Lesion Formation and Antibody Response Induced by Papillomatous Digital Dermatitis-Associated Spirochetes in a Murine Abscess Model

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1 INFECTION AND IMMUNITY, Sept. 2007, p Vol. 75, No /07/$ doi: /iai Lesion Formation and Antibody Response Induced by Papillomatous Digital Dermatitis-Associated Spirochetes in a Murine Abscess Model Margaret K. Elliott,* David P. Alt, and Richard L. Zuerner Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Ames, Iowa Received 4 January 2007/Returned for modification 19 February 2007/Accepted 17 June 2007 Papillomatous digital dermatitis (PDD), also known as hairy heel wart, is a growing cause of lameness of cows in the U.S. dairy industry. Farms with PDD-afflicted cows experience economic loss due to treatment costs, decreased milk production, lower reproductive efficiency, and premature culling. While the exact cause of PDD is unknown, lesion development is associated with the presence of anaerobic spirochetes. This study was undertaken to investigate the virulence and antigenic relatedness of four previously isolated Treponema phagedenis-like spirochetes (1A, 3A, 4A, and 5B) by using a mouse abscess model with subcutaneous inoculation of 10 9,10 10, and spirochetes. Each of the PDD isolates induced abscess formation, with strain 3A causing cutaneous ulceration. Lesion development and antibody responses were dose dependent and differed significantly from those seen with the nonpathogenic human T. phagedenis strain. Strains 3A, 4A, and 5B showed two-way cross-reactivity with each other and a one-way cross-reaction with T. phagedenis. Strain 5B showed one-way cross-reactivity with 1A. None of the isolates showed cross-reactivity with T. denticola. In addition, distinct differences in immunoglobulin G subclass elicitation occurred between the PDD strains and T. phagedenis. From these data, we conclude that spirochetes isolated from PDD lesions have differential virulence and antigenic traits in vivo. Continuing investigation of these properties is important for the elucidation of virulence mechanisms and antigenic targets for vaccine development. Bovine papillomatous digital dermatitis (PDD) is a disease involving inflammation and lesion development of the hoof in cattle. PDD begins as an ulcerative lesion of the epidermis and often progresses to a painful, proliferative, nodular mass on the caudal interdigital space below the heel bulb. PDD is responsible for a significant percentage of lameness reported on dairy farms in the United States (36). Dairy farmers suffer economic loss due to PDD because of treatment costs, lower milk production, decreased reproductive efficiency, and early culling (31). Although the epidemiology of the disease is unknown, it is thought to be passed from cow to cow in the paddock or barn alleyway. In many instances, the occurrence of PDD is linked to the introduction of replacement cows into the herd (20, 28). PDD lesions generally do not heal spontaneously and may progress to a severe state involving the horny tissue junction of the foot resulting in loss of the hoof in erosive lesions (3). Parenteral and topical antibiotics and footbaths are effective treatments for this disease. However, lesions reoccur in a large percentage of treated cows. Dairy cattle do mount both a serological and a cell-mediated immune response to PDD-associated spirochetes (10, 34, 37), but the effectiveness of this response has not been determined. Evidence exists that PDD lesions contain a variety of bacteria, including Fusobacterium, Bacteroides, Mycobacterium, Campylobacter, and Treponema species (4, 7, 8, 21). Cruz et al. evaluated greater than 500 lesions from cattle sent to slaughter * Corresponding author. Mailing address: USDA, ARS, National Animal Disease Center, 2300 N. Dayton Ave., P.O. Box 70, Ames, IA Phone: (515) Fax: (515) margaret.elliott@ars.usda.gov. Published ahead of print on 25 June and found that spirochetes were the most prevalent microorganisms observed (8) and a number of Treponema species have been isolated from diseased tissue (8, 11, 21, 33, 38). Spirochetes detected in PDD lesions, and the isolates obtained thus far, are a diverse group. Isolates from California cluster most closely to the human-associated oral treponemes Treponema denticola, T. medium/t. vincentii, and T. phagedenis, a genital commensal (37). Punch biopsies from German cattle contained treponemes related to those listed above and T. maltophilum (5). A novel treponeme, T. brennaborense, was also isolated from a dairy cow in Germany (30). By using immunohistochemistry, Walker et al. demonstrated that spirochetes found in PDD lesions from dairy cattle in the United States show similarities to PDD-associated spirochetes in 16 other countries (39). Edwards et al. isolated a treponeme from PDD lesions in dairy cattle from the United Kingdom that was related to T. vincentii not only genetically but phenotypically as well, sharing an outer membrane profile and binding of extracellular matrix proteins with this treponeme (14). Demirkan et al. successfully isolated a PDD-associated treponeme by using polyclonal antibodies to T. denticola and T. vincentii (9), indicating that this particular isolate shares antigenic determinants with these two treponemes. Polyclonal antiserum to T. pallidum has also been shown to bind treponemes in PDD lesion sections, suggesting antigenic similarity between T. pallidum and some PDD-associated treponemes (8). Previous work using lesion material obtained from cattle on a dairy farm in Iowa resulted in the isolation of four different spirochete strains designated 1A, 3A, 4A, and 5B (34). These spirochetes were shown to have a 16S rrna gene sequence most similar to that of T. phagedenis, a nonpathogenic member of the resident genital microflora (34, 42), and were described 4400

2 VOL. 75, 2007 PDD SPIROCHETE VIRULENCE IN A MOUSE ABSCESS MODEL 4401 as T. phagedenis like. Sera from cows having PDD lesions were tested for antigen reactivity to each isolate by Western blotting. Reactivity to each isolate varied, with the strongest reactivity occurring with isolate 4A (34). The role these spirochetes play in lesion initiation or development is not known. Although Read and Walker showed the transient development of PDD lesions in calves inoculated directly with lesion material (26), no well-characterized animal model of this disease has been reported to date. Therefore, we evaluated the pathogenicity of PDD-associated spirochetes by using a previously described mouse abscess model (18). We compared the lesions and antibody responses induced by PDD spirochetes to each other and to those induced by T. denticola, a pathogenic spirochete associated with dental caries previously evaluated by using this model (18). We found that PDD spirochetes differed in the ability to produce lesions in mice. Each strain possessed differing levels of serological cross-reactivity with each other and with T. phagedenis. In addition, the subclasses of antibodies elicited in response to the PDD spirochetes were variable between isolates. MATERIALS AND METHODS Mice. Female BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) at 6 to 10 weeks of age and maintained in a standard barrier facility in microisolator cages. Mice were acclimated to the facility for 1 week prior to the start of exposure and received standard rodent chow and water ad libitum for the entire course of the study. All of the experimental protocols were reviewed and approved by the Animal Care and Use Committee of the National Animal Disease Center and conformed to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Bacterial strains and media. PDD-associated treponeme strains 1A, 3A, 4A, and 5B were used in this study (34). The PDD isolates were grown in prereduced, anaerobic oral treponeme isolation broth containing 10% inactivated fetal bovine serum without antibiotics that was prepared as previously described (34). T. denticola (ATCC 35405) was grown in modified new oral spirochete broth (ATCC medium 1494). T. phagedenis (ATCC 27087) was grown in PY medium with cocarboxylase and serum (ATCC medium 1828). All of the bacteria were incubated under anaerobic conditions in a Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) under an atmosphere consisting of 85% N, 5% CO 2, and 10% H. All bacterial manipulations were carried out under anaerobic conditions. Virulence model. Spirochete cultures were harvested at an optical density at 620 nm of 0.6 during the late exponential phase of growth. Culture viability was confirmed by the observation of active motility and the absence of spherical bodies by dark-field microscopy. Cell counts were performed with a Petroff- Hausser bacterial counting chamber prior to harvesting by centrifugation (6,000 g for 20 min). Spirochetes were resuspended in their respective broths at concentrations of 10 9,10 10, and 10 ll spirochetes per inoculum (spi) in a volume of 0.2 ml. A mixed inoculum was prepared that contained all four of the PDD isolates at a ratio of 1:1:1:1 for each concentration. For example, spirochetes of each isolate were pooled, mixed, pelleted by centrifugation, and resuspended in 0.2 ml for one 10 9 mixed inoculum. The mixed inoculum contained spirochetes of each isolate, etc. Additionally, formalin-killed (FK) preparations of each isolate were prepared as previously described (13). FK spirochetes were inoculated at a concentration of spi and served as a negative control for each isolate and the mixed inoculum. T. denticola and T. phagedenis served as pathogenic and nonpathogenic controls, respectively. Six to 10 mice were inoculated for each group. The posterior dorsolateral surface of each mouse was shaved, swabbed with 70% ethanol, and inoculated with a single subcutaneous injection of a bacterial suspension. Separate groups of mice received injections of each of the uninoculated bacterial media used to ensure that the resulting lesions were not due to medium components. All inoculations of live spirochetes were performed within 15 min of inoculum preparation. Mice were observed approximately 1 and 6 h following inoculation and then once a day for 34 days. Lesion assessment. Lesion development was monitored daily and recorded every other day with an electronic caliper gauge. During abscess formation, the length, width, and height were measured and lesion size was expressed in cubic millimeters. Ulcers or abscesses 1 mm tall were expressed as the product of the length and width multiplied by 1 mm and also expressed in cubic millimeters. Time course lesion measurements are the average lesion size for each mouse on that particular day. At the end of the 34-day observation period, lesion material was collected from those mice still having intact abscesses and examined for the presence of spirochetes. Immediately following euthanasia, mice were transferred to a biosafety hood. The area containing the abscess was swabbed with 70% ethanol. A sterile needle and syringe were used to aseptically aspirate material from the lesion. Sterile phosphate-buffered saline (PBS) was then drawn into the syringe to dilute the lesion material, and then the syringe contents were expelled into a sterile Eppendorf tube. The tube was capped and vortexed. Two aliquots from each sample were then placed on a slide with a coverslip and examined for the presence of intact, motile spirochetes by dark-field microscopy. PDD spirochete ELISAs. Mice were bled from the orbital plexus prior to day 0 and on day 14 postinoculation. Blood was clotted at 25 C for 1 h. Sera were collected and stored at 80 C until use. Standard enzyme-linked immunosorbent assay (ELISA) methods were used for determination of antibody production. Briefly, 96-well microtiter plates (Immulux; Dynex, Chantilly, VA) were coated with FK PDD isolates (either 1A, 3A, 4A, or 5B), FK T. denticola, orfkt. phagedenis at 10 8 spirochetes/ml in a volume of 100 l of 0.1 M carbonate buffer (ph 9.6) and incubated overnight at 4 C. The coating solution was removed, and the plates were blocked with 1% bovine serum albumin (BSA) in PBS for 2hat room temperature (RT). Plates were washed three times with PBS containing 0.5% Tween 20 (PBS-t). All sera were diluted in PBS-t containing 1% BSA for analysis. Serum from each mouse was tested individually beginning with a 1:400 dilution and then diluted 1:3 down columns of the plate. Plates were incubated for 2 h at RT, followed by washing three times with PBS-t. Secondary antibodies were then added to the plate. Horseradish peroxidase-conjugated goat antimouse antibodies against murine immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 (Southern Biotech, Birmingham, AL) were diluted 1:1,000 in PBS-t 1% BSA for determination of the antibody isotypes produced. Total IgG was determined with horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL) antibodies diluted 1:4,000 in PBS-t 1% BSA. Plates were incubated for 2 h at RT. Following incubation, plates were washed three times with PBS-t and 3,3,5,5 -tetramethylbenzidine (Sigma) was used to visualize bound antibody. The reaction was stopped with 0.18 M sulfuric acid, and the absorbance was read at 450 nm. Control and reference sera were run for each set, and the number of relative ELISA units for each sample was calculated by the reference line method (27) to ensure reproducibility of data for assays run for the same set on different days for the determination of cross-reactivity. The use of different reference sera for each spirochete produced a different y-axis scale for each graph and does not necessarily indicate differences in antibody titer elicited by each spirochete. Sera were considered cross-reactive when significant heterologous reactivity was measured. Cross-reactivity was considered minimal when heterologous reactivity occurred but was not significant. Sera were not considered cross-reactive if only homologous reactivity was detected. For the determination of antibodies produced by mixed infection, ELISA units represent the optical density value times the dilution factor of the highest dilution to fall on the linear portion of the graphed data. Statistical analysis. Lesion size and mixed-infection antibody response data were analyzed by one-way analysis of variance (GraphPad Prism) with the Tukey- Kramer test as a posttest. Antibody cross-reactivity data were analyzed with a two-tailed, unpaired t test (GraphPad Prism). A 0.05 value of significance was used for all data. RESULTS Gross pathology. It is not known if the spirochetes found in PDD lesions possess pathogenic potential or if the potential differs between strains. We used a murine abscess model to determine if PDD-associated spirochetes would produce abscesses in mice and to determine if visible or measurable differences would result between strains. All of the inoculums showed a clear dose response with regard to lesion size, with PDD isolate 1A producing the largest lesions (Table 1). With the exception of 3A, all of the spirochetes induced localized abscess formation consistent with previous reports for this model using oral treponemes (18). By the second day postinoculation, the challenge site showed gross evidence of hyperemia and diffuse edema. These progressed into a more pro-

3 4402 ELLIOTT ET AL. INFECT. IMMUN. TABLE 1. Largest mean lesion size for each isolate at each inoculum size Spirochete(s) Mean lesion size (mm 3 ) SEM at inoculum size a of: FK b T. denticola ND f T. phagedenis d ND 1A , e A A B Mixed c a Number of spirochetes per inoculum in 0.2 ml. b FK spirochetes were inoculated at FK versus for all isolates, P c All four PDD isolates at a ratio of 1:1:1:1 for the total number of spirochetes indicated for each column. d Versus 1A and 4A, P 0.001; versus 5B and T. denticola, P There were 6 to 10 mice per group. e Versus 3A, T. phagedenis, and mixed infection, P 0.001; versus 5B and T. denticola, P f ND, not done. nounced swelling over the next 24 to 48 h, with abscess formation becoming evident shortly thereafter. Lesions either remained closed until the time of resolution or eventually developed a small necrotic patch that subsequently opened, followed by drainage and healing. PDD isolate 3A induced the development of cutaneous necrosis involving most of the apical surface of the lesion by day 2. The necrotic skin sloughed off by day 7 or 8, leaving an ulcer exposing underlying tissues. This observed response to strain 3A may contribute to an increased ability to resolve the infection, leading to smaller lesion size. Inoculation with a mixture of equal numbers of PDD isolates produced lesions similar to those produced by strain 3A. The mean abscess size varied among the PDD isolates and the ATCC strains (see below). At the end of the 34-day observation period, lesion material was collected from those mice still having intact abscesses as described in Materials and Methods and examined for the presence of spirochetes. Intact spirochetes were found in lesions from mice challenged with T. denticola spirochetes and all four PDD isolates at spi, with active motility of spirochetes noted from lesions containing PDD isolates 4A and 1A. At spi, intact but nonmotile spirochetes were only seen in lesions from mice inoculated with strain 5B (data not shown). Mice receiving inoculations of medium only failed to develop lesions or pathology of any kind (data not shown). Time course of lesion development. PDD isolates differed in the ability to induce abscesses with regard to lesion size and development time. T. denticola and T. phagedenis were included as pathogenic and nonpathogenic controls, respectively. In addition, we evaluated T. phagedenis because of the genetic similarity of its 16S rrna gene to that of the PDD isolates. Inoculation of T. denticola spirochetes induced a mean abscess size at day 5 of mm 3 (standard error of the mean [SEM]), while spi induced lesions of half that size (Fig. 1A). Lesion resolution occurred in some of the mice inoculated with T. denticola spirochetes as early as day 15. However, approximately half of the mice in this group had unresolved lesions by day 34. T. phagedenis produced significantly smaller lesions than T. denticola ( [SEM] mm 3 )at10 11 spi, with lesion size peaking on day 6 and lesion resolution by day 15 (Fig. 1B). At spi, PDD isolate 1A produced lesions larger than those seen with T. denticola and all of the other strains, but peak size was not reached until day 14, with resolution occurring in all mice by day 28 (Fig. 1C). Isolate 4A also had the largest lesion size over the longest course of time at spi, with only one mouse having an unresolved lesion by day 34 (Fig. 1E). The peak lesion size for spi of PDD isolate 3A was less than that seen for all of the other isolates and occurred on day 6, with all of the mice healing by day 28 (Fig. 1D). PDD isolate 5B at spi produced the latest-occurring peak lesion size at 20 days with the largest mean measurement, similar to that obtained with isolate 3A (Fig. 1F). The mixed inoculum containing all four strains at spi induced lesions smaller than those obtained with the individual isolates and had a time course of development similar to that of PDD isolate 3A (Fig. 1H). Reducing the inoculums to spi resulted in a lesion size reduced to approximately one-half to one-third of that obtained with spi in all groups, with the exception of strain 3A, which produced peak lesion sizes that were similar in mice receiving and spi. It is interesting that only T. phagedenis had complete resolution of lesions on all of the mice inoculated with spirochetes on day 34, while all of the other treponemes induced a consistent mean lesion size throughout the length of the observation period. Lesion size. Differences were seen in the peak lesion size as measured on the day the lesion was largest for each individual mouse. Within groups (rows, Table 1), the lesion size was dose dependent and significantly larger with increasing inoculum size for all of the strains (P 0.05, P 0.01, and P 0.001), except isolate 3A. Formalin treatment of the spirochetes reduced lesion formation compared to that produced by the same number of live spirochetes of each of the PDD isolates inoculated separately (P for all isolates). Although the mice receiving spirochetes in the mixed inoculum developed a mean lesion size greater than that obtained with the mixed formalin-treated preparation, the difference was not significant. Comparing the groups on the basis of inoculum size, (columns, Table 1), PDD isolate 1A at spirochetes induced the formation of a significantly larger abscess than that seen with any other spirochete (versus 3A, T. phagedenis, and the mixed inoculum, P 0.001; versus 5B and T. denticola, P 0.01), with the exception of 4A. Lesions produced by T. phagedenis were significantly smaller than those produced by the

4 VOL. 75, 2007 PDD SPIROCHETE VIRULENCE IN A MOUSE ABSCESS MODEL 4403 FIG. 1. Time course of lesion development. Mice received treponemes subcutaneously at inoculums of 10 9 (closed square, solid line), (open square, dashed line), (closed circle, solid line), and formalin-treated (open circle, dashed line) spi. Lesion development was monitored for 34 days. T. denticola (A) was used as a pathogenic control. T. phagedenis (B) served as a nonpathogenic control. PDD isolates 1A (C), 3A (D), 4A (E), and 5B (F) differed in peak lesion size, days to peak lesion size, and lesion duration. A mixed inoculum (H) composed of all of the PDD isolates together (1A, 3A, 4A, and 5B at 1:1:1:1) induced lesions smaller than those induced by the individual isolates. In all of the groups, formalin killing (f-k) of the spirochetes severely hampered lesion development. There were 6 to 10 mice per group.

5 4404 ELLIOTT ET AL. INFECT. IMMUN. Downloaded from FIG. 2. Antibody response and cross-reactivity. Antibody response and cross-reactivity of antibodies produced were measured by ELISA. Bars indicate the organism against which the serum run on the plate was raised, as follows: 1A, clear open bar; 3A, small gray dots; 4A, clear bar with horizontal stripes; 5B, black filled bar; T. denticola, cross-hatched bar; T. phagedenis, clear bar with left diagonal stripes. Each value on the x axis is the number of spirochetes in the inoculum for each serum. FK (f-k) spirochetes were inoculated at spi. The label inside the graph in the top corner indicates the organism coating the ELISA plate. Top left, T. denticola (T dent). Top right, T. phagedenis (T phag). Middle left, PDD isolate 1A. Middle right, PDD isolate 3A. Bottom left, PDD isolate 4A. Bottom right, PDD isolate 5B. There were 6 to 10 mice per group. Differences in the y-axis scale do not indicate differences in the antibody titers induced by the different spirochetes. See Materials and Methods. other spirochetes (versus 1A and 4A, P 0.001; versus 5B and T. denticola, P 0.05), with the exception of the mixed-infection group and isolate 3A. Of the PDD isolates, 3A produced the smallest abscesses, on average, at spi. When the inoculums were reduced to spi, PDD isolates 1A and 3A produced almost identical-size abscesses while T. denticola produced a slightly smaller mean abscess size. PDD isolates 4A and 5B, the mixed inoculum, and T. phagedenis all produced a much smaller mean lesion size, with T. phagedenis lesions again being the smallest. PDD isolate 3A produced the largest mean lesion size in the 10 9 inoculum group, but it was only significantly larger than lesions produced by isolate 1A and T. denticola (P 0.05), with the remaining groups having similar-size lesions at 10 9 spi. Data showing the peak lesion size are the mean of the largest lesion measured on each mouse, regardless of the day on which the largest lesion was recorded. For this reason, the mean lesion sizes shown in Table 1 are slightly, but not significantly, larger than those in Fig. 1. Antibody response and cross-reactivity. The production of antibodies to each isolate was measured by ELISA on day 14 to determine if mice would mount a serological response to the PDD strains and to determine antibody cross-reactivity to each strain. We found that the antibody response to each spirochete strain was typically dose dependent and reduced by formalin treatment of the bacteria (Fig. 2). Strains 3A, 4A, and 5B showed two-way cross-reactivity with each other and a one-way cross-reaction with T. phagedenis. Strain 5B showed one-way cross-reactivity with 1A. None of the isolates showed crossreactivity with T. denticola. It is interesting that antibodies produced to PDD isolate 5B cross-reacted with all of the other PDD isolates at all of the inoculum doses (Fig. 2, isolate 1A, middle left; isolate 3A, middle right; and isolate 4A, lower left) with no significant cross-reactivity of antibodies against any of the other PDD isolates to isolate 5B (Fig. 2, lower right). Antibody subclass. Antibody subclass production was evaluated to determine if profiles would differ among the PDD on May 22, 2018 by guest

6 VOL. 75, 2007 PDD SPIROCHETE VIRULENCE IN A MOUSE ABSCESS MODEL 4405 FIG. 3. Antibody subclass production. The relative amounts of IgG1 (gray right diagonal lined bar), IgG2a (gray hatched bar), IgG2b (clear right diagonal lined bar), and IgG3 (clear open bar) produced in response to each isolate were determined by ELISA. Each value on the x axis is the number of spirochetes in the inoculum for each serum. FK (f-k) spirochetes were inoculated at spi. Upper left, T. denticola. Upper right, T. phagedenis. Middle left, PDD isolate 1A. Middle right, PDD isolate 3A. Lower left, PDD isolate 4A. Lower right, PDD isolate 5B. N.D. not done. Data are the mean ( SEM) of 6 to 10 mice per group. Differences in the y-axis scale do not indicate differences in the antibody titers induced by the different spirochetes. See Materials and Methods. isolates. Mice inoculated with T. denticola produced mainly IgG3 for 10 9 and inoculums, shifting to a higher percentage of IgG1 with (Fig. 3, top left). These results are consistent with previous reports (17). Antibodies produced in response to T. phagedenis consisted mostly of IgG1 at 10 9 spi and shifted to a greater percentage of IgG3 overall as the number of spirochetes in the inoculum increased to (Fig. 3, top right). PDD isolate 1A induced the production of IgG1 at a higher percentage than all of the other subclasses at all of the inoculum levels tested. Although IgG2a was not produced at high levels in response to 1A, formalin killing of the spirochetes abrogated its production (Fig. 3, middle left). Mice challenged with PDD isolate 3A produced IgG1 at the highest percentage compared to all of the other subclasses at all of the inoculum levels, although not as high as that seen with isolate 1A. At spirochetes, isolate 3A induced the most balanced subclass response of all of the isolates (Fig. 3, middle right). Formalin killing of isolate 3A shifted the antibody response to

7 4406 ELLIOTT ET AL. INFECT. IMMUN. FIG. 4. Mixed-infection antibody response. Sera from mice receiving inoculations of all of the isolates together were tested for reactivity to individual isolates. For all of the inoculum sizes, sera were most reactive with 5B, followed by 1A. Binding to isolates 3A and 4A was seen but at a lower level. Statistical significance of differences: a, versus 1A, P 0.01; b, versus 3A and 4A, P 0.001; c, versus 1A, P 0.001; e, versus 3A, P 0.05; f, versus 4A, P There were 6 to 10 mice per group. f-k, FK spirochetes. almost exclusively an IgG1 response. PDD isolate 4A induced a mostly IgG3 response at 10 9 spi, shifting to IgG1 at and spi (Fig. 3, bottom left). As with isolate 3A, formalin treatment of 4A limited the response to IgG1. PDD isolate 5B induced almost equal percentages of IgG3 and IgG1, with formalin treatment once again limiting the response to IgG1 (Fig. 3, bottom right). Mixed-infection antibody response. PDD lesions often contain a variety of bacterial types including different spirochetes. Therefore, we sought to determine the outcome of using an inoculum containing equal numbers of each isolate. The humoral response to each of the different isolates was evaluated for the mice receiving a mixed inoculum (Fig. 4). Animals receiving mixed inoculums produced antibodies to all of the isolates. At 10 9 spi, the antibody response to 5B was significantly greater to than the response to 1A (P 0.01), 3A, or 4A (P 0.001). The mean response to 1A was greater than that to 3A and 4A, although the difference was not statistically significant. At spi, the response to 5B was again the greatest (versus 3A and 4A, P 0.001) although not significantly higher than that to 1A. At spi, the response to 5B was significantly higher than those to all of the other isolates (P 0.001), with the mean response to 1A being the next highest. There was no significant difference in the antibody responses to PDD isolates 3A and 4A at any inoculum level. DISCUSSION Although a number of bacterial species are associated with the presence of lesions in bovine PDD (7, 21), spirochetes are abundant, are found deep in the tissues (8, 21), and can be isolated from lesion material (11, 34, 38). The role of treponemes in lesion development in PDD has not been definitively proven, but consistent association of these spirochetes with lesions implies that these bacteria have an important role in the disease process. In this report, we used a mouse abscess model to determine if PDD-associated treponemes (1A, 3A, 4A, and 5B) differ in lesion induction and antibody elicitation. In addition, we compared the relatedness of the antigenicity of the isolates to each other, T. denticola, and T. phagedenis. All four of the PDD isolates produced lesions in mice comparable to those produced by T. denticola (pathogenic control) and much larger and of longer duration than those produced by T. phagedenis (nonpathogenic control) in this study, indicating that these isolates possess pathogenic potential. Differences in lesion development were apparent for some of the isolates. For example, PDD isolate 1A produced significantly larger lesions compared with the other isolates and T. denticola, while isolate 3A was the only spirochete to produce cutaneous necrosis early in lesion induction. These data give evidence that, along with the genetic differences in these isolates (34), functional and/or antigenic differences involved in lesion induction are present. PDD spirochetes induce abscess formation in this model without the need for potentiating agents, as needed in experiments with Bacteroides fragilis and Escherichia coli (15). It was found that the potentiating agent used in those experiments interfered with complement activation and opsonization of the bacteria, allowing B. fragilis and E. coli to persist and initiate abscess formation. Because the PDD-associated isolates induce abscess formation without the need for such agents, it is likely that they possess complement evasion strategies. Digital dermatitis lesions are associated with severe inflammatory infiltration (8). The induction of inflammatory mediators in response to microbes inhabiting PDD lesions may contribute to tissue destruction. We cannot rule out the possibility that lesion development in this study was solely due to the immune reaction of the mouse. Cutaneous necrosis following a subcutaneous challenge can be linked to the major histocompatibility complex in mice. For example, mice having the H-2 d and H-2 k haplotype are prone to the development of cutaneous necrosis when challenged with Mycoplasma arthritidis (6). As BALB/c mice have the H-2 d haplotype, this may contribute to necrosis of the dermis and the subsequent formation of ulcers, suggesting that necrosis may be due to immune pathology. However, the lack of lesion development in the mice receiving FK isolate 3A suggests that viable spirochetes are needed for lesion induction and is consistent with the possibility that strain 3A produces toxigenic proteins that contribute to destruction of the dermal tissue. The process of fixing the spirochetes in formalin may change antigen structure, remove the outer envelope, or damage the protoplasmic cylinder (24, 40), possibly interfering with T-cell help (2, 35). This may explain why mice challenged with spirochetes fixed in formalin did not develop abscesses of significant size and had reduced serological responses. Formalin treatment prior to challenge has been shown to reduce the serological response as a whole and to eliminate the response to certain antigens, compared to the serological response to a challenge with live spirochetes, bacteria, and some viruses (2, 12, 16, 25, 41). Our study shows that this is true for the PDDassociated spirochetes (Fig. 1), despite the fact that, when viewed under an electron microscope, the majority of the formalin-treated spirochetes appeared to possess intact outer membranes and flagella (data not shown). The formation of smaller abscesses in response to FK spirochetes likely indicates

8 VOL. 75, 2007 PDD SPIROCHETE VIRULENCE IN A MOUSE ABSCESS MODEL 4407 that active, live spirochetes play a role in the pathogenesis of lesion development in this model. The only isolate that did not produce the largest peak lesion size at spi was isolate 3A. The abscess size for isolate 3A at spi is small because of the development of necrosis and sloughing of the skin. This is likely the main reason that the lesion development at is the same as at peak size. Interestingly, for all of the isolates, spi induced lesions that remained fairly constant in size throughout the length of the study period. The spi dose was the only one for each isolate in which live spirochetes could be detected in lesion material at the end of the observation period, suggesting more favorable conditions for survival at this population level. On average, the lesion size for mice receiving the mixed inoculum was smaller than that for mice receiving inoculums containing a single strain of spirochete. The reason for this is unknown. The reduced lesion size may indicate an antagonistic relationship between the PDD-associated spirochetes. A negative association has been shown for Prevotella intermedia/p. nigrescens and Actinobacillus actinomycetemcomitans in periodontal disease. A. actinomycetemcomitans has been observed in the presence of Porphyromonas gingivalis but never in the presence of P. intermedia/p. nigrescens in early-onset periodontitis (22). Moter et al. have shown that different phylotypes of treponemes exist at different levels in PDD-diseased tissues. T. denticola-related treponemes occupied the superficial layers of the epidermis, while T. vincentii-, T. maltophilum-, and T. phagedenis-like treponemes were located deep in the stratum spinosum (21). This is also seen in periodontal disease with oral treponemes in subgingival plaque. T. denticola and T. medium were detected at pocket depths of up to 8 mm, whereas T. vincentii was located mainly in shallow pockets 4 to 5 mm deep (1). Differences in location within the lesion may be due to an inability of the different strains to exist in close proximity to each other, in addition to differences in pathogenic mechanisms. In the case of the mixed inoculum, the presence of isolate 3A determined the lesion type while the antibody response was largely directed at isolate 5B. These data provide evidence of the complexity of polyclonal infections, even among closely related organisms, and may shed some light on the difficulty in determining which bacterium involved in PDD is actually responsible for infection resulting in the development of chronic lesions. Possibly, the serological response is not very effective at clearing the infection in PDD because less-pathogenic organisms are targeted. The differences in the lesions produced by these PDD isolates give evidence that they may contribute to disease development in different ways. In the initial investigation by Trott et al., Western blot analysis of pooled sera from cows having PDD lesions indicated that the immunogenicity of the isolates varied, with 4A eliciting the strongest reaction, especially with sera from cows having erosive lesions. On the basis of this finding, the authors concluded that the isolates were antigenically diverse. It is important to note that the sera used in this study were not obtained from the same cows as the lesion material. Therefore, it is not known if the cows from which serum was obtained were exposed to all, or any, of the treponemes isolated, making determination of antigenic differences between the isolates inconclusive. In other words, we do not know if cattle that did not react strongly to 5B were infected with 5B, although exposure to 5B and the other isolates was assumed. However, given the number of different spirochetes that have been associated with PDD lesions, it is likely that differences in pathogenicity and antigenic structure exist. Using the mouse model allows us to obtain polyclonal serum known to be specific for each isolate and to compare the cross-reactivity of each isolate and the serum s cross-reactivity to other Treponema species. We found that antibodies produced to the isolates possessed variable cross-reactivity to each other and to T. phagedenis. No crossreactivity was noted with T. denticola as measured by ELISA. This indicates variation in antigenicity among the isolates and similar antigens with T. phagedenis, while T. denticola is antigenically distinct from the PDD isolates. The variation in the serological cross-reactivity between isolates suggests that an effective vaccine may require a complex mixture of strains. The antigenic diversity of the treponemes in the lesions is likely higher than that seen in this study. Assuming that treponemes play an important role in lesion induction and persistence, not having as a component of the vaccine treponemes with the right composition of antigens that cross-react with all or a large portion of the pathogenic treponemes in PDD allows the treponemal component of the disease to persist unaffected. In addition, other bacteria contributing to lesion development and progression will not be targeted. All subclasses of IgG were elicited by each PDD isolate, indicating the induction of both T-dependent and T-independent responses. The range and approximate percentage of each subclass produced in response to T. denticola are consistent with a previously published report for the spi dose (17). A significantly higher percentage of IgG1 was induced by isolates 1A and 4A compared to 3A and 5B, indicating the presence of soluble proteins with antigenic properties and a T-dependent response dominating for these isolates (29, 32) in this model. These data differ from those of Trott et al., in which the bovine antibodies reactive to the isolates retained reactivity with protease-treated sonicates (34). Isolate 5B induced the highest percentage of IgG3, which may indicate a greater number or accessibility of, or to, polysaccharide antigens (23, 32). Over all of the inoculum sizes, IgG1 was the subclass produced most by each of the isolates, suggesting a possible defense mechanism against complement activation as IgG1 antibodies are poor activators of complement (19). Inhibition of antibodymediated complement activation would enhance survival in vivo. Trott et al. described the PDD-associated treponemes as T. phagedenis like. The data in this study suggest that they share antigenic determinants with T. phagedenis while, unlike T. phagedenis, they possess pathogenic potential. Most importantly, this study gives evidence that the spirochetes found in PDD are pathogenically and antigenically diverse, giving us a hint at the complexity of this disease just within the spirochetal element. ACKNOWLEDGMENTS We thank Richard Hornsby and Renae DeVries for expert technical assistance. We are grateful to Thad Stanton and Alexandra Scupham for critical readings of the manuscript and Ron Limberger for providing T. denticola for these studies.

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