Bartonella spp. are etiological agents of life-threatening zoonotic diseases in dogs worldwide. Due to the poor sensitivity of immunofluorescent-antibody assays (IFAs), a reliable serodiagnostic test for canine bartonelloses is of clinical importance. The utility of Western blotting (WB) for the serodiagnosis of canine bartonelloses has not been critically investigated. The objective of this study was to characterize WB immunodominant proteins that could be used to confirm a serodiagnosis of bartonelloses.
KEYWORDS: serology, zoonosis, vector borne, Western blotting, antigen, Bartonella henselae
ABSTRACT
Bartonella spp. are etiological agents of life-threatening zoonotic diseases in dogs worldwide. Due to the poor sensitivity of immunofluorescent-antibody assays (IFAs), a reliable serodiagnostic test for canine bartonelloses is of clinical importance. The utility of Western blotting (WB) for the serodiagnosis of canine bartonelloses has not been critically investigated. The objective of this study was to characterize WB immunodominant proteins that could be used to confirm a serodiagnosis of bartonelloses. Using agar-grown Bartonella henselae San Antonio type 2 (SA2) whole-cell proteins, sera derived from four dog groups were tested by WB to assess immunodominant protein recognition patterns: group I consisted of 92 serum samples (10 preexposure and 82 postexposure serum samples) from 10 adult beagles experimentally inoculated with Bartonella spp., group II consisted of 36 serum samples from Bartonella PCR-positive naturally infected dogs, group III consisted of 26 serum samples from Bartonella PCR-negative and IFA-negative dogs, and group IV consisted of serum samples from 8 Brucella canis IFA-positive and 10 Rickettsia rickettsii IFA-positive dogs. Following experimental inoculation, 9 (90%) group I dogs were variably seroreactive to one or more of six specific immunodominant proteins (13, 17, 29, 50, 56, and 150 kDa). There was a strong but variable recognition of these proteins among 81% of group II dogs. In contrast, 24/26 group III dogs were not reactive to any immunodominant protein. In this study, the sensitivity and diagnostic accuracy of B. henselae SA2 WB were higher than those of B. henselae SA2 IFA testing. Some B. henselae SA2 immunodominant proteins were recognized by dogs experimentally and naturally infected with Bartonella spp. other than B. henselae. Additional research is necessary to more fully define the utility of WB for the serodiagnosis of canine bartonelloses.
INTRODUCTION
Species of Bartonella, a genus of Gram-negative bacteria, are etiological agents of acute, chronic, and potentially life-threatening infectious diseases in animals and humans throughout the world. Bartonella-associated disease manifestations in humans include cat scratch disease (CSD), endocarditis, myocarditis, bacillary angiomatosis, epistaxis, vasculitis, thromboembolism, polyarthritis, granulomatous lymphadenitis, and peliosis hepatis and a spectrum of neurological symptoms (1–4). Canine bartonelloses are associated with similar pathologies, serious morbidity, and, at times, mortality (5–7). Serology is the primary modality used for the diagnosis of bartonelloses in dogs and human patients (8, 9). Historically, serological methods, such as immunofluorescent-antibody assays (IFAs), have provided the most practical and least invasive means of deriving a clinical diagnosis. However, poor IFA sensitivity often leads to false-negative serological results in dogs and humans, in whom infection can be confirmed by culture or PCR testing (9–11). For example, antibodies were not detected by IFAs in the majority (75%) of Bartonella henselae-infected dogs (11). In addition, testing of serum samples from Bartonella PCR-positive dogs (n = 34) against a panel of eight Bartonella IFA antigens, including three B. henselae strains, did not substantially improve the sensitivity (10). Thus, in the clinical setting, negative IFA results should be interpreted with caution.
Western blotting (WB) has been used for the serodiagnosis of bartonelloses in humans; however, the utility of WB for the serodiagnosis of canine bartonelloses has not been critically investigated. A study investigating the utility of B. henselae WB using serum samples from 92 human patients clinically suspected of cat scratch disease (CSD) documented a higher sensitivity of WB (53.5%) than of IFA (28.3%) (12). Investigators reported a 100% sensitivity of WB when it was used to diagnose blood culture-negative endocarditis in humans (3).
We hypothesized that B. henselae WB would be more sensitive than the B. henselae IFA for the serodiagnosis of bartonelloses in dogs. Our three specific objectives were (i) to define Bartonella immunodominant proteins recognized by sera from dogs experimentally and naturally infected with Bartonella spp., (ii) to characterize the WB patterns that could be used for the serodiagnosis of canine bartonelloses, and (iii) to compare the sensitivity and the specificity of B. henselae WB and B. henselae IFA.
MATERIALS AND METHODS
Group I (10 dogs experimentally infected with Bartonella spp.).
Sera from dogs that were inoculated intradermally with blood agar plate-grown Bartonella spp. as described previously (13) were provided by Bruno Chomel, School of Veterinary Medicine, University of California, Davis, CA. This study was approved by the UC Davis Institutional Animal Care and Use Committee (13). In brief, 6 adult beagles were inoculated with B. henselae (strain 94022, isolated from a cat experimentally infested with fleas). Two dogs each were intradermally infected with inocula of 1.1 × 109 CFU/ml, 6.3 × 107 CFU/ml, and 2.4 × 106 CFU/ml. Two dogs receiving 6.3 × 107 CFU/ml were reinoculated with B. henselae strain 94022 at 6.6 × 107 CFU/ml at 40 days after the initial B. henselae inoculation. Two beagles were inoculated with Bartonella vinsonii subsp. berkhoffii type II (strain Coyote 15). After 7 months, two of the B. henselae-inoculated dogs were inoculated with Bartonella rochalimae (strain Coyote 004). Prior to the initial inoculation, all dogs were Bartonella PCR negative and seronegative and seroconverted within 2 weeks postinoculation. Dogs inoculated with B. vinsonii subsp. berkhoffii type II and B. rochalimae, Bartonella spp. for which dogs may be reservoirs, became bacteremic, whereas blood cultures were negative for B. henselae-inoculated dogs (13). A total of 92 serum samples sequentially collected from each dog prior to and following inoculation of the Bartonella spp. were used in this study. Sera from eight time points were tested (Table 1).
TABLE 1.
Patterns of Bartonella henselae SA2 protein recognition among 10 group I dogsa
| Dog | Strain/inoculum dose (CFU/ml) |
B. henselae SA2 WB banding pattern(s) (kDa) in dogs experimentally inoculated with Bartonella spp. |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| Preexposure sera | p.i. wk 1 | p.i. wk 2 | p.i. wk 3 | p.i. wk 4 | p.i. wk 5–6 | p.i. wk 7–8 | p.i. wk 9–10 | ||
| 6GCB10 | B. henselae/1.1 × 109 | None | None | 29, 27, 25 | 29,27, 25 | 68, 29, 27, 25, 17, 13, 10 | 29, 27, 25 | NA | 27 |
| 6GCB11 | B. henselae/1.1 × 109 | None | 28 | 56, 29, 27, 25 | 62, 56, 29, 27, 25, 15, 13 | 62, 56, 29, 27, 25, 15, 13 | 62, 29, 27 | NA | 62, 29, 27 |
| 6GCB4 | B. henselae/6.3 × 107 | 183, 27, 25, 17, 13 | NA | 168, 27, 25, 17 | 168,27, 25, 17 | 168, 27, 25, 17 | 168, 27, 25, 17 | 168, 62, 56, 50, 45, 30, 27, 25, 17, 15 | 168, 138, 62, 56, 50, 45, 30, 27, 25, 17, 15 |
| 6GCB7 | B. henselae/6.3 × 107 | None | NA | 168, 150, 56, 50, 35, 27, 25, 17, 13 | 56, 35, 27, 25 | None | 35 | 75, 62, 56, 35, 17 | 75, 62, 56, 35, 17 |
| 6GCB5 | B. henselae/2.4 × 106 | None | None | None | 13 | 13 | 13 | None | NA |
| 6GCB1 | B. henselae/2.4 × 106 | None | None | None | None | None | None | None | NA |
| 6GCB6 | B. vinsonii subsp. berkhoffii type II/7.6 × 107 | 29 | 29 | 29 | 41, 37, 29 | 233, 124, 94, 56, 41, 37, 29, 22, 13 | 233, 124, 94, 56, 41, 37, 29, 22, 13 | NA | 94, 56, 41, 37, 29, 13 |
| 6GCB9 | B. vinsonii subsp. berkhoffii type II/7.6 × 107 | None | None | 13 | 183, 56, 29, 13 | 183, 56, 29, 13, 10 | 183, 56, 29, 13, 10 | NA | 183, 56, 29, 13 |
| 6GCB10b | B. rochalimae/9.2 × 106 | None | <10 | <10 | <10 | <10 | 56, <10 | 56, <10 | 56, <10 |
| 6GCB11b | B. rochalimae/9.2 × 106 | None | 56, 15, 13 | 150, 138, 56, 15, 13 | 56, 15 | 150, 138, 56, 15 | 150, 138, 56, 15 | 150, 138, 56, 43, 15, 13 | 56, 15 |
Group I dogs were experimentally inoculated with Bartonella spp. Entries for dogs inoculated with B. henselae are unshaded, entries for dogs inoculated with B. vinsonii subsp. berkhoffii genotype II are shaded light gray, and entries for dogs inoculated with B. rochalimae are shaded dark gray. Two dogs (dogs 6GCB4 and 6GCB7) were reinoculated with the same B. henselae strain, strain 94022, at 6.6 × 107 CFU/ml at 40 days after the initial B. henselae inoculation. p.i., postinoculation; NA, not available.
Dogs that had previously been inoculated (7 months earlier) with B. henselae.
Group II (36 Bartonella PCR-positive naturally infected dogs).
Serum samples from 36 naturally exposed Bartonella PCR-positive dogs, 34 of which were reported on in a previous study (10), were tested by B. henselae WB. Based upon PCR amplification/DNA sequencing results, these dogs were infected with B. henselae (n = 22), B. vinsonii subsp. berkhoffii (n = 8), B. rochalimae (n = 2), Bartonella koehlerae (n = 2), Bartonella quintana (n = 1), and Bartonella clarridgeiae (n = 1).
Group III (26 Bartonella PCR-negative and IFA-negative dogs).
Sera from group III dogs, reported on in a previous study of the IFA (10), were selected from the North Carolina State University (NCSU) College of Veterinary Medicine (CVM) Vector Borne Diseases Diagnostic Laboratory (VBDDL) archives. In an effort to identify dogs with minimal or no vector exposure, these sera were selected on the basis of negative serological and PCR assay results. All 26 dogs had previously tested IFA negative (titers, ≤1:16 screening dilution) for three Bartonella spp. (B. henselae, B. vinsonii subsp. berkhoffii, and B. koehlerae). In addition, these dogs were not seroreactive to Anaplasma, Babesia canis, Borrelia burgdorferi, Rickettsia rickettsii, or Ehrlichia antigens by IFA or a commercial enzyme-linked immunosorbent assay (ELISA; SNAP 4Dx+; Idexx Laboratories, Westbrook, MI) and were PCR negative for Bartonella, Babesia, Ehrlichia, Anaplasma, Rickettsia, Mycoplasma, and Leishmania (NCSU, VBDDL, Raleigh, NC). Therefore, these dogs were presumably considered to be unexposed to Bartonella spp.
Group IV (8 Brucella canis IFA-positive and 10 Rickettsia rickettsii IFA-positive dogs).
To further assess specificity, Brucella canis (n = 8)- or R. rickettsii (n = 10)-seroreactive dog sera were tested by WB. Sera from Brucella canis IFA- and ELISA-positive dogs, kindly provided by Andrew Johnson, Veterinary Medical Research and Development, Inc., or R. rickettsii IFA-positive dogs, obtained from the NCSU VBDDL, were tested, as these are two important intracellular pathogens belonging to the class alphaproteobacteria, the members of which are phylogenetically related to the genus Bartonella. These sera were screened for the presence of antibodies to B. henselae San Antonio type 2 (SA2), B. vinsonii subsp. berkhoffii type I, and B. koehlerae by IFA using cell culture-grown B. henselae SA2, B. vinsonii subsp. berkhoffii type I, and B. koehlerae antigens and by B. henselae SA2 WB at NCSU.
IFA.
Group I dogs’ sera were tested for the presence of Bartonella antibodies using an immunofluorescent-antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA) in a previous study (13). In brief, Bartonella antibody titers were determined for 6 B. henselae- and 2 B. rochalimae-inoculated dogs by IFA using the inoculation strain type, as described previously (13). For B. vinsonii subsp. berkhoffii type II-inoculated dogs, an ELISA was performed to assess seroconversion, as described previously (13). To compare WB and IFA results for group I dogs, serum samples from six B. henselae-inoculated dogs were tested for the presence of Bartonella antibodies at the NCSU laboratory by IFA using as the antigen cell culture-grown B. henselae SA2, the same strain that was used for WB analysis in this study. Due to inadequate serum volumes, NCSU B. henselae SA2 IFA results were not available for B. vinsonii subsp. berkhoffii type II- and B. rochalimae-inoculated dogs.
IFA results for group II (PCR-positive) and group III (PCR-negative and IFA-negative) dogs were reported in a previous study from the NCSU laboratory (10). These sera were tested by the IFA using eight Bartonella antigens. IgG titers of ≥1:64 were considered positive for Bartonella exposure. Based on an IFA using eight Bartonella antigens, 58% (21/36) of Bartonella PCR-positive group II dogs were seroreactive to at least one of the eight Bartonella antigens, whereas 25% (9/36) were seroreactive to at least one of three B. henselae antigens (B. henselae California-1, B. henselae Houston-1, and B. henselae SA2). Only 8 (22%) group II dogs were IFA positive for B. henselae SA2. Twenty-six group III dogs were negative on initial diagnostic IFA screening for B. henselae SA2, B. vinsonii subsp. berkhoffii type I, and B. koehlerae antibodies, and 4 of these dogs were IFA positive for one or more of the eight Bartonella antigens: one dog each was IFA positive for B. vinsonii subsp. berkhoffii type II, B. vinsonii subsp. berkhoffii type III, or B. quintana, and the fourth dog was B. vinsonii subsp. berkhoffii type II, B. vinsonii subsp. berkhoffii type III, B. henselae California-1, B. henselae Houston-1, and B. henselae SA2 seroreactive.
PCR amplification, BAPGM enrichment blood culture, and DNA sequencing.
For group I dogs, a Bartonella PCR targeting the gltA gene was performed using a previously described PCR methodology (14) on DNA extracted from Bartonella blood agar plate isolates (Bruno Chomel, personal communication). Bartonella PCR was not performed on whole blood for group I dogs (13). EDTA-anticoagulated samples from group I and IV dogs were not available for enrichment culture testing on Bartonella alphaproteobacterium growth medium (BAPGM).
For group II and III dogs, blood samples were processed in BAPGM for up to 21 days as previously described (8). Genomic DNA was extracted from EDTA-anticoagulated blood specimens using an automated robotic workstation (BioRobot M48; Qiagen, Valencia, CA) according to the manufacturer’s protocol. DNA extracted from blood and BAPGM subcultures was tested using Bartonella genus-specific PCR primers targeting the intergenic transcribed spacer (ITS) region as previously described (8). Contig Express software and Align X software (Vector NT Suite 10.1; Invitrogen Corp., CA, USA) were used for chromatograph evaluation and sequence alignment. In silico analyses of DNA sequences were performed using the NCBI BLAST program (version 2.0) for the identification of bacterial species and strain.
Preparation of B. henselae SA2 whole-cell proteins for WB.
An isolate of B. henselae SA2 (NCSU strain 95 FO-099), obtained from a naturally infected cat (15), was used for the WB antigen preparations in this study. The B. henselae SA2 stock used for WB antigen preparation had not been passaged more than 3 times. The B. henselae SA2 strain has been the most frequently identified B. henselae strain isolated or PCR amplified from clinical specimens from sick dogs tested in the NCSU CVM VBDDL. B. henselae SA2 was cultivated on Trypticase soy agar plates supplemented with 5% defibrinated rabbit blood (BBL, Cockeysville, MD). The plates were incubated for 5 to 7 days at 35°C in the presence of 5% CO2 and 99% relative humidity. B. henselae SA2 whole-cell proteins were extracted by harvesting 5- to 7-day-old colonies in 300 μl of the B-PER reagent (Pierce, Rockford, IL). The contents were vortexed vigorously until the cell suspension was homogeneous. Soluble proteins were collected by centrifugation at 13,000 rpm (13,793 × g) for 5 min. The protein concentrations of B. henselae SA2 lysates were determined by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL), with bovine serum albumin used as a standard. The whole-cell protein lysates were stored in 50 μl aliquots at −80°C until use.
SDS-PAGE and WB.
B. henselae SA2 whole-cell proteins (1.5 mg/ml) from frozen stocks were suspended in an equal volume of 1× sample buffer (62.5 mM Tris hydrochloride [pH 8.0], 2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue; Bio-Rad, Hercules, CA) and heated for 5 min at 100°C. The denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in Criterion precast gels, using 4 to 15% gradient polyacrylamide Tris-glycine precast midigels (Bio-Rad, Hercules, CA), at a constant current (100 V) for 1 h 50 min in 1× running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). A prestained broad-range (10- to 250-kDa)-molecular-weight protein marker (Bio-Rad, Hercules, CA) was used as a standard.
For SDS-PAGE analysis, fractionated proteins were visualized by staining the gel overnight with Bio-Safe Coomassie brilliant blue (Bio-Rad, Hercules, CA). Each batch of B. henselae SA2 proteins, which were used at concentrations of 15 and 30 μg per lane, was tested by SDS-PAGE to evaluate the reproducibility of immunogenic protein separations. B. henselae SA2 whole-cell protein profile analysis by SDS-PAGE revealed numerous proteins, with their molecular sizes ranging from 12 kDa to 250 kDa. Based on analyses of Coomassie brilliant blue-stained SDS-PAGE gels, proteins of 13, 22, 25, 31, 39, 41, 56, 62, 68, 75, 84, 94, and 250 kDa were present at a higher intensity, as indicated by the blue arrows in Fig. S1 in the supplemental material.
For WB, fractionated proteins were electrophoretically transferred to 0.2-mm-pore-size polyvinylidene fluoride (PVDF) membranes at 100 V for 1 h 30 min at 4°C in an electrophoretic transfer cell (Criterion blotter; Bio-Rad). The membranes were transiently stained with ponceau S (Sigma-Aldrich, St. Louis, MO) to verify adequate protein transfer. After transfer, the membranes were blocked overnight at 4°C with 5% nonfat dry milk in 1× Tris-buffered saline containing 0.05% Tween 20 (1× TBS-T). After overnight blocking, the membranes were incubated with dog serum diluted 1:200 in 1× TBS-T containing 3% nonfat dry milk at room temperature for 1 h. The blots were washed five times with 1× TBS-T. Bound antibodies were detected with alkaline phosphatase (AP)-conjugated goat anti-dog whole IgG (Abcam, Cambridge, MA) diluted at 1:35,000 in 1× TBS-T containing 3% nonfat dry milk and incubated at room temperature for 1 h. After washing 5 times with 1× TBS-T (as described above), a colorimetric detection method was used with the addition of a commercially available substrate solution containing nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP; Pierce, Rockford, IL). The color development time was optimized to be between 35 and 50 s. The blots were rinsed for 15 min using sterile distilled water and imaged. Image acquisition was performed using a ChemiDoc imaging system (Bio-Rad, Hercules, CA). Size estimates of B. henselae SA2 immunogenic proteins and blots were analyzed with Image Lab software (version 4.1; Bio-Rad, Hercules, CA).
WB assay optimization.
Antigen and antibody concentrations were optimized using a checkerboard titration assay. Briefly, 5 μl of B. henselae SA2 antigen at a concentration of 15 μg was spotted onto PVDF membranes. The spots were allowed to dry at room temperature. After drying, the membranes were sliced into individual strips. The strips were then blocked overnight with 5% nonfat dry milk in 1× TBS-T. After overnight blocking, each strip containing antigen (15 μg) was treated with different dilutions (1:100, 1:200, 1:500, 1:1,000) of primary antibody (a positive-control serum sample from a naturally exposed dog with a B. henselae IgG titer of 1:1,024) in 1× TBS-T containing 3% nonfat dry milk. A negative-control dog serum sample (B. henselae, B. vinsonii subsp. berkhoffii, and B. koehlerae IgG IFA titers, <1:16) was chosen from the VBDDL archives. These serum samples were also seronegative for other canine vector-borne pathogens included in the VBDDL diagnostic panel described above. The strips were incubated at room temperature for an hour. After incubation, the strips were washed five times for 10 min each time with 1× TBS-T. After washing, secondary antibody (alkaline phosphatase [AP]-conjugated goat anti-dog whole IgG) was added at serial dilutions (1:5,000, 1:10,000, 1:35,000, and 1:50,000) in 1× TBS-T containing 3% nonfat dry milk. The strips were then incubated at room temperature for an hour. After incubation, the strips were washed as described above with 1× TBS-T. Additionally, a commercially available mouse monoclonal antibody (monoclonal antibody H2A10) to B. henselae (Abcam, Cambridge, MA) diluted 1:500 in 1× TBS-T containing 3% milk was used as an additional positive control, with horseradish peroxidase (HRP)-conjugated goat anti-mouse whole IgG (Jackson Immunoresearch Laboratories, West Grove, PA) being used as the secondary antibody. After the washings with secondary antibody, the membrane strips were developed using a commercially available BCIP-NBT solution. Image acquisition was performed using a ChemiDoc imaging system (Bio-Rad, Hercules, CA). The blots were analyzed with Image Lab software (version 4.1; Bio-Rad, Hercules, CA). The checkerboard titration assay was also repeated using 25 μg and 50 μg of antigen.
Based on the checkerboard optimization results (data not shown), WB testing was performed using 15 μg/lane of B. henselae SA2 whole-cell proteins, primary antibody at 1:200, and secondary antibody at a 1:35,000 dilution in 3% 1× TBS-T milk solution to obtain an acceptable signal precision with minimal or no background.
Statistical analysis.
To compare the serological results obtained from B. henselae SA2 WB and B. henselae SA2 IFA for the three groups, the positive, negative, and overall percent agreements between WB and IFA were calculated as described previously (16). Due to a lack of residual serum volumes, B. henselae SA2 IFA serology results were not available for 4 group I dogs, 2 each inoculated with B. vinsonii subsp. berkhoffii type II or B. rochalimae. Therefore, only 6 B. henselae-inoculated group I dogs were included in the WB and IFA comparison.
To propose WB criteria, the sensitivity, specificity, and positive predictive values for six B. henselae immunodominant proteins were calculated as described previously (16), using group II and III dogs’ WB results. Also, for calculation of the sensitivity, specificity, and diagnostic accuracy for B. henselae SA2 WB and B. henselae SA2 IFA, only group II and group III dogs were used since comparable Bartonella PCR (on blood), BAPGM enrichment culture, and B. henselae SA2 IFA results were not available for group I and group IV dogs. The results for group IV dogs were not analyzed statistically.
RESULTS
WB seroreactivity among group I dogs.
Despite being IFA or ELISA negative, 2 group I dogs (10 dogs experimentally infected with Bartonella spp.) were B. henselae WB seroreactive prior to inoculation (Table 1). Sera from one dog (dog 6GCB4) reacted with 13-, 17-, 25-, 27-, and 183-kDa proteins, whereas the other dog (dog 6GCB6) was reactive only with a 29-kDa protein (Table 1). Following inoculation of the Bartonella spp., all but one dog developed variable WB seroreactivity, recognizing B. henselae proteins of 10, 13, 15, 17, 22, 25, 27, 29, 30, 41, 45, 50, 56, 62, 68, 75, 94, 124, 138, 150, 168, 183, and 233 kDa (Fig. 1). One dog (dog 6GCB1) failed to recognize any B. henselae WB proteins prior to or after B. henselae inoculation.
FIG 1.
Representative images of WB results for group I dogs (dogs experimentally inoculated with Bartonella henselae), group II dogs (naturally infected Bartonella PCR-positive dogs), and group III dogs (Bartonella PCR-negative and IFA-negative dogs). Whole-cell proteins extracted from agar-grown Bartonella henselae San Antonio type 2 were used as WB antigens. WB was performed using alkaline phosphatase-conjugated goat anti-dog IgG (H+L). (A) Image of WB results for a group I dog. Lanes 1 to 5, image of blotting results for a dog (6GCB11) experimentally inoculated with B. henselae for preexposure serum (lane 1) and serum collected at postinoculation days 6, 10, 13, and 24 (lanes 2 to 5, respectively). (B) Image of WB results for selected group II dogs. Lanes 1 to 5, each lane represents the blot image for one of five group II dogs. (C) Image of WB results for group III dogs. Lanes 1 and 2, WB images for two group III dogs. The gel images in panels A, B and C are from three separate gels. The white space within each image differentiates different areas of the same gel to show the variation in WB seroreactivity over the experimental study period (for the group I dog) or for group II dogs infected with different Bartonella spp. Images were selected and are depicted by group designations I to III. IgG IFA titers of ≥1:64 were considered positive for Bartonella exposure. Bh, B. henselae; Bvb I, B. vinsonii subsp. berkhoffii genotype I; Bcl, B. clarridgeiae; NEG, negative; lanes L, Bio-Rad Precision Plus Kaleidoscope protein standard.
The WB reactivity patterns using sera from dogs inoculated with high, medium, and low doses of B. henselae inoculum were compared. Four dogs inoculated with high or medium doses recognized multiple B. henselae proteins (≥5 proteins; Table 1). In contrast, of the two dogs (dogs 6GCB5 and 6GCB1) receiving a low-dose inoculum, 6GCB5 was reactive to a single protein (13 kDa) and 6GCB1 did not seroconvert (Table 1). Two dogs (dogs 6GCB4 and 6GCB7), reinoculated with B. henselae strain 94022 at 6.6 × 107 CFU/ml at 40 days after initial infection reacted with at least 4 additional B. henselae proteins by postinoculation weeks 8 and 9 (Table 1).
To examine the WB banding pattern variability among dogs inoculated with the same or different Bartonella spp., we compared the WB seroreactivity among/between B. henselae-, B. vinsonii subsp. berkhoffii type II-, and B. rochalimae-inoculated dogs. Bartonella henselae 13- and 56-kDa proteins were most often recognized by B. henselae-, B. vinsonii subsp. berkhoffii type II-, and B. rochalimae-inoculated dogs. Nine of 10 dogs recognized either the 13- or 56-kDa protein, and 5 of these 9 dogs were reactive to both the 13- and 56-kDa proteins. Other common WB-reactive bands detected among group I dogs were those for the 15-, 25-, 27-, or 29-kDa protein. Two dogs inoculated with B. vinsonii subsp. berkhoffii type II and two dogs inoculated with B. rochalimae recognized up to 9 B. henselae proteins following the inoculation (Table 1). Two dogs used for B. rochalimae inoculation had been inoculated with B. henselae 7 months earlier. These dogs did not react with any B. henselae protein immediately prior to B. rochalimae inoculation (Table 1). There was variable seroreactivity across time and among dogs following the inoculation with each Bartonella spp.
WB seroreactivity among group II dogs.
Sera from group II dogs (36 Bartonella PCR-positive naturally infected dogs) were variably reactive to B. henselae proteins with molecular sizes of 13, 15, 17, 25, 28, 31, 35, 37, 41, 43, 50, 56, 60, 68, 75, 94, 115, 124, 138, 150, 168, and 233 kDa (Fig. 2). Of the 36 group II dogs, 27 recognized three or more of these B. henselae SA2 proteins. Five dogs were seroreactive to only a 31-, 37-, 50-, or 56-kDa protein. One dog each was seroreactive to 17- and 68-kDa, 31- and 41-kDa, and 37- and 150-kDa proteins. The remaining dog was not B. henselae WB seroreactive.
FIG 2.
Frequency of WB bands among group I dogs (dogs experimentally inoculated with Bartonella spp.) and group II dogs (Bartonella PCR-positive naturally infected dogs). The percentage of dogs recognizing each B. henselae protein on WB is represented as WB seroreactivity (in percent) in the histogram.
Among the group II dogs, reactivity to the 17-, 29-, 37-, 50-, 56-, 68-, and 124-kDa B. henselae proteins predominated (Fig. 2). Twenty-four, 22, and 15 dogs recognized the 124-, 17-, and 68-kDa proteins, respectively. These were the most consistently recognized B. henselae proteins among the group II dogs, regardless of the Bartonella spp. identified by PCR (Fig. 2 and Table 2). Eighteen dogs were WB seroreactive to the 50- or 56-kDa protein, and of these 18 dogs, 11 dogs reacted with both the 50- and 56-kDa proteins.
TABLE 2.
Comparison of Bartonella henselae SA2 WB banding patterns among group II dogs PCR positive for B. henselae, B. vinsonii subsp. berkhoffii, and other Bartonella spp.a
| WB band (kDa) | Frequency (%) of WB bands among group II dogs PCR positive for: |
|||
|---|---|---|---|---|
| B. henselae (n = 22) | B. vinsonii subsp. berkhoffii (n = 8) | Other Bartonella spp.b (n = 6) | Total | |
| 13 | 14 | 38 | 50 | 25 |
| 17 | 57 | 88 | 50 | 61 |
| 25 | 23 | 25 | 33 | 25 |
| 29 | 38 | 38 | 50 | 39 |
| 43 | 19 | 50 | 50 | 31 |
| 50 | 29 | 63 | 33 | 36 |
| 56 | 29 | 50 | 50 | 36 |
| 68 | 43 | 25 | 67 | 42 |
| 94 | 24 | 50 | 50 | 33 |
| 124 | 52 | 100 | 83 | 67 |
| 138 | 5 | 25 | 17 | 11 |
| 150 | 29 | 38 | 33 | 31 |
| 168 | 0 | 13 | 0 | 3 |
| 233 | 19 | 0 | 0 | 11 |
| Nonec | 0 | 0 | 17 | 3 |
Group II dogs consisted of 36 Bartonella PCR-positive naturally infected dogs.
Of the 6 dogs naturally infected with other Bartonella spp., 2 dogs were PCR positive for B. koehlerae, 2 dogs were PCR positive for B. rochalimae, 1 dog was PCR positive for B. clarridgeiae, and 1 was PCR positive for B. quintana.
None represents the number of group II dogs that were not seroreactive to any of the following B. henselae SA2 proteins: proteins of 13, 17, 25, 29, 43, 50, 56, 68, 94, 124, 138, 150, 168, and 233 kDa.
As B. henselae and B. vinsonii subsp. berkhoffii were the two most prevalent organisms confirmed by PCR testing among the group II dogs, we compared the B. henselae WB banding patterns for each of these organisms to the patterns found among the dogs for the remaining Bartonella spp. (Table 2). The 17-kDa protein was the most often recognized protein among the B. henselae PCR-positive dogs. Dogs naturally infected with B. vinsonii subsp. berkhoffii or other Bartonella spp. were most often reactive by WB to the 124-kDa protein. All 8 B. vinsonii subsp. berkhoffii PCR-positive dogs recognized the 124-kDa protein, whereas 11 of 22 B. henselae-infected dogs (50%) and 5 of 6 (83%) dogs infected with other Bartonella spp. recognized the 124-kDa protein (Table 2). Four of 6 dogs infected with other Bartonella spp. were seroreactive to a 68-kDa protein. Four of 22 B. henselae PCR-positive dogs recognized a 233-kDa protein, whereas this band was not visualized for dogs infected with B. vinsonii subsp. berkhoffii or other Bartonella spp. Of the 36 group II dogs, only 1 dog (a B. vinsonii subsp. berkhoffii PCR-positive dog) reacted with a 168-kDa protein and 4 dogs (1 B. henselae PCR-positive dog, 2 B. vinsonii subsp. berkhoffii PCR-positive dogs, and 1 other Bartonella PCR-positive dog) recognized a 138-kDa proteins. Thus, the 138-, 168-, and 233-kDa proteins are unlikely to be useful for the serodiagnosis of bartonelloses. Definitive WB banding pattern differences were not observed among B. henselae, B. vinsonii subsp. berkhoffii, and other Bartonella PCR-positive group II dogs.
WB seroreactivity among group III dogs.
Of the 26 group III dogs (26 Bartonella PCR-negative and IFA-negative dogs) that were presumed to be unexposed to Bartonella spp., based upon prior testing by IFA and PCR, no WB bands were visualized on the blots for 9 (34%) dogs (Table 3). Sera from 9 other dogs (34%) recognized only a single protein band at either 11 (n = 1 dog), 27 (n = 2), 68 (n = 2), 37 (n = 2), 124 (n = 1), or 233 (n = 1) kDa. Of the remaining eight (30%) PCR-negative/IFA-negative dogs that recognized more than two B. henselae proteins, only two dogs were reactive to the 17-kDa protein. One dog was reactive to 17-, 24-, 68-, and 124-kDa proteins, and another dog recognized 15-, 17-, 25-, 27-, 50-, 56-, 62-, and 124-kDa proteins. With the exception of those 2 dogs’ WB seroreactivity, group III dogs did not recognize proteins with molecular masses of between 41 and 63 kDa or proteins with masses of greater than 124 kDa and less than 233 kDa (Table 3).
TABLE 3.
Comparison of Bartonella henselae SA2 WB seroreactivity to proteins of various molecular weights among dogs in groups I to IVa
| WB protein recognized (kDa) | No. of dogs in: |
||||
|---|---|---|---|---|---|
| Group I (n = 10) |
Group II (n = 36) | Group III (n = 26) | Group IV (n = 18) | ||
| Preexposure serum | Postexposure serum | ||||
| 150 | None | 2 | 11 | 0 | 0 |
| 56 | None | 7 | 13 | 1 | 4 (3b ) |
| 50 | None | 2 | 13 | 1 | 0 |
| 29 | None | 4 | 14 | 1 | 0 |
| 17 | 1 | 3 | 22 | 2 | 0 |
| 13 | 1 | 7 | 9 | 0 | 1 |
| 168 | None | 2 | 1 | 0 | 0 |
| 138 | None | 2 | 4 | 0 | 3b |
| 75 | None | 1 | 8 | 1 | 0 |
| 62 | None | 3 | 6 | 1 | 0 |
| 43 | None | 1 | 11 | 0 | 3b |
| 35 | None | 1 | 4 | 0 | 0 |
| 31 | None | 1 | 9 | 0 | 0 |
| 233 | None | 1 | 4 | 2 | 0 |
| 124 | None | 1 | 24 | 5 | 0 |
| 94 | None | 1 | 12 | 3 | 0 |
| 68 | None | 1 | 15 | 3 | 1 |
| 41 | None | 1 | 9 | 0 | 3 |
| 37 | None | 1 | 13 | 4 | 0 |
| 25–27 | 1 | 4 | 9 | 7 | 4 |
| 15 | None | 4 | 6 | 3 | 5b |
| Nonec | 8 | 1 | 1 | 9 | 5 (2b ) |
Group I dogs were experimentally inoculated with Bartonella spp., group II dogs were Bartonella PCR-positive naturally infected dogs, group III dogs were Bartonella PCR negative and IFA negative, and group IV dogs consisted of 8 Brucella canis IFA-positive and 10 Rickettsia rickettsii IFA-positive dogs. No shading indicates B. henselae San Antonio 2 diagnostically relevant immunodominant proteins, light-gray shading indicates other potential immunodominant proteins, and dark-gray shading indicates nonspecific or cross-reactive proteins.
Dogs IFA positive for Brucella canis recognizing the corresponding B. henselae SA2 protein.
None represents the number of dogs that were not seroreactive to any B. henselae SA2 proteins on the blots.
WB seroreactivity among group IV dogs.
For the group IV dogs (8 Brucella canis IFA-positive and 10 Rickettsia rickettsii IFA-positive dogs), based on B. henselae SA2, B. vinsonii subsp. berkhoffii type I, and B. koehlerae IFAs, no R. rickettsii-seropositive dog was seroreactive to B. henselae SA2, B. vinsonii subsp. berkhoffii type I, or B. koehlerae antigens. Of the eight Brucella canis-seropositive dogs, three were B. henselae SA2 and B. vinsonii subsp. berkhoffii type I seroreactive (IFA titers, 1:64), two dogs were only B. vinsonii subsp. berkhoffii type I seroreactive (IFA titers, 1:64), and no dog was B. koehlerae seroreactive (IFA titers, <1:16).
The WB seroreactivity among the 8 Brucella canis-seropositive and 10 R. rickettsii-seropositive dogs is summarized in Table 3. Two of the Brucella canis-seropositive dogs and three of the R. rickettsii-seropositive were not seroreactive to any B. henselae protein. Three of 18 dogs were seroreactive to only a single protein. Among eight Brucella canis-seropositive dogs, the 15-, 43-, and 56-kDa proteins were the most frequently recognized (Table 3), whereas four R. rickettsii-seropositive dogs recognized a 25-kDa protein. Despite the WB variability among these subgroups, no dog was seroreactive to the 17-, 29-, 50-, and 150-kDa proteins.
Comparison of WB seroreactivity among the four dog groups.
To further assess specific versus nonspecific Bartonella species WB seroreactivity and to define B. henselae-specific immunodominant antigens, the WB patterns among the three groups were compared (Table 3). The B. henselae proteins recognized by at least 20% of group I dogs and 25% of group II dogs but not recognized by more than 8% of group III dogs were considered B. henselae immunodominant antigens. Based upon WB analyses of sera from experimentally inoculated and naturally infected dogs, 6 B. henselae proteins with molecular masses of 13, 17, 29, 50, 56, and 150 kDa were considered Bartonella-relevant immunodominant proteins. With the exception of 2 dogs (reactive to the 17-, 50-, or 56-kDa protein), none of these 6 immunodominant proteins were recognized by sera from the 26 group III dogs. Of the 18 group IV dogs, one R. rickettsii IFA-positive dog each recognized the 13- or 56-kDa protein. Three Brucella canis-seropositive dogs (2 of which were also B. henselae IFA positive) recognized the 56-kDa protein. The diagnostic scores for these immunodominant proteins are provided in Table 4. With a >95% specificity and a ≥95% positive predictive value, WB reactivity to any two or more of six B. henselae-immunodominant proteins yielded the highest diagnostic sensitivity (53%) for canine bartonelloses.
TABLE 4.
Evaluation of Bartonella henselae SA2 WB criterion interpretation for diagnosis of canine bartonellosesa
| WB criterion (protein [kDa] recognized) | Sensitivity (%) | Specificity (%) | PPV (%) |
|---|---|---|---|
| 13 | 25 | 100 | 100 |
| 17 | 61 | 92 | 92 |
| 29 | 39 | 96 | 93 |
| 50 | 36 | 96 | 93 |
| 56 | 36 | 96 | 93 |
| 150 | 31 | 100 | 100 |
| Any 1 or more proteinsb | 81 | 92 | 93 |
| Any 2 or more proteinsb | 53 | 96 | 95 |
| Any 3 or more proteinsb | 39 | 96 | 93 |
WB results from group II dogs (36 dogs naturally infected with Bartonella spp.) and group III dogs (26 Bartonella PCR-negative and IFA-negative dogs) were used for calculation of sensitivity, specificity, and positive predictive value (PPV) of the B. henselae SA2 immunodominant protein(s) to assess utility of these proteins as diagnostic candidates for documentation of Bartonella exposure in dogs. The WB criterion in bold was interpreted as a positive WB criterion in this study.
Proteins of 13, 17, 29, 50, 56, and 150 kDa.
Proposed WB interpretation criteria.
Despite substantial variation in the B. henselae protein recognition patterns between and within the four groups of dogs, we propose that dogs are Bartonella WB positive if they are seroreactive to two or more of 6 B. henselae-relevant immunodominant proteins with molecular sizes of 13, 17, 29, 50, 56, and 150 kDa. Based on these WB criteria, 70% (7/10) of group I dogs and 53% (19/36) of group II dogs would have satisfied the criteria for WB positivity. One group I dog (dog 6GCB4), reactive to 13-, 17-, 24-, 27-, and 183-kDa proteins prior to inoculation, was potentially exposed to a Bartonella sp. prior to study entry. In the context of specificity, sera from 24/26 (92%) and 25/26 (96%) Bartonella IFA-negative and PCR-negative group III dogs did not react to any of the six B. henselae immunodominant proteins and to two of the six B. henselae immunodominant proteins, respectively (Table 4). No group IV dog was seroreactive to two or more B. henselae immunodominant proteins. One group III dog (which was B. vinsonii subsp. berkhoffii type III IFA positive when tested against 8 Bartonella spp. antigens) was seroreactive to the 17-, 50-, and 56-kDa B. henselae proteins, likely reflecting prior Bartonella species exposure. We consider B. henselae proteins of 15, 25 to 27, 37, 41, 68, 94, 124, and 233 kDa (highlighted in dark gray in Table 3) to be nonspecific, potentially reflecting cross-reactive epitopes shared among other bacteria to which these dogs had been exposed. As the 31-, 35-, 43-, 62-, 75-, 138-, and 168-kDa B. henselae proteins were not recognized by 50% of PCR-positive group II dogs, these proteins are of questionable diagnostic utility. As discussed below, WB sensitivity and specificity will vary depending upon laboratory interpretation criteria.
Comparison of IFA and WB among the three dog groups.
Since IFA is the most frequently used “gold standard” method for screening anti-Bartonella antibodies in serum samples from dogs and humans, we compared the diagnostic accuracy of and agreements between B. henselae SA2 IFA and B. henselae SA2 WB results. Based on the results of the B. henselae SA2 IFA and B. henselae SA2 WB analyses (with our WB criteria) of group II and III dogs, the sensitivity (53%) and the diagnostic accuracy (71%) of WB were better than the 22% sensitivity and 53% diagnostic accuracy of IFA (Tables 5 and 6). Our results indicate that the overall agreement between these two modalities was 71%, 64%, and 92% for group I, group II, and group III dogs, respectively (Table 5). All 6 B. henselae-inoculated dogs were IFA positive for the B. henselae inoculating strain (13), whereas only 1 dog was B. henselae SA2 IFA seroreactive. During the experimental infection study, 4 of 6 B. henselae-inoculated group I dogs became WB positive (Table 5). For group II, 7 (88%) of 8 IFA-positive dogs were WB positive, whereas 63% of WB-positive dogs were IFA negative. Among the 22 B. henselae PCR-positive group II dogs, 90% (9/10) of WB-positive dogs were B. henselae SA2 IFA negative and all WB-negative dogs (n = 12) were also B. henselae SA2 IFA negative (Table 5). For group II dogs that were B. henselae and B. vinsonii subsp. berkhoffii PCR positive, WB was more sensitive than IFA (Table 6). Although WB was more sensitive than IFA, both modalities had a specificity of 96% (Table 6).
TABLE 5.
Comparison of Bartonella henselae SA2 WB and B. henselae SA2 IFA results for dogs in groups I to IIIa
| Group (no. of dogs) | B. henselae SA2 WB result (no. of dogs) | No. (%b
) of dogs with the following B. henselae SA2 IFA result: |
Agreement between B. henselae SA2 WB and IFA |
|||
|---|---|---|---|---|---|---|
| POS | NEG | PPA | NPA | OA | ||
| Comparison of results for group I to III dogsc | ||||||
| Group I (56) | POS (14) | 0 | 14 (100) | 0 | 74 | 71 |
| NEG (42) | 2 (5) | 40 (95) | ||||
| Group II (36) | POS (19) | 7 (37) | 12 (63) | 88 | 57 | 64 |
| NEG (17) | 1 (6) | 16 (94) | ||||
| Group III (26) | POS (1) | 0 | 1 (100) | 0 | 96 | 92 |
| NEG (25) | 1 (4) | 24 (96) | ||||
| Comparison among group II dogs (36)d | ||||||
| B. henselae PCR POS (22) | POS (10) | 1 (10) | 9 (90) | 100 | 57 | 59 |
| NEG (12) | 0 | 12 (100) | ||||
| B. vinsonii subsp. berkhoffii PCR POS (8) | POS (6) | 3 (50) | 3 (50) | 100 | 40 | 63 |
| NEG (2) | 0 | 2 (100) | ||||
| Other Bartonella speciese PCR POS (6) | POS (3) | 3 (100) | 0 | 75 | 100 | 83 |
| NEG (3) | 1 (33) | 2 (67) | ||||
Group I dogs were dogs experimentally inoculated with Bartonella spp., group II dogs consisted of 36 Bartonella PCR-positive naturally infected dogs, and group III dogs consisted of 26 Bartonella PCR-negative and IFA-negative dogs. PPA, positive percent agreement; NPA, negative percent agreement; OA, overall percent agreement; POS, positive; NEG, negative.
The percentage of WB-positive or -negative dogs that were B. henselae SA2 IFA positive and negative, respectively.
Comparison of B. henselae SA2 WB and B. henselae SA2 IFA results for group I, group II, and group III dogs. Since B. henselae SA2 IFA serology results were not available for 4 group I dogs, 2 each inoculated with B. vinsonii subsp. berkhoffii type II or B. rochalimae, only 6 B. henselae-inoculated group I dogs were included in the comparison of IFA and WB results. For comparison of group I B. henselae SA2 IFA and B. henselae SA2 WB results, 56 serum samples (1 preexposure serum sample and at least 7 postexposure serum samples) from each of 6 B. henselae-inoculated dogs were tested. In all 6 group I dogs, the preexposure sera were negative by both B. henselae SA2 WB and B. henselae SA2 IFA testing. Discrepant results occurred in all 6 dogs: 4 dogs had samples that were IFA negative but WB positive, 1 dog had samples that were IFA positive but WB negative, and the remaining dog was WB negative and IFA negative at all time points.
Comparison of B. henselae SA2 WB and B. henselae SA2 IFA results among group II dogs, based on the infecting Bartonella spp.
Of the 6 dogs naturally infected with other Bartonella spp., 2 dogs were PCR positive for B. koehlerae, 2 dogs were PCR positive for B. rochalimae, 1 was PCR positive for B. clarridgeiae, and 1 was PCR positive for B. quintana.
TABLE 6.
Sensitivity, specificity, and diagnostic accuracy of Bartonella henselae SA2 WB and B. henselae SA2 IFA with Bartonella PCR as the reference standarda
| Test | Test result | No. (%) of group II and III dogs with the following PCR result: |
Sensitivity (%) | Specificity (%) | Accuracy (%) | |
|---|---|---|---|---|---|---|
| Positive | Negative | |||||
| Results based on the assay usedb | 36 | 26 | ||||
| B. henselae SA2 WB | Positive | 19 | 1 | 53 | 96 | 71 |
| Negative | 17 | 25 | ||||
| B. henselae SA2 IFA | Positive | 8 | 1 | 22 | 96 | 53 |
| Negative | 28 | 25 | ||||
| Results based on the Bartonella spp.c | ||||||
| B. henselae PCR | 22 | 40 | ||||
| B. henselae SA2 WB | Positive | 10 | 10 | 45 | 75 | 65 |
| Negative | 12 | 30 | ||||
| B. henselae SA2 IFA | Positive | 1 | 8 | 5 | 80 | 53 |
| Negative | 21 | 32 | ||||
| B. vinsonii subsp. berkhoffii PCR | 8 | 54 | ||||
| B. henselae SA2 WB | Positive | 6 | 14 | 75 | 74 | 74 |
| Negative | 2 | 40 | ||||
| B. henselae SA2 IFA | Positive | 3 | 6 | 38 | 89 | 82 |
| Negative | 5 | 48 | ||||
| Other Bartonella speciesd PCR | 6 | 56 | ||||
| B. henselae SA2 WB | Positive | 3 | 17 | 50 | 70 | 68 |
| Negative | 3 | 39 | ||||
| B. henselae SA2 IFA | Positive | 4 | 5 | 67 | 91 | 89 |
| Negative | 2 | 51 | ||||
For calculation of sensitivity, specificity, and diagnostic accuracy, B. henselae SA2 WB and B. henselae SA2 IFA results from group II (36 Bartonella PCR-positive naturally infected dogs) and group III (26 Bartonella PCR-negative and IFA-negative dogs) dogs were used. POS, positive.
Comparison of the results of B. henselae SA2 WB and B. henselae SA2 IFAs to those of the Bartonella PCR.
Comparison of the results of B. henselae SA2 WB and B. henselae SA2 IFAs based on the Bartonella spp. infecting the dog (Bartonella PCR result).
Of the 6 dogs naturally infected with other Bartonella spp., 2 dogs were PCR positive for B. koehlerae, 2 dogs were PCR positive for B. rochalimae, 1 was PCR positive for B. clarridgeiae, and 1 was PCR positive for B. quintana.
DISCUSSION
To define Bartonella species immunodominant proteins, we evaluated WB seroreactivity using 172 serum samples derived from four dog groups. Subsequently, we used the data to propose IgG WB criteria for the serodiagnosis of canine bartonelloses. Following experimental inoculation of dogs with Bartonella spp., there were strong but variable patterns of seroreactivity to six B. henselae SA2 immunodominant proteins across time. The reactivity patterns among B. rochalimae-inoculated dogs could also have been due to the stimulation of B. henselae-specific long-lived plasma and memory cells generated prior to B. rochalimae infection. The antigenic protein patterns varied among the 9 seroreactive group I dogs. Similarly, 81% of naturally infected dogs variably recognized one or more of the six immunodominant proteins, supporting the diagnostic utility of these immunodominant proteins for the documentation of exposure to Bartonella spp. in dogs. No combination of immunodominant proteins was consistent among the sera obtained from experimentally inoculated or naturally infected dogs. Remarkably, individual, naturally infected PCR-positive dogs were either IFA positive/WB positive, IFA positive/WB negative, IFA negative/WB positive, or IFA negative/WB negative. Thus, neither IFA, WB, nor both assays combined were 100% sensitive in documenting exposures to Bartonella spp. However, the specificity of WB in this study was at least 96%.
We purposefully elected to examine sera from dogs that were experimentally inoculated with B. henselae at different doses and sera from dogs infected with other Bartonella spp. Various inoculum doses and infection with three Bartonella spp. most likely contributed to some variability in antigenic protein recognition among dogs during the study time course. Variability in antigenic recognition among naturally infected dogs was an expected finding, as the route (vector versus scratch or bite inoculation), strain, and duration of Bartonella infection were likely variables among dogs. Also, the immunological host response of infected dogs could vary due to breed, age, sex, and other factors. In addition, inherent genetic differences among individual beagles, not examined in this study, may have contributed to the variability in B. henselae protein recognition (17–19). Importantly, based upon the WB results, at least 1 group I dog (dog 6GCB4) had been previously exposed to a Bartonella sp. prior to experimental inoculation with B. henselae. Recently, our NCSU-CVM laboratory reported ear tip vasculitis in association with B. henselae SA2 infection in a specific pathogen-free beagle (not a group I dog) maintained in our laboratory animal facility (20).
Our laboratory previously reported on the recognition of 19-, 33-, 42-, 56-, and 76-kDa B. vinsonii subsp. berkhoffii immunodominant proteins in dogs naturally exposed to B. vinsonii subsp. berkhoffii (determined by IFA seroreactivity) or experimentally infected with culture-grown B. vinsonii subsp. berkhoffii (21). Proteins of similar molecular weights were identified in this study using B. henselae SA2 WB, further suggesting that some immunodominant proteins common across Bartonella spp. could be reliable diagnostic targets. Another study (22) testing experimentally infected cat sera by WB identified several B. henselae immunodominant antigens, including antigens of 11.3, 16.9, 51, 54, 57, 65, 69, and 97 kDa, which may correspond to the immunodominant B. henselae proteins that were recognized by the dogs in this study. Another study evaluating the association between Bartonella serology and PCR results in cats with and without fever identified several B. henselae H-1 immunodominant proteins (8, 20, 39, 48, 57, 62, 69, 73, and 82 kDa) that were variably recognized by sera from experimentally inoculated cats (23). Similar to the findings described in previously published studies (22–24), the antigenic responses varied among individual experimentally infected cats, as was found with inoculated dogs in this study.
The lack of IFA and WB seroreactivity in a subset of B. henselae PCR-positive dogs could be due to the variability of Bartonella membrane proteins, testing during an early stage of infection, Bartonella’s ability to downregulate and evade the host humoral response, a lack of immunogenicity, or elimination by the host (25, 26). Collectively, these observations support the need for additional immunological characterization of the consequences of persistent Bartonella bacteremia in dogs and people. Taken together, variability in WB seroreactivity patterns among naturally infected sick dogs might suggest a dynamic interplay among the infecting Bartonella spp. and strain, the duration of infection, the influence of antibiotic or immunosuppressive drug therapies, and each dog’s unique immune response following Bartonella infection (25, 26).
Due to low IFA sensitivity, Bartonella diagnostic and epidemiological prevalence were likely underestimated in previously published studies from our group and others (3, 8, 9, 27). Forty-three percent (n = 12) of 28 group II IFA-negative dogs were WB positive, indicating the improved diagnostic sensitivity of WB for documenting Bartonella exposure. The variability in diagnostic parameters between the B. henselae SA2 WB and B. henselae SA2 IFA modalities for group I, II, and III dogs can be attributed to several factors, such as differences in antigen preparation, differences in the fixation processes used, and differences in antigen-antibody interaction when testing by IFA versus WB.
In concordance with the findings of previous studies (28), no group I dogs inoculated with B. henselae became bacteremic, suggesting either low levels of bacteria in blood following experimental challenge, rapid immune clearance, or a latent period of bacterial localization at the inoculation site. Despite intradermal inoculation with a high dose (bacterial load) of the B. henselae inoculum, bacteremia was not documented in 6 of 6 dogs experimentally infected with B. henselae, despite B. henselae seroconversion by week 2 postinoculation (13). One of the limitations of this study is that EDTA-anticoagulated blood samples from group I dogs were not processed in BAPGM enrichment culture, which could have resulted in an increased Bartonella PCR sensitivity or subculture isolation (11, 29).
In conclusion, six B. henselae SA2 proteins (proteins of 13, 17, 29, 50, 56, and 150 kDa) appear to represent Bartonella immunodominant antigens. Based on our study, low- and high-molecular-weight B. henselae SA2 proteins, including proteins of 15, 25 to 27, 37, 41, 68, 94, 124, and 233 kDa, represent nonspecific or cross-reactive epitopes. Although WB and IFA were 96% specific, WB was more sensitive than B. henselae IFA (53% versus 22%, respectively) for the serodiagnosis of bartonelloses in dogs. The findings from this study will be used to assess specific B. henselae SA2 immunodominant proteins by proteomic analysis to further improve diagnostic sensitivity. Although the results are promising, evaluation of additional dogs infected with one or more Bartonella spp. or infected with other bacteria is needed to further assess the sensitivity and the specificity of the B. henselae SA2 immunodominant antigens in the clinical diagnostic setting. In addition, further study will be required to determine if any Bartonella antigen recognition pattern is associated with clinical signs of bartonelloses in dogs.
Supplementary Material
ACKNOWLEDGMENTS
We are grateful to the NCSU CVM VBDDL for providing the characterized canine serum samples, without which this study would not have been possible. We thank Julie Bradley and Kaye Gore for technical assistance. We would also thank Gerard P. Andrews and Erin Lashnits for their helpful comments and discussion of the study results.
This study was performed at the North Carolina State University (NCSU) College of Veterinary Medicine (CVM) Vector Borne Diseases Diagnostic Laboratory (VBDDL) and the North Carolina State University College of Veterinary Medicine Intracellular Pathogens Research Laboratory (IPRL).
This research was funded by a grant from the American Kennel Club Canine Health Foundation (grant number 02287, Enhanced Serological Testing Modalities for the Diagnosis of Bartonellosis in Dogs).
The data presented in this article have not been presented at any meetings.
There are no conflicts of interest to report.
Footnotes
Supplemental material is available online only.
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