New insights on the Golgi complex of Tritrichomonas foetus.

Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

First description of naturally acquired Tritrichomonas foetus infection in a Persian cattery in Spain.

Tritrichomonas foetus has been identified as the causative agent of feline intestinal trichomonosis, characterized by clinical signs of chronic large bowel diarrhoea. This disease has been reported in cats from the USA, Europe and Australia. However, its epidemiology is still unclear. The aim of the present study was to describe T. foetus infection in a Persian cattery in Spain. T. foetus infection was sequentially diagnosed in 20 cats by direct faecal smear examined under the microscope, specific culture (In Pouch TF medium) and PCR. A standard coprological sedimentation method was also performed in order to screen for other intestinal parasites in all the cats included. In addition, sera were tested for IgG antibodies against Leishmania infantum, Toxoplasma gondii, and for the detection of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). Five out of 20 cats were positive for T. foetus (25%), two of them by microscopy, culture and PCR and three by culture and PCR. No association was found between T. foetus infection and age or sex. L. infantum and T. gondii seroprevalence rates were 15% and 10%, respectively. The prevalence of FeLV p27 antigen and of FIV antibodies in the study population was zero. Cystoisospora spp. oocysts were detected in one cat. These preliminary results show that the transmission of T. foetus infection in cluster conditions may occur between asymptomatic cats and young or immunocompromised animals.

Comparative histopathology and antibody responses of non-Tritrichomonas foetus trichomonad and Tritrichomonas foetus genital infections in virgin heifers.

The potential pathogenicity of non-Tritrichomonas foetus trichomonads (NTfTs) recently isolated from the prepuce of virgin bulls is not known. The purpose of this study was to determine the ability of these NTfTs to cause disease in the female reproductive tract relative to T. foetus. Forty-four virgin heifers were experimentally infected intravaginally with either one of two NTfTs (Pentatrichomonas hominis or Tetratrichomonas spp.), T. foetus, or sterile media and cultured weekly from 0 time until slaughter at 8 weeks. Serum and vaginal antibody responses during infection were assessed, and the reproductive tracts were histologically examined, scored, and compared based on numbers of neutrophils, eosinophils, lymphocytes, and plasma cells as well as the qualitative appearance of the reproductive tract. The NTfTs did not persist in the reproductive tract, while T. foetus persisted for at least 6-8 weeks. Further, no vaginal IgA response to infection was found in NTfT-infected and control heifers, but a vaginal IgA response was present in the T. foetus-infected group. Heifers infected with NTfT or controls showed little mucosal inflammatory response compared to T. foetus-infected heifers. Among the trichomonads studied, persistent infection by T. foetus alone seems responsible for uterine inflammatory lesions usually associated with pregnancy loss. The NTfTs studied in this work only transiently infected the vagina and were associated with strictly mild inflammatory changes, which probably do not cause significant disease, i.e., pregnancy loss.

Systemic humoral immunity in beef bulls following therapeutic vaccination against Tritrichomonas foetus.

The utility of therapeutic vaccination of bulls against Tritrichomonas foetus has been advocated in previous studies, but anecdotal reports suggest this practice does not clear infections and may additionally confound diagnostic testing by reducing parasite burdens below detectable limits. The objective of this study was to characterize the systemic humoral immune response to therapeutic vaccination in T. foetus-infected bulls over a period of four months using an indirect ELISA and to compare the dynamics of this response to culture and PCR results to establish the existence of a relationship (or lack thereof) between immunization and infection status. A study population of 4- to 6-year-old T. foetus-infected beef bulls (n = 20) was divided equally into a treatment group and a control group. The treatment group received two doses of commercially prepared whole cell killed vaccine 2 weeks apart while the control group received injections of vaccine diluent. Blood samples were collected at each injection and at 4 subsequent dates every 4 weeks thereafter (i.e. 0, 2, 6, 10, 14, and 18 wks) to measure IgG1 and IgG2 antibody subisotype response via an indirect ELISA. Preputial smegma samples were collected at the four monthly intervals following vaccination for diagnosis of infection via InPouch™ culture, Modified Diamond's Medium (MDM) culture, and PCR. Humoral response for both IgG isotypes from week 2 through week 18 were significantly increased in vaccinates compared to controls. No significant decrease in infection prevalence was detected in the treatment group for any of the diagnostic methods used. The apparent lack of pathogen clearance during a stimulated immune response suggests that therapeutic vaccination may not be a useful T. foetus management practice.

Evaluation of Tritrichomonas foetus infection clearance in heifers immunized with a single intravaginal dose of formaldehyde fixed strain B1 cells.

Vaccines against Tritrichomonas foetus have been shown to reduce the time of infection after natural or experimental exposure. The object of this study was to assess the protection against T. foetus infection conferred by a single vaginal instillation of formaldehyde fixed T. foetus cells. Aberdeen Angus virgin heifers were randomly allocated to 3 groups of 12 individuals to receive placebo or formaldehyde fixed T. foetus cells prepared following one of two procedures (formalin or freshly prepared solution) and six weeks later they were challenged with 106T. foetus trophozoites. The median time for clearance among control heifers was 93.75 days while in animals immunized with formaldehyde fixed T. foetus it was 45 days. A single vaginal dose of cells fixed with fresh formaldehyde solution gave a rate of decay of infection per unit of time of 2.54 (CI 95% = 1.07;6.01).

Neutrophils and inducible nitric-oxide synthase are critical for early resistance to the establishment of Tritrichomonas foetus infection.

Tritrichomonas foetus is the cause of trichomoniasis in cattle. Severe infection is often associated with heavy neutrophil and macrophage accumulation, although it is not known how this response protects during early parasite colonization. The goal of this study was to examine the effects of an early host response upon initial T. foetus colonization within the murine reproductive tract. Mice depleted of neutrophils before T. foetus infection had a significantly higher parasite burden within the reproductive tract compared with mock-depleted control mice. Additionally, gp91(phox-/-)/ iNOS(-/-), and iNOS(-/-) mice had substantially larger parasite burdens than C57BL/6 control mice, whereas gp91l(Phox-/-) mice had similar parasite burden to C57BL/6 control mice. Interestingly, phorbol 12-myristate 13-acetate-stimulated neutrophils and macrophages isolated from all groups of mice were unable to kill T. foetus in vitro. However, macrophages isolated from gp91l(phox-/-) and C57BL/6 mice stimulated with interferon-gamma and lipopolysaccharide were able to kill T. foetus in vitro, whereas macrophages isolated from gp91(phox(-/-)/ iNOS(-/-) and iNOS(-/-) mice were unable to kill T. foetus, suggesting the ability of macrophages to produce reactive nitrogen species but not reactive oxygen species (ROS) is critical for parasite killing during early infection in vivo and in vitro. Additionally, neutrophils seem to control early dissemination of T. foetus throughout the reproductive tract, although production of ROS is not critical for this process.

Serum, uterine, and vaginal mucosal IgG antibody responses against Tritrichomonas foetus after administration of a commercial killed whole T foetus vaccine in beef cows.

The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus-purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus-specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of beef cows with a commercial vaccine containing T foetus induced significant increase in the levels of IgG to the T foetus TF1.17 surface antigen in serum, vaginal secretions, and uterine fluid, which remained elevated through Days 43, 75, and 182 in uterine fluids, vaginal secretions, and serum, respectively. Since purified TF1.17 antigen has been shown to protect against experimental T foetus infection in heifers, the vaccine-induced TF1.17-specific IgG response is likely to be important in the prevention of trichomoniasis in beef cattle.

Impact of a killed Tritrichomonas foetus vaccine on clearance of the organism and subsequent fertility of heifers following experimental inoculation.

Tritrichomonas foetus is a sexually transmitted reproductive pathogen of cattle that causes transient infertility, early embryonic death, metritis, pyometra, and sporadic abortions. The objective of this research was to assess the impact on reproductive health of vaccinating naïve heifers with a killed T. foetus vaccine (TrichGuard) before experimental exposure followed by breeding. A total of 40 beef heifers were randomly assigned into two treatment groups. Heifers where then vaccinated with two doses of TrichGuard or sham vaccinated with 0.9% sterile saline according to their respective groups. Sixty days following vaccination or sham vaccination, heifers were intravaginally inoculated with 2 × 106 organisms of a cloned isolate of T. foetus of bovine origin (CDTf-4) during synchronized estrus. Three days following inoculation of T. foetus, bulls free of T. foetus were introduced for natural breeding. Three bulls were maintained with the 40 heifers (20 vaccinated; 20 sham vaccinated) for a 49-day breeding season. Cervical mucous samples were obtained from each heifer at Day 0 and at 29 additional time points throughout the study for T. foetus culture. Pregnancy assessments were performed routinely by using transrectal palpation and ultrasonography. Pregnancies were detected in 19/20 (95%) vaccinated heifers and 14/20 (70%) sham-vaccinated heifers (P = 0.046). Only 4/20 (20%) of the sham-vaccinated heifers gave birth to a live calf compared with 10/20 (50%) of the vaccinated heifers (P = 0.048). Thus, embryonic or fetal loss was detected in 9/19 (47%) vaccinated heifers and 10/14 (71%) sham-vaccinated heifers (P = 0.153). The interval of time between inoculations with T. foetus and conceptions of pregnancies that were maintained until birth did not differ significantly between groups (vaccinated = 18.7 days; sham-vaccinated = 17.3 days; P = 0.716). The infectious challenge in this study proved to be very rigorous as a positive culture was detected from all heifers. The culture-positive results on the last culture day did not differ significantly (P = 0.115) between vaccinated heifers (63.9 days) and sham-vaccinated heifers (79.2 days). All uterine culture samples collected from the 26 nonpregnant heifers on Day 207 postinoculation did not result in the detection of T. foetus. These findings indicate that the killed, whole cell vaccine used in this study (TrichGuard) was effective in improving reproductive health evidenced by significantly reducing losses associated with T. foetus infections.

Evaluation of surface antigen TF1.17 in feline Tritrichomonas foetus isolates.

Tritrichomonas foetus (T. foetus) is a flagellated protozoa that infects the distal ileum and proximal colon of domestic cats, as well as the urogenital tract of cattle. Feline trichomonosis is recognized as a prevalent cause of chronic diarrhea in cats worldwide. The suspected route of transmission is fecal-oral, with cats in densely crowded environments at highest risk for infection. Thus, the recommended strategy for minimizing spread of infection is to identify and isolate T. foetus-positive cats from the general population. Rapid identification of infected cats can be challenging due to the inability to accurately and quickly detect the organism in samples at point of care facilities. Thus, identification of targets for use in development of a novel diagnostic test, as well as a vaccine or therapy for T. foetus infection is a significant area of research. Despite a difference in organ tropism between T. foetus genotypes, evidence exists for conserved virulence factors between feline and bovine T. foetus. The bovine T. foetus surface antigen, TF1.17, is an adhesin that is conserved across isolates. Vaccination with the purified antigen results in amelioration of cytopathogenicity and more rapid clearance of infection in cattle. We previously showed that three feline isolates of T. foetus were positive for TF1.17 antigen so we further hypothesized that TF1.17 is conserved across feline T. foetus isolates and that this antigen would represent an attractive target for development of a novel diagnostic test or therapy for feline trichomonosis. In these studies, we used monoclonal antibodies previously generated against 1.15 and 1.17 epitopes of the bovine T. foetus TF1.17 antigen, to evaluate for the presence and role of TF1.17 in the cytopathogenicity of feline T. foetus. A previously validated in vitro co-culture approach was used to model feline T. foetus infection. Immunoblotting, immunofluorescence assays, and flow cytometric analysis confirmed the presence and surface localization of antigen TF1.17 across all feline T. foetus isolates tested. Antigen TF1.17 was notably absent in the presumably nonpathogenic intestinal trichomonad, Pentatrichomonas hominis, a parasite that can be confused microscopically with T. foetus. Similar to bovine trichomoniasis, TF1.17 was found to promote T. foetus adhesion to the intestinal epithelium. These results support further characterization and development of the TF1.17 antigen as a possible target for the diagnosis and prevention of feline T. foetus infection.

Clearance of Tritrichomonas foetus in experimentally infected heifers protected with vaccines based on killed-T. foetus with different adjuvants.

Tritrichomonas foetus is a flagellated protozoan that causes a sexually transmitted disease in cattle. Trichomonosis is characterized by early abortions, subfertility and a significant decrease in productivity. Vaccine preparations containing whole T. foetus can reduce the time of residence of the pathogen in the host cervix after experimental infection. Here, T. foetus vaccines prepared with different adjuvants were tested, in parallel with a commercial vaccine, for their efficacy to clear the infection. The median time for clearance of infection was 69days in non-immunized animals, 55days in animals treated with aluminum hydroxide, 41days with oil-in-water or saponin based vaccines or with a commercial vaccine and 27days in animals treated with saponin plus aluminum hydroxide. A slight increase in the risk of T. foetus clearance from the genital tract was found with the saponin based vaccine (hazard ratio, 2.52; 95% confidence interval, 1.03-6.17) or the commercial vaccine (hazard ratio, 2.61; 95% confidence interval, 1.07-6.38). A significant increase in the risk of T. foetus clearance was found with the combination of saponin plus aluminum hydroxide based vaccine (hazard ratio, 5.12; 95% confidence interval, 2.04-12.83).

Host-parasite interaction in bovine infection with Tritrichomonas foetus.

Tritrichomonas foetus is a parasite of particular veterinary importance causing bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. The present review summarizes the current knowledge on potential mechanisms of pathogenicity of T. foetus, the immunology of host-parasite interaction in bovine tritrichomonosis, and the experimental model systems of this parasitic disease.

Polypeptides of hydrogenosome-enriched fractions from rumen ciliate protozoa and trichomonads: immunological studies.

The evolution of hydrogenosomes, energy-generating organelles of rumen ciliate protozoa and the flagellate trichomonads has been the subject of much speculation. Polypeptides of the hydrogenosome-enriched fractions from the rumen ciliates, Dasytricha ruminantium, Isostricha spp., Polyplastron multivesiculatum and Eudiplodinium maggii were separated by SDS-PAGE and compared to analogous polypeptide preparations from Tritrichomonas foetus. Immunoblotting with antisera specific to the hydrogenosomes of T. foetus identified common immunoreactive polypeptides present at estimated molecular masses of 28, 35, 38, 44, 48, 58, 100 and 120 kDa. That at 120 kDa corresponds to a single subunit of the purified pyruvate:ferredoxin oxidoreductase from the hydrogenosome of Trichomonas vaginalis.

Heifers immunized with whole-cell and membrane vaccines against Tritrichomonas foetus and naturally challenged with an infected bull.

The performance of a whole-cell vaccine and the other vaccine with cellular membranes of Tritrichomonas foetus applied to heifers naturally challenged by mating with an infected bull was determined. Forty heifers were divided into three groups: a control group (n=16) without immunizing, another group (n=12) immunized with whole cells (10(8)/dose) and a third group (n=12) immunized with cellular membranes (300 micro g of membranes/dose protein). The females were subcutaneously vaccinated at 3-week on two occasions and received a third intravaginal booster dose. After 3 weeks of the last vaccinal doses, the heifers were served by a T. foetus infected bull over 90-day period. The mean duration of infection for membrane-vaccinated heifers was 60 days +/-25, compared with 63 days +/-35.8 of infection for whole-cell-vaccinated heifers and 79 days +/-41.3 for control heifers. Calving rates were 6/12 for membrane-vaccinated heifers, 3/12 for whole-cell-vaccinated animals, and 2/16 for control animals. Fetal mortality rates were 3/12 for membrane-vaccinated animals, 4/12 for those vaccinated with whole cells and 10/16 for control animals. These reproductive parameters were significantly different (P<0.05) between heifers vaccinated with membranes and control heifers. The hemolytic test and enzyme-linked immunoabsorbent assay (ELISA) with T. foetus antigen showed that serum immunoglobulins peaked before and during the breeding period. The heifers vaccinated with membranes developed an important response during the critical period of fetal loss, second and third month of the breeding time, and another month after the same period. The ELISA method was more sensitive and more reliable than the hemolytic test for the evaluation of the systemic immune response in females infected and/or vaccinated with T. foetus.

Genital and systemic immune responses in a murine model of Tritrichomonas foetus infection.

A reliable laboratory animal model would be useful for the study of immune responses to trichomoniasis, a sexually transmitted disease of human beings and cattle. Murine models are available, but pretreatment with estrogen is used, which may influence immune responses. To evaluate whether vaginal trichomoniasis could be established in nonestrogenized mice and to define the immune responses associated with the infection, CD1 and BALB/c mice were studied with or without estrogen treatment prior to inoculation with Tritrichomonas foetus. Tritrichomonas Foetus was cultured from the vagina and uterus of both estrogen-treated and untreated control mice for up to 26 wk. The infection was sustained better in BALB/c than in CD1 mice, suggesting that the former strain was most susceptible. In CD1 mice, infection was sustained less well in estrogen-treated than in untreated control mice, but there was no difference between treatment groups of BALB/c mice. IgA and IgG antibodies in vaginal secretions, uterine secretions, and serum specific for a surface antigen of T. foetus (TF1.17) were measured by enzyme-linked immunosorbent assay. In infected CD1 mice, vaginal IgA and IgG antibodies were detected by 8 wk postinoculation (PI). In infected BALB/c mice, vaginal IgA and IgG antibodies were detected by 12 wk PI. Uterine IgG responses predominated over IgA in estrogen-treated and untreated CD1 and BALB/c mice. There were high levels of IgG, but relatively no IgA in the sera of CD1 and BALB/c mice. Overall, the highest IgA response was in the vaginal secretions of infected CD1 mice, and some animals of this strain cleared the infection. These results show that a chronic trichomonad infection was established in mice without prior treatment with estrogen. The infection was associated with antibody responses in reproductive secretions and serum. This animal model will be useful in studying immunization to protect against trichomoniasis in mice not immunocompromised by estrogen.

Immune and inflammatory responses to reproductive tract infection with Tritrichomonas foetus in immunized and control heifers.

Histopathologic changes and local antibody responses were studied in immunized and control heifers after intravaginal challenge with 10(6) Tritrichomonas foetus. Animals were given 3 intramuscular inoculations of immunoaffinity-purified superficial antigen, TF 1.17, in 2 different adjuvant combinations (incomplete Freund's adjuvant or dextran sulfate plus IFA-8 animals each) or adjuvant alone at 3-wk intervals and were challenged with T. foetus 2 wk later. Histologically, a nonsuppurative endometritis with nodular lymphoid aggregates in the stratum spongiosum was present in 9 of 24 heifers. Twice as many control heifers as immunized had moderate to severe endometritis at 10 wk and the rate of clearance of the organism was significantly faster in immunized than in control heifers. Furthermore, time of clearance was statistically correlated with severity of endometritis at 10 wk postinfection, when necropsies were done (P < 0.02). Because 9-10 wk postinfection is thought to be the critical period for determining fetal loss associated with endometritis, this correlation with early clearance is important to protection against disease. In heifers with moderate to severe infiltration of mononuclear cells in the endometrium, lymphoid nodules and some secondary follicles were detected. In the subgroup of 12 animals from which uterine secretions were collected. IgA antibody responses to antigen were detected by 6 wk in infected animals with increases in mean responses at 8 and 10 wk, but not in uninfected animals. A rationale is presented for consideration of the lymphoid nodules as a possible inductive site for this local antibody response to T. foetus.

Immunological and biochemical analysis of glycosylated surface antigens and lipophosphoglycan of Tritrichomonas foetus.

Immunoaffinity-purified TF1.17 adhesin antigen was compared biochemically and antigenically to Tritrichomonas foetus (TF) lipophosphoglycan (LPG) and a soluble glycosylated antigen (SGA) released from T. foetus and implicated in pathogenesis and immunity. The monoclonal antibodies (Mabs TF1.15 and TF1.17) specific for a glycosylated TF1.17 antigen were previously shown to prevent adhesion of the T. foetus parasites to bovine vaginal epithelial cells and to mediate killing by bovine complement. SGA was isolated from T. foetus-conditioned buffer and purified by octyl-Sepharose hydrophobic column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SGA showed a major SGA1 component (approximately 190 kDa) and a minor SGA2 component (50-70 kDa), which migrated close to TF-LPG and TF1.17. The carbohydrate and lipid compositional analyses of affinity-purified TF1.17 and SGA2 by high-performance liquid chromatography (HPLC) and gas-liquid chromatography revealed the presence of monosaccharides and fatty acids as found in TF-LPG. All antigens contained terminal fucose as determined by alpha-fucosidase digestion followed by HPLC. ELISA and western blots were used to further characterize these glycosylated antigens and to analyze their relationships. The Mabs TF1.15 and TF1.17 reacted very strongly to TF-LPG and SGA2. as well as TF1.17 antigen, indicating that these molecules share common epitopes. These Mabs did not react with the SGA1 component either in ELISA and western blot analyses. Also, the monosaccharide composition of SGA1 was very different from the other three antigen, suggesting SGA1 was different from LPG, SGA2 and TF1.17. Although LPG reacted with Mabs to native TF1.17 antigen, LPG did not induce an immune response in cattle with the same route and adjuvant used to produce strong antibody responses to the native antigen. The latter response suggests that the tightly bound peptide present in the immunoaffinity-purified antigen is necessary for induction of a response to (an) epitope(s) in TF-LPG and TF1.17. Furthermore, vaginal fluid from T. foetus-infected heifers and serum from a cow with a T. foetus-associated pyometra recognized both TF1.17 and TF-LPG in western blots. These results suggest that T. foetus LPG and SGA2 are related to TF1.17 antigen, which was previously shown to play an important role in the pathogenesis and host response in bovine trichomoniasis.

Characterization of antigenic proteins from Tritrichomonas foetus recognized by antibodies in rabbit serum, bovine serum and bovine cervicovaginal mucus.

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract and is recognized as 1 of the more common infectious agents causing decreased reproductive efficiency in beef cattle. Infections result in reproductive failure and produce considerable economic loss. Vaccination of heifers with vaccines containing T. foetus induces elevated serological responses to many T. foetus antigens, decreases the rate and/or length of infection with T. foetus, and decreases fetal loss caused by infection. Because T. foetus infections are usually limited to lumen and mucosal surfaces of the reproductive tract, it has been assumed that protection from infection and abortion is partially mediated by immunoglobulins in the uterus and vagina. The objective of this study was to identify and characterize specific antigens of T. foetus that show promise for use in a recombinant vaccine that will generate a protective mucosal immune response in cattle. Surface proteins were identified by using polyclonal rabbit anti-trichomonal sera eluted from paraformaldehyde-fixed cells. Analyses of these proteins, utilizing mucosal antibodies from vaccinated and convalescent cows, have identified proteins involved in generating a local immune response. Western immunoblot analysis indicates that these proteins are well conserved and are excellent candidates for incorporation into a recombinant vaccine.

Rapid internalization and degradation of surface-bound antibodies by Tritrichomonas foetus.

Tritrichomonas foetus is a protozoan parasite of cattle that can be cultured axenically. Three monoclonal antibodies specific for surface antigens of T. foetus were found to be rapidly internalized and degraded by these cells after binding. Degradation was not due to secreted or artificially liberated proteases but depended on targeting to internal degradative compartments. Radiolabeled catabolites of the antibodies were subsequently incorporated into the parasite's own proteins. Antibody degradation could be inhibited by certain protease inhibitors or lowered temperatures; a sharp reduction in degradation between 20 C and 15 C was similar to a well documented block in endocytic transport to degradative compartments of mammalian cells. Growth and proliferation of T.foetus in the continuous presence of the antibodies appeared unhindered, but there was a general shift toward expression of both more and less of each epitope among cells within each population. Subclones of these populations always exhibited striking variability in epitope expression levels, with patterns similar to the parent cultures. These findings may lead to a better understanding of how T. foetus resists host immune responses.

Antigen-specific T-cell responses in cattle immunized with antigens of Tritrichomonas foetus.

The cellular immune responses of cattle immunized with antigens of Tritrichomonas foetus were investigated. Subcutaneous injections of antigen preparations primed bovine peripheral blood mononuclear cells (PBMC) by 30 days of immunization as demonstrated by antigen-specific proliferation and by cytokine production upon antigen challenge of PBMC. Antigen-specific T-cells derived from PBMC responded by production of interferon (IFN)-gamma message detected by reverse transcriptase polymerase chain reaction, secreted IFN-gamma detected by enzyme-linked immunosorbent assay, and intracellular IFN-gamma detected by flow cytometry. Phenotypic analysis of PBMC responding in vitro to parasite antigen demonstrated a shift from a mixed CD4+, CD8+, gammadelta+, to predominantly CD4+, CD8-, gammadelta- phenotype in the Tf190-primed PBMC. In conclusion, systemic immunization of cattle with parasite antigen results in priming of bovine T-cells that are antigen specific and can produce an anamnestic IFN-gamma response to subsequent stimulation with antigens of T. foetus.