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

Accepted Manuscript Title: Evaluation of surface antigen TF1.17 in feline Tritrichomonas foetus isolates Authors: E.N. Gould, L.B. Corbeil, S.A. Kania, M.K. Tolbert PII: DOI: Reference:

S0304-4017(17)30337-0 http://dx.doi.org/doi:10.1016/j.vetpar.2017.08.001 VETPAR 8433

To appear in:

Veterinary Parasitology

Received date: Revised date: Accepted date:

26-4-2017 18-7-2017 2-8-2017

Please cite this article as: Gould, E.N., Corbeil, L.B., Kania, S.A., Tolbert, M.K., Evaluation of surface antigen TF1.17 in feline Tritrichomonas foetus isolates.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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

Gould EN1, Corbeil LB2, Kania SA2, *Tolbert MK1

University of Tennessee College of Veterinary Medicine, Departments of Small Animal Clinical Sciences and Biomedical and Diagnostic Sciences, Knoxville, TN

1. The University of Tennessee College of Veterinary Medicine, Knoxville, TN; Department of Small Animal Clinical Sciences (Gould, Tolbert) and Biomedical and Diagnostic Sciences (Kania).

2. The University of San Diego School of Medicine, San Diego, CA; Department of Pathology.

*Corresponding author: M. Katherine Tolbert DVM PhD DACVIM ([email protected]); Department of Small Animal Clinical Sciences, 2407 River Dr., University of Tennessee College of Veterinary Medicine, Knoxville, TN. Tel.:+1 865 974 8387; fax: +1 865 974 5554.

Highlights



A bovine Tritrichomonas foetus (TF) surface antigen is identified in feline TF.



The antigen, TF1.17, is conserved across all feline TF evaluated.



This antigen contributes to feline TF adhesion to in vitro porcine intestinal epithelial cells.



Feline TF1.17 is a novel diagnostic target for diagnosis of feline trichomoniasis.

Abstract

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 trichomoniasis 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 trichomoniasis. 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, TF1.17was 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.

Abbreviations mAb Monoclonal antibody IPEC-J2 Porcine intestinal epithelial cell CFSE Carboxyfluorescein succinimidyl ester

Keywords: Tritrichomonas foetus; 1.15-1.17; epithelium; protozoa; adhesion; epitopes

1. Introduction Tritrichomonas foetus (T. foetus) is a protozoal pathogen responsible for chronic diarrhea in domestic cats as well as infertility, abortions, and early embryonic death in cattle. Feline T. foetus is a global enteric pathogen with infections recognized in North America, Europe, and Australia, among others (Bissett et al., 2008; Gookin et al., 2004; Gunn-Moore et al., 2007; Hosein et al., 2013; Miro et al., 2011; Xenoulis, 2011). The suspected route of transmission is fecal-oral, with cats in densely crowded environments such as shelters, catteries, and cat shows

at highest risk for infection (Hosein et al., 2013). Thus, the recommended strategy for minimizing spread of infection is to identify and isolate T. foetus-positive cats from the general population. Rapid and accurate identification of infected cats can be challenging, particularly at point of care facilities, where in-clinic detection is currently limited to fecal cytology and InPouch™ culture. The sensitivities of these tests are low, 14 and 55%, respectively, (Foster et al., 2004; Gookin et al., 2003) and may be associated with misdiagnosis as a result of the presence of other intestinal protozoal pathogens that infect cats (e.g. Giardia spp and Pentatrichomonas hominis [P. hominis]). The best available diagnostic test, polymerase chain reaction (PCR), is not available as an in-clinic assay. Although PCR carries moderately good sensitivity when performed by experienced diagnosticians (Gookin et al., 2002), results are not available bedside, which results in a delay in diagnosis. Therefore, PCR may not be a viable option for shelters needing to frequently test suspect cats. Safe and effective therapies for the treatment of feline trichomononiasis are also limited. Only one drug, ronidazole, is available to treat feline T. foetus infection; however, this drug is used off-label for treatment of cats, has been associated with increasing resistance, and has a narrow margin of safety (Gookin et al., 2010; LeVine et al., 2011; Rosado et al., 2007). Thus, identification of targets for use in development of a novel diagnostic test, vaccine, or therapy for feline T. foetus infection is a significant area of research. Despite a difference in organ tropism, bovine and feline T. foetus are genetically similar and evidence exists for conserved virulence factors between genotypes (Morin-Adeline et al., 2014; Slapeta et al., 2012; Sun et al., 2012; Tolbert et al., 2014). The bovine T. foetus surface antigen, TF1.17, has been identified as an adhesin that facilitates parasite attachment to the urogenital epithelium (Hodgson et al., 1990). This surface

antigen, later identified to be part of the T. foetus protein/lipophosphoglycan (LPG) complex, was found to be conserved across 37 bovine isolates tested (Ikeda, 1993; Singh, 1994). The nature of the LPG glycosylation and relationship to TF1.17 has been previously elucidated and is well described (Singh, 1994); thus, deglycosylation studies were not pursued here. Treatment of bovine T. foetus with monoclonal antibodies directed against 1.15 and 1.17 epitopes of TF1.17significantly reduced adhesion of bovine T. foetus to bovine vaginal epithelial cells (Hodgson et al., 1990). Given the similarities previously identified between the two genotypes, and the previous identification of TF1.17 in three feline isolates of T. foetus, (Corbeil et al 2008), we hypothesized that feline T. foetus also express antigen TF1.17, which, based on the function of this antigen in bovine T. foetus, may represent a novel diagnostic and therapeutic target for feline trichomonosis.

2. Materials and Methods

2.1 Feline T. foetus isolates

Twelve axenized feline T. foetus isolates (Isolates A, B, C, D, F, Ja, JT, K, Ma, Mo, Sta and Sti) were used for all assays to account for variability amongst trichomonads. Isolates were harvested at the University of Tennessee and North Carolina State University from fecal samples obtained from naturally infected cats geographically distributed throughout the USA. Passage numbers 3-10 were used for all assays. An isolate of the nonpathogenic feline intestinal trichomonad, Pentatrichomonas hominis, harvested from the feces of a naturally infected cat, and two bovine T. foetus isolates, one from the American Type Culture Collection ([ATCC® 30003™], Manassas, KS) and one isolated from a naturally infected bull from Auburn

University (Bull #40204), were used in all assays. Feline P. hominis and bovine T. foetus isolates served as negative and positive controls, respectively. As only one P. hominis isolate has been harvested from a naturally infected cat, this was the only P. hominis isolate utilized. All isolates were cultivated as previously described (Gookin et al., 2001; Tolbert et al., 2012). The Aub bovine T. foetus isolate was donated from Dr. Chance Armstrong (Auburn University).

2.2 IPEC-J2 cells

The porcine jejunal epithelial cell line (IPEC-J2), a non-transformed primary cell line originally isolated from neonatal piglet jejunum, was cultured as previously described (Tolbert et al., 2014; Tolbert et al., 2013). IPEC-J2 cells were used at passage numbers 45-61. This line was originally isolated from neonatal piglet jejunum and was obtained as a gift from Helen M. Berschneider. Briefly, IPEC-J2 monolayers were cultivated in culture media that included Advanced Dulbecco’s Minimal Essential Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 5 g/ml each of insulin, transferrin, and selenium, EGF (5 ng/ml), penicillin (50,000 IU/ml), streptomycin (50,000 mg/ml) and 5% fetal bovine serum and incubated at 37°C in 5% CO2. All cell culture reagents were supplied by Thermo Fisher Scientific (Thermo Scientific, Rockford, IL). Prior to co-culture studies with feline T. foetus isolates, IPEC-J2 were seeded on 24-well polystyrene plates and allowed to reach confluence as previously described (Tolbert et al., 2014; Tolbert et al., 2013). Immediately prior to infection studies, culture media was replaced with co-culture media, which contained the same constituents as culture media but was devoid of serum to prevent replication of trichomonads and epithelial cells during infection studies. Uninfected IPEC-J2 monolayers and monolayers infected with isotype control (mouse IgM; Southern Biotech, Birmingham, AL) and DPBS-treated T. foetus served as negative,

isotype, and positive controls, respectively, for all co-culture assays using monoclonal antibodytreated T. foetus.

2.3 Monoclonal antibodies Monoclonal antibodies (mAbs) targeting bovine T. foetus surface epitopes 1.15 and 1.17, were used for all assays. Both mAbs had been prepared by standard methods using hybridomas prepared with splenocytes from BALB/c mice immunized with 0.8 x 106 live bovine trichomonads. (Hodgson et al., 1990) Ascetic fluid antibodies and cell culture supernatant were quantified via either radial immunodiffusion (RID) or enzyme-linked immunosorbent assay (ELISA). Ascetic fluid antibodies were quantified using murine RID kits (Rockland Antibodies and Assays, Limerick, PA) with IgM antiserum-infused agar. Immunoglobulin M quantities were determined using manufacturers’ instructions, with a standard curve created from the provided calibration controls and an unlabeled mouse IgM isotype (Southern Biotech, Birmingham, AL) as a reference control. Two individuals each scored all RID ring diameters and the average of the two readings were used, with all scores differing by a maximum of only 0.2 mm. Supernatant was quantified according to manufacturers’ instructions using a commercial ELISA (Abcam, Cambridge, MA) for murine IgM. All antibodies were stored as aliquots at -20°C to prevent antibody degradation from repetitive freezing and thawing.

2.4 Western blot Western blot analysis was used to evaluate for the presence of bovine TF 1.17 antigen in feline T. foetus isolates. Trichomonads at mid-logarithmic growth phase were washed twice with 1X Dulbecco’s Phosphate Buffered Saline (DPBS), lysed in 400 µL radioimmunoprecipitation assay (RIPA) buffer containing 1X phosphate buffered saline (PBS) (pH 7.4), 1% IGEPAL,

0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS), then sonicated twice for 15 seconds each prior to incubation for 30 min at 4°C. Lysates were centrifuged at 13,793 x g for 10 min at 4°C, with supernatant recovered and diluted in lithium dodecyl sulfate (LDS) buffer (Novex® by Life Technologies, Carlsbad, CA). Protein concentrations were determined with a bicinchoninic acid (BCA) assay according to manufacturer’s instructions (Thermo Scientific, Rockford, IL). Lysates were reduced in SDS reducing buffer (Novex® by Life Technologies, Carlsbad, CA) and heated at 70°C for 10 minutes prior to SDS-PAGE analysis. Proteins (i.e. equivalent of 180 µg lysate per lane) were electrophoretically separated at a voltage of 200V for 45 minutes using 4-12% Bis-Tris gels (Novex® by Life Technologies, Carlsbad, CA), and transferred to a nitrocellulose membrane at 30V for 60 minutes. Nitrocellulose membranes were blocked overnight in blocking buffer (Starting Block T20 buffer; Thermo Fisher Scientific, Rockford, IL) at 4°C. Immunoblotting was performed using the supernatant of anti-1.15 and 1.17 primary antibodies diluted 1:500 in Tris-Buffered Saline Tween-20 (TBST) for 4 hours at 22°C (room temperature [RT]) under gentle agitation. Membranes were washed three times with TBST for 15 minutes each under gentle agitation prior to addition of horseradish peroxidase-conjugated F(ab’)2 goat anti-mouse IgG and IgM secondary antibody (Jackson Immuno, West Grove, PA) diluted at 1:500 in blocking buffer for 30 minutes at RT. Electrophoresis using murine IgM in the place of primary antibody was performed to account for any non-specific binding. All membranes were washed in TBST six times for 15 minutes each prior to developing. Immunoblots were exposed to a chemiluminescent agent for 5 minutes (Thermo Fisher Scientific, Rockford, IL) and evaluated with ImageQuant™ LAS 4000 software. 2.5 Indirect immunofluorescence

Immunofluorescence (IF) was used as an additional qualitative assay to evaluate for the presence of TF1.17in feline T. foetus isolates. Mid-log phase whole organism trichomonads were pelleted at 1500 x g for 5 minutes at RT, washed once with 1X PBS, and re-suspended to 1 x 107 trichomonads/mL in 1X PBS. A hydrophobic barrier pen (Vector Laboratories, Burlingame, CA) was used to create barriers on slides pre-treated for electrostatic adherence (Thermo Fisher Scientific, Rockford, IL) in order to ensure trichomonads would remain contained to specified areas of the slide. 3 x 106 whole organism trichomonads were applied to slides pre-treated for electrostatic adherence (ThermoFisher Scientific) via direct pipetting to areas already denoted by the barrier pen and allowed to adhere at RT for 1 hour. All samples were fixed and permeabilized in acetone at -20°C for 15 min and then allowed to air dry for up to 3 minutes. Both adherence and fixation steps were optimized from an original protocol that utilized a cytospin to adhere trichomonads to slides without a permeabilizing agent, thus allowing surface localization of the antigen via IF. At centrifugation speeds required to successfully adhere whole T. foetus to slides, damage to protozoal membranes was observed. Thus, hand application with fixation was necessary to preserve the shape of trichomonads. Slides were rinsed briefly in 1X PBS for 5 minutes and incubated for 1 hr at RT in blocking buffer (1X PBS, 5% goat serum and 2% BSA). Slides were then incubated with either mAb (1.15 or 1.17) ascetic fluid or isotype control IgM diluted 1:100 in blocking buffer for 3 hours in a humidified chamber at RT. Following incubation with primary antibody or isotype control, slides were rinsed three times in 1X PBS for 5 minutes each followed by incubation in fluorescein isothiocyanate (FITC)conjugated F(ab’)2 goat anti-mouse IgG and IgM (Jackson Immuno Research, West Grove, PA) diluted 1:50 in block buffer for 1 hour at RT. Following three rinses with 1X PBS for 5 minutes each at RT, the nuclear counterstain, DAPI (i.e. 4’6-diamidino-2phenylindole) (Vectashield,

Vector Laboratories, Burlingame, CA) was applied for visualization of epithelial and trichomonad nuclei. Both confocal microscopy and differential interference contrast (DIC) imaging techniques were employed for imaging of IF assays. For confocal microscopy, specialized 0.17 mm coverslips (Thermo Fisher Scientific, Rockford, IL) were used. A Leica SP8 epifluorescence confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) was utilized for acquisition of all confocal and DIC images. 2.6 Flow cytometry Flow cytometry was performed for semi-quantitative analysis and confirmation of surface localization of TF1.17. Trichomonads, harvested at mid-logarithmic phase of growth, were pelleted at 1500 x g for 5 minutes, washed once in 1X PBS and re-suspended to 5 x 106 trichomonads/mL in flow buffer containing 60 mL of 0.5% sodium azide solution, 87 mL of 1X PBS, and 3 mL of fetal bovine serum. This flow buffer preserved trichomonad membrane structure while preventing permeabilization of the cells, thus allowing for the detection of epitopes 1.15 and 1.17 on the surface of feline isolates of T. foetus. The use of microcentrifuge tubes was avoided for all flow cytometric analyses as shear forces from a microcentrifuge lysed the protozoa, rendering it impossible to evaluate for the presence of surface antigens. Conical tubes (15 mL) preserved the shape of mid-log phase trichomonads and were used for all flow assays. Three isolates (A, Sti, Ja) were evaluated for the presence of both epitopes with flow cytometric analysis. Each isolate was divided into 1 mL aliquots for the following treatment groups: (1) T. foetus receiving both primary and secondary antibody, (2) T. foetus receiving equivalent concentrations of murine IgM (isotype control) or negative control (i.e. lacking application of any primary antibody or murine IgM) and, (3) T. foetus receiving no antibodies (auto-fluorescence). Isolates were pelleted, and 10 µL of one of two primary antibodies (i.e.

mAb 1.15 or 1.17 supernatant or ascetic fluid preparations) or an equivalent concentration of murine IgM was added to the pellet for a final dilution of 1:100 per 1 mL of re-suspended trichomonads. All samples were incubated on ice for 30 minutes and washed once with 1 mL of 1X PBS. Fluorescein isothiocyanate (FITC)-conjugated F(ab’)2 goat anti-mouse IgG and IgM antibody was applied at the same concentration of 1:100 directly to the pellet, incubated for 30 min on ice, washed once more, and re-suspended to a final volume of 1 mL in 1XPBS for analysis. A total of 10,000 events (i.e. trichomonads) were analyzed for each treatment population, and each isolate was analyzed in triplicate. All data were acquired on an Attune® acoustic focusing flow cytometer (Applied Biosystems, Thermo Fisher Scientific, Rockford, IL). 2.7 Flow cytometric analyses for evaluation of surface capping To account for any effect of surface capping, mid-log phase trichomonads (isolate A) were prepared as described above under flow cytometry (2.6) and treated with either 1:100 primary antibody (i.e. ascetic fluid mAb 1.15 or 1.17) or an equivalent concentration of isotype control (mouse IgM). Flow cytometric analysis on the same isolate was performed at t=0, 30 minutes, 45 minutes, and hourly for a period of 4 hours to evaluate for a change in mean fluorescence. All trichomonads were maintained in non-serum co-culture IPEC-J2 media in order to mimic the conditions of co-culture assays, until the respective time points of analysis. At each time point, trichomonads were fixed with sodium azide buffer immediately prior to incubation with the respective treatment and the remainder of the assay carried out just as detailed above in flow cytometry (2.6). 2.8 Co-culture adhesion assays

To quantitatively evaluate the effect of mAbs 1.15 and 1.17 on the adhesion of feline T. foetus to the intestinal epithelium, adhesion assays were performed using uninfected monolayers and monolayers infected with isotype control-treated (mouse IgM, Southern Biotech, Birmingham, AL) or mAb (i.e. anti-1.15 or 1.17)-treated T. foetus. Carboxyfluorescein succinimidyl ester (CFSE) (CellTrace™ CFSE cell proliferation kit, Thermo Fisher Scientific, Rockford, IL) was used to fluorescently label viable trichomonads as previously described (Tolbert et al., 2013) prior to treatment with antibody or isotype control groups. IPEC-J2 monolayers were cultivated to confluence at a previously established concentration of approximately 5 x 104 cells/well in 24-well polystyrene plates prior to infection with T. foetus as previously described (Tolbert et al., 2014; Tolbert et al., 2013). IPEC-J2 monolayers were inoculated with 10 x 106 T. foetus per well as previously described (Tolbert et al., 2014; Tolbert et al., 2013), with all trichomonad groups pre-treated with either one of four concentrations of ascetic fluid mAb (anti-1.15 or 1.17) or a volume of mouse IgM equivalent to the same µg of mAb used, prior to infection. IPEC-J2 monolayers were infected with trichomonads within a few minutes following application of the respective treatment (i.e. antibody or isotype control) to each group. Any volume differences between the isotype control and mAb-treated groups were accounted for with addition of DPBS. A third group containing only ascetic fluid mAb (1.15 or 1.17) at the appropriate dilution but lacking T. foetus was used as an additional control to evaluate for nonspecific binding of antibody to the IPEC-J2 cells. Infected and uninfected groups of IPEC-J2 cells were maintained at 37°C in 5% CO2. Each respective treatment was re-applied at hourly intervals to account for the effect of surface capping (i.e. diminishing effect of antibody due to antigen-antibody complex internalization). After 6 hours of co-culture, an established time for adhesion based on a previously validated co-culture model (Tolbert et al., 2013), the cells were

washed twice gently with 37°C DPBS and counterstained with DAPI (Vectashield, Vector Laboratories, Burlingame, CA). Adherent trichomonads in individual wells were counted in six high power fields (HPFs) using an epifluorescence microscope (Nikon® Digital Sight DS, Melville, NY), with the average count representative of one replicate. Two feline T. foetus isolates (A, Sti) were utilized for adhesion experiments. Assays were performed with a minimum of four replicate cultures per treatment group and repeated for a minimum of two experiments per isolate. Mean or median adherence were compared among all groups with either parametric or nonparametric distributions, respectively. 2.9 Crystal violet cytotoxicity assays To provide a quantitative analysis of the effect of mAb 1.17 on T. foetus-induced epithelial cytotoxicity, crystal violet (CV) assays were performed, as previously described (Tolbert et al., 2014), using uninfected monolayers and monolayers infected with one of four concentrations of ascetic fluid mAb (i.e. anti-1.17) or isotype control (mouse IgM) -treated T. foetus. Monolayers were infected as described above for adhesion assays, with the exclusion of labeling with CFSE. Each respective treatment was re-applied at hourly intervals, as described above in the methods for adhesion assays (2.8). After 24 hours of co-culture (time based on previously established cytotoxicity model), wells were washed twice with DPBS and stained with crystal violet as previously described (Tolbert et al., 2014). IPEC-J2 monolayers were gently washed twice with dH20 and allowed to air dry. Monolayers were immediately observed by light microscopy using a Nikon inverted light phase-contrast microscope (Nikon® Digital Sight DS-U3, Melville, NY), then were solubilized in 100 L 1% SDS in 50% ethanol for spectrophotometric analysis. Solubilized cells were transferred to 96 well plates and the intensity of staining was quantified using a spectrophotometer at a wavelength of 570 nM. Assays were performed in a minimum of

4 replicate cultures per treatment group. Spectrometric means or medians were compared among all groups (T. foetus with mAb or isotype control [murine IgM] treatment and uninfected monolayers) for either parametric or nonparametric distributions, respectively. 2.11 Statistical Analysis Data were analyzed for normality (Shapiro-Wilk) and variance (Brown-Forsythe) using a statistical software package and tested for significance using parametric or non-parametric tests as appropriate (Sigma Plot13, Systat Software, Inc.). Parametric data were analyzed using a Student’s t-test or one way ANOVA, and non-parametric data were analyzed using a MannWhitney rank sum test or one way ANOVA on ranks. Each assay performed had a separate statistical analysis, so that different isolates and replicate assays for the same isolate on different days were still individually analyzed. Each significant result reported was confirmed with a minimum of two additional duplicate assays which also achieved statistical significance. n = number of replicates. Results are reported as mean ± standard deviation. For all analyses, P  0.05 was considered significant.

3. Results 3.1 Bovine TF1.17 is present in feline T. foetus isolates Western blotting was performed to evaluate for the presence of antigen TF1.17 across multiple feline T. foetus isolates. Protein lysates from twelve feline and two bovine T. foetus isolates were found to possess a diffusely migrating band, characteristic of a glycosylated antigen (e.g. lipophosphoglycan protein complex), ranging from approximately 36 to 64 kDa following incubation with mAb 1.15 (Fig. 1A). Diffuse bands of approximately the same molecular weight were also seen in the same isolates following application of TF1.17 (Fig. 1B). Both epitopes

were identified to be absent in P. hominis (Fig. 1A and B). Non-specific binding was not appreciated following substitution of mAbs with isotype control, denoted by lack of a positive band. Although equivalent concentrations of protein lysate, based on the previously performed BCA assay, were evaluated in SDS-PAGE analysis, no acceptable protein loading control is available for trichomonads. Thus, densitometric quantification of antigen was not performed. Indirect immunofluorescence confocal microscopy assays were used as an additional qualitative methodology to confirm presence of TF1.17 in feline T. foetus. Immunofluorescence was first confirmed using a bovine T. foetus isolate (Aub) obtained from a naturally infected bull for both 1.15 and 1.17 (Fig. 2A). Feline T. foetus isolates displayed patchy positive fluorescence following application of mAb 1.17 (Fig. 2B). No fluorescence was observed following application of either mAb to P. hominis (Fig. 2C). All trichomonad populations were negative for fluorescence following omission of all antibodies (i.e. auto-fluorescent controls) and after application of either isotype control or secondary antibody alone (i.e. negative controls). 3.2 TF1.17 is expressed on the surface of feline T. foetus Flow cytometry was used to confirm surface localization of TF1.17. Figure 3 graphically represents the positive fluorescence of one bovine and one feline T. foetus isolate following treatment with mAb 1.15 or 1.17 supernatant, and the relative negative fluorescence of the respective auto, isotype and/or negative control populations. The use of sodium azide fixative, rather than a permeabilizing agent such as formalin, prevented surface capping (i.e. inversion of the cell membrane) and confirmed positive fluorescence was due to the presence of a surface localized, rather than intracellular, antigen. Sodium azide is a fixative agent that has been shown to produce repeatable results when used for flow cytometry (Cuchens and Buttke, 1984), and might do so in trichomonads via inhibition of ATP and ADP metabolism (de Aguiar Matos et al.,

2001). Feline T. foetus isolates demonstrated surface expression of T. foetus (isolate Sti) epitopes 1.15 (Fig. 3A) and 1.17 (Fig. 3B). The average mean fluorescence of all bovine T. foetus treatment groups for auto fluorescence, negative control, and trichomonads treated with either mAb 1.15 (Fig. 3C) or 1.17 (Fig. 3D) supernatant were 1.7, 6.1, 15.7 and 22.9 mean fluorescent units (MFUs), respectively. Average mean fluorescence of bovine controls following addition of mAbs 1.15 and 1.17 were 2.9 and 3.8 times greater than the negative control, respectively. The average fluorescence of auto-fluorescent, negative control, and isotype control populations (i.e. treated with equivalent concentrations of murine IgM) of feline T. foetus isolate A were 29.5, 100, and 730 (equivalent to mAb 1.15) or 130 (equivalent to mAb 1.17) MFUs (Fig 4). Subsequent treatment of the same populations with mAb 1.15 and 1.17 yielded 1,359 and 972 MFUs (Fig.4). Isolate A had average MFUs 8.5 and 1.4 times greater than that of isotype control populations following addition of mAbs 1.15 and 1.17, respectively. Similarly, feline T. foetus isolate Sti had auto-fluorescent and negative control MFUs of 17.5 and 63.3, which increased to 82.7 and 128.3 MFUs with addition of mAbs 1.15 and 1.17, respectively. This resulted in average MFUs that were 1.3 and 2 times greater than negative control populations for isolate Sti. Although some of the increases in MFUs compared to isotype control were subjectively minimal (e.g. 1.3-1.4 fold increases), these values were averaged from multiple replicates. The single feline P. hominis isolate had auto-fluorescent and negative MFUs that were equivalent, with negative control populations only 4% more fluorescent than the auto control. Addition of mAbs 1.15 and 1.17 to P. hominis resulted in MFUs equivalent to those of the negative control populations, with populations that were only 9% and 17% less fluorescent, respectively. Mean fluorescence units of mAb-treated groups were compared to either isotype (receiving equivalent concentrations of murine IgM and FITC-conjugated secondary antibody) or negative (lacking

addition of primary antibody) control populations to provide an accurate reflection of fluorescence secondary to the presence of epitopes 1.15-1.17 rather than background fluorescence and non-specific binding of secondary antibody. Some positive fluorescence was seen when evaluating isotype controls in comparison to auto-fluorescent populations, indicating some non-specific binding of murine IgM to feline T. foetus isolates. 3.3 Internalization of surface antigen-antibody complexes occurs within 2 hours of complex formation Mean fluorescent units of isolate A treated with either ascetic fluid mAb or isotype control murine IgM consistently increased from baseline to 1 hour, then declined between one and two hour time points. Figure 5 illustrates the changes seen in MFUs over time for isolate A following application of mAb 1.15, 1.17 or corresponding isotype control murine IgM. The MFUs for isolate A treated with 1:25 mAb 1.17 at baseline, 30 minutes, 45 minutes and one hour were 82, 96, 98 and 123. This translated to a fluorescence at one hour that was 1.5, 1.3 and 1.2 times more fluorescent than baseline, 30 and 45 min groups, respectively. Between hours one and two, the MFUs were 123 and 84 for mAb 1.17, respectively. The MFUs for isolate A treated with 1:25 mAb 1.15 at baseline, 30 minutes, 45 minutes and one hour were 1,187, 1,226, 1,260 and 1,299. This translated to a fluorescence at one hour that was 1.1 times more fluorescent than baseline, and equivalent to the 30 and 45 min groups, respectively. Between hours one and two, the MFUs were 1,299 and 985 for mAb 1.15. This indicated internalization of surface antigenantibody complexes (i.e. surface capping) and diminishing fluorescence between 1 and 2 hours following application. Similarly, murine IgM at an equivalent concentration to mAb 1.17 was found to have relatively static MFUs for the four time points from baseline to 1 hour (59, 84, 86 and 90), which then decreased from 90 back down to 52 between hours one and two. When

applied at equivalent concentrations to mAb 1.15, the MFUs of the isotype control were 950, 1,010, 1,100 and 1,080 for the first hour, then decreased to 800. At all time points, motility was assessed to be normal in greater than 90% of the trichomonads in each respective group. Subjectively, agglutination was not appreciated. 3.4 TF1.17 participates significantly in feline T. foetus adhesion to but not cytotoxicity towards the intestinal epithelium To investigate the role of TF1.17 in facilitating adhesion and cytotoxicity of feline T. foetus to the intestinal epithelium, adhesion and cytotoxicity assays were performed.. A significant decrease in the average number of 1:25 mAb 1.15- treated feline T. foetus adhered following 6 hours of co-culture was observed (P = 0.01; Fig. 6A). Similarly, there was a significantly decreased number of 1:25 mAb 1.17 treated-feline T. foetus adhered to IPEC-J2 cells (P < 0.01) when compared to monolayers infected with equivalent concentrations of isotype control-treated feline T. foetus at the 6 hour time point (Fig. 6B). A dose response curve was used to determine the lowest effective concentration of mAb 1.17 effective at decreasing T. foetus adhesion to IPEC-J2 cells, and found that inhibition occurred at a 1:150 dilution but a dilution of 1:200 did not significantly affect adhesion (Fig. 7). In order to assess for any possible cytotoxicity directly secondary to application of isotype control to IPEC-J2 cells, crystal violet assays were initially performed independent of T. foetus infection. After 10 hours, a significantly greater amount of cytotoxicity was seen in the group treated with 1:25 isotype control compared to the uninfected DPBS control (P=0.04). This led to the conclusion that the high concentrations of murine IgM might be inducing toxicity to the IPEC-J2 cells. Given that lower dilutions of antibody (e.g. 1:100, 1:150) also significantly decreased adhesion, crystal violet assays were performed using these concentrations to determine

if they were effective at modulating cytotoxicity. Neither 1:100 or 1:150 concentrations of mAb 1.17 were effective at significantly reducing cytotoxicity to IPEC-J2 cells (P > 0.05). A dose response curve was performed to assess the highest concentration of murine IgM that was not cytotoxic to IPEC-J2 cells, with the goal being to find a concentration that did not directly induce cytotoxicity to epithelial cells but was also effective in diminishing T. foetus induced toxicity to IPEC-J2 cells. Three additional concentrations were assessed (1:50, 1:75 and 1:100), and compared to equal volumes of DPBS as a control. The 1:50 concentration caused significant cytotoxicity to epithelial cells, whereas 1:75 and 1:100 did not. Based on this, final cytotoxicity assays were performed with four treatment groups: (1) 1:75 mAb 1.17-treated T. foetus, (2) equivalent concentrations of murine IgM isotype control- treated, (3) DPBS-treated T. foetus and (4) uninfected IPEC-J2 cells treated with DPBS only (i.e. lacking protozoa). Groups of IPEC-J2 cells infected with 1:75 mAb-treated T. foetus displayed no significant differences in cytotoxicity when compared to either isotype control murine-IgM (P=0.156) or DPBS-treated (P=0.198) groups. The isotype control murine IgM-treated group showed a significant decrease in cytotoxicity compared to the DPBS-treated (P=0.002) control. When compared to the uninfected control, the DPBS-treated T. foetus group showed significantly more cytotoxicity (P=0.042), whereas there was no significant difference in cytotoxicity between mAb-treated (P=0.425) or IgM-treated T. foetus (P=0.426) and uninfected controls (Fig. 8).

Discussion Despite the difference in organ tropism, bovine and feline T. foetus are genetically similar and utilize analogous virulence factors in their pathogenicity (Morin-Adeline et al., 2014; Slapeta et al., 2012; Tolbert et al., 2014). Given the role of the previously discovered bovine T. foetus

surface antigen in mediating T. foetus adhesion to the intestinal epithelium, TF1.17 represented an appealing marker for exploration as a diagnostic, preventative, or therapeutic target in feline trichomonosis. This antigen has also been identified in the soluble fraction (SF) of bovine T. foetus protein lysate (Bondurant et al., 1993), which, if also present in feline T. foetus SF, could open the future possibility of a copro-antigen assay. Our first study objective was to determine if TF1.17 antigen was conserved across multiple feline T. foetus isolates. Using immunoblotting and confocal microscopy immunofluorescence, we demonstrated that TF1.17 was expressed by all twelve feline T. foetus isolates tested, which confirmed our preliminary findings in three feline isolates of T. foetus (Corbeil et al., 2008). Moreover, Pentatrichomonas hominis, a nonpathogenic feline intestinal trichomonad that can be confused for T. foetus using in-clinic diagnostics such as fecal smear and In-Pouch™ TF culture (Ceplecha et al., 2013; Gookin et al., 2003), did not express the TF1.17 antigen, as we showed previously for P. hominis from the genital tracts of cattle (Corbeil et al., 2008). While immunofluorescence is often used to denote localization of an antigen (i.e. surface versus intracellular), acetone was used as a fixative in these assays. Given that acetone acts as a simultaneous permeabilizing agent, results confirmed presence of TF1.17 but did not distinguish between intracellular and surface localization. Flow cytometric evaluation was necessary to demonstrate that, similar to the bovine genotype, this antigen was located on the surface of feline T. foetus. The culmination of our initial results suggests that the feline TF1.17 antigen is conserved across multiple isolates and surface localized, similar to bovine T. foetus. The TF1.17 antigen is also recognized to be an important virulence factor for bovine T. foetus where it functions as an adhesin, facilitating attachment of the parasite to the urogenital epithelium.

Given the precedential role established for TF1.17 antigen in bovine cytopathogenicity, a second objective of the present study was to evaluate if TF 1.17 antigen also functioned as a virulence factor in feline trichomoniasis. Assays for our second objective were performed with a previously validated co-culture model system (Tolbert et al., 2014; Tolbert et al., 2013) using porcine jejunal epithelial (IPEC-J2) cells as no nontransformed feline intestinal epithelial cell line is available. Moreover, feline T. foetus and porcine T. foetus, formerly known as Tritrichomonas suis, are genetically similar and both have a tropism for the gastrointestinal tract (Mostegl et al., 2011; Slapeta et al., 2012; Tachezy et al., 2002). To characterize the role of this antigen in feline T. foetus infection, feline isolates were pre-treated with mAbs directed against epitope 1.17 of TF1.17 antigen prior to co-culture with IPEC-J2 monolayers. Similar to bovine T. foetus, inhibition of epitope 1.17 significantly reduced adhesion of feline T. foetus to the intestinal epithelium in a dose-dependent manner. These findings suggest that TF1.17 antigen promotes adhesion of feline T. foetus to the intestinal epithelium in vitro, which might have important implications for induction of cytotoxicity in feline trichomononiasis given that adhesion has been found to play an integral role in this protozoa’s induction of intestinal epithelial cell cytotoxicity (Tolbert et al., 2013). Despite this, it has also been shown that pathogenic protozoa such as bovine T. foetus, Giardia and Entamoeba sp. utilize additional virulence factors that contribute to host epithelial cell damage (GarciaRivera et al., 1999; Olivos-Garcia et al., 2015; Singh et al., 2005). Thus, is it not surprising that although antibody to TF1.17 antigen decreased adhesion to the intestinal epithelium, the same concentration of antibody did not significantly mitigate cytotoxicity to host cells. Higher concentration of antibody might be necessary to effectively decrease cytotoxicity compared to that necessary to decrease adhesion, as seen in previous work with TF1.17 (Hodgson et al.,

1990). Although a concentration of 1:25 monoclonal antibody was chosen for flow cytometric evaluation of surface capping in order to mimic the highest concentration of antibody used in crystal violet assays, it is also possible that the flow cytometry data does not accurately reflect capping when trichomonads are co-cultured with epithelial cells for prolonged periods of time (i.e. > 20 hours), as is required for observation of cytotoxicity. If capping were to occur more rapidly, the amount of available antibody might be diminished to the point where an effect would not be apparent. Another conclusion from our work was that, while mAb 1.17 did not ameliorate in vitro cytotoxicity towards intestinal epithelial cells in these experiments, murine IgM-treated T. foetus had significantly less cytotoxicity compared to other treatment groups. This effect may be due to non-specific binding of IgM on the surface of T. foetus isolates, similar to bovine IgM (Corbeil et al., 1991). We postulate this because, in our cytotoxicity assays, murine IgM-treated groups had significantly less cytotoxicity than both mAb and DPBS-treated groups. While further study would be necessary to prove this result repeatable, one conclusion would be that murine IgM decreases the ability of feline T. foetus to attach to host cells by non-specifically binding or by cross-reactive natural antibody binding to surface antigens necessary for attachment. This is supported by our flow cytometry data that show that while murine IgM-treated trichomonads are less fluorescent than those treated with monoclonal antibody, their mean fluorescent units exceed that of the auto-fluorescent populations. This signifies that there is some non-specific or crossreactive binding of murine IgM by feline T. foetus. In conclusion, the present work confirms that feline T. foetus possess the previously identified bovine TF1.17 antigen, and it is shared across all feline isolates, harvested from the feces of cats residing in the continental United States, tested thus far. Documentation of this

antigen in 12 feline isolates provides continued evidence of similarities between feline and bovine genotypes. Also similar to the bovine genotype, the TF1.17 antigen significantly facilitated adhesion of T. foetus to the intestinal epithelium in an in vitro model. Moreover, TF1.17 was absent in the non-pathogenic feline intestinal trichomonad, Pentatrichomonas hominis, which might make it a good candidate marker for a novel, on-site diagnostic test.

Acknowledgments This project was supported by the Winn Feline Foundation (Tolbert W15-011). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the views of Winn. We would like to thank Drs. Jody Gookin (North Caroline State University), Rebecca Wilkes (University of Tennessee) and Chance Armstrong (Auburn University) for their generous donation of antibodies and trichomonad isolates. We would also like to acknowledge John Dunlap (University of Tennessee) and Jonathan Wall (University of Tennessee Medical Center) for their assistance in acquisition of immunofluorescence and CFSE-adhesion images, as well as Dianne Trent, Mabre Brand, and Rachel Dickson (University of Tennessee) for their assistance with immunoblotting and flow cytometry assays.

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Figure Captions

Figure 1. Western blot confirms presence of TF1.17 in feline T. foetus (Tf) isolates and absence in Pentatrichomonas hominis (P. hominis). Panel A reacted with mAb 1.15 and Panel B reacted with mAb 1.17. Protein lysates were harvested from 2 bovine Tf isolates (ATCC, Aub; lanes 3-4), 12 feline Tf isolates (5 depicted here are: A, Sti, Ja, Mo and F isolates; lanes 59) and 1 feline P. hominis isolate (lane 10). Immunoblotting confirms the presence of epitopes 1.15 (1A) and 1.17 (1B). P. hominis was negative for the presence of both epitopes. The diffusely migrating appearance of the band is characteristic for glycosylated antigens.

Figure 2. Indirect immunofluorescence confirms presence of TF 1.17 in bovine (Aub) and feline (A isolate) T. foetus (Tf). Patchy fluorescence was noted following addition of 1:100 mAb 1.17 to bovine (A) and feline (B) Tf isolates. Auto-fluorescent (i.e. lacking application of all antibody), and negative (i.e. lacking addition of primary antibody only) controls were also evaluated when testing all isolates. (C) No green fluorescence was observed following addition of mAb 1.17 in a feline P. hominis isolate. Auto-fluorescent (i.e. lacking application of all antibody), and negative (i.e. lacking primary antibody only) controls were negative in all isolates. 620X magnification.

Figure 3. Flow cytometry confirms surface localization of TF1.17 in a feline T. foetus (Tf) isolate. Histograms denote positive fluorescence in one feline Tf isolate (Isolate Sti; A, B), one bovine Tf isolate (C, D), and negative fluorescence in a P. hominis isolate (E, F) following addition of mAb 1.15 or 1.17 and FITC-conjugated goat anti-mouse F(ab’)2 IgG + IgM secondary antibody. For all histograms, the X axis represents the mean fluorescence intensity (FITC) on a logarithmic scale and the Y axis the cell count. Each peak, moving from left to right along the X axis, represents the mean fluorescence of the following groups: auto fluorescence (i.e. gray peaks for A, B, E and F, red peaks for C,D; lacking addition of any antibody), isotype control (i.e. blue peaks for A,B,E and F, gray peaks for C,D; equivalent concentrations of murine IgM and 1:100 FITC-conjugated secondary antibody) and fluorescence following application of either mAb 1.15 (A, C, E) or mAb 1.17 (B, D, F) with FITC-conjugated secondary antibody (i.e. all black peaks). Assays were performed in triplicate with 10,000 events counted for each treatment population.

Figure 4. Mean fluorescent units of feline T. foetus increases with application of mAbs 1.15 and 1.17 compared to controls. Mid-log phase feline T. foetus (isolate A) were treated with either 1:100 preparation of mAb 1.17 or 1.15. The Y axis represents the mean fluorescent units (MFUs) for a particular treatment group. Groups are denoted as follows: 1) Auto fluorescent control (lacking primary and secondary antibody; grey bar), 2) Negative control (lacking primary antibody; white bar), 3) Isotype control 1.15 (murine IgM at equivalent concentration to mAb 1.15; first hatched bar), 4) Monoclonal antibody 1.17 (1:100 mAb 1.15; first black bar), 5) Isotype control 1.17 (murine IgM at equivalent concentration to mAb 1.17; second hatched bar), 6) Monoclonal antibody 1.17 (1:100 mAb 1.17; second black bar).

Figure 5. Surface antigen-antibody complexes of both mAb 1.15 and 1.17, as well as murine IgM controls, undergo membrane internalization between 1 and 2 hours. Mid-log phase feline T. foetus (isolate A) were treated with either 1:25 ascetic fluid preparation of mAb 1.17 (5A) or 1.15 (5B) and incubated for 30, 45, 60 and 120 minutes in 5% CO2 at 37°C in nonserum co-culture media. Figures 5C and 5D represent equivalent concentrations of isotype control IgM applied to match either mAb 1.17 (5C) or 1.15 (5D). One milliliter aliquots from each treatment group were taken at the respective time points and flow cytometric analyses were performed. The Y axis represents the mean fluorescent units (MFUs) for a particular time point on the X axis. Surface capping (i.e. internalization of surface antigen-antibody complexes), indicated by a decrease in MFUs between hours 1 and 2 for all treatment groups, was observed between 60 and 90 minutes.

Figure 6. TF1.17 promotes adhesion of feline T. foetus to intestinal epithelial cells. The mean number of adhered trichomonads to IPEC-J2 monolayers after 6 hours of co-culture in the presence of 10 x 106 feline T. foetus (isolate A) treated with either an equivalent concentration of isotype control (white circle; mouse IgM) or 1:25 mAb (black circle; 1.15 (A) or 1.17 (B)). Data represent n=5-6 cultures per treatment and are reported as mean + SD (A) or median + the range (B). ** P = 0.01 between groups. P  0.05 was considered significant. Determined by a Student’s t-test (A) or Mann-Whitney rank sum test (B). All assays were performed in triplicate.

Figure 7. The effect of mAb 1.17 on feline T. foetus adhesion to IPEC-J2 cells is dose dependent. The mean number of adhered trichomonads to IPEC-J2 monolayers after 6 hours of co-culture in the presence of 10 x 106 feline T. foetus (isolate A) treated with either an equivalent volume of isotype control (white circles; mouse IgM) or 1:100, 1:150 or 1:200 mAb (black circles; 1.17). Data represent n=4 cultures per treatment and are reported as mean + SD. **P < 0.001 and *P<0.01 between groups. P  0.05 was considered significant. All comparisons determined by a Student’s t-test. All assays were performed in duplicate.

Figure 8. Murine IgM, but not mAb 1.17, exhibits a protective effect to IPEC-J2 cells compared to a DPBS treated control group. The average crystal violet absorbance (i.e. living epithelial cells) following 24 hours co-culture of confluent IPEC-J2 cells in the presence of 10 x 106 feline T. foetus (isolate A) treated with either 1:75 monoclonal antibody (black circle; mAb 1.17), or an equivalent volume of isotype control (white circle; mouse IgM at equivalent concentration to mAb 1.17) or DPBS (downward triangle). The upward triangle represents an uninfected DPBS control (i.e. lacking T. foetus). Data represent n=8 cultures per treatment and are reported as mean + SD. **P = 0.002 between groups. P  0.05 was considered significant. Comparisons determined with a one-way ANOVA.