Clinical Relevance of Annual Screening Using a Commercial Enzyme-Linked Immunosorbent Assay (SNAP 3Dx) for Canine Ehrlichiosis
Eighty-six dogs were selected based upon Ehrlichia (E.) canis SNAP 3Dx positive results to determine clinical relevance of annual E. canis screening. Immunofluorescence assay showed 72 (84%) of 86 dogs were seroreactive for E. canis. Polymerase chain reaction (PCR) revealed that 12 (14%) of 86 dogs had Ehrlichia deoxyribonucleic acid; seven had E. canis, four had E. ewingii, and one was coinfected with E. chaffeensis and E. ewingii. Thrombocytopenia (<164,000 platelets/μL) was found in 28 (39%) of 72 dogs. In this study, thrombocytopenia was frequently detected in healthy Ehrlichia SNAP 3Dx-positive dogs, whereas active infection was infrequently confirmed by PCR. Therefore, treatment based upon screening results alone is not recommended.
Introduction
Ehrlichiosis, caused by Ehrlichia (E.) canis, E. chaffeensis, and E. ewingii, is an important tick-borne disease of dogs in the United States and throughout tropical and subtropical regions of the world.1–4 Ehrlichia infection can contribute to a spectrum of clinical, hematological, and biochemical abnormalities in dogs. If left untreated, these organisms can induce a chronic disease process that in the later stages of infection is more difficult to treat successfully.5–7
For over two decades, preventive healthcare for dogs has included annual heartworm screening to monitor for occult infection, owner compliance, and efficacy of heartworm prevention products in areas that are endemic for Dirofilaria (D.) immitis. To increase the versatility and usefulness of the D. immitis antigen assay, additional antigens have been included to facilitate screening for other vector-borne pathogens in simple in-hospital kits.8 One such screening kit, the IDEXX SNAP 3Dx,a detects Borrelia (B.) burgdorferi and Ehrlichia species antibodies. 9 Subsequent to this study, a fourth antigen has been added in the SNAP 4Dx kit to detect exposure to Anaplasma (A.) species.
Before the introduction of annual screening kits, most veterinarians tested only sick dogs that had clinical (e.g., lymphadenopathy, bleeding, weight loss), hematological (e.g., anemia, thrombocytopenia), or biochemical (e.g., hyperglobulinemia) abnormalities consistent with ehrlichiosis. They looked for serological evidence of infection with an Ehrlichia species. Now, while monitoring for evidence of heartworm infection (based upon antigen detection), veterinarians may determine that a healthy dog has had prior exposure to and potentially may be infected with B. burgdorferi, E. canis, or an Anaplasma species. When prior exposure to a tick-borne organism is detected using an antibody-based annual screening test, it becomes the veterinarian’s responsibility to determine if the dog is actively infected and if treatment is warranted.
The manufacturer of the SNAP 3Dx kit currently recommends that a complete blood count (CBC) be assessed for all dogs in which Ehrlichia antibodies have been detected.10 Based upon CBC results, particularly documentation of thrombocytopenia (reference interval 160,000 to 510,000/μL), anemia (hematocrit <37%), or hyperglobulinemia (serum globulin >3.8 g/dL), antibiotic treatment may be warranted. If no hematological abnormalities are found, treatment would not be advised unless an active infection status could be established by polymerase chain reaction (PCR) testing. The PCR amplifies organism-specific deoxyribonucleic acid (DNA) sequences from patient blood or tissue sample; therefore, a positive test result should reflect active infection with the target organism.11
The SNAP 3Dx kit utilizes synthetic peptides derived from the major immunodominant E. canis proteins, P30 and P30-1.9 With the SNAP 3Dx test, dogs that have been exposed to or are actively infected with E. canis would test positive. Sera from E. chaffeensis-positive dogs can cross react in the SNAP 3Dx test, whereas dogs infected with E. ewingii or A. phagocytophilum develop antibodies that are not believed to cross react with P30 and P30-1 E. canis synthetic peptides used in this kit.9 To maintain a higher level of specificity, commercial peptides are applied to the enzyme-linked immunosorbent assay (ELISA) in a manner that consistently detects immunofluorescence assay (IFA) antibody titers ≥1:160. The IFA titers <1:160 are less likely to induce a visible (i.e., positive) color reaction.9 Anecdotally, technicians processing test kits record as “weak positives” those color reactions that are lighter in color than the control dot. It is unknown if a test read as “weak positive” is associated with a specific antibody titer. Studies in healthy pet dogs are needed to determine the agreement between positive SNAP 3Dx results, IFA seroreactivity, and PCR amplification of organism-specific DNA sequences. In addition to supporting active E. canis bloodborne infection, PCR (depending upon design) can also identify dogs that are coinfected with other Ehrlichia or Anaplasma species, specifically E. chaffeensis, E. ewingii, A. platys, and A. phagocytophilum.4,12
The purpose of this study was to examine the agreement between a commercially-available ELISA (containing two synthetic peptides), an indirect IFA (using cell-cultured E. canis organisms), and a 16S ribosomal ribonucleic acid (rRNA) gene PCR assay designed to detect Ehrlichia and Anaplasma species DNA in dogs naturally exposed to tickborne infections.
Materials and Methods
Case Selection
Thirty clinics located in the eastern, upper midwest, southeastern, and southwestern regions of the United States that were currently utilizing SNAP 3Dx as an annual screen for exposure to D. immitis, B. burgdorferi, and E. canis were asked to enroll Ehrlichia SNAP 3Dx-positive dogs as participants in this study. Entry criteria included first-time Ehrlichia-positive dogs tested with SNAP 3Dxa by primary care veterinary hospitals. Dogs were excluded from the study if treated with a tetracycline derivative within the previous 6 months. As dogs were identified as reactive to SNAP 3Dx E. canis peptides during routine screening, the attending veterinarian would request owner permission to obtain additional blood samples for research purposes, at no cost to the owner. From each dog, 2 mL of ethylenediaminetetraacetic acid (EDTA) anticoagulated blood and at least 1 mL of serum were collected prior to treatment with antibiotics; the samples were sent to North Carolina State University (NCSU) Vector Borne Disease Diagnostic Laboratory (VBDDL). Personnel in the VBDDL retested all serum samples using the SNAP 3Dx kit according to manufacturer’s instructions, and they also performed IFA testing to endpoint titers using E. canis (also known as NCSU strain “DJ”) whole-organism antigens grown in DH82 cells. All EDTA anticoagulated blood samples were tested by personnel in the Intracellular Pathogens Research Laboratory (IPRL) at NCSU on two independent occasions using a previously described 16S rRNA PCR. At the time of sample collection, veterinarians were asked to send a paired EDTA anticoagulated blood sample to IDEXX Reference Laboratories for a CBC.
Immunofluorescence Assay
Ehrlichia canis (NCSU canine-origin isolate DJ) was grown in DH82 cells to 80% infectivity. Harvested infected cells were centrifuged to pellet, and the pellet was then resuspended in phosphate-buffered saline and overlain on Teflon-coated 30 well slides.b Slides were air-dried and acetone-fixed before being stored frozen until use. Serum was titrated from 1:16 in phosphate-buffered saline containing 0.05% Tween 20, 0.5% nonfat dry milk, and 1% normal goat serum in 10 twofold dilutions to 1:8,192. Serum dilutions were incubated on antigen slides at 37°C for 30 minutes, washed, and incubated with fluorescein-conjugated canine immunoglobulin G for another 30 minutes before a final wash and counterstaining with Eriochrome black. Endpoint titers were determined as the last dilution giving bright fluorescence of E. canis morulae under ultraviolet light. Titers ≥1:64 were defined as seroreactive using cutoff titers established by the VBDDL diagnostic service. A subset of samples were tested against E. chaffeensis antigens grown in DH82 cells and A. phagocytophilum antigens grown in HL60 cells prepared and stained as described above.
Extraction of DNA and Quality Control
Using a commercial kit,c DNA was manually extracted from 200 μL of canine EDTA-whole blood samples. Concentration of DNA was quantified by spectrophotometryd and the absence of PCR inhibitors demonstrated by the amplification of a fragment of the Glyceraldehyde 3-phosphate dehydrogenase gene.13
Polymerase Chain Reaction Assay
In the Ehrlichia and Anaplasma species amplification, 16S rRNA PCR was performed for E. canis, E. chaffeensis, and E. ewingii detection, and GroEL gene PCR was performed for A. platys as described previously.14 For A. phagocytophilum, GroEL gene was targeted with new primers manually designede at the annealing temperature of 62°C: Gro-Ap67s 5′-TTA TGC TAC GGT TGT TTG TTC TAT TG-3′ and Gro-Ap594as 5′-TCT TAC TTC CTA TGT TCT TGT CTC CAT-3′. Conventional PCR was performed in an Eppendorf Mastercycler EPf as described.14 Positive controls included DNA from the following organisms: E. canis (CP000107), E. chaffeensis (AF416764), E. ewingii (U96436), A. platys (AF478129), and A. phagocytophilum (DQ680012). The limit of detection observed in PCR amplifications was 10 copies per reaction.
Statistical Analysis
Significance tests were done with Fisher’s exact test (for dichotomous variables) and the Mann-Whitney U test (for continuous variables); a P value of <0.05 was considered significant. Analyses were done using SigmaStat software for Windows version 3.5.g
Results
SNAP 3Dx
Between April 2004 and April 2006, 13 clinics provided 86 sample sets from SNAP 3Dx-positive dogs. Seventy-five (87%) of the 86 samples were received from clinics located in the southeast; 10 (12%) of the 86 samples were received from clinics located in the upper northeast, and one sample was from the southern midwest. Upon repeat testing with SNAP 3Dx, all samples were again positive for E. canis, with 13 out of 86 noted as “weak positives.”
Immunofluorescence Assay
Ehrlichia canis reciprocal IFA antibody titers ranged from <16 (n=1) to >8192 (n=6) [Table 1]. The remaining reciprocal IFA titers were 16 (n=3), 32 (n=10), 64 (n=7), 128 (n=17), 256 (n=12), 512 (n=17), 1024 (n=11), 2048 (n=1), and 4096 (n=1). Using 1:64 as the cutoff, 72 (84%) of the 86 samples were diagnostically positive for exposure, whereas 14 (16%) would be considered diagnostically seronegative. Subjective ratings of “weak positives” were distributed over a range of antibody titers from <16 to 512 [Table 1]. When comparing the frequencies of IFA-positive results among states and regions, no differences were found (P=0.20 and P=0.18, respectively).
Polymerase Chain Reaction Assay
Twelve of 86 EDTA-anticoagulated blood samples contained Ehrlichia DNA [Table 2]. Using species-specific primers, seven dogs were infected with E. canis; four were infected with E. ewingii, and one dog was coinfected with E. chaffeensis and E. ewingii. Using species-specific primers, A. phagocytophilum and A. platys DNA was not detected in any of the 86 samples tested. No differences were found in the frequencies of PCR-positive results among the states or regions (P=0.13 and P=0.36, respectively). Eleven of the 12 PCR-positive samples had reciprocal IFA antibody titers ≥64. One E. canis PCR-positive dog had an E. canis IFA reciprocal titer of 32. No statistical agreement was seen between the IFA- and PCR-positive frequencies (P=0.28).
Thrombocytopenia
Complete blood count results were unavailable for 14 dogs, including four Ehrlichia PCR-positive cases. Thrombocytopenia (IDEXX Laboratory normal reference interval 164,000 to 510,000 platelets/μL) was found in 28 (39%) of 72 dogs tested. Five thrombocytopenic dogs were PCR positive (four were infected with E. canis, and one was infected with E. ewingii). Three Ehrlichia PCR-positive dogs (one with E. canis and two with E. ewingii) had platelet counts that were above the lower end of the laboratory reference interval (>164,000/μL). Of the 74 Ehrlichia PCR-negative dogs, 23 (31%) were thrombocytopenic; however, no differences were seen in the frequencies of thrombocytopenia among PCR-positive versus PCR-negative groups (P=0.47) or thrombocytopenia among IFA results with a cutoff of 1:64 (P=0.077). Also, no differences were seen in the frequencies of thrombocytopenia based upon state or region of sample origin (P=0.55 and P=0.59, respectively).
Discussion
When using an annual screening test that detects Ehrlichia species antibodies in a simple, in-clinic assay, the veterinarian gains potentially valuable information. To utilize this information, the veterinarian must recognize the test limitations and know what further information must be obtained to appropriately manage an individual case. In this study population, all samples were SNAP 3Dx positive on two independent tests. The tests were performed by two different individuals—one at the clinic of origin, and the other at NCSU-VBDDL. Further information such as endpoint titers by IFA, CBCs, and PCR were examined to elucidate the clinical relevance of SNAP 3Dx E. canis antibody results.
Using the cutoff titer established by the VBDDL diagnostic service, the majority of dogs included in this study were IFA reactive with titers ≥64. Interestingly, reciprocal titers <64 were found in 14 (16%) samples. Seven of these 14 samples had “weak positive” results reported by SNAP 3Dx testing compared to six “weak positive” results with IFA titers of 64 to 512; however, the density of the color reaction is a subjective assessment and should not be considered predictive of the antibody titer. When the kit is loaded and activated according to manufacturer’s instructions, any color development should be considered positive. Low titers can be interpreted several ways: they can be indicative of early infection as antibody develops; they can result from the decrease in antigenic stimulation that is expected over time following effective immunological or antibiotic clearance of the pathogen; they can reflect cross reactions caused by other Ehrlichia or Anaplasma species that have common immunological epitopes; or they can reflect a false-positive SNAP 3Dx result. Determining the rate of false positives in a natural setting is difficult; however, as a frame of reference, the SNAP 3Dx E. canis-positive rate in a nonendemic area, such as Canada during 2006, was 0.0004% (20 out of 53,511 dogs tested) according to unpublished IDEXX, Inc., data.
One case with a reciprocal IFA titer of 32 was PCR positive for E. canis (indicative of acute infection), although this dog was not thrombocytopenic (platelet count 289,000/μL). Follow-up inquiries into the 14 low-titer cases from this study uncovered two cases that converted to negative on the SNAP test by the next year. When seroreactivity was tested against E. chaffeensis and A. phagocytophilum antigens by IFA, six of the 14 samples that had reciprocal titers of <16 to 32 against E. canis in this study would have been considered seroreactive at 64 or 128 for E. chaffeensis, and two of the six also reacted to A. phagocytophilum at 128 (data not shown). This led to the hypothesis that another closely related species may have been responsible for some of the low titer results in this study.
Manufacturer’s researchers maintain that the SNAP 3Dx does not detect E. ewingii antibodies (O’Connor unpublished data). Ehrlichia canis, E. chaffeensis, and E. ewingii coinfections have been reported previously.2,4,15,16 The four E. ewingii PCR-positive dogs, which were SNAP 3Dx positive with E. canis IFA reciprocal titers of 128 to 1024, may have been previously exposed to and immunologically or therapeutically cleared of the E. canis infection. As demonstrated by others,17 some dogs can naturally eliminate infection or suppress the degree of E. canis parasitemia to a level not detectable by PCR. Alternatively, the dogs could have been concurrently infected with E. canis and E. ewingii at the time of PCR testing, with only E. ewingii DNA detected in the blood sample.
Ehrlichia PCR prevalence was 14% when preselection was based upon SNAP 3Dx as compared to 4.5% prevalence for 1337 diagnostic samples submitted to the NCSU VBDDL during the same time period (between April 2005 and April 2006). Although the NCSU VBDDL diagnostic samples were submitted from dogs with a broad spectrum of clinical signs and hematological abnormalities that could be consistent with ehrlichiosis, one might have expected a higher PCR prevalence in sick dogs (i.e., diagnostic samples) as compared to healthy dogs being screened annually. Treatment history is not routinely provided for VBDDL diagnostic samples undergoing PCR evaluation; therefore, the lower PCR positive results among diagnostic samples could be related to post-treatment screening for elimination of active infection.
Two recent studies from the authors’ laboratory indicate that the E. canis PCR assay used in this study is highly sensitive and detects 10 gene copies/reaction, amplifying E. canis DNA in 154 (77.7%) of 198 naturally infected dogs and in 15 (100%) of 15 experimentally infected dogs.18,19 This sensitivity level is as good as or even better than some described real-time PCR reactions for the detection of these pathogens.20 Failure to detect organism-specific DNA by PCR in a small quantity of extracted peripheral blood cannot definitively rule out active Ehrlichia species infection.21 Results in this study showed a strong agreement between PCR-positive samples (11 of 12 samples tested) and IFA titers ≥1:64.
Veterinarians attempting to decide whether to treat an E. canis seroreactive dog with doxycycline or other tetracycline derivatives should obtain a CBC and consider submitting a blood sample for PCR testing to confirm active infection. A positive E. canis SNAP 3Dx result with thrombocytopenia in conjunction with a positive E. canis PCR result confirms active Ehrlichia infection. This combination of data would warrant treatment to avoid chronic infection and the potential of serious clinical and hematological abnormalities induced by E. canis, E. chaffeensis, or E. ewingii.3,22,23 However, it is important to recognize that positive E. canis SNAP 3Dx or IFA serology in conjunction with thrombocytopenia alone will not reliably confirm active infection in all dogs, as determined by PCR analysis. Experimentally, dogs infected with both E. canis and E. ewingii develop chronic intravascular infections that can persist for years.24–27 A SNAP 3Dx positive result in conjunction with a normal CBC and a negative PCR test result could suggest that the dog may have immunologically cleared the infection and not need treatment. Important to recognize, however, is that dogs can be SNAP 3Dx positive, IFA seroreactive, and have normal platelet numbers and yet be actively infected. This was demonstrated in three PCR-positive dogs that fit the criteria of this study.
The finding that 39% of E. canis SNAP 3Dx-positive healthy dogs screened in this study population were thrombocytopenic could support a high degree of subclinical illness, which is a well-recognized aspect of canine ehrlichiosis (whether the infection is E. canis24,25 or E. ewingii).26,27 Also, the E. canis SNAP 3Dx positive results in thrombocytopenic PCR-negative dogs might reflect iatrogenic thrombocytopenia associated with platelet aggregation during sample collection. Additionally, dogs with prior therapeutic or immunological elimination of Ehrlichia species could be infected with other vector-borne organisms that induce thrombocytopenia, such as Babesia canis or Bartonella vinsonii subspecies berkhoffii that are not eliminated by treatment with tetracycline or doxycycline.28,29 Finally, the thrombocytopenia detected in these E. canis SNAP 3Dx-positive dogs possibly was not related to a vector- borne organism. Although all thrombocytopenia could not be directly attributable to ehrlichiosis in this study, occult thrombocytopenia indicates an active disease process in some of these healthy dogs.
Detection of active infection, followed by therapeutic elimination of chronic infections, are the primary basis for annual screening for vector-borne infections in dogs. The fact that PCR did not detect an Ehrlichia species in 23 of the 28 thrombocytopenic samples may reflect infection below the sensitivity level for PCR or the immunological or therapeutic elimination of ehrlichial infection prior to sample acquisition. Using peptides of a more transient nature, which would fade with time after clearance of infection, would be a technological advancement allowing veterinarians to discern between cleared infections and new or repeat infections.
The SNAP 3Dx kit is designed in its simplicity and ease of use to screen for exposure rather than function as a diagnostic modality for clinically affected dogs. Restricting the use of kits to cases in which there is suspicion of disease based upon clinical signs or exposure probability would increase their positive-predictive value. Noting test results in a dog’s medical record, educating owners as to the benefits and limitations of the test results, and continuing to screen for tick-borne disease exposure on an annual or semiannual basis are reasonable approaches for utilizing the information obtained with this screening tool when a dog is seroreactive but not thrombocytopenic or PCR positive. No one test can reliably dictate a course of action, but simple screening kits can greatly assist veterinarians in determining the need for and choice of more time-consuming and costly tests.
Conclusion
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Kit results were predictive of IFA reactivity in 84% of dogs included in this study.
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Ehrlichia species PCR prevalence was 14% with preselection by SNAP 3Dx, as compared to 4.5% PCR prevalence in samples submitted to an established diagnostic service.
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Occult thrombocytopenia that was not attributed to ehrlichiosis indicated active disease in some of these healthy dogs.
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The use of peptides of a more transient nature, which would fade with time after clearance of infection, would be a technological advancement allowing veterinarians to discern between cleared infections and new or repeat infections.
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No one test can reliably dictate a course of action, but annual E. canis screening can assist veterinarians in determining the need for and choice of more time-consuming and costly tests.
IDEXX Laboratories, Inc., Westbrook, ME 04092
Cel-Line Brand Slides, Thermo Fisher Scientific, Inc., Waltham, MA 02454
Qiagen, Inc., Valencia, CA 91355
Nanodrop ND1000, Thermo Fisher Scientific, Inc., Waltham, MA 02454
Primer position based on sequences U96728 and AY077621, respectively
Eppendorf North America, Westbury, NY 11590
Systat Software, Inc., Chicago, IL 60606
Contributor Notes
