Vol. 58, No. 9

INFECTION AND IMMUNITY, Sept. 1990, p. 3078-3083 0019-9567/90/093078-06$02.00/0

Characterization of Tritrichomonas foetus Antigens by Use of Monoclonal Antibodies JENNIFER L. HODGSON,1 DAVID W. JONES,' PHILLIP R. WIDDERS,lt AND LYNETTE B. CORBEIL2* Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040,1 and Department of Pathology H81 IF, University of California, San Diego Medical Center, San Diego, California 921032 Received 25 June 1990/Accepted 28 June 1990

The specificity for and function of monoclonal antibodies against Tritrichomonas foetus were characterized. Four monoclonal antibodies generated by immunization of mice with live T. foetus were selected on the basis of enzyme-linked immunosorbent assay reactions. The approximate molecular masses of the predominant proteins were determined by Western blotting (immunoblotting). Monoclonal antibody TF3.8 recognized a predominant band at -155 kilodaltons, whereas TF3.2 reacted with several bands. Monoclonal antibodies TF1.17 and TF1.15 recognized broad bands between 45 and 75 kilodaltons. The first two antibodies (TF3.8 and TF3.2) did not react with the surface of T. foetus, as determined by live-cell immunofluorescence, agglutination, and immobilization, whereas two other monoclonal antibodies (TF1.17 and TF1.15) did react with surface epitopes, as determined by these criteria. The latter two monoclonal antibodies also mediated complementdependent killing of T. foetus and prevented of adherence of organisms to bovine vaginal epithelial cells. One antibody, TF1.15, also killed in the absence of complement. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may induce protective immunity.

report a study of antibody specificities and functions which should correlate with protection.

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract. In cows it is associated with vaginitis, endometritis, abortion and, rarely, pyometra. Endometritis usually results in transient infertility, whereas pyometra can result in permanent sterility (17). In bulls, infections are mild but generally result in a preputial carrier state (25). Infection is spread by venereal transmission, so herd problems result when natural breeding is practiced. Considerable economic loss occurs because of reproductive failure (14, 27). Furthermore, the recorded prevalence of trichomoniasis in the United States has increased over the last few years (4). It is not clear whether this apparent increase is due to an increased prevalence of the disease or merely an increased awareness, reflected in an increased diagnosis. Nonetheless, trichomoniasis is now recognized as one of the most common infections causing reproductive inefficiency in beef cattle and occasionally dairy herds (14). Despite the increased prevalence of bovine trichomoniasis with accompanying economic loss, treatment and control are problematic. Control of the disease is difficult because there are no fully effective therapeutic measures approved by the Food and Drug Administration (4, 29). Furthermore, the management practices required for effective control of this

MATERIALS AND METHODS Trichomonads. The T. foetus isolate used in this study was obtained from a clinical sample submitted to the Washington Animal Disease Diagnostic Laboratory. Organisms were isolated in Diamond Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-maltose medium (11) with 10% bovine serum (4). The organisms were serially passaged in Trypticase-yeast extract-maltose medium with 10% fetal calf serum (FCS) but no agar. For this study, trichomonads were grown to the mid-logarithmic growth phase at 37°C. Before use, the trichomonads were washed three times by centrifugation at 250 x g for 10 min in phosphate-buffered saline (PBS; 0.01 M; pH 7.2; with 0.15 M NaCl), unless otherwise specified. Monoclonal antibody production. BALB/c mice were immunized intraperitoneally three times at 2- to 3-week intervals with 0.5 ml of freshly prepared, washed, live trichomonads (0.8 x 106 organisms). The final inoculation was given 72 h prior to the fusion. Splenocytes of immunized mice were fused with X-63 Ag8.653 myeloma cells (19). Hybridomas were produced by standard methods (20, 23) with a thymocyte feeder layer. Supernatants were tested for antibody by an enzyme-linked immunosorbent assay (ELISA), as described below, with whole T. foetus as the antigen. Positive cell lines were transferred to 12-well plates, retested by the ELISA for activity, and cloned by limiting dilution at least twice, until the cell lines were stable (as exhibited by 90% of the wells showing positive supernatants in the ELISA with whole T. foetus as the antigen). Positive clones were expanded and injected into mice primed with 1 ml of pristane (2,6,10,14-tetramethylpentadecane, 96%; Aldrich Chemical Co., St. Louis, Mo.) 2 weeks prior to obtaining ascites fluid.

disease often are not economically feasible. Protective immunization would be a very useful method of control. However, experimental killed whole-cell vaccines have only afforded partial protection (4, 7, 17, 21). A recent preliminary study indicated that T. foetus membrane glycoproteins were promising immunogens in bulls (8). To identify specific surface membrane antigens, we generated monoclonal antibodies to whole T. foetus. Here we *

Corresponding author.

t Present address: Attwood Veterinary Research Laboratory, Attwood, Victoria 3049, Australia. 3078

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ELISA. Antibody titers of bovine serum and ascites fluid were determined by the ELISA. Plates were coated with whole T. foetus fixed in 1% Formalin in 0.9% sodium chloride as previously described (10). In brief, 105 organisms per well were incubated in microtiter plates overnight at 4°C. Antibody was diluted in PBS with 0.05% Tween 20, 2% polyvinylpyrrolidone, and 0.2% ovalbumin. After 1 h of incubation at 37°C and three washes, reactions were developed with antibovine isotype-specific monoclonal antibodies (from A. Giudry, U.S. Department of Agriculture, Beltsville, Md., or from W. Davis, Washington State University, Pullman). This step was followed by treatment with peroxidase-labeled goat anti-mouse immunoglobulin G (IgG) plus IgM (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.). For assays to detect monoclonal antibodies to T. foetus, only goat anti-mouse IgG plus IgM was used. The endpoint for titer determination was three times the absorbance of eight negative control wells without antibody. Titers were corrected for daily variation by comparison to a positive control on each plate. The immunoglobulin class and subclass of the monoclonal antibodies were determined with a mouse immunoglobulin subtype identification ELISA kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Electrophoresis and Western blot (immunoblot) analysis. The specificity of antibodies to T. foetus was further defined by Western blot analysis after sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Whole cells were suspended at 3 x 107 organisms per ml of PBS and boiled for 3 min in sample buffer containing 5% P-mercaptoethanol and SDS as described by Laemmli (22). Approximately 106 trichomonads per well were loaded per lane of a 1.5-mm-thick slab gel. Discontinuous SDS-PAGE was performed with 5% acrylamide in the stacking gel and 7.5 to 15% acrylamide in the separating gel. After electrophoresis at a constant current of 60 mA (two gels), proteins were electrotransferred to nitrocellulose as described by Towbin et al. (31). The nitrocellulose was washed in Tris-buffered saline (pH 7.5) containing 0.05% Tween 20 and blocked for 1 h in Tris-buffered saline-Tween with 0.03% gelatin. Strips were incubated for 1 h in bovine immune serum or mouse ascites fluid (1:1,000 in Tris-buffered saline-Tween-gelatin) at room temperature. After three washes in Tris-buffered saline-Tween-gelatin, bound antibody was detected by incubation in a 1: 2,000 dilution of goat anti-bovine IgGperoxidase conjugate (ICN Immunobiologicals, Costa Mesa, Calif.) or a 1: 1,000 dilution of goat anti-mouse IgG plus IgM-peroxidase conjugate (Kirkegaard and Perry) for 1 h at room temperature. A second washing step with Tris-buffered saline alone was followed by color development in a combination of hydrogen peroxide and 4-chloro-1-naphthol as previously described (10). Periodate oxidation. Antigens were subjected to SDSPAGE and electroblotted to nitrocellulose as described above. Blots were treated with sodium periodate before reactions with antibodies. Conditions of mild periodate oxidation were essentially as described by Woodward et al. (32). In brief, nitrocellulose blots were incubated in 20 mM sodium periodate in 50 mM sodium acetate buffer (pH 4.5) for 1 h in the dark at room temperature. Untreated and periodate-treated strips were washed in the acetate buffer and blocked with 1% glycine in 0.15 M NaCl for 30 min at room temperature. Then immunoreactivity was detected as described above for Western blotting. Reactivity with peroxidase-conjugated concanavalin A (Sigma Chemical Co., St. Louis, Mo.) served as a control for carbohydrate oxida-

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3079

tion, and amido black staining of parallel blots served as a control for possible protein denaturation. Thymol stain. Glycoproteins were detected by the method of Gander (12). In brief, proteins were separated by discontinuous SDS-PAGE as described above. After electrophoresis, the gel was washed twice for at least 2 h each time in isopropanol-acetic acid-water (25:10:65) to fix the proteins and remove the low-molecular-weight molecules. The gel was washed for 2 h in the above-described solvent plus 0.2% thymol (wt/vol), followed by incubation in concentrated H2SO4-absolute ethanol (80:20) at 35°C with shaking until the opalescent appearance of the gel disappeared. Zones that contained glycoprotein stained red. Inhibition of attachment. Bovine vaginal epithelial cells (VEC) were collected by gently scraping the vaginal surface. Cells from a minimum of three cows were suspended in PBS, pooled, and washed twice in PBS. Washed trichomonads were suspended at a concentration of 1.25 x 105 per ml of bovine serum or ascites fluid diluted in PBS with 1% FCS (PBS-F) or in 1 ml of PBS-F alone (control). After incubation for 15 min at 37°C, T. foetus cells were centrifuged at 13,000 x g for 1 min in a microcentrifuge and suspended with 5 x 104 washed VEC in 0.5 ml of PBS-F at pH 6.5. After 30 min of incubation at 37°C, the cells were pelleted, suspended in 150 ,ul of PBS-F at pH 7.2, and evaluated for adherence of T. foetus to bovine squamous VEC by phase-contrast microscopy.

Agglutination and immobilization. The ability of antibodies to agglutinate and/or immobilize T. foetus was determined in a microtiter plate assay. Serial dilutions of heat-inactivated (56°C, 30 min) bovine immune serum or ascites fluid were mixed with equal volumes (100 ,ul) of washed T. foetus cells (5 x 105/ml) in PBS-F at pH 7.2. After 30 min of incubation at 37°C, agglutination and immobilization were evaluated in slide preparations by phase-contrast microscopy. The titer was defined as the point at which approximately 50% of the cells were agglutinated or immobilized. Complement-mediated killing. Aliquots of neonatal colostrum-deprived calf serum were frozen at -70°C within 4 h of collection as a source of bovine complement. The neonatal colostrum-deprived calf serum was shown to lack immunoglobulins by radial immunodiffusion. Bovine immune serum, ascites fluid, FCS (control), and half of sample of complement-preserved calf serum were heat inactivated by incubation in a 56°C water bath for 30 min. Heat-inactivated bovine immune serum or ascites fluid was used at the lowest dilution giving no agglutination and no immobilization in PBS-F at 30 min. In a microtiter plate, 50 ,ul of the diluted antibody solution was added to 50 ,ul of complement-containing (the

non-heat-inactivated half) or heat-inactivated neonatal calf serum. The latter was included to determine agglutination and complement-independent killing. The antibody-complement mixture was incubated at 37°C for 30 min with 100 ,ul of washed mid-logarithmic-growth-phase T. foetus cells (105) in PBS-F (final volume, 200 pAl). Controls included in each assay were heat-inactivated fetal bovine serum at the same

dilution as the test serum plus either complement-containing or heat-inactivated calf serum. The organisms were examined in a Neubauer hemacytometer, and the numbers of motile, teardrop-shaped cells (live) and nonmotile, rounded or disintegrating cells (dead) were counted. Indirect fluorescent-antibody test. Washed mid-logarithmic-growth-phase T. foetus cells (1 ml) were centrifuged at 13,000 x g for 1 min in a microcentrifuge. The pellets were suspended in monoclonal antibody-containing ascites fluid diluted to a final concentration of 1:100 in 150 ,ul of Hanks

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INFECT. IMMUN.

TABLE 1. Titers for bovine immune serum and monoclonal antibody ascites fluid Antibody source

Bovine preimmunization seruma Bovine postimmunization seruma TF1.17 ascites fluid TF1.15 ascites fluid TF3.8 ascites fluid TF3.2 ascites fluid

ELISA titer

Functional titer

IgM

Aggluti-

Immobilization

IgGl

IgG2

200

200

3,200

4

4

6,400

1,600

3,200

32

32

3,200 6,400 640 2,560

256 128 4 2

128 128 2 2

nation

a Bovine serum source, cow 64. Preimmunization serum was collected before immunization. Postimmunization serum was collected 10 days after the third biweekly intramuscular immunization with live T. foetus.

balanced salt solution without phenol red and containing 10% FCS (pH 7.2). After incubation for 10 min at 37°C, samples were washed twice in Hanks balanced salt solution as described above. Samples were incubated for 10 min at 37°C with fluorescein-conjugated goat anti-mouse IgG plus IgM (Kirkegaard and Perry) diluted 1:50. After another wash, the pellets were suspended in 50 ,ul of Hanks balanced salt solution and the fluorescence of the trichomonads was determined by examination with a Zeiss microscope equipped for epifluorescence. Statistics. In the adherence inhibition assay, differences between antibody treatments were calculated with the z statistic. A one-way analysis of variance was used to determine if there was a significant difference in the amount of killing by each of the antibody sources in the complementmediated killing assay. An arcsine transformation of the percentage of cells killed was performed before statistical analysis (24). Since there was a significant F value at a = 0.01, a Fisher protected least-significant-difference test was performed to compare the amount of killing by the various immune sera with the appropriate control. RESULTS Four monoclonal antibodies were chosen for the characterization of T. foetus antigens on the basis of a high absorbance in the ELISA against Formalin-fixed T. foetus cells and no reactivity with wells containing no T. foetus. Each of the four cloned hybridomas produced antibodies of the IgM class (Table 1). In the Western blot analysis, TF1.17 recognized a diffuse band ranging from approximately 50 to 74 kilodaltons (kDa), TF1.15 reacted with a similar diffuse band (approximately 45 to 65 kDa), and TF3.8 reacted predominantly with a 155-kDa band but also slightly with a diffuse area below 100 kDa (Fig. 1). Multiple bands reacted with TF3.2, but the predominant bands had approximate molecular masses of 46 and 70 kDa. A control monoclonal antibody generated against Haemophilus influenzae (H.I. 4.3) did not react with any of the trichomonad proteins in the ELISA or Western blot analysis. Bovine serum collected before immunization did not react in Western blots at the lowest dilution used (1:1,000), whereas immune serum at a 1:1,000 dilution produced several major bands, including diffuse bands in the areas recognized by TF1.17 and TF1.15. The nature of the antigens was further studied by thymol staining of SDS-PAGE gels for the presence of carbohydrates. These gels demonstrated a high-molecular-weight band and a lower-molecular-weight broad band which cor-

205

116-6. 6 6-

2 9-

6 4

1

1

1

1

3 3 8 2

5 7 FIG. 1. Western blot of T. foetus whole-cell antigens. Sizes of molecular mass markers (in kilodaltons) are on the left. Lane 64 was reacted with immune serum from cow 64, and subsequent lanes were reacted with each of the monoclonal antibodies (TF1.15, TF1.17, TF3.8, and TF3.2).

responded to the general region that was recognized by TF1.17 and TF1.15. However, mild peroxidate oxidation of Western blots did not decrease the reactivity of these two monoclonal antibodies with either whole-cell or membrane preparations. In parallel experiments, treated and nontreated blots reacted with amido black at similar intensities, indicating that proteins were unaffected by periodate oxidation. In parallel experiments, periodate-treated blots did not react with concanavalin A-peroxidase, whereas untreated blots demonstrated multiple bands after incubation with concanavalin A-peroxidase. Control periodate-treated or untreated strips stained with amido black were identical, showing that proteins were unaffected by the mild periodate oxidation, as expected. To determine whether the four antibodies recognized surface-exposed epitopes, we performed indirect immunofluorescence assays with live T. foetus (Fig. 2). When T. foetus was incubated with monoclonal antibody TF1.15, approximately half of the intact organisms fluoresced. The distribution of binding was patchy over the surface of the parasite, although in some cases polar distribution of the fluorescence resembled capping. Substantial debris was present in these preparations. Monoclonal antibody TF1.17 also resulted in patchy fluorescence of approximately half of the organisms, but little debris was observed. Neither TF3.8 nor TF3.2 resulted in fluorescence of T. foetus. Functional assays showed that bovine immune serum, monoclonal antibody TF1.17, and monoclonal antibody TF1.15 were able to both immobilize and agglutinate T. foetus even at relatively high dilutions (Table 1). These same antibody preparations (at nonagglutinating and nonimmobilizing concentrations) mediated the killing of T. foetus in the presence of bovine complement, as did TF3.8, but not TF3.2 (Table 2). Interestingly, bovine complement in the absence of any antibody killed some trichomonads. Since the complement source was colostrum-deprived calf serum which

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3081

FIG. 2. Fluorescent-antibody reactivities. (A) Patchy distribution over the surface of the parasite with monoclonal antibody TF1.17. (B) Possible capping and much debris with monoclonal antibody TF1.15.

lacked immunoglobulin, this killing in the absence of monoclonal or polyclonal antibodies was not due to antibodymediated complement killing. There was also some killing by TF1.15 in the absence of complement, decreasing the viable T. foetus counts significantly more than FCS without complement (a = 0.025). However, when killing occurred it was always significantly greater in the presence of complement and antibody than in the presence of antibody alone or complement alone (agammaglobulinemic colostrum-deprived calf serum). TABLE 2. Killing of T. foetus by antibody plus complement

Studies of immune inhibition of trichomonad adherence to VEC showed that monoclonal antibodies TF3.8 and TF3.2 did not inhibit adherence. Monoclonal antibodies TF1.17 and TF1.15 inhibited adherence at least as efficiently as did immune serum (Table 3), even though the dilutions of monoclonal antibodies were higher than the dilutions of immune serum. DISCUSSION In this study we characterized monoclonal antibodies which reacted with antigens of T. foetus. Two of the antibodies, TF1.17 and TF1.15, recognized antigens which mi-

% Dead (-SD)

Antibody source

With

Without

complement

complement

Bovine preimmunization seruma 42.61 (0.43)b,C 2.84 (0.56) Bovine postimmunization seruma 62.33 (5.40)b.C 6.25 (2.16) TF1.17 ascites fluid 38.74 (6.17)b.c 13.54 (5.86) TF1.15 ascites fluid 73.06 (10.01)b c 26.07 (10.53)d TF3.8 ascites fluid 33.37 (9.10)bC 8.98 (3.62) TF3.2 ascites fluid 6.85 (1.88) 6.65 (1.36) FCSe 13.38 (5.68)c 6.75 (3.98) a Bovine serum was collected from cow 64 before (preimmunization) and 10 days after (postimmunization) the final immunization with live T. foetus. b Significantly greater than value for control with FCS plus complement (a = 0.025). c Complement-dependent killing was significantly greater than complementindependent killing (a = 0.025). d Significantly greater than value for control with FCS plus no complement

(a

0.025). Control, with no immunoglobulin.

= e

TABLE 3. Antibody-mediated inhibition of adherence of

T. foetus to bovine squamous VEC Antibody source

Dilutiona

Rate of adherence'

Bovine postimmunization serumd TF1.17 ascites fluid TF1.15 ascites fluid TF3.8 ascites fluid TF3.2 ascites fluid

1:500 1:2,000 1:2,000

0.044e (38) 0.031e (23) 0.037e (27) 0.110 (97) 0.094 (69)

1:500

1:2,000

a Chosen on the basis of ELISA titers (Table 1). b Ratio of number of squamous VEC with attached T. foetus to total number of squamous VEC; values were used for statistical analysis. c Calculated as follows: (rate of adherence with immune serum or ascites fluid)/(rate of adherence with FCS control) x 100. d Collected from cow 64 10 days after the final immunization with live T. foetus. e Significantly different from value for FCS control at P = 0.001.

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HODGSON ET AL.

grated as diffuse bands in Western blots, as is characteristic of glycoproteins because of heterogeneity in the degree of glycosylation. Thymol staining of these diffuse bands in SDS-PAGE was further evidence for glycosylation of the antigens. However, the reactivity of these two monoclonal antibodies was not decreased after mild periodate oxidation of blots. Thus, antibodies TF1. 17 and TF1.15 probably react with protein epitopes of the glycoprotein rather than carbohydrate epitopes. The abolition of concanavalin A reactivity in control blots after periodate oxidation but no change in protein staining was evidence that periodate oxidation altered carbohydrate but not protein epitopes, as expected. Antibody TF3.2 reacted with antigens of several molecular masses in Western blots. This result could have been due to cross-reacting epitopes on these proteins, multiple subunits of a high-molecular-weight antigen, or nonspecific binding of the monoclonal antibody to these proteins in this assay. Although antibody TF3.8 reacted predominantly with a high-molecular-mass antigen (-155 kDa) it also reacted slightly with a diffuse area below 100 kDa. This result may have been related to the functional activities discussed below. Previous studies demonstrated many T. foetus antigens reacting with bovine immune serum in Western blots (10, 15, 18). In our previous study, serum from cattle immunized with live T. foetus recognized bands within the 45- to 75-kDa range, similar to the reactivity of TF1.17 and TF1.15 (10). Huang et al. (18) also found a predominant surface glycoprotein antigen which migrated as a diffuse band between 45 and 65 kDa. The antigen was conserved in isolates from five western states and was detected by serum from immunized cattle. Since a similar immunization showed some protection (21), it is likely that this antigen will be an important immunogen for cattle. Surface exposure of antigens was demonstrated by several assays. Monoclonal antibodies TF1.17 and TF1.15 are apparently directed to surface epitopes, based on the results of the live-cell immunofluorescence assay, immobilization, and agglutination. Since all these assays showed negative results with monoclonal antibodies TF3.8 and TF3.2, we concluded that these antibodies were not directed to surface-exposed epitopes. With the two surface-reactive antibodies, the lack of fluorescence of some live cells may have been due to capping of a surface antigen cross-linked by an antibody, followed by internalization of complexes before reaction with the fluorescein conjugate. Alternatively, it is possible that not all of the trichomonads express these epitopes at all stages of the growth cycle or that there is a subpopulation which never expresses the antigen. The ability of antibodies to immobilize and agglutinate T. foetus may be protective. This conclusion is based on the pathogenesis of infertility. The initial site of infection in cows is the vagina. The organism subsequently migrates to the uterus (26, 30), where it can persist for many months and commonly results in prolonged infertility because of chronic endometritis (29). Thus, immobilization and/or agglutination should help prevent migration from the vagina and so prevent uterine infection, endometritis, and infertility. Furthermore, agglutination may deter penetration into the mucus layer and so decrease migration. Since TF1.17 and TF1.15 agglutinated T. foetus to higher titer than immune serum, antibodies to these antigens may prevent the migration of T. foetus in vivo and subsequent infertility. Complement-dependent killing of T. foetus should also be related to protection. The killing of some trichomonads by bovine complement in the absence of antibody may be due to

INFECT. IMMUN .

alternative pathway killing, as has been reported for Trichomonas vaginalis (13, 16) and T. foetus (3). The ability of bovine preimmune serum to produce an intermediate amount of complement-mediated killing was thought to be associated with cross-reacting IgM antibodies present in the serum of animals which have not been exposed to T. foetus. These antibodies were probably cross-reactive antibodies to either gut protozoa or gram-negative bacteria, as reported by Reece et al. (28). Similar cross-reacting antibodies of the IgM subclass were detected in the ELISA and Western blot analysis. Since IgM is very efficient in mediating complement killing, a low titer of IgM antibody should be sufficient to give the detected amount of killing. While these crossreacting serum IgM antibodies may protect against invasion of the bloodstream, they do not prevent mucosal infection, as indicated by the susceptibility of animals with serum IgM antibodies to genital infections, including trichomoniasis. This result is expected, since IgM antibodies are not transferred from serum to uterine secretions in cattle (9). Immune serum and antibodies TF1.17 and TF1.15 all initiated complement-mediated killing, confirming their surface specificity. Thus, if antigens recognized by TF1.17 and TF1.15 were used to stimulate an active systemic IgG response in cattle, the antibodies should be transferred to genital secretions and would be likely to play a role in protection. Antibody TF3.8 also produced an intermediate degree of complement-mediated killing which may have been related to the slight reactivity of this antibody with a faint diffuse band below 100 kDa in Western blots. Apparently, the reactivity was weak, since it not only produced a faint band in Western blots but also did not result in agglutination, immobilization, or immunofluorescence, even at high concentrations of antibody. Not only was complement-mediated killing detected in this study, but complement-independent killing was also observed with TF1.15. Although less killing was seen than in the presence of complement, complement-independent killing was significantly different from the control. Thus, whether complement is active in the presence of genital mucus or not, antibodies of the same specificity as TF1.15 may kill T. foetus. Earlier, Burgess (5) demonstrated complement-mediated killing by monoclonal antibodies against a 150-kDa surface antigen of T. foetus which was widely distributed among strains of T. foetus (6). None of the monoclonal antibodies, however, also mediated complement-independent killing. Alternatively, Alderete and Kasmala (2) demonstrated complement-independent lysis of T. vaginalis by monoclonal antibodies to a major glycoprotein antigen. The lysis may be similar to the complement-independent killing of T. foetus by monoclonal antibody TF1.15. Finally, we demonstrated the ability of antibodies to prevent the adherence of the trichomonads to bovine VEC. This ability may be similar to the inhibition of cytoadherence of T. vaginalis by monoclonal antibodies (1). However, Alderete and Garza (1) showed that an antibody inhibiting T. vaginalis cytadherence reacted with an antigen of .21 kDa, whereas the T. foetus antibodies involved in the inhibition of adherence reacted with diffuse bands of higher molecular masses. Adherence is an important virulence factor because it should promote vaginal colonization, persistent infection, and subepithelial inflammatory cell infiltration, which are characteristic of trichomoniasis. Furthermore, since T. foetus is first deposited in the vagina by the bull, inhibition of initial colonization by antibodies should prevent ascension to the upper reproductive tract, with accompanying infertility. Thus, the prevention of adherence by monoclonal anti-

VOL. 58, 1990

bodies TF1.17 and TF1.15 indicates that bovine antibodies to the same antigen(s) should prevent infection. In summary, we have characterized four monoclonal antibodies and compared their potentially protective functions with those of polyclonal serum antibodies to T. foetus. Two monoclonal antibodies, TF1.17 and TF1.15, were both directed to surface epitopes, as determined by agglutination, immobilization, and immunofluorescence of live T. foetus. They also caused complement-mediated killing, caused complement-independent killing (TF1.15), and prevented adherence of the organism to bovine VEC. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may be useful for protection against bovine trichomoniasis. ACKNOWLEDGMENTS We are grateful to Jeanine Kleeman and Nancy Hollingsworth for technical assistance and to Sharon McFarlin for preparation of the manuscript. This study was supported in part by U.S. Department of Agriculture grants 85-CRCR-1-1915 and 85-CRSR-2-2736. LITERATURE CITED 1. Alderete, J. F., and G. E. Garza. 1988. Identification and properties of Trichomonas vaginalis proteins involved in cytadherence. Infect. Immun. 56:28-33. 2. Alderete, J. F., and L. Kasmala. 1986. Monoclonal antibody to a major glycoprotein immunogen mediates differential complement-independent lysis of Trichomonas vaginalis. Infect. Immun. 53:697-699. 3. Aydintug, M. K., R. W. Leid, and P. R. Widders. 1990. Antibody enhances killing of Tritrichomonas foetus by the alternative bovine complement pathway. Infect. Immun. 58: 944-948. 4. BonDurant, R. H. 1985. Diagnosis, treatment and control of bovine trichomoniasis. Comp. Cont. Ed. Pract. Vet. 7:51795188. 5. Burgess, D. E. 1986. Trichomonasfoetus: preparation of monoclonal antibodies with effector function. Exp. Parasitol. 62:266274. 6. Burgess, D. E. 1988. Clonal and geographic distribution of a surface antigen of Tritrichomonas foetus. J. Protozool. 35:119122. 7. Clark, B. L., J. M. Dufty, and I. M. Parsonson. 1983. Immunization of bulls against trichomoniasis. Aust. Vet. J. 60:178-179. 8. Clark, B. L., D. L. Emery, and J. H. Dufty. 1984. Therapeutic immunization of bulls with the membranes and glycoproteins of Tritrichomonas foetus var. brisbane. Aust. Vet. J. 61:65-66. 9. Corbeil, L. B., J. R. Duncan, G. G. D. Schurig, C. R. Hall, and A. J. Winter. 1974. Bovine venereal vibriosis: variations in immunoglobulin class of antibodies in genital secretions and serum. Infect. Immun. 10:1084-1090. 10. Corbeil, L. B., J. L. Hodgson, D. W. Jones, R. R. Corbeil, P. R. Widders, and L. R. Stephens. 1989. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells. Infect. Immun. 57:2158-2165. 11. Diamond, L. S. 1983. Lumen dwelling protozoa: Entamoeba, Trichomonas and Giardia, p. 65-109. In J. B. Jensen (ed.), In vitro cultivation of protozoan parasites. CRC Press, Inc., Boca Raton, Fla. 12. Gander, J. E. 1984. Gel protein stains: glycoproteins. Methods Enzymol. 104:417-451.

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