated with virulence and cytotoxicity in other microorganisms and T. foetus is known to have a variety of proteases, we examined the possible association of protease activity and cytotoxicity. 10 0 n0






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Effector.Target FIG. 3. Kinetics of the lysis of BL-3 target cells by T. foetus at 4 h (solid bars) and 6 h (hatched bars) of incubation. Parasites and targets were incubated at the indicated E/T ratios in round-bottom microtiter plates.

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FIG. 5. Resistance of the hemolytic effect of T. foetus-conditioned medium to protease inhibitors. T. foetus (1 x 106 cell equivalents per well) and 1 x 106 51Cr-labeled BRBC per well were incubated without inhibitors (solid bar) or with PMSF (open bars), EDTA (hatched bars), or iodacetamide (cross-hatched bars) at the indicated concentrations for 4 h in a final volume of 200 ,ul.







Ex 2 FIG. 6. Reduction of cytotoxicity of T. foetus toward HeLa targets by protease inhibitors. Labeled HeLa and T. foetus cells (E/T ratio, 10:1) were incubated without inhibitors (heavily hatched bars) or with leupeptin (0.5 ,uglml) (open bars) or 1 mM PMSF (solid bars) at 37°C as described in the legend to Fig. 2. Controls (HeLa plus leupeptin [lightly hatched bars] and HeLa plus PMSF [crosshatched bar]) without T. foetus were included. Exp 1

To test this hypothesis, protease inhibitors were added to the cytotoxicity assay mix, and preliminary experiments were done to establish levels of each inhibitor which were not toxic to T. foetus by incubation for 20 h followed by assessment of parasite viability (i.e., motility) (data not shown). Two protease inhibitors, leupeptin and PMSF, were selected for further study. When leupeptin was added to the cytotoxicity assay mix, it reduced the specific release to 64% of control release without inhibitors, while the addition of PMSF reduced specific release to 28 to 45% of control release (Fig. 6). When DTT was added to cultures containing PMSF and parasites, the cytotoxicity was enhanced over that in cultures with PMSF and parasites (data not shown). Addition of DTT did not restore full cytotoxicity in the presence of leupeptin, however. In order to determine the proteases of T. foetus which were inhibited by leupeptin and PMSF, extracts of whole T. foetus were electrophoresed on protein substrate gels, and the protease activities were assessed in the presence and absence of these inhibitors. Whole-parasite extracts showed protease activity (Fig. 7, lane 1) at low (below 24,000) and



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FIG. 7. Protease

activity present in whole-cell extracts of T. gelatin substrate gel. A total of 4.5 x 104 cells of line MT 85-330 was applied to each lane of a 12.5% polyacrylamide gel containing 0.1% gelatin I in potassium phosphate buffer (pH 6.0) and electrophoresed. The gel was then processed as described in Materials and Methods and reference 20. Lane 1, Parasites without additions; lane 2, parasites plus 5 mM DTT; lane 3, parasites plus 1 mM PMSF; lane 4, parasites plus 1 mM PMSF plus 5 mM DTT; lane 5, parasites plus 1 ,uM leupeptin; lane 6, parasites plus 1 ,uM leupeptin plus 5 mM DTT. MW standards (at left, in thousands) were (top to bottom) phosphorylase b, bovine serum albumin, ovalbumin, glyceraldehyde 3-phosphate dehydrogenase, and trypsinogen.


as demonstrated on


high (above 60,000) relative molecular weights (MWs). The low-MW protease(s) was particularly sensitive to PMSF inhibition (Fig. 7, lane 3) as well as to inhibition by leupeptin (Fig. 7, lane 5). The higher-MW protease(s) (e.g., above 60,000), while sensitive to inhibition by PMSF (lanes 3 and 4, Fig. 7), was not inhibited by leupeptin (lanes 5 and 6, Fig. 7). When DTT was added in the presence of PMSF (Fig. 7, lane 4) or leupeptin (Fig. 7, lane 6) the activity of the low-MW protease(s) was restored and enhanced to a level near the activity observed in the absence of protease inhibitors plus DTT (Fig. 7, lane 2). Parasite-conditioned medium was also tested for the presence of proteases (5) and found to contain protease activity (data not shown). Since leupeptin and PMSF reduced cytotoxicity by whole, intact T. foetus, we tested whether PMSF, EDTA, or iodoacetamide at concentrations which inhibited protease activity in the fluorometric assay (5) would reduce the lytic activity in medium conditioned by T. foetus. No reduction of hemolysis by conditioned medium in the presence of PMSF, EDTA, or iodoacetamide occurred (Fig. 5), suggesting that released lytic factors do not require the activity of proteases inhibited by these inhibitors. DISCUSSION In order to understand the pathogenesis of bovine trichomoniasis, it is crucial to determine the mechanism(s) of damage of host tissue and cells at the cellular and molecular level. To assess damage at the cellular level, we have used an in vitro procedure in which target cells are exposed to T. foetus in culture and the effects of T. foetus can be evaluated spectrophotometrically or by a 51Cr release assay. Cytotoxicity was shown against HeLa targets with four lines of T. foetus, although different levels of cytotoxicity were demonstrated between different lines of T. foetus (Fig. 2) by both assays. Thus, the relative ability of T. foetus to be cytotoxic for a mammalian target varies among different parasite isolates and lines. Similar results have been reported for the human parasite T. vaginalis, and this difference in cytotoxicity has been correlated with virulence (1, 3, 25). Trichomonads may or may not change their cytotoxic capacity over time in culture (1, 3), depending on the presence or absence of certain antibody-defined phenotypes (1). For example, the JH31A line of T. vaginalis was shown to have comparable levels of cytotoxicity for several months of culture when assayed repeatedly, although other lines changed their relative cytotoxicity over time (3). We have observed that one line of T. foetus which has been in continuous culture since 1985 has not appeared to change its cytotoxicity for 2 years (Burgess, unpublished results). We have, however, observed different levels of cytotoxicity in different lines of T. foetus (Fig. 2) and clones from the same line (Burgess, Lancto, and Daugherty, unpublished data), suggesting that this difference in cytotoxicity is probably a stable phenotypic characteristic in a particular line of T. foetus but may differ from one line to another. In addition to differences in cytotoxicity between parasite lines, we also observed differences in the susceptibility of several target cell lines to the same cytotoxic line of T. foetus (Fig. 1 and 3). No clear species-related target susceptibilities were found, however, since both human cells (HeLa) and bovine cells (BL-3) were highly susceptible to lysis while other bovine cells (MDBK) were more resistant to damage (Fig. 1). The reason that some target cells are more susceptible than others to damage by T. foetus is not clear at present. It does not appear to be due to differences in

VOL. 58, 1990

adherent versus nonadherent cells, since nonadherent BL-3 cells were destroyed more easily than adherent MDBK cells. In several situations in which microorganisms display cytotoxicity, cell-target contact (25, 32, 35) and soluble factors (12, 18, 23, 39) have been implicated in the mechanism of this cytotoxicity. Whether adherence of the T. foetus to the target (13) is a major determinant in target cell damage is unclear, although such adhesion could be important for triggering contact-dependent cytotoxicity as well as release of toxic factors by T. foetus. We have observed adherence of BRBC and nucleated targets to T. foetus (Lancto, Daugherty, and Burgess, unpublished results) and are investigating the relationship of the levels of adherence of clones of T. foetus with different cytotoxic capacities to their adhesion properties toward targets. The presence of soluble lytic factors elaborated by the parasite (Fig. 5) does not rule out contact-dependent cytotoxicity as an additional issue in the mechanism of host cell damage, as has been shown for Entamoeba histolytica (8, 32) and T. vaginalis (2, 25). Recently, Filho and de Souza reported that T. vaginalis and T. foetus could cause Madin-Darby canine kidney cells to substantially detach from their substrate by 3 days of culture at parasite-to-target cell ratios of 5:1 (16). We have observed detachment of MDBK cells within 20 h at T. foetus-target cell ratios of 10:1 (Fig. 1) as well. The possible importance of contact-dependent host cell damage is also suggested by a recent report indicating that T. foetus can adhere to primary bovine vaginal epithelial cells (13), although this report did not address target cell damage by adhering parasites. Regarding soluble mediators of target cell damage, lytic molecules with protease activities (17, 26, 36) and without protease activities (12, 39) have been reported. We (this report) and others (26) have demonstrated at least three MW classes of proteases in whole-parasite extracts of T. foetus, and some proteases do seem to be involved in cytotoxicity caused by intact T. foetus. Several other microorganisms, notably protozoa (38, 39) and bacteria (23), have been shown to produce cytotoxic factors associated with or composed of proteases. Protease activities such as cathepsin B (27) and sulfhydryl-dependent proteases (7) have been described for E. histolytica, and the levels of cathepsin B activity correlated with virulence (27). Collagenase activity associated with the plasma membrane and released into conditioned medium has also been suggested to correlate with increased virulence of E. histolytica (17). The protease activity secreted by T. foetus (26; Daugherty and Burgess, unpublished data) could well function in assisting in tissue invasion (33) by attacking the extracellular matrix of placental tissue, thus allowing access to the deeper placental tissue and the amnionic spaces (11, 33). A variety of bacteria have been shown to produce extracellular molecules which are hemolytic and cytotoxic (18, 23, 36). A metalloprotease has been described for Legionella pneumophila (36) which was active on gelatin and casein, was inhibited by EDTA (but not PMSF), and had a pH optimum of 6 to 7. This L. pneumophila protease was subsequently shown to have an MW of 38,000 and to be cytotoxic to CHO cell targets (23). Pasteurella hemolytica has been reported to release a leukotoxin, devoid of enzyme activity, which is cytotoxic against the BL-3 lymphosarcoma line (12), the same cell line used in this work with T. foetus. Components of the cytotoxic mechanisms of E. histolytica also include the production of the pore-forming protein amoebapore, which is incorporated spontaneously into tar-



get membranes, forming ion channels (38, 39), and does not appear to have protease activity. It is unclear at this point whether the lytic molecules of T. foetus act in a manner similar to amoebapore, but such a mechanism cannot be ruled out at present. Hemolytic activity was shown by T. foetus cells and in medium conditioned by hemolytic T. foetus as well. Whether this hemolytic activity proves to be an indicator of virulence for isolates of T. foetus, as has been shown for bacteria such as Shigella flexneri (34) and the trichomonad T. vaginalis (24), must await further investigation. We have examined the cytotoxicity of T. foetus towards mammalian targets and developed a system to examine some of the parameters necessary for host cell damage. Our results indicate that factors released by the parasite are involved in the cytotoxic mechanism and do not appear to have protease activities. As pointed out by Gitler and Mirelman (19), a variety of factors such as contact-dependent killing, soluble lytic factors, and collagenase or other secreted proteases are likely to be involved in the mechanisms of virulence of E. histolytica. Likewise, we expect several elements, such as adhesion (13, 16), secretion of proteases (26), and release of lytic factors, to function in the mechanism of tissue damage of T. foetus. Future studies will be designed to characterize these molecules and the molecular basis of host cell damage by T. foetus. ACKNOWLEDGMENTS We appreciate the excellent technical assistance of Patti Havstad and Sarah Crisafulli in the preparation of cell cultures. This work was supported by U.S. Department of Agriculture Hatch Project 428 and Special Grant 88-34116-3494. LITERATURE CITED 1. Alderete, J. F., P. Demes, A. Gambosora, M. Valent, A. Yonoska, H. Fabusoua, L. Kasmala, G. E. Garza, and E. C. Metcalfe. 1987. Phenotypes and protein-epitope phenotype variation among fresh isolates of Trichomonas vaginalis. Infect. Immun. 55:1037-1041. 2. Alderete, J. F., and G. E. Garza. 1985. Specific nature of Trichomonas vaginalis parasitism of host cell surfaces. Infect. Immun. 50:701-708. 3. Alderete, J. F., L. Kasmala, E. Metcalfe, and G. E. Garza. 1986. Phenotypic variation and diversity among Trichomonas vaginalis isolates and correlation of phenotype with trichomonal virulence determinants. Infect. Immun. 53:285-293. 4. Alderete, J. F., and E. Pearlman. 1984. Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers. Br. J. Vener. Dis. 60:99-105. 5. Aschom, J. D., and L. A. Jacobson. 1989. Self-quenched fluorogenic protein substrate for the detection of cathepsin D and other protease activities. Anal. Biochem. 176:261-264. 6. Ashkenazi, S., T. G. Cleary, B. E. Murray, A. Wanger, and L. K. Pickering. 1988. Quantitative analysis and partial characterization of cytotoxin production by Salmonella strains. Infect. Immun. 56:3089-3094. 7. Avila, E. E., M. Sanchez-Garza, and J. Calderon. 1985. Entamoeba histolytica and E. invadens: sulfhydryl-dependent proteolytic activity. J. Protozool. 32:163-166. 8. Bos, H. J., and R. J. van de Griend. 1977. Virulence and toxicity of axenic Entamoeba histolytica. Nature (London) 265:341-343. 9. Burgess, D. E. 1986. Tritrichomonas foetus: preparation of monoclonal antibodies with effector function. Exp. Parasitol.


10. Burgess, D. E. 1988. Clonal and geographic distribution of a surface antigen of Tritrichomonas foetus. J. Protozool. 35:119122. 11. Burgess, D. E., and K. F. Knoblock. 1989. Identification of Tritrichomonasfoetus in sections of bovine placental tissue with



monoclonal antibodies. J. Parasitol. 75:977-980. 12. Clinkenbeard, K. D., D. A. Mosier, and W. W. Confer. 1989. Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella hemolytica leukotoxin. Infect. Immun. 57:420-425. 13. Corbeil, L. B., J. L. Hodgson, D. W. Jones, R. R. Corbeil, P. P. Widders, and L. R. Stephens. 1989. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells. Infect. Immun. 57:2158-2165. 14. Cropyren, M., H. von Nicolai, and F. Zilliken. 1979. Properties and substrate specification of two neuraminidases from Tritrichomonas fetus. Hoppe-Seyler's Z. Physiol. Chem. 360:17031712. 15. Diamond, L. S. 1957. The establishment of various trichomonads of animals and man in axenic culture. J. Parasitol. 43:488490. 16. Filho, F. C. S., and W. de Souza. 1988. The interaction of Trichomonas vaginalis and Tritrichomonas foetus with epithelial cells in vitro. Cell Struct. Funct. 13:301-310. 17. Gadasi, H., and E. Kessler. 1983. Correlation of virulence and collagenolytic activity in Entamoeba histolytica. Infect. Immun. 39:528-531. 18. Gentry, M. K., and J. M. Dalrymple. 1980. Quantitative microtiter cytotoxicity assay for Shigella toxin. J. Clin. Microbiol. 12:361-366. 19. Gitler, C., and D. Mirelman. 1986. Factors contributing to the pathogenic behavior of Entamoeba histolytica. Annu. Rev. Microbiol. 40:237-261. 20. Henssen, C., and E. B. Dowdle. 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102:196-202. 21. Hollander, P. H. 1976. Colonial morphology of Trichomonas vaginalis in agar. J. Parasitol. 62:826-828. 22. Honigberg, B. M. 1978. Trichomonads of veterinary importance, p. 164-275. In J. P. Krier (ed.), Parasitic protozoa, vol. 2. Academic Press, Inc., New York. 23. Keen, M. G., and P. S. Hoffman. 1989. Characterization of a Legionella pneumophila extracellular protease exhibiting hemolytic and cytotoxic activities. Infect. Immun. 57:732-738. 24. Krieger, J. N., M. A. Poisson, and M. F. Rein. 1983. Betahemolytic activity of Trichomonas vaginalis correlates with virulence. Infect. Immun. 41:1291-1295. 25. Krieger, J. N., J. I. Ravdin, and M. F. Rein. 1985. Contactdependent cytopathic mechanisms of Trichomonas vaginalis. Infect. Immun. 50:778-786. 26. Lockwood, B. C., M. J. North, K. I. Scott, A. F. Bremer, and G. H. Coombs. 1987. The use of a highly sensitive electropho-


27. 28.


30. 31.

32. 33. 34.


36. 37.



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Cytotoxic and hemolytic effects of Tritrichomonas foetus on mammalian cells.

Geographically distinct lines of Tritrichomonas foetus were assayed for their ability to cause cytotoxicity in nucleated mammalian cells and lysis of ...
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