Identification of immunogenic proteins in Treponema phagedenis-like strain V1 from digital dermatitis lesions by phage display

Veterinary Microbiology 153 (2011) 315–322

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Identification of immunogenic proteins in Treponema phagedenis-like strain V1 from digital dermatitis lesions by phage display Anna Rosander a,*, Bengt Guss b, Lars Frykberg b, Camilla Bjo¨rkman c, Katarina Na¨slund d, Ma¨rit Pringle a a

Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7009, SE-750 07 Uppsala, Sweden Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden Department of Clinical Sciences, Swedish University of Agricultural Sciences, Box 7054, SE-750 07 Uppsala, Sweden d Department of Virology, Immunobiology and Parasitology, National Veterinary Institute, SVA, SE-751 89 Uppsala, Sweden b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 January 2011 Received in revised form 19 May 2011 Accepted 7 June 2011

Digital dermatitis (DD) is a contagious claw disease causing lameness in cattle, affecting both animal welfare and economics. In this study, shotgun phage display was used to identify immunogenic proteins in a strain (V1) of the Treponema phylotype closely related to Treponema phagedenis, indicated as a key agent in the pathogenesis of DD. A genomic phage library was constructed and selected against antibodies from a rabbit immunized with live strain V1 bacteria. A homolog to the immunogenic protein TmpA of Treponema pallidum subsp. pallidum was identified, as well as a putative phage tail tape measure protein (Ttm), and a putative proline-rich repeat lipoprotein (PrrA). The complete amino acid sequences of these proteins were predicted from a genomic sequence of strain V1 generated by 454 SequencingTM. The presence of these genes in ten Treponema spp. field isolates was investigated by PCR. The tmpA and ttm genes were detected in all T. phagedenis-like isolates while prrA was detected in four out of seven. None of the genes were detected in the three Treponema pedis isolates investigated. Recombinant proteins were produced and used in indirect ELISAs. For all three proteins, a majority of serum samples from cattle with DD (n = 8) showed higher optical density values than samples from cattle without DD (n = 7). ß 2011 Elsevier B.V. All rights reserved.

Keywords: Treponema phagedenis-like Immunogenic Phage display TmpA Digital dermatitis

1. Introduction Digital dermatitis (DD) is a contagious claw disease causing lameness in cattle, most commonly seen in intensive dairy production. The occurrence of DD is connected to wet/dirty claw environments (Rodrı´guezLainz et al., 1996), for example in cubicle systems where accumulation of feces and urine on the alleys is a typical hygienic problem. Besides being an animal welfare problem, DD is also associated with economic losses due to reduced milk production and weight loss (Bruijnis

* Corresponding author. Tel.: +46 18 67 23 87; fax: +46 18 67 33 34. E-mail address: [email protected] (A. Rosander). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.06.005

et al., 2010; Cha et al., 2010). There is strong circumstantial evidence that Treponema spp. are central in the etiology of DD. At least 17 different phylotypes of genus Treponema have been found in DD lesions (Jensen et al., 2009). However, in several studies the Treponema phagedenis-like (Tpl) phylotype has been indicated to be a key agent in the pathogenesis of DD (Klitgaard et al., 2008; Nordhoff et al., 2008; Yano et al., 2009). A humoral response against Treponema spp. has been shown in cattle with DD and whole cell lysate ELISAs have been used in research (Walker et al., 1997; Demirkan et al., 1999; Trott et al., 2003; Vink et al., 2009; Moe et al., 2010). Antibody response to, and cross-reactivity between, Tpl isolates have also been evaluated in a murine abscess model (Elliott et al., 2007). However, little is known about the

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individual antigens that evoke an immune response in affected cattle. Since the first report on phage display (Smith, 1985), this method has been widely used to identify and characterize protein–protein interactions. In shotgun phage display, phage libraries made from randomly fragmented bacterial DNA contain phage particles displaying bacterial polypeptides. Theoretically, polypeptides derived from all genes on the bacterial chromosome are represented (Jacobsson and Frykberg, 1995; Jacobsson et al., 2003). Such libraries can be used for identification of ligand-binding domains of bacterial proteins by affinity selection. The objective of this study was to identify immunogenic proteins in a Tpl strain (V1) from DD through phage display selection against polyclonal antibodies. To obtain the complete genes for the detected proteins a genomic sequence of V1 generated by 454 SequencingTM was used. 2. Materials and methods 2.1. Bacterial strains, culture conditions, and helper phage The isolation and characterization of Tpl isolate V1 from a dairy cow with DD has been described previously (Pringle et al., 2008). The spirochetes were grown in flasks with 10 ml FABGS (fastidious anaerobe broth, LAB 71, LabM) with 2.0 g D-glucose per liter and 25% fetal calf serum (S 0115, Biochrom AG), and incubated at 37 8C in anaerobic jars on a shaker (90 rpm). Escherichia coli TG1 (Stratagene) was used as host in all experiments involving phages or phagemids and grown in Luria–Bertani broth (LB) or on Luria–Bertani agar (LA). When appropriate, 50 mg/ml ampicillin (C16H18N3O4SNa, Roche) was added. Incubations were at 37 8C. Phage R408 (Promega) was used as helper phage. 2.2. DNA-techniques Unless stated otherwise, standard DNA-techniques were used (Sambrook et al., 1989). Restriction and modification enzymes were from MBI Fermentas AB and used according to the manufacturer’s instructions. Genomic DNA for PCR was prepared with the DNeasy Blood & Tissue Kit (QIAGEN) following the protocol for Gramnegative bacteria. Plasmid DNA was prepared using the QIAprepTM Miniprep kit (QIAGEN) and sequenced on a 3730xl DNA Analyzer (Applied Biosystems) at Uppsala Genome Centre. Sequence analysis was performed using the CLC Main Workbench software (CLC bio). Homology searches were performed using the BLAST algorithm (Altschul et al., 1990) at the National Center for Biotechnology Information. 2.3. Genome sequencing The chromosomal DNA of Tpl strain V1 was sequenced and assembled at the KTH Genome Center at the Royal Institute of Technology, Stockholm, Sweden, using the Genome Sequencer FLX System, with long-read GS FLX Titanium chemistry (single-end) and the 454 de novo

assembler Newbler (454 Life Sciences, Branford, CT, USA). An additional De Novo assembly of the reads was made with CLC Genomics Workbench 3 (CLC bio) and for further sequence editing CLC Main Workbench 5 (CLC bio) was used. 2.4. Immunization and purification of polyclonal antibodies To obtain a defined serum against Tpl spirochetes with as little background as possible, a New Zealand white rabbit was immunized subcutaneously with a live culture of the Tpl strain V1. This part of the study was approved by the ethical committee on animal experiments in Uppsala (C 300/8). A dose of approximately 109 bacteria, washed twice and suspended in a volume of 0.5 ml isotonic saline, was injected twice with 20 days in between. Serum from the final bleed at day 38 post first immunization was used for purification of antibodies (IgG) for this study. Ten ml serum was sterile filtered through a 0.45 mm syringe filter and applied to a 5 ml HiTrap Protein G HP column (GE Healthcare). Rabbit IgG was purified according to the manufacturer’s instructions using the Ab Buffer Kit (GE Healthcare). Buffer exchange was performed on the eluates using Zeba Spin Desalting columns (Pierce) and the purified antibodies were stored in phosphate buffered saline pH 7.4 (PBS) at 20 8C. 2.5. Construction of the phage display library and selection of binding phages (panning) The phage library was constructed in the pG8SAET phagemid vector (Jacobsson and Frykberg, 2001). Library construction and panning was performed essentially as described by Jacobsson and Frykberg (1995) and according to detailed protocols in Jacobsson et al. (2003). Chromosomal DNA from Tpl strain V1 was used for construction of the phage library. Broth cultures were washed three times in isotonic saline (pH 6.3), followed by one wash in PBS. The Treponema DNA was prepared by conventional phenolchloroform extraction and fragmented by sonication until the majority of the fragments were between 0.4 and 1.5 kb in length. The fragments were made blunt-ended by T4 DNA polymerase and T4 DNA kinase treatment and then ligated into SnaB1-digested and dephosphorylated phagemid vector pG8SAET using Ready-To-GoTM T4 DNA ligase tubes (GE Healthcare). The phage library was generated by electrotransformation of the ligated material into E. coli TG1 cells (2.5 kV, 25 mF, 360 V), infection with helper phage, and proliferation of phage particles. This procedure yielded 4  107 transformants, considered as unique clones, 86% of which carried an insert as determined by colony PCR on 14 randomly selected clones. The final library had a titer of 1  1011 colony forming units per ml. Phages displaying immunogenic polypeptides were isolated by panning against rabbit anti-Tpl strain V1 IgG (described above). Three panning experiments were performed. Microwells (MaxiSorpTM, Nalge Nunc International) were coated with Zymed recombinant Protein G (Invitrogen) at a concentration of 10 mg in 200 ml 50 mM sodium carbonate, pH 9.5. Thereafter, the wells were blocked with PBS with 0.05% Tween 20 (PBS-T). Rabbit anti-

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Tpl strain V1 IgG was added at a concentration of 85 mg in 200 ml PBS (1st panning experiment) or 85 mg or 215 mg in 100 ml PBS (2nd and 3rd panning experiment, respectively) + 100 ml sterile filtered crude E. coli lysate (for blocking). After washing, 200 ml of the phage library was added. The wells were incubated for 3 h at room temperature or over night at 2 8C, after which they were washed 25 times before phages were eluted by addition of 50 mM Na-citrate/ 140 mM NaCl pH 2.0. The eluate was immediately neutralized with 2 M Tris-buffer pH 8.0 and used to infect E. coli TG1, which were plated on LA-plates with ampicillin (LAamp). In the 3rd panning experiment, elution was also carried out by direct infection by the bound phages of TG1 cells added to the well. Fifty ml of a TG1 over night culture + 150 ml LB were added to the well and incubated at room temperature for 30 min before plating on LAamp. After incubation of the LAamp plates overnight, colonies were counted and 100 colonies transferred to another LAamp-plate. These colonies were then transferred to nitrocellulose-filters for screening of E-tag expression using mouse anti-Etag antibodies (GE Healthcare) and secondary horse radish peroxidase-labeled sheep anti-mouse antibodies (GE Healthcare). The remaining colonies were washed off the plates and superinfected with helper phage to make an enriched library/phage stock, which was used in the second enrichment cycle (repanning) according to the same protocol. In total, two repannings were performed in each panning experiment. More than 200 E-tag positive colonies were chosen for plasmid preparation and sequence determination of the inserts using primer SAsekv (Table 1). Analyses of the inserts revealed nine, nine, and eight overlapping partial sequences, respectively, from three different genes. The complete sequences of these genes were extracted from the genomic sequence of Tpl strain V1. Based on BLAST searches and sequence analyses on the deduced amino acid sequences they were identified as a Treponema membrane protein A (TmpA) homolog, a putative phage tail tape measure protein (designated Ttm), and a putative proline-rich repeat lipoprotein (designated PrrA).

Table 1 (nos. 1–3), 1.25 U Pfu DNA polymerase (Fermentas) and 50 ng genomic DNA, was prepared. The thermal cycling conditions were 95 8C for 1 min, 30 cycles of 95 8C for 30 s, 50 8C for 30 s, and 72 8C for 3 min and a final extension at 72 8C for 5 min. PCR products were analyzed by agarose gel electrophoresis and purified with the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). Purified amplicons were digested with BamHI and XhoI and purified as described above. The digested amplicons were ligated into the BamHI and XhoI digested vector pGEX-6P-1 (GE Healthcare) using the ReadyToGo T4DNA Ligase (GE Healthcare). Ligated material was electrotransformed into competent E. coli strain BL21(DE3)pLysS (Novagen) and spread on LAamp. The presence of inserts in a number of colonies was analyzed by PCR using the vector sequencing primers pGEX 50 and pGEX 30 (Table 1). Clones with a correct size insert were further analyzed by DNA sequencing. The bulk GST purification module (GE Healthcare) was used for production of recombinant immunogenic Treponema proteins according to the manufacturer’s instructions. Recombinant clones were grown at 37 8C in LB media supplemented with ampicillin. At an optical density (OD600 nm) 0.7–1.0, the growth medium was supplemented with IPTG (final concentration 0.2 mM) after which the cultures continued to grow at 37 8C for another 3 h. After incubation, the cells were harvested and suspended in PBS-T whereupon the cells were lysed by freezing and thawing. After centrifugation, the supernatants were sterile filtrated and batch purified using glutathione-sepharose beads. After extensive washing using PBS-T the fusion proteins were treated with PreScission Protease (GE Healthcare) to release the produced proteins. Buffer exchange of the eluted proteins was performed by dialysis using Slide-A-Lyzer dialysis cassettes (Pierce) according to the manufacturer’s instructions. Finally, the amounts of proteins obtained were determined using spectrophotometry and the quality analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie staining. The proteins were stored at 20 8C.

2.6. Construction of clones for protein expression and purification

2.7. Enzyme-linked immunosorbant assay (ELISA)

A 50 ml reaction mixture of 5 ml 10 Pfu Buffer with MgSO4 (Fermentas), 0.2 mM of each deoxynucleotide, 0.2 mM of forward and reverse primer as indicated in

The assays were performed with sera from eight dairy cows with acute DD from the herd from which Tpl strain V1 was isolated, two cows from another herd with no known history of DD, and five calves 6–7 months of age.

Table 1 Deoxyoligoribonucleotides. No.

Name of forward (fwd) and reverse (rev) primer pair

Sequence, 50 to 30 , of each primer. Restriction enzyme cleavage sites are indicated in bold.

1

GSTTmpAF1 (fwd) GSTTmpAR1 (rev) GSTKallaF1 (fwd) GSTKallaR1 (rev) GSTPGKEEF1 (fwd) GSTPGKEER1 (rev) pGEX 50 (fwd) pGEX 30 (rev) SASEKV (fwd)

GGT GGT GGA TCC AAA GCG GAA CAA GAA GCT CA GGT GGT CTC GAG TCA TTG TAC ACC TCC CTC TA GGT GGT GGA TCC AAG AAA GAG CTG TTA GAT TT GGT GGT CTC GAG TTA TTT ATC AAT TTC TGC CAA GGT GGT GGA TCC CAA GGT CCA GCT AAC CCC ACA GGT GGT CTC GAG TTA GAG CTT CTC TAG CAC AAA GGG CTG GCA AGC CAC GTT TGG TG CCG GGA GCT GCA TGT GTC AGA GG TAT CTG GTG GCG TAA CAC CTG CT

2 3 4 5

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Digital dermatitis diagnosis was made by visual examination. Microplates (PolySorpTM, Nalge Nunc International) were coated with recombinant proteins at concentrations of 1 mg/ml TmpA, 0.8 mg/ml Ttm or 0.02 mg/ml PrrA in 100 ml 50 mM sodium carbonate, pH 9.5, overnight at 2 8C. Wells were washed twice with PBS-T and blocked with PBS-T for 1 h at room temperature. One hundred ml serum diluted 1:100 in PBS-T was added to each well. The microplates were incubated at 37 8C for 1 h and then washed with PBS-T. Horse-radish peroxidase (HRP) conjugated rabbit anti-bovine IgG antibodies (Sigma) diluted 1:8000 were added to the wells and plates were incubated for 1 h at 37 8C. The wells were washed three times with PBS-T after which 100 ml solution consisting of 1 mM tetramethylbenzidine and 0.006% H2O2 in 0.1 M potassium citrate pH 4.25, was added. The plates were incubated for 10 min at room temperature. To stop the reaction 50 ml 10% sulfuric acid was added. Optical density was measured at 450 nm and the readings were subtracted by the optical density of a sample buffer blank giving the corrected optical density (COD). 2.8. PCR for detection of genes encoding for the immunogenic proteins in field isolates of Treponema spp. DNA from isolates of Tpl and T. pedis from clinical submissions were used (Table 2). Bacterial cells were washed twice in PBS, boiled for 10 min in sterile water and cell debris removed by centrifugation. For detection of the genes encoding the immunogenic proteins in clinical isolates, primer pairs nos. 1–3 (Table 1) were used. A 50 ml reaction mixture of 5 ml GeneAmp 10 PCR buffer II (Applied Biosystems), 2.0 mM MgCl2, 0.2 mM of each deoxynucleotide, 0.2 mM of each primer, 1 U AmpliTaq DNA polymerase (Applied Biosystems) and 2 ml genomic DNA, was prepared. The thermal cycling conditions were 96 8C for 20 s, 30 cycles of 96 8C for 20 s, 50 8C for 30 s, and 72 8C for 60 s and a final extension at 72 8C for 2 min. PCR products were analyzed by agarose gel electrophoresis. 2.9. Western blot for detection of PrrA in field isolates of Treponema spp. One half mg of bulk GST purified PrrA, according to the protocol above, was coupled to a 1 ml HiTrap NHS-

activated HP column (GE Healthcare) according to the instructions provided by the manufacturer. Ten ml of final bleed rabbit serum obtained from immunization with Tpl strain V1 (described above) was passed through the column. The column was washed with 15 ml of PBS-T followed by washing with 10 ml of PBS before antibodies were eluted in 5 ml of 0.1 M glycine–HCL (pH 3.0). The pH of the 0.5 ml fractions was neutralized using Tris–HCl (pH 8.2) after which buffer exchange was performed on the eluates using Zeba Spin Desalting columns (Pierce). The purified antibodies were stored in phosphate buffered saline pH 7.4 (PBS) at 20 8C. For Western blot, isolates of Treponema spp. from clinical submissions were used (Table 2). Proteins were prepared from bacterial cells washed twice in PBS, boiled in sample buffer (62.5 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS, 0.1 M dithiothreitol, and 0.01% bromophenol blue) for 5 min, and centrifuged to remove cell debris. Released proteins were subjected to SDS-PAGE using the PhastSystem with precast 8–25% gradient gels (GE Healthcare). Proteins were diffusion blotted (PhastSystem at 65 8C for 45 min) onto nitrocellulose membranes (Hybond-ECL, GE Healthcare). Thereafter, the membranes were blocked for 30 min in PBS-T containing ovine albumin at a concentration of 0.1 mg/ml. The presence of PrrA was detected using the PrrA-antibodies described above followed by HRPlabeled anti-rabbit antibodies (Dako). Bound HRP-labeled antibodies were visualized when filters were developed with 4-chloro-1-naphtol according to standard procedure. 3. Results 3.1. Construction of the phage display library and selection of binding phages (panning) More than 200 selected phagemid inserts were analyzed of which 121 were unique. In shotgun phage display, the identification of a true interaction between proteins is characterized by the isolation of a number of clones with overlapping inserts (encoding parts of the same gene). Nine, nine, and eight overlapping partial sequences, respectively, from three different genes were detected (Fig. 1), all other inserts encoded genes only represented once in the panning. The contigs obtained from the 454 sequence were used to predict the full open reading frames

Table 2 Detection of genes encoding for three immunogenic proteins (tmpA, ttm, and prrA) in Treponema spp. field isolates by PCR. Isolate

Origina

V1 V2 T 413 T 603 T 2378 T 551B T 1126 T A4 T M1 T B683

DD Herd DD Herd DD Herd DD Herd DD Herd DD Herd DD Herd PEN PG PSU

a b c

A A B C D E F

Speciesb

tmpA

ttm

prrA

Ref

Tpl Tpl Tpl Tpl Tpl Tpl Tpl T. pedis T. pedis T. pedis

+ + + + + + +   

+ + + + + + +   

+ +   + +    

Pringle et al. (2008) This studyc Pringle et al. (2008) Pringle et al. (2008) Pringle et al. (2008) Pringle et al. (2008) This studyc Pringle et al. (2009) Pringle et al. (2009) Pringle and Fellstro¨m (2010)

DD, digital dermatitis; PEN, porcine ear necrosis; PG, porcine gingiva; PSU, porcine shoulder ulcer. Species determination based on 16S rRNAsequences; Tpl, Treponema phagedenis-like. Species identification was made by ISR2 PCR and sequencing according to Stamm et al. (2002).

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Fig. 1. Schematic presentation of three immunogenic proteins in Treponema phagedenis-like strain V1, TmpA, Ttm, and PrrA, aligned against translated inserts from phagemid particles binding rabbit-anti Treponema phagedenis-like strain V1 antibodies. The gray boxes cover regions of possible epitopes. Protein and translated insert lengths (aa) are shown to the right of each alignment.

and the corresponding amino acid (aa) sequences of the three immunogenic proteins. A homolog to the well characterized immunogenic lipoprotein TmpA of Treponema pallidum subsp. pallidum (hereafter referred to as T. pallidum), a putative phage tail tape measure protein (designated Ttm), and a putative proline-rich repeat lipoprotein (designated PrrA) were identified (GenBank acc. nos. HQ632795–HQ632797). The number of nucleotides (and aa:s) was predicted to be 1032 (344), 3543 (1181), and 753 (251), respectively. The TmpA homolog of Tpl strain V1 was almost identical to the earlier described TmpA in T. phagedenis (Yelton et al., 1991; GenBank acc. no. AAA27482), differing at only three aa positions. Both these TmpA homologs have an aa sequence identity of 44–45% to T. pallidum TmpA (GenBank acc. no. NP_219205) and were predicted by SpLip (Setubal et al., 2006) to be lipoproteins as well. The N-terminal third of Ttm was most similar to a bacteriophage protein belonging to the TP901 family while for the remaining part, no significant similarity was found in GenBank. No signal peptide was predicted for this protein according to the Signal P algorithm (Bendtsen et al., 2004). The PrrA protein was predicted as a lipoprotein by SpLip. A KAEEKKPE aa motif repeated three times was located at the N-terminal of the mature protein followed by a (PGKEE)7 aa repeat (aa 79–113). Immediately

following the proline-rich repeat region were two almost identical 67 aa domains, separated by four aa:s and differing at only three aa positions. The protein in total was rich in glutamic acid residues (approximately 22%). The only similarity found in GenBank for PrrA was within the proline-rich repeat region, e.g. in a Branchiostoma floridae hypothetical protein (GenBank acc. no. XP_002612362) and a Bacillus clausii cell surface protein (GenBank acc. no. YP_176917). There were no identical proline-rich repeats (PGKEE) among the Blast hits. 3.2. Genome sequencing The 454 sequence run generated 306,592 individual reads with an average length of 346 nucleotides and 35 coverage of the draft sequence. From the Newbler de novo assembly, 309 contigs were obtained and 197 large contigs represented 98.8% of the draft sequence (3,025,333 bp) which had a GC content of 39.9%. This assembly generated contigs containing the genes encoding TmpA and Ttm however, no contig contained the complete prrA gene. Another assembly was made with the CLC bio de novo assembler and default settings from which 1168 contigs were obtained. From this assembly the complete repetitive prrA gene sequence could be extracted.

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COD 3,6 3,2 2,8 2,4 2,0 1,6 1,2 0,8 0,4 0,0 TmpA

Ttm

PrrA

Fig. 2. Enzyme-linked immunosorbant assay with recombinant Treponema phagedenis-like strain V1 immunogenic proteins TmpA, Ttm, and PrrA as antigens. The assays were performed with sera from eight dairy cows with acute digital dermatitis (black bars), two cows with no known history of digital dermatitis and five calves 6–7 months of age (gray bars). Horse-radish peroxidase (HRP) conjugated rabbit anti-bovine IgG antibodies (Sigma) were used as secondary antibodies. Corrected optical density (COD) was measured at 450 nm.

3.3. Enzyme-linked immunosorbant assay (ELISA)

4. Discussion

The three immunogenic proteins identified in Tpl strain V1 were produced recombinantly; PrrA as a fulllength mature protein from aa +1 relative the cysteine residue of the predicted lipoprotein signal peptide to the last aa before the stop codon (aa:s 22–251), TmpA from aa +7 relative the cystein residue of the predicted lipoprotein signal peptide to the last aa before the stop codon (aa:s 29–344), and Ttm as a partial polypeptide covering aa:s 689–970, which are the aa:s constituting the consensus sequence of the overlapping Ttm sequences from the panning experiments. These proteins/polypeptides were used as antigens in indirect ELISAs where serum samples from cattle with and without DD were analyzed for presence of antibodies against the antigens. Different concentrations of antigen, sera, and different secondary antibodies were tested in pilot experiments (data not shown). The conditions under which the best discrimination between cattle with and without DD was achieved were used in the final experiment. For the TmpA antigen, the optical density for three samples from cattle with DD was lower than the highest value for the samples from clinically healthy cattle, while the tests with Ttm and PrrA were discriminatory in all cases but one (Fig. 2).

To date, diagnosis of DD is performed by visual examination of the claw. Due to the character of the infection (poly-microbial, including fastidious bacteria) conventional diagnostic methods such as culturing and PCR are not routinely used. A diagnostic test for identification of healthy herds would be of use to control the disease. In this study, three immunogenic proteins, TmpA, Ttm, and PrrA, were identified by selection of a Tpl phage display library against antibodies from a rabbit immunized with live bacteria. Additionally, the presence of antibodies against these proteins was detected in sera from cattle with DD by indirect ELISA. The T. pallidum immunogenic lipoprotein TmpA was described in 1985 (Hansen et al., 1985) and is used for syphilis serology (Tsang et al., 2007). In addition to T. phagedenis and Tpl strain V1, homologs to TmpA can also be found in Treponema vincentii (GenBank acc. no. ZP_05623496) and T. denticola (GenBank acc. no. AAS12950). When analyzed in the Tpl TmpA ELISA, sera from cattle with DD showed greater individual variation compared to in tests with the other two antigens. Except for normal individual variation, this may be an effect of the stage of the disease in the tested animals at sampling. The tmpB gene has been shown to be coordinately expressed with tmpA from a single operon in T. pallidum and the encoded TmpB protein has also shown immunogenic properties (Hansen et al., 1985). A tmpB homolog was identified in the genome sequence of Tpl strain V1 with the same gene organization as in T. pallidum (data not shown) but has not yet been evaluated as an immunogenic protein. The gene encoding the protein with homology to the TP901 family of phage tail tape measure proteins is situated within a segment of the genome containing a potentially intact prophage. Phage particles have been detected in electron microscope preparations of a Tpl culture (Demirkan et al., 2006) and have been induced in T.

3.4. PCR for detection of genes encoding for the immunogenic proteins and Western blot for detection of PrrA in field isolates of Treponema spp. The genes encoding for the TmpA homolog and Ttm were detected in all T. phagedenis-like field isolates but not in T. pedis isolated from pigs. The prrA gene was detected in four out of seven T. phagedenis-like isolates, confirmed by results from Western blot using purified PrrA-antibodies (PCR results are summarized in Table 2, Western blot data are not shown).

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phagedenis biotype Reiter (Masuda and Kawata, 1979). A biofilm-induced prophage has also recently been reported in Treponema denticola ATCC 35405 (Mitchell et al., 2010). The presence of phage particles has not been investigated for Tpl strain V1 but the antibody production in both the immunized rabbit and cattle with DD indicate that Ttm is expressed in Tpl spirochetes. The phagemid Ttm inserts aligned only to the C-terminal part of the protein, which had no homology to any protein in the GenBank database. All inserts but one covered aa:s 858–970 and one deviant insert extended into this region but started at aa 689 (Fig. 1). It has been suggested that tail tape measure proteins may be processed and cleaved (Pedersen et al., 2000), as has been shown for other phage tail proteins (Arisaka et al., 2003). Such cleavage products may have functional and immunogenic properties other than the precursor protein. Proline-rich repeats generally form extended structures and flexible regions (Williamson, 1994) and are found in both prokaryotic and eukaryotic proteins, most with unknown function. These were the only kind of proteins showing any homology to PrrA in the BLAST search and the homology shared was only within the proline-rich repeat region. At least two separate regions containing possible epitopes flanking the (PGKEE)7 repeats were indicated by the alignment of the PrrA phagemid inserts to full-length PrrA. However, because inserts covered the entire mature protein no region could be ruled out as harboring epitopes (Fig. 1). The prrA gene and gene product could not be detected in all Tpl field isolates (from herds B, C, and F, Table 2) and thus the use of this protein as a single antigen for detection of Tpl spirochetes in DD could give false negative results. The sera used in the ELISA tests were from the same herd as Tpl strain V1 and gave high COD values for PrrA. If PrrA-positive clones dominate in this herd one could expect positive results in the PrrA ELISA. However, Tpl isolates from a single farm have been shown to represent distinct clonal groups (Trott et al., 2003) why it is possible that both PrrA positive and PrrA negative Tpl spirochetes may be present within one herd. Because no sera were available from herds B, C, and F it was not possible to determine if cattle in these herds had any antibody response to PrrA. 5. Conclusion In this study, sera from cattle with DD have high levels of antibodies against the three Tpl proteins identified and used as antigens in indirect ELISAs. Advantages using purified recombinant proteins produced in E. coli compared to whole cell lysates in an ELISA are the possibilities of large-scale production, optimization and standardization of the test, e.g. different combinations of proteins may give less background due to higher specificity. Further testing of a larger number of individuals and herds will confirm whether the proteins identified in this study are suitable antigens in ELISA tests useful to declare herds as free from DD. Acknowledgement The study was supported by the Swedish Research Council Formas (221 – 2006-2254).

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