Humoral Immune Response in Dogs Naturally Infected with Borrelia burgdorferi sensu. lato and in Dogs after Immunization with a Borrelia Vaccine

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1 CVI Accepts, published online ahead of print on 0 March 00 Clin. Vaccine Immunol. doi:0./cvi.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Humoral Immune Response in Dogs Naturally Infected with Borrelia burgdorferi sensu lato and in Dogs after Immunization with a Borrelia Vaccine 0 0 Michael W. Leschnik *, Georges Kirtz, Gelas Khanakah, Georg Duscher, Ernst Leidinger, Johann G. Thalhammer, Anja Joachim, and Gerold Stanek Running title: canine immune response to borrelia antigen Medical Clinic for Internal Medicine and Infectious Diseases, Veterinary Parasitology Vienna, Veterinary University Vienna, A-0 Vienna, Austria InVitro Laboratory for Veterinary Diagnostic and Hygiene, A-00 Vienna, Austria Department for Hygiene and Applied Immunology, Medical University of Vienna, A-0 Vienna, Austria * Corresponding author. Mailing address: Medical Clinic for Internal Medicine and Infectious Diseases, Veterinärplatz, Veterinary University Vienna, A-0 Vienna, Austria Phone: (00)-00. Fax: (00) michael.leschnik@vetmeduni.ac.at Downloaded from on December, 0 by guest

2 0 0 ABSTRACT Lyme arthritis in dogs can be induced under experimental and natural conditions. However, the veterinary relevance of canine borreliosis is still under extensive investigation. The prevalence of symptoms is clearly low although the risk of tick exposure is high. Current research focuses on case definitions, methods for diagnosing clinical disease in dogs, and discrimination between an immune response to a natural infection versus vaccination. In this experimental study, dogs raised under tick-free conditions were allocated to two groups: dogs were vaccinated with a commercial borrelia vaccine and subsequently developed detectable antibody titers; were walked in a tick-endemic area on two consecutive days. On day five after exposure engorged ticks were removed from the dogs and analyzed for Borrelia DNA in real-time PCR assay. Blood samples were taken before exposure/vaccination and at defined time points thereafter. Antibody responses were evaluated using IFAT and Western blot. Seven dogs from which Borrelia-positive ticks were removed seroconverted and developed an individual immune response. Blood and urine samples taken from the tick-exposed group at weeks and for real-time PCR analysis and culture were always negative for bacterial DNA. In conclusion, despite serological evidence of infection/immunization no clinical signs of disease were observed. The antibody patterns in a single Western blot did not permit differentiation between the different sources of antigen (vaccine vs. natural infection). However repeated Western blots may be useful for confirmation of infection or vaccination status as time course of specific antibodies levels seem to be different. Downloaded from on December, 0 by guest

3 0 0 INTRODUCTION After more than 0 years' research on canine borreliosis, diagnosis of the infection remains difficult and often unsatisfactory when interpreting laboratory results and correlating them with a dog s symptoms (). When the first cases of canine borreliosis were published in the 0s it was assumed that the disease was similar to human Lyme disease. In recent years these presumptions have had to be corrected as studies have failed to correlate some clinical symptoms (neurological symptoms, renal failure, heart failure) and tissue analysis with definite confirmation of Borrelia burgdorferi sensu lato (s.l.) as the causative agent (,,, ). Thus far, Lyme arthritis is the only confirmed clinical outcome of infection with Borrelia spp. in dogs (, ). Identification of Borrelia spp. in infected dogs has been documented with varying success. Straubinger et al. () was able to regularly detect Borrelia spp. by PCR or culture from tissue specimens taken at the known site of tick attachment under experimental conditions, whereas this was impossible () or only possible in a low percentage of naturally infected dogs (, ). B. burgdorferi DNA was rarely detected in kidneys (), heart muscle, and joints () by PCR, immunohistochemistry (IHC) or modified silver staining, and fluorescence in situ hybridization. Most authors describe the isolation of spirochetes in blood samples from dogs as insensitive (, 0), although others reported detection of Borrelia DNA in one-third of dogs with suspected natural infection (). Canine immune response to B. burgdorferi s.l. has been tested by ELISA and Western blot assays based on recombinant or whole cell antigen. Sensitivities of % to.% and specificities of 0% to.% were reported by Štefančíková et al. () for ELISA assays based on three different strains, demonstrating the importance using local strains for serodiagnosis. Levy et al. (0) reported a sensitivity of.% and specificity of.% in a C antigen ELISA, Jacobson et al. () reported a sensitivity of.% and a specificity of Downloaded from on December, 0 by guest

4 0 0.% in a kinetic ELISA. All values were calculated by comparing the test results to other ELISA tests or Western blots. Cross-reactivity with other spirochetes (Leptospira spp., Treponema spp.) impairs the specificity of tests for Lyme borreliosis as Western blot bands at the level of p, p0-, and p were detectable in canine sera containing antibodies to Leptospira spp. (, ). Antibody response to B. burgdorferi s.l. is common in both symptomatic and asymptomatic animals in endemic areas, leading to the conclusion that only a very small percentage of dogs naturally infected by B. burgdorferi s.l. become symptomatic after a typical incubation period of a few weeks (). In previous studies the occurrence of clinical symptoms in experimentally infected dogs ranged from 0-% (,, ). In Europe the seroprevalence for B. burgdorferi s.l. in dogs (.%.%) has been documented in several studies (,,, ). Following the introduction of a commercially available vaccine for canine borreliosis (Merilyme, Merial, France), the percentage of dogs testing seropositive has increased considerably. In Austria seropositivity increased from % to % of all tested dogs (). The identification of Western blot patterns specific for infection with Borrelia spp. and the differentiation between naturally infected, vaccinated, or vaccinated and subsequently infected dogs are still major goals in the diagnostic procedures for canine borreliosis. There are indications to test vaccinated dogs and differentiate antibodies derived from vaccination from those induced after infection: ) There is strong evidence for only minimal cross-protection of vaccinal antibodies towards heterologous Borrelia spp. making infection and clinical symptoms possible (). ) Dogs may be vaccinated during incubation time causing clinical symptoms weeks thereafter. ) Dogs may develop clinical signs similar to borreliosis after vaccination without natural infection (). Downloaded from on December, 0 by guest

5 0 0 Previous studies identified specific Western blot bands as markers for infection or vaccination (, 0,, ) and reported different patterns for symptomatic and asymptomatic dogs (). Greene et al. () compared Western blot patterns of experimentally infected dogs to results of naturally exposed dogs concluding that fewer bands in experimentally infected dogs represent an acute immune response after single infection whereas a higher number of bands in naturally infected dogs reflect a chronic response to repeated exposure. However, because of differing experimental techniques (different strains, recombinant and whole cell Western blots), blot patterns vary greatly across the studies, making comparability and consequently diagnostic conclusions in single cases difficult. Levy et al. () demonstrated that serological examinations of apparently healthy dogs had no predictive value for the subsequent development of limb or joint disorder. When testing immune responses in dogs, in many cases lack of information on the exact time of natural infection, and consequently the stage of infection, makes the interpretation even more difficult. The aim of this study was to compare canine antibody responses to vaccination and natural infection with B. burgdorferi s.l. under controlled conditions, and to investigate possible hematogenous spread and urinary excretion in these dogs. MATERIALS AND METHODS Animals. Twenty-three adult Beagle dogs ( months of age) were included in this study. The experiments were approved by the institutional ethics committee (Veterinary University Vienna) and the Austrian Ministry for Science and Research (GZ.0/0-C/GT/00). All dogs were born and raised at the Veterinary University of Vienna under tick-free environmental conditions. The dogs were under continuing veterinary medical care and were examined for side effects of vaccination or symptomatic evidence of infectious disease. All dogs tested negative for Borrelia antibodies in an immunofluorescence antibody test (IFAT) prior to the study. Downloaded from on December, 0 by guest

6 0 0 Vaccination study. Eleven healthy, adult Beagle dogs were vaccinated with a commercially available whole-cell lysate Borrelia vaccine (B. burgdorferi s.l., Merilyme, Merial, France) according to the manufacturer s recommendations ( vaccinations weeks apart and vaccination a year later). Blood was sampled 0 times in each dog at intervals of weeks and twice more in six dogs a year after the first vaccination (Fig. ). Borrelia antibodies were evaluated in an IFAT (MegaScreen FLUOBORRELIA, MegaCor, Diagnostic Gmb H, Austria; cut-off titer :; sensitivity 0%, specificity.%) before the first and after the second vaccination (n=), then after weeks (n=) and after the third vaccination a year later (n=). Serum samples from all dogs were tested in two Western blots for detection of IgG and IgM antibodies (Fig. ). The recombinant recomblot (Microgen, Germany) provides specific bands for p00, VlsE, p (flagellin), p, OspA (kd), three bands of OspC (kd; B. garinii strain T, B. garinii strain 00, a combined band for B. burgdorferi sensu stricto and B. afzelii), two bands of a specific internal part of p antigen (p/i of B. garinii and p/i B. afzelii) and p (decorin binding protein A). The second Western Blot (MegaBlot, MegaCor, Diagnostic Gmb H, Austria; IgG and IgM) derived from B. garinii (strain VS0) was used for detection of additional bands (,,, and kda) and also served as an internal control. Band intensities were classified in a 0 scoring system, where intensity 0 corresponds to no band and intensity to the highest intensity. The cut-off for positivity was intensity, which was the same strength as the weak positive control provided by the manufacturer. Infection study. Eleven dogs were walked on a long leash for a few hours in highly endemic tick areas in Burgenland (autumn) and Vienna (spring) and exposed to possibly infected ticks on two consecutive days in October 00 (n=) and in April 00 (n=). One dog in the autumnal session had to be excluded from the study after three months because of developing a behavioral incompatibility, therefore only five remaining dogs and six new ones were walked in spring 00. Five days after the end of tick exposure the dogs were carefully Downloaded from on December, 0 by guest

7 0 0 examined for engorged ticks; these were subsequently removed and stored. Sites of tick bites were documented. All dogs received anti-tick spot-on permethrin treatment (Exspot, Essex, Germany) immediately after tick removal and thereafter no further ticks were found. Ticks were stored at -0 C until further examination. After being thawed they were classified for species, sex and developmental stage before dissection with sterile scalpel blades for DNA extraction with the DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany). Blood samples were collected three times at intervals of weeks in autumn 00 and seven times at intervals of - weeks in spring 00 (Fig. ). Borrelia antibodies were detected in IFATs, as above, at two time points (before tick exposure and at the end of the observation period) in autumn 00 and two in spring 00 (Fig. ). Samples from all dogs were tested in both Western blots as described for the vaccination study. In spring, urine samples were collected from the dogs one and two weeks after tick exposure (Fig. ). Urine and blood samples collected simultaneously were analyzed for Borrelia spp. in real-time PCR and in culture. PCR and culture. Ticks, samples of EDTA blood and urine specimens were analyzed in realtime PCR assays. A two-step procedure was used with two types of kit for each sample. First, the presence of Borrelia DNA was investigated by using part of the Borrelia flagellin gene () and part of the OspA gene of a commercially available real-time PCR assay (Ingenetix, Vienna, Austria). The primer and the TaqMan probe for fluorescent online detection were designed to react with all Borrelia species. In the second step, samples were typed using the ABI/Prism 00 Sequence Detector (Applied BioSystems, Foster City, CA) with three different probes for the spacer regions and with different kinds of dye. The reaction cycles were as follows: min at 0 C and min at C, followed by 0 cycles of s at C and min at 0 C. All preparations with positive signals were submitted to sequencing (Ingenetix, Vienna, Austria). Downloaded from on December, 0 by guest

8 For cultivation of B. burgdorferi s.l., -ml samples of each specimen (blood and urine) were placed in vials containing ml BSK II medium (Sigma-Aldrich, St. Louis, MO) and incubated at C. The presence of spirochetes was checked by dark field microscopy every week for eight weeks. 0 0 Statistics. Concurrent band intensities in vaccinated and infected dogs as well as the number of positive bands were compared using the unpaired Student s t-test. RESULTS Throughout the examination period of 0 weeks none of the vaccinated dogs showed any symptoms suggesting a vaccination side effect. All seven dogs infested with Borrelia spp. infected ticks remained healthy. Vaccination study. All eleven vaccinated dogs seroconverted after the second vaccination. Titers of dogs tested weeks after the second vaccination returned to negative (Table ). Six dogs were vaccinated a third time and were tested positive in the IFAT again weeks thereafter. Infection study. A total of 0 ticks, attached and engorged on the dogs, were collected and analyzed (Table ). It cannot be excluded that a small number of ticks were missed when collecting them from the dogs, because some ticks may have been removed by the dogs and some, especially larvae and nymphs, may have fallen off earlier. In real-time PCR analysis out of 0 ticks were positive for Borrelia burgdorferi s.l.: six female Ixodes ricinus, one I. ricinus nymph and two female I. canisuga. Of these Borreliae only four could be typed at geno-species level, namely B. afzelii, B. garinii and a mixed infection with B. afzelii and B. spielmani from three female I. ricinus, and one B. afzelii from an I. ricinus nymphal tick. Downloaded from on December, 0 by guest

9 0 0 Among the dogs in the infection study, out of seroconverted after autumnal exposure and out of seroconverted or had increased titers after spring exposure (Table ). Two out of these dogs had repeated infections (autumn and spring). All dogs that were infested with infected ticks showed a specific immune response (rising titers by IFAT). Development of seropositivity in the IFAT and detectable Borrelia DNA in attached ticks identified dogs for further evaluation by Western blot. Blood and urine samples tested negative for B. burgdorferi s.l. by PCR and culture in all dogs in the infection study. Comparison of Western blot results before/after vaccination and before/after tick exposure in dogs: ) Baseline Western blot results were obtained from all dogs before the first vaccination or first exposure to ticks. Results (IgG and IgM, recomblot and Megablot ) are displayed in Figure, indicating that several bands in variable frequency appear even before Borrelia vaccination or infection in dogs. Major differences (>0%) in the results of both IgM blots were detected when comparing frequencies of the p00, p, and the OspC band. ) The frequency of positive bands after vaccination or infection is displayed separately for Western blots early and late after vaccination/infection in Fig. a (IgG blots) and b (IgM blots). After vaccination frequencies of positive bands in the IgG Western blots decrease (from early to late), in particular p00, OspC, and pint./garinii more than 0%, which is in contrast to the immune response after infection, where band frequencies increase or stay stable in all bands but VlsE. In general IgM response after vaccination is of shorter duration than after infection and only bands are visible late after vaccination (p, p, p, OspA). Comparing IgM immune response in both study groups, bands for p, OspA, and pint./garinii occur solely after vaccination and pint./afzelii occures only after infection.. Downloaded from on December, 0 by guest

10 0 0 0 ) Specific mean IgG band intensities are displayed over time contrasting the results of the vaccinated dogs with the naturally infected dogs (Fig. ). All mean band intensities increased after each consecutive vaccination or infection; only the IgG p and p bands were seen solely after vaccination. Intensities for OspA remained high between the second and the third vaccination, whereas all other bands responsive to vaccination decreased in intensity to negative levels within four months. Mean band intensities after the second and/or third vaccination became positive or were classified as rising band score within weeks in p00, VlsE, p, p, p, p, OspA, OspC, and pint./garinii. Most band intensities increased even more after the third vaccination compared to the second vaccination (Fig. ). In the subclinically infected dogs, mean band intensities became positive or were classified as rising band score in p00 (after to weeks), p (after to weeks), p (after weeks), p (after weeks), and OspC (after weeks). In some individual dogs Western blots band intensities for p00, p, p, p, OspC, and pint./afzelii even increased within weeks after infection. When comparing consecutive band intensities in both groups, significant higher scores were evaluated in vaccinated dogs after the first vaccination/infection in p, and p; after the second vaccination/infection in p00, p, p, OspA, OspC (within to weeks), and pint./garinii. Significant higher scores in the infected dogs were seen after the first vaccination/infection in p00, VlsE, and p; after the second vaccination/infection in p, p, and in OspC only after weeks. When comparing the mean standard deviation of band intensities as a parameter for heterogeneity of Western blot results in each group we calculated a mean standard deviation of band intensities > in the vaccination group for OspA, and int./garinii (.0 and.); in the infection group for p00, VlsE, p, OspC, and pint./afzelii (range.0.). Downloaded from on December, 0 by guest

11 ) The mean percentage of positive IgG bands in each Western blot (recomblot and Megablot ) is displayed over time contrasting the results of vaccinated dogs and naturally infected dogs (Fig. ) showing significant higher values in the vaccinated group to weeks after second vaccination/infection. 0 0 DISCUSSION Natural infections with B. burgdorferi s.l. or vaccination with a Borrelia whole-cell lysate vaccine resulted in an individual and specific humoral immune response in each of the vaccinated or infected dogs. IFAT titers of seropositive dogs were similar irrespective of the groups to which the animals were allocated. Although there were differences between the Western blot patterns of vaccinated vs. infected dogs, individual variations within each group were considerable, and a single Western blot analysis may be unsuitable for differentiation between infection and vaccination. Even the baseline patterns prior to vaccination or tick exposure were highly individual and displayed positive results in individual dogs without history of tick infestation. Thus for proper interpretation of test results it is therefore necessary that clinical history and physical examination are taken into account for a definitive diagnosis. In the present study the infection pressure exerted by infected ticks on dogs was low. Despite the short exposure time seven of dogs became infected. This is a strong indication for a high infection risk in dogs that are walked in areas highly endemic for ticks. The infection risk is also based on the prevalence of B. burgdorferi s.l. in ticks, which is approximately 0% in both areas (). Although multiple transmissions increase infection stress for the host and may consequently increase the probability of clinical signs, this could not be observed in our study. Downloaded from on December, 0 by guest

12 0 0 In dogs, the incidence of clinical symptoms after a Borrelia-infected tick bite is estimated as being very low (< %) (, ). In an experimental setting, ticks were attached to the dogs on the thoracic wall and prevented from moving to another part of the dog s body. The joints next to the site of the tick bite developed more severe signs of arthritis (, ). In our natural setting of exposure, ticks were attached at different sites. Most ticks were collected from the head, neck, and ventral abdomen, which might influence bacterial distribution and give the host s immune system more time to eliminate the bacteria. Identifying the infectious agent in vivo is a major goal in the diagnosis of infectious diseases. In canine borreliosis it is particularly difficult and unproductive to attempt isolation of the bacteria or amplification of DNA from blood and urine samples (,, ), although some research groups have described successful identification of Borrelia DNA in canine blood and urine (, ). Even under controlled conditions and with precise knowledge of the time of infection, thus indicating the most promising time for identification of bacteria in blood and urine, we were not able to detect Borrelia spp. by culture and PCR assay. No correlation between the absence of clinical signs and the lack of detectable Borrelia DNA can be drawn from the given results. Detection of B. burgdorferi DNA in tissue samples of infected dogs was mostly successful after experimental infection (0), but was much less effective in examination of paraffin-embedded tissue sections from dogs with presumptive and naturally acquired Lyme borreliosis (). Thus it will be more promising to test for B. burgdorferi DNA in synovia or synovial membrane than in blood or urine in dogs with clinical evidence for arthritis. Serology has been considered as a major part of diagnostic criteria for canine borreliosis beside tick exposure, clinical symptoms, and response to antibiotics (). Quantitiative detection of specific antibodies in dogs in our study was highly sensitive as all vaccinated dogs and all dogs infested by infected ticks seroconverted. Downloaded from on December, 0 by guest

13 0 0 Commercial Western blot test systems for dogs utilize interpretation patterns for humans regarding the occurrence of specific bands in the time course of disease. This must be reconsidered in light of the present study, which shows that the time course of pattern development in dogs is different from that of humans, also documented by Lovrich et al. () for OspC. Specificity of single bands will have to be demonstrated for canine samples as many bands were detected in some dogs even before vaccination or infection (Fig.). This phenomenon might be explained by cross-reactivity of specific bacterial flora in the gastrointestinal tract of dogs. In previous studies antibodies directed against bands at the level of p, of the 0 kda range, as well as p, OspC, and pint./garinii showed detectable levels of crossreactivity (, ). In addition to the choice of bands, the combination and number of positive bands is another challenge in the diagnostic work-up of canine borreliosis. A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis in humans showed remarkable variation in results from different test systems (). It was reported that by including more bands defining positivity the resulting specificity increased and the sensitivity decreased. Test-specific scoring systems for individual bands, resulting in a cut-off sum for distinguishing positive from negative results, are commonly used in commercial test systems. Our results show the problem of these scoring systems as 0% of IgG and % of IgM baseline Western blots would have been interpreted as positive on the basis of a single specific band (p00, p or OspC). The mean percentage of total positive bands in dogs is significantly higher at to weeks after vaccination compared to dogs after infection. Later on the number of positive bands in the Westen blot does not allow discriminating between vaccinated and naturally infected dogs (Fig. ). In general, band patterns change in the time course after vaccination and infection. Downloaded from on December, 0 by guest

14 0 0 Intensities of OspA and p bands remain high after vaccination over a much longer period than bands for p00, OspC, and p/i. antigens. This is in contrast to the vaccination study by Töpfer and Straubinger (), who found that specific OspA antibodies decreased similarly to total antibody levels a few months after vaccination. After experimental infection, the first episode of lameness occurred after an incubation time of 0 days after tick exposure (0), which might be similar the time to test for canine borreliosis in veterinary practice. The corresponding Western blots in our study ( to weeks after infection, Fig. ) show that infected dogs had significantly lower mean band intensities for OspA and higher intensities for p and to some extent OspC, which is in concordance with Barthold et al. (). Nevertheless, it was nearly impossible to assign individual immunoblots to the vaccination group or infection group by the occurrence of a single band, as demonstrated by high standard deviation (>) even in the most promising bands (p00, VlsE, p, OspA, OspC, and both p/int. antigens; Fig. ). Repeating the Western blot within this critical period after the onset of clinical signs may assist the interpretation. A decrease of p00, OspC, and pint./garinii band intensities and stable high OspA bands is indicative for post vaccinal immune response. Stable or rising intensities in the p00, p, OspC, and pint./afzelli band gives strong indication for ongoing immune response after natural infection (Fig. a and ). The VlsE protein and its synthetic C peptide are supposedly not expressed in ticks, cell culture or Lyme vaccines and are therefore recommended as specific markers for infection in humans and dogs (). In the present study, however, VlsE was not helpful in distinguishing humoral responses to vaccination from humoral responses to infection. The IgM recombinant blot detected the VlsE band in some baseline blots as well as in presumed uninfected dogs; the IgG recombinant blot showed the VlsE band as a target of a strong immune response in Downloaded from on December, 0 by guest

15 both groups. These results highlight the difficulty of reproducing findings under differing test procedures and field conditions. 0 0 In general, the IgM responses in the dogs were less intense than IgG responses and appeared to develop within the same time period. Differing test results in the IgM recomblot and the IgM Megablot show insufficient reproducibility for the p00, p, and OspC band, and low specificity for several bands as evident in the baseline blots (Fig. ). The bands p00, VlsE, p, OspC, and pint./garinii bands did never occur in the late blots after vaccination and p, OspA and pint./garinii were only seen after vaccination (Fig. a and b). Due to general low frequency of specific bands testing for IgM in diagnosing canine Lyme disease is not recommended, a view also shared by Jacobson et al. (). In conclusion, the present study has highlighted some of the difficulties in diagnosis of canine borreliosis and the need to validate existing assays in dogs as well as developing more accurate criteria for defining canine borreliosis. Veterinarians should follow strict guidelines in the diagnostic work up of canine borreliosis. Detecting specific canine antibody response is one major diagnostic criterion for canine borreliosis but first arthritis should be confirmed by clinical findings and laboratory results. As Borrelia spp. are certainly of minor importance in the aethiology of canine polyarthritis differentials have to be ruled out. Serology is easy to perform but it should not replace the attempt to detect antigen (by culture and/or PCR) in synovial specimens in symptomatic dogs although sensitivity is known to be low. Serology alone can be misleading in the work up of a lame dog without any further investigation. Downloaded from on December, 0 by guest

16 0 0 One single Western blot test is insufficient in the differentiation between vaccinated and infected dogs. However repeated Western blots may be useful for confirmation of infection or vaccination status as time course of specific antibodies levels seem to be different. Western blot analysis is recommended for confirmation of screenings test systems, but individual immunoblots have several limitations. The interpretation aids supplied with commercial test kits reflect the situation in human and not canine borreliosis. The latter is certainly overdiagnosed because Western blots seem to be non-specific in several cases, as demonstrated in the present work where baseline blots appeared to be positive even though tick infestation and thus previous exposure to Borrelia spp. was extremely unlikely. The exact time point of infection is unknown in the majority of canine cases, and therefore differentiation of single Western blot patterns as early or late infection does not seem appropriate, particularly as dogs have a high incidence of tick infestation, making multiple transmissions of pathogens likely. Assays for detection of canine immune response should be validated and adapted to the knowledge of canine borreliosis. ACKNOWLEDGEMENTS This work was supported by Novartis Animal Health, Switzerland. The IFAT slides (MegaScreen FLUOBORRELIA c.) and Western blots (MEGABlot (IgM, IgG) BORRELIA canis) were in part provided by MegaCor Diagnostik GmbH, Austria. The Western Blots (recomblot Borrelia canis (IgM, IgG), Mikrogen, Germany) were in part provided by Biomedica Medizinprodukte GmbH & Co KG, Austria. Downloaded from on December, 0 by guest

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18 . Chou, J., A. Wünschmann, E. Hodzic, and D.L. Borjesson. 00. Detection of Borrelia burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis. J. Am. Vet. Med. Assoc. : Egenvall, A., B.N. Bonnett, A. Gunnarsson, A. Hedhammar, M. Shoukri, S. Bornstein, and K. Artursson Sero-prevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs -. Scand. J. Infect. Dis. :-.. Greene, R.T., R.L. Walker, W.L. Nicholson, H.W. Heidner, J.F. Levine, E.C. Burgess, M. Wyand, E.B: Breitschwerdt, and H.A. Berkhoff.. Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs. J. Clin. Microbiol. :-. 0. Guerra, M.A., E.D. Walker, and U. Kitron Quantitative approach for the serodiagnosis of canine Lyme diseases by the immunoblot procedure. J. Clin. Microbiol. :-.. Hovius, J.W.R., K.E. Hovius, A. Oei, D.J. Houwers, and A.P. van Dam Antibodies against specific proteins of and immobilizing activity against three strains of Borrelia burgdorferi sensu lato can be found in symptomatic but not in infected asymptomatic dogs. J. Clin. Microbiol. :-.. Hovius, K.E., L.A.M. Stark, N.M.C. Bleumink-Pluym, I. van de Pol, N. Verbeek-de Kruif, S.G.T. Rijpkema, L.M. Schouls, and D.J. Houwers.. Presence and distribution of Borrelia burgdorferi sensu lato species in internal organs and skin of naturally infected Downloaded from on December, 0 by guest

19 symptomatic and asymptomatic dogs, as detected by polymerase chain reaction. Vet. Quart. : Hovius, K.E., S.G. Rijpkema, P. Westers, B.A.M. van der Zeijst, F.J.A.M. van Asten, and D.J. Hopuwers.. A serological study of cohorts of young dogs, naturally exposed to Ixodes ricinus ticks, indicates seasonal reinfection by Borrelia burgdorferi sensu lato. Vet. Quart. :-0.. Hutton, T.A., R.E. Goldstein, B.L. Njaa, D.Z. Atwater, Y.-F. Chang, and K.W. Simpson. 00. Search for Borrelia burgdorferi in kidneys of dogs with suspected Lyme Nephritis. J. Vet. Intern. Med. :0-.. Jacobson, R.H., Y.-F. Chang, and S.J. Shin.. Lyme disease: Laboratory diagnosis of infected and vaccinated symtomatic dogs. Semin. Vet. Med. Surg. :-.. Jäderlund, K.H., A. Egenvall, K. Bergström, Å. Hedhammar. 00. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in dogs with neurological signs. Vet. Rec. 0:-.. Leschnik, M., G. Kirtz, R. Skerlak, and P. Ludwig. 00. Cross-reactivity of leptospiral antibodies in Borrelia Western blot in acute stage of leptospirosis in dogs but not in leptospiral vaccinated dogs. P-, p. 0. Abstr. VII. Internat. Potsdam Sympos. on Tickborne Diseases, Potsdam, Germany.. Leschnik, M.W., and G. Kirtz. 00. Borreliosis in dogs: current procedure of diagnosis and prevalence of antibodies against Borrelia burgdorferi sensu lato after 0 months of the Downloaded from on December, 0 by guest

20 0 introduction of a dog vaccine against borreliosis in Austria. P-, p.. Abstr. Sympos. on Tick-transmitted Diseases, Ljubljana, Slovenia Levy, S.A., L.A. Magnarelli.. Relationship between development of antibodies to Borrelia burgdorferi in dogs and the subsequent development of limb/joint borreliosis. J. Am. Vet. Med. Assoc. :-. 0. Levy, S.A., T.P. O Connor, J.L. Hanscom, and P. Shields. 00. Utility of an in-office C ELISA test kit for determination of infection status of dogs naturally exposed to Borrelia burgdorferi. Vet. Ther. :0-.. Littman, M.P. 00. Canine borreliosis. Vet. Clin. Small. Anim. :-.. Littmann, M.P., R.E. Goldstein, M.A. Labato, M.R. Lappin, and G.E. Moore. 00. ACVIM small animal consensus statement on Lyme disease in dogs: diagnosis, treatment, and prevention. J. Vet. Intern. Med. 0:-.. Lovrich, S.D., R.L. La Fleur, D.A. Jobe, J.C. Johnson, K.E. Asp, R.F. Schell, and S.M. Callister. 00. Borreliacidal OspC antibody response of canines with Lyme disease differs significantly from that of humans with Lyme disease. Clinical and Vaccine Immunology :-.. Pejchalová, K., A. Žakovská, K. Fučik, and P. Schánilec. 00. Serological confirmation of Borrelia burgdorferi infection in dogs in the Czech Republic. Vet. Res. Comm. 0:-. Downloaded from on December, 0 by guest

21 . Robertson, J., E. Guy, N., Andrews, B. Wilske, P. Anda, M. Granström, U. Hauser, Y. Moosmann, V. Sambri, J. Schellekens, G. Stanek, and J. Gray A European multicenter study of immunoblotting in serodiagnosis of Lyme borreliosis. J. Clin. Microbiol. : Skotarczak, B., B. Wodecka, A. Rymaszewska, M. Sawczuk, A. Maciejewska, M. Adamska, T. Hermanowska-Szpakowicz, and R. Świerzbińska. 00. Prevalence of DNA and antibodies to Borrelia burgdorferi sensu lato in dogs suspected of Borreliosis. Ann. Agric. Environ. Med. :-0.. Speck, S., B. Reiner, W.J. Streich, C. Reusch, and M.M. Wittenbrink. 00. Canine borreliosis: A laboratory diagnostic trial. Vet. Microbiol. 0:-.. Speck, S., K. Failing, B. Reiner, and M.M. Wittenbrink. 00. Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs. Vet. Microbiol. :-0.. Štefančíková, A., G. Tresová, B. Pet ko, I. Škardová, and E. Sesztáková.. ELISA comparison of three whole-cell antigens of Borrelia burgdorferi sensu lato in serological study of dogs from area of Košice, Eastern Slovakia. Ann. Agric. Environ. Med. : Straubinger, R.K PCR-based quantification of Borrelia burgdorferi organisms in canine tissue over a 00-day postinfection period. J. Clin. Microbiol. :-. Downloaded from on December, 0 by guest

22 . Straubinger, R.K., A.F. Straubinger, B.A. Summers, R.H. Jacobson, and H.N. Erb.. Clinical manifestations, pathogenesis, and effect of antibiotic treatment on Lyme borreliosis in dogs. Wien. Klin. Wochenschr. 0: Töpfer, K.H., and R.K. Straubinger. 00. Characterization of the humoral immune response in dogs after vaccination against the Lyme borreliosis agent. A study with five commercial vaccines using two different vaccination schedules. Vaccine :-.. Turk, N., A. Marinculić, and Z. Modrić Serologic studies of canine Lyme borreliosis in the Zagreb area (Croatia). Vet. Archiv 0:-.. Wallich, R., S.E. Moter, M.M. Simon, K. Ebnet, A. Heiberger, and M.D. Kramer. 0. The Borrelia burgdorferi flagellum-associated -kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect. Immun. :-.. Weber, A., U. Heim, and R. Schäfer.. Zum Vorkommen von Antikörpern gegen Borrelia burgdorferi bei Hunden einer Kleintierpraxis in Nordbayern. Berl. Münch. Tierarztl. Wochenschr. 0:-.. Wieler, L.H., C. Szattelberger, R. Weiß, R. Bauerfeind, P. Kutzer, K. Failing, and G. Baljer.. Serum antibodies against particular antigens of Borrelia burgdorferi sensu stricto and their potential in the diagnosis of canine Lyme borreliosis. Berl. Münch. Tierarztl. Wschr. :-. Downloaded from on December, 0 by guest

23 TABLES TABLE. Borrelia IFAT titers in dogs before and after vaccination. 0 Dog no. Titer before vaccination Titer weeks after nd vaccination Titer weeks after nd vaccination Titer weeks after rd vaccination n : : n : : n : : n : n n : n n : : n : : n : : n : n 0 n : n n : n n, negative. Downloaded from on December, 0 by guest

24 TABLE. Number of engorged ticks collected from dogs after natural exposure, number of Borrelia PCR-positive ticks, and Borrelia IFAT-titers before and after natural tick exposure Dog no. No. of ticks (no. PCR-positive) Titer - before infection Titer - weeks after st infection Titer - before nd infection Autumn Spring Autumn Spring Titer - weeks after nd infection (0) 0 (0) n n n n () () n : n : 0 (0) () n n n : () (0) n : n : () () n : : : (0) excluded n n excluded excluded - () n : - () n : 0 - () n : - (0) n n - (0) n n - (0) n n total () () Downloaded from on December, 0 by guest n, negative.

25 FIGURE LEGENDS FIG.. Time schedule for the vaccination and infection studies in dogs. i= infection study group; v= vaccination study group. 0 0 FIG.. Frequency of Borrelia Western blot bands in baseline blots (recomblot +MegaBlot ; IgG and IgM) in dogs. FIG. a, b. Frequency of Borrelia Western blot bands (recomblot +MegaBlot ; IgG, IgM) in dogs: after vaccination (early: weeks after nd and rd vaccination; late: weeks after nd vaccination) and after infection (early: weeks after infection; late: weeks after infection). FIG.. Mean intensities (± standard deviation) of IgG Western blot bands in dogs after B. burgdorferi vaccination and natural infection (at each week 0). Significance value: * p<0.0 and ** p<0.0. FIG.. Mean percentage of positive bands in IgG Western blots in dogs after B. burgdorferi vaccination and natural infection (at each week 0). Significance value: ** p<0.0. Downloaded from on December, 0 by guest

26 FIGURES FIG.. group v (n=) examination time schedule tick exposure ( days) vaccination tick exposure ( days) ** i (n=) FIG.. frequency (%) p00 VlsE time (weeks) baseline blots (IgM and IgG) p p p p band p * urine / blood sampling for PCR Borrelia -IFAT IgG Western blot IgM Western blot OspA vaccination baseline recomblot IgM baseline Megablot IgM baseline recomblot IgG baseline Megablot IgG OspC pint./g. Downloaded from on December, 0 by guest

27 FIG. a. frequency (%) FIG. b. frequency (%) p00 p00 VlsE VlsE p p p p frequency of bands (IgG) p p p band p OspA frequency of bands (IgM) p p p band p OspA OspC OspC pint./g. pint./g. early after vaccination late after vaccination early after infection late after infection pint./a. pint./a. p early after vaccination late after vaccination early after infection late after infection p Downloaded from on December, 0 by guest 0

28 FIG.. band intensity vaccinated infected * ** p00 * band intensity vaccinated infected ** ** VlsE band intensity weeks after vaccination / infection vaccinated infected ** kda weeks after vaccination / infection band intensity weeks after vacination / infection vaccinated infected * kda ** * weeks after vaccination / infection ** * * Downloaded from band intensity band intensity 0 0 vaccinated infected ** p ** * ** * ** weeks after vaccination / infection vaccinated infected ** kda weeks after vaccination / infection band intensity band intensity 0 0 vaccinated infected p weeks after vaccination / infection vaccinated infected OspA ** * * ** ** ** ** ** * weeks after vaccination / infection on December, 0 by guest

29 band intensity band intensity 0 0 vaccinated infected OspC ** * * weeks after vaccination / infection vaccinated infected pint/afzelii weeks after vaccination / infection band intensity 0 pint/garinii vaccinated infected ** ** ** weeks after vaccination / infection Downloaded from on December, 0 by guest

30 0 FIG. mean percentage of positive bands in IgG Western-blots percentage (%) vaccinated infected ** ** week Downloaded from on December, 0 by guest