Performance of a commercially available in-clinic ELISA for detection of antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii and Dirofilaria immitis antigen in dogs

Brett A. Stillman IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Michael Monn IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Jiayou Liu IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Brendon Thatcher IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Paulette Foster IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Blaine Andrews Town and Country Veterinary Clinic, 201 N Thorne Ave, Green Forest, AR 72638.

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Susan Little Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078.

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Matt Eberts Lakeland Veterinary Hospital, 7372 Woida Rd N, Baxter, MN 56425.

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Edward B. Breitschwerdt Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695.

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Melissa J. Beall IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Ramaswamy Chandrashekar IDEXX Laboratories, 1 IDEXX Dr, Westbrook, ME 04092.

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Abstract

Objective—To evaluate the performance of an in-clinic ELISA designed for detection of heartworm antigen and antibodies against 5 tick-borne pathogens.

Design—Validation study.

Sample—1,601 serum or matched serum, plasma, and blood samples from dogs.

Procedures—Samples were tested for Dirofilaria immitis (heartworm) antigen and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by means of an in-clinic ELISA. Evaluation of assay sensitivity and specificity, agreement of results among sample types, and cross-reactivity of E canis antigens in the assay with anti–Ehrlichia chaffeensis antibodies in stored samples from experimentally infected dogs were performed at a reference laboratory. Field tests of the in-clinic ELISA were performed at 6 veterinary facilities. Results were compared with confirmatory test results.

Results—Sensitivity and specificity of the in-clinic ELISA were > 89% for detection of antibodies against A phagocytophilum (93.2% and 99.2%, respectively), A platys (89.2% and 99.2%, respectively), B burgdorferi (96.7% and 98.8%, respectively), E canis (97.8% and 92.3%, respectively), and E ewingii (96.5% and 93.9%, respectively). Sensitivity of the assay for detection of D immitis was 98.9%, with 99.3% specificity. The in-clinic ELISA identified exposure to > 1 vector-borne pathogen in 354 of 1,195 samples. Cross-reactivity of E canis antigens with anti–E chaffeensis antibodies was confirmed. Results of field evaluations confirmed that the in-clinic ELISA could be reliably used under typical clinical conditions to identify dogs exposed to the pathogens of interest.

Conclusions and Clinical Relevance—The in-clinic ELISA provided a comprehensive in-house serologic screening test for all vector-borne pathogens evaluated.

Abstract

Objective—To evaluate the performance of an in-clinic ELISA designed for detection of heartworm antigen and antibodies against 5 tick-borne pathogens.

Design—Validation study.

Sample—1,601 serum or matched serum, plasma, and blood samples from dogs.

Procedures—Samples were tested for Dirofilaria immitis (heartworm) antigen and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by means of an in-clinic ELISA. Evaluation of assay sensitivity and specificity, agreement of results among sample types, and cross-reactivity of E canis antigens in the assay with anti–Ehrlichia chaffeensis antibodies in stored samples from experimentally infected dogs were performed at a reference laboratory. Field tests of the in-clinic ELISA were performed at 6 veterinary facilities. Results were compared with confirmatory test results.

Results—Sensitivity and specificity of the in-clinic ELISA were > 89% for detection of antibodies against A phagocytophilum (93.2% and 99.2%, respectively), A platys (89.2% and 99.2%, respectively), B burgdorferi (96.7% and 98.8%, respectively), E canis (97.8% and 92.3%, respectively), and E ewingii (96.5% and 93.9%, respectively). Sensitivity of the assay for detection of D immitis was 98.9%, with 99.3% specificity. The in-clinic ELISA identified exposure to > 1 vector-borne pathogen in 354 of 1,195 samples. Cross-reactivity of E canis antigens with anti–E chaffeensis antibodies was confirmed. Results of field evaluations confirmed that the in-clinic ELISA could be reliably used under typical clinical conditions to identify dogs exposed to the pathogens of interest.

Conclusions and Clinical Relevance—The in-clinic ELISA provided a comprehensive in-house serologic screening test for all vector-borne pathogens evaluated.

A commercially available in-clinic ELISAa,b has been developed to detect heartworm antigen and antibodies against 5 tick-borne pathogens in a single sample of canine serum, plasma, or whole blood. The assay detects antibodies against Ehrlichia ewingii by use of a peptide derived from the E ewingii p28 outer membrane protein family, which binds these antibodies with high specificity.1–3 The assay also detects antibodies against Anaplasma platys by cross-reactivity with the immunodominant p44 protein of Anaplasma phagocytophilum.4,5 Similar to an earlier-generation ELISA device, the new in-clinic ELISA detects antibodies against peptides derived from the major immunodominant p30 and p30–1 proteins of E canis5,6 and from the VlsE protein–derived C6 peptide of B burgdorferi.5,7 To test for heartworm infection, a monoclonal antibody is used to capture circulating antigens from Dirofilaria immitis and a labeled polyclonal antibody against soluble antigens of D immitis adult worms is used for detection.5,b

Anaplasma phagocytophilum, transmitted by Ixodes spp ticks, infects neutrophils of dogs and causes granulocytotropic anaplasmosis.8 Clinical disease in dogs (with signs including lameness, lethargy, fever, anorexia, and thrombocytopenia) is usually associated with the acute phase of infection when morulae can be seen in the blood smears. The duration of the acute phase can vary greatly and can last from 1 to several days.4,8 In addition to detecting morulae in smears, PCR assays can be used to detect DNA from organisms and confirm reinfection or carrier status.9 Antibodies against Anaplasma spp can also be detected by serologic testing.8

Dogs are the primary reservoir host for A platys, and the brown dog tick, Rhipicephalus sanguineus, has been proposed as the vector for transmission of this organism.10 Although efforts have been made to experimentally infect dogs via exposure to R sanguineus ticks that fed on dogs with acute A platys infection, transmission has not been conclusively demonstrated.11 Anaplasma platys is a gram-negative obligate intracellular bacterium that infects platelets and causes infectious cyclic thrombocytopenia.10,12 Dogs infected with A platys are generally found in the geographic regions where E canis infection is prevalent, and exposure to both organisms may often be detected in the same dog.13,14 Clinical cases of confirmed coinfection with A platys and E canis have been described.13–16 Dogs experimentally coinfected with A platys and E canis have more severe thrombocytopenia than dogs infected with either organism alone.17

Borrelia burgdorferi (transmitted by Ixodes spp) is the causative agent of Lyme borreliosis in North America. Dominant clinical features of the disease in dogs include recurrent lameness due to inflammation of the joints, lack of appetite, and signs of depression.18 Traditional methods of diagnosing Lyme disease have relied on detecting antibodies against extracts from whole B burgdorferi organisms (commonly referred to as whole-cell antigens). These methods have limited utility for identifying dogs infected with B burgdorferi because antibodies against whole-cell antigens are present after exposure to or vaccination against the organism and, in the case of Lyme borreliosis, can remain high after successful treatment of the disease.18 Newer methods that involve detection of antibodies against C6 peptide have been shown to be specific for natural infection with B burgdorferi and do not cross-react with antibodies produced in response to commercial vaccines in dogs.7,19,20

Ehrlichia canis is transmitted by R sanguineus. The pathogenesis of E canis infection in dogs involves an incubation period of 8 to 20 days, followed by acute, subclinical, and sometimes chronic disease phases.8 The common clinical signs of the chronic disease are weakness, signs of depression, anorexia, chronic weight loss, pale mucous membranes, fever, and peripheral edema.21 Diagnosis of E canis is confirmed by microscopic detection of its morulae in circulating monocytes, detection of an increase in antibody titers against E canis (4-fold change between acute and convalescent samples),22 or detection of E canis DNA by PCR assay.23 Immunofluorescence assays with whole-cell E canis antigen have been widely used, but these assays may detect cross-reactive antibodies in samples from dogs exposed to E ewingii, Ehrlichia chaffeensis, and potentially other Ehrlichia spp.24–27

Canine granulocytic ehrlichiosis is caused by the bacterium E ewingii, which replicates in neutrophils and occasionally eosinophils in naturally infected dogs. Clinical signs associated with this disease in dogs include fever, lethargy, anorexia, lameness, neutrophilic polyarthritis, and severe thrombocytopenia.28–30 The lone star tick, Ambylomma americanum, is the only confirmed vector for transmission of E ewingii in North America.31,32 Ambylomma americanum is found throughout the southeastern and south-central United States, and its distribution is rapidly expanding northward.33–36 Ehrlichia ewingii has not been cultured in vitro, and therefore, a specific serologic test to detect antibodies against the organism in dogs has not been available.36 Prior to the availability of PCR testing, diagnosis of E ewingii infection relied on the microscopic identification of morulae in circulating neutrophils in Giemsastained blood smears during the acute phase of infection30; however, diagnosis of infection by this method lacks specificity because A phagocytophilum also induces morulae in neutrophils and the range of these 2 organisms (and their respective tick vectors) can overlap.

Heartworm disease is transmitted by mosquitoes, is widely distributed throughout the United States,37–40 and can be fatal if left untreated.39 Clinical signs may include coughing, lethargy, and exercise intolerance, although some infected dogs have no clinical signs of infection. Most commercially available heartworm antigen tests detect circulating carbohydrate antigens from mature female heartworms and are used to diagnose active canine heartworm infections.39 In general, these tests accurately detect the antigens in serum from dogs infected with D immitis if ≥ 1 mature female heartworm (7 or 8 months old) is present but do not detect infections consisting of only male worms or infections of < 5 months’ duration in dogs, owing to the lack of circulating antigens.39

The purpose of the study reported here was to evaluate the performance of the described in-clinic ELISAa for detection of heartworm antigen and antibodies against A phagocytophilum, A platys, B burgdorferi, E canis, and E ewingii in canine serum samples. In addition to laboratory performance tests, we performed field tests of the same ELISA at 4 veterinary clinics and 2 academic veterinary medical centers to further assess diagnostic performance.

Materials and Methods

Study design and sample—The study included 1,601 serum or matched serum, plasma, and blood samples from dogs. For laboratory testing, individual canine serum samples (n = 1,195) used to assess assay sensitivity and specificity were obtained from the sample bank of a commercial reference laboratoryc and from individual veterinary clinics in various regions of the United States and Caribbean Islands. For A platys sp identification, a subset of these samples (n = 123) was sourced from dogs living in A platys–endemic areas (ie, from Native American reservations in Arizona and from the Turks and Caicos)13–15 where infestations with Ixodes spp that transmit A phagocytophilum have not been reported to occur. Samples were aliquoted and stored at −20°C until tested with the in-clinic ELISAa (BT, MM, and PF). The tests were performed in a blinded manner.

An additional 20 samples were obtained from 2 female 12- to 24-month-old specific-pathogen–free mixed-breed dogs that had previously been experimentally infected with E chaffeensis in an unrelated study.1 These samples were used for temporal analysis of cross-reactivity of E canis p-30–p31 peptides in the in-clinic ELISA with antibodies against E chaffeensis. The protocol for that study was approved by the Institutional Animal Care and Use Committee of The Ohio State University. Briefly, both dogs were inoculated on day 0 by IV injection of 5 × 106 E chaffeensis (Arkansas)–infected DH82 cells at near 100% cell culture infectivity. Blood samples were collected from the dogs every 7 days for 8 weeks; one dog had 1 additional sample collected on day 82, and the other dog had 3 additional samples collected (on days 63, 77, and 96) after inoculation. Serum was collected via centrifugation at 5,000 × g and stored at −20°C until use. An IFA was performed at each time point to determine whether the dogs seroconverted after treatment with E chaffeensis–infected DH82 cells,25 and each of the samples was tested with the in-clinic ELISA.

To validate assay performance with different sample types, 246 matched individual canine sample sets (whole blood, plasma, and serum) obtained at the commercial reference laboratoryc and from a commercial vendord were tested in random order with 3 different kit lots of the in-clinic ELISA by personnel blinded to sample origin (BT and PF). The sample sets were tested with multiple ELISA kit lots to detect the impact, if any, on the performance of various sample types among kit lots.

Field trials (ie, evaluations under typical clinical conditions where the test would be used to screen for infection with various organisms) were conducted at 4 veterinary clinics and 2 academic veterinary medical centers in regions where the in-clinic ELISA was anticipated to have clinical utility (Maine, Minnesota, North Carolina, Oklahoma, Arkansas, and Missouri). Each trial consisted of testing 2 types of samples. Twenty serum samples were obtained at each individual location from dogs of unknown infection status that had a blood sample collected as part of a routine clinical evaluation; 0.5-mL aliquots of these 20 serum samples were submitted to the reference laboratory,c and confirmatory (reference method) testing was performed for comparison with the in-clinic ELISA results. Aliquots of another 20 serum samples from dogs of known infection status (sourced from the reference laboratoryc sample bank) were additionally provided to each of the participating veterinary hospitals. All samples were tested sequentially with 3 independent test kit lots by participants at each test site, and results for the in-clinic ELISA were subjectively evaluated across all facilities.

In-clinic ELISA kit—The in-clinic ELISAa was used for detection of heartworm antigen and antibodies against A phagocytophilum, A platys, B burgdorferi, E canis, and E ewingii. The in-clinic assay uses a proprietary device that provides reversible chromatographic flow of sample and automatic, sequential flow of wash and enzyme substrate; positive test results are detected visually as blue-colored spots. The assay was performed in accordance with the manufacturer's instructions for all samples tested in the study.

Confirmatory (reference) tests for sensitivity and specificity evaluation—To evaluate sensitivity and specificity of the in-clinic ELISA for detection of antibodies against Anaplasma spp, E canis, and B burgdorferi, results were compared with the results of IFAs for Anaplasma spp, E canis, and B burgdorferi, respectively, performed at the reference laboratory.c Because the in-clinic ELISA and the IFA do not differentiate between A phagocytophilum and A platys, species was inferred on the basis of the sample's geographic origin. A recombinant protein ELISA for E ewingii1,35 was used as the reference test in determining sensitivity and specificity of the in-clinic ELISA for detecting antibodies against this organism.

Samples used to assess sensitivity of the in-clinic ELISA for detection of D immitis antigen were obtained from dogs confirmed as positive for heartworm infection at necropsy. Because of ethical concerns associated with obtaining necropsy data for heartworm-negative animals, specificity of the in-clinic ELISA was assessed with samples from dogs that tested negative by means of a commercially available D immitis antigen teste in accordance with the manufacturer's instructions.

Statistical analysis—Data analysis included sensitivity and specificity calculations performed by use of statistical software.f Sensitivity and specificity were calculated as described elsewhere.41 The concordant and discordant results for various tests were assessed by computing κ statistics. The κ statistic measures the degree of agreement between findings (eg, results of the in-clinic ELISA vs the confirmatory test for the same analyte), with κ < 0.40 considered poor, 0.50 to 0.75 considered good, and > 0.75 considered excellent agreement.42

Results

Sensitivity and specificity analysis—Sensitivity and specificity data for in-clinic ELISAa results (as compared with reference test results) were tabulated (Table 1). Samples were preselected for this analysis on the basis of confirmatory test results; thus, sensitivity and specificity for each analyte were not evaluated for every sample.

Table 1—

Mean (95% confidence interval) sensitivity and specificity of a commercially available in-clinic ELISAa for the detection of antibodies against various tick-transmitted pathogens and Dirofilaria immitis antigen in 1,195 canine serum samples.

 Test results    
PathogenTrue positiveFalse negativeFalse positiveTrue negativeTotalSensitivity (%)Specificity (%)κ
Anaplasma phagocytophilum*13610223838693.2 (87.8–96.7)99.2 (97.06–99.9)0.93
Anaplasma platys*11514224237389.2 (82.5–93.9)99.2 (96.7–99.8)0.90
Borrelia burgdorferi*1194324937596.7 (92.0–96.7)98.8 (96.6–99.9)0.96
Ehrlichia canis*13131821736997.8 (93.6–99.2)92.3 (88.2–95.4)0.97
Ehrlichia ewingii10941015427796.5 (91.2–98.9)93.9 (89.1–97.0)0.98
D immitis§941226936698.9 (94.3–99.8)99.3 (97.4–99.9)0.98

True-positive or true-negative test results were identified when a given sample had positive or negative results, respectively, for both the in-clinic ELISA and the reference test. A negative in-clinic ELISA result for a sample that tested positive by the reference method was designated as false negative, and a positive in-clinic ELISA result for a sample that tested negative via the reference method was designated as false positive. The total number of samples tested for exposure to each pathogen varied because samples tested with the in-clinic ELISA were preselected on the basis of availability of confirmatory test results. The κ value reflects the degree of agreement between the in-clinic ELISA and the reference test.

Reference test was IFA.

The in-clinic ELISA and reference test did not distinguish between Anaplasma spp, and these were identified on the basis of geographic location where the tested dog resided.

Reference test was an ELISA.1

Reference tests were necropsy (for positive results) and a commercial heartworm antigen teste (for negative results).

With identification of the Anaplasma spp based on geographic location of the sample source, 136 of 146 canine serum samples that tested positive for antibodies against A phagocytophilum by IFA tested positive by means of the in-clinic ELISA, and 238 of 240 samples that tested negative by IFA had negative results with the in-clinic ELISA. Sensitivity and specificity of the in-clinic ELISA for the detection of antibodies against A phagocytophilum were 93.2% and 99.2%, respectively. One hundred fifteen of 129 samples that tested positive for anti–A platys antibodies via IFA had positive test results with the in-clinic ELISA (89.2% sensitivity). Of the 244 samples that tested negative for these antibodies by IFA, 242 had negative results with the in-clinic ELISA (99.2% specificity).

For 119 of 123 samples that tested positive for antibodies against B burgdorferi by means of IFA, the results of the in-clinic ELISA were also positive. Two hundred forty-nine of 252 samples with negative results via IFA tested negative with the in-clinic ELISA. Sensitivity and specificity of in-clinic ELISA for detection of anti–B burgdorferi antibodies was 96.7% and 98.8%, respectively.

For anti–E canis antibody testing, 131 of 134 samples with positive results by IFA tested positive with the in-clinic ELISA, with 217 of 235 samples that had negative IFA results testing negative via in-clinic ELISA. Sensitivity and specificity for detection of antibodies against E canis were 97.8% and 92.3%, respectively. Similarly, sensitivity and specificity of the in-clinic assay for antibodies against E ewingii were 96.5% and 93.9%, respectively (with 109/113 samples that tested positive with the reference ELISA having positive results and 154/164 reference test–negative samples having negative results via in-clinic ELISA).

Serum samples for 94 of 95 dogs with necropsy-confirmed heartworm infection tested positive, and 269 of 271 samples confirmed negative by use of another commercially available assaye tested negative, for D immitis antigen with the in-clinic ELISA. Sensitivity and specificity of the in-clinic ELISA for detection of the antigen in these samples were 98.9% and 99.3%, respectively.

Results of κ tests for agreement ranged from 0.90 to 0.98 for all analytes evaluated. This indicated excellent agreement between results of the in-clinic ELISA and the confirmatory tests used.

Temporal testing—Serum samples collected from the 2 dogs experimentally infected with E chaffeensis tested negative for antibodies against the organism via IFA and had negative results for the E canis spot on the in-clinic ELISA prior to inoculation on day 0. On days 7 and 14, antibody titers (1:160 and 1:640, respectively, for one dog and 1:80 and 1:320, respectively, for the other) were detectable with IFA, but in-clinic ELISA results were negative for both dogs. All samples obtained on or after day 21 tested positive with the in-clinic ELISA; these corresponded to IFA-determined anti–E chaffeensis antibody titers of 1:640 (day 21) to 1:1,280 (day 82) for one dog and 1:640 (day 21) to 1:5,120 (day 96) for the other. These results indicated that the E canis antigens in the assay cross-reacted with antibodies against E chaffeensis.

Exposure to multiple organisms—Three hundred fifty-four of 1,195 dogs tested positive for exposure to ≥ 1 pathogen via in-clinic ELISA tests of a single serum sample. Samples from 112 dogs tested positive for antibodies against A phagocytophilum and B burgdorferi; 6 of these samples also had positive results for D immitis antigen or antibodies against E canis or E ewingii. Samples from 85 dogs tested positive for antibodies against A platys and E canis; of these, 1 had anti–B burgdorferi antibodies detected, and 26 tested positive for D immitis antigen. Sixty-two samples tested positive for D immitis antigen and anti–E ewingii antibodies (9 of these also had positive results for exposure to a third organism [either B burgdorferi or A platys]).

Assay performance in multiple sample types—Proportions of the 246 matched sample sets with agreement among in-clinic ELISA results for a given test across all 3 sample types (serum, whole blood, and plasma) were summarized for each of 3 test kit lots (Table 2). Because of sample volume limitations, not every sample was tested with all 3 lots. Results of tests for exposure to Anaplasma spp (A phagocytophilum and A platys), B burgdorferi, and Ehrlichia spp (E canis and E ewingii) were examined. Agreement among sample types was > 95% for all pathogens with each test kit lot. Data were not analyzed statistically; however, subjective evaluation suggested there was no substantial influence of sample type on the results for any analyte.

Table 2—

Results of an in-clinic ELISA for antibodies against various tick-transmitted pathogens and for D immitis antigen in 246 matched sample sets (serum, plasma, and whole blood) from 246 dogs.

PathogenLotNo. of animals with results for 3 sample typesNo. (%) with agreement across all sample types
Anaplasma spp*A109106 (97.2)
 B109107 (98.2)
 C246241 (98.0)
B burgdorferiA7777 (100.0)
 B7777 (100.0)
 C124124 (100.0)
Erlichia sppA9999 (100.0)
 B9995 (96.0)
 C237236 (99.6)
D immitisA122117 (95.9)
 B122117 (95.9)
 C232225 (97.0)

Sample sets were tested with ELISA kits from 3 lots (designated A, B, or C), and the proportion of sets with results in agreement across all sample types was determined for each lot. Sample numbers varied among kits because of sample size limitations.

Anaplasma spp results include both A phagocytophilum and A platys.

Ehrlichia spp results include both E canis and E ewingii.

Field trials—Results of the 6 field trials evaluating performance of the in-clinic ELISA under typical clinical conditions were summarized (Table 3). In-clinic ELISA and reference method results were in agreement for 340 of the 360 tests (representing triplicate ELISAs [use of 3 kit lots] for each of 120 serum samples collected on-site across all locations). The highest proportion of discordant results (6 false-negative and 2 false-positive results) was found for A phagocytophilum. Three samples that tested positive for antibodies against Anaplasma spp via the reference method were interpreted as having negative results with 2 in-clinic ELISA lots by an operator who had limited experience using the in-clinic test kit. The host veterinarian, who was a more experienced user of the in-clinic test kit, performed the third replicate of tests (with the third lot) at that facility and correctly interpreted the results for the same 3 samples as Anaplasma-positive. Furthermore, on repeat analysis (also in a blinded manner) by an experienced operator at the reference laboratory using the in-clinic ELISA, all 3 of the samples that had false-negative results were confirmed as having positive results when tested in duplicate with the same test kit lots used at the veterinary facility (data not shown).

Table 3—

Results of field evaluations of an in-clinic ELISA for samples collected from canine patients at 6 veterinary facilities.

 In-clinic ELISA results (No. of tests)
PathogenTrue positiveFalse negativeFalse positiveTrue negativeTotal
A phagocytophilum*3062322360
A platys*300357360
B burgdorferi*1203345360
E canis*3810321360
E ewingii6244290360
D immitis§300357360

At each location, blood samples were collected from 20 dogs with unknown exposure status for the organisms of interest; serum was obtained, and each sample was tested on-site in triplicate by use of in-clinic ELISA kits from 3 different lots. Results were compared with reference method test results for the same samples.

See Table 1 for remainder of key.

In-clinic ELISA results for laboratory-sourced samples from the 20 dogs with known infection status were summarized for all participating veterinary facilities (Table 4). All reference test–positive samples tested positive for the same pathogens in triplicate (3 kit lots) in-clinic ELISA tests across all facilities. All samples that tested negative for detection of antibodies against B burgdorferi, E canis, and E ewingii and for D immitis antigen via reference methods had negative results for the respective in-clinic ELISAs in all facilities. For A phagocytophilum, 1 replicate in 288 tests of confirmed negative samples had a false-positive result and for A platys, 1 replicate in 270 tests of confirmed negative samples had a false-positive result.

Table 4—

Results of in-clinic ELISA field evaluations performed at 6 veterinary facilities with samples provided by the reference laboratory.

 In-clinic ELISA results (No. of tests)
PathogenTrue positiveFalse negativeFalse positiveTrue negativeTotal
A phagocytophilum*7201287360
A platys*9001269360
B burgdorferi*7200288360
E canis*7200288360
E ewingii9000270360
D immitis§10800252360

Twenty serum samples collected from 20 dogs with known exposure status for organisms of interest were sourced from the reference laboratory sample bank. Identical sets of the 20 samples were tested in triplicate by use of 3 different kit lots at each location.

See Table 1 for remainder of key.

Discussion

In the present study, we evaluated the performance of an in-clinic ELISAa for the detection of antibodies against A phagocytophilum, A platys, B burgdorferi, E canis, and E ewingii and antigen from D immitis in samples from dogs. The in-clinic test kit uses a monoclonal and polyclonal antibody-based heartworm antigen detection system and synthetic peptides for detection of antibodies against Anaplasma spp, B burgdorferi, E canis, and E ewingii.

Conventional assays used to diagnose ehrlichiosis, such as IFA, have limitations related to the nature of antigens used in the tests. Because E ewingii has not been successfully cultured, IFAs for antibodies against other Ehrlichia spp (E canis and E chaffeensis) have been widely used for detecting anti–E ewingii antibodies in dogs.27 Serologic cross-reactivity among E canis, E ewingii, and E chaffeensis has been reported,43 and in areas where multiple ehrlichial diseases are endemic, diagnosis on the basis of whole-cell ELISA or IFA results is limited. In the present study, a species-specific recombinant antigen-based assay1 was used as the reference test in evaluating sensitivity and specificity of the in-clinic ELISA for detection of anti–E ewingii antibodies. The in-clinic ELISA had a sensitivity and specificity of 96.7% and 93.9%, respectively, for detecting this analyte, and it is likely that the reference test may detect antibodies that fail to bind the E ewingii p28 protein-derived linear peptide epitopes in the in-clinic assay. However, to our knowledge, the in-clinic ELISA is the only commercial test available to identify E ewingii–exposed dogs. Results of a study35 to evaluate (by species-specific ELISA) 8,662 canine blood samples submitted from 14 veterinary colleges, 6 private veterinary practices, and 4 diagnostic laboratories across the south and central regions of the United States revealed that dogs were more commonly exposed to E ewingii (5.1% of all samples in the study) than to E canis and E chaffeensis, (0.8% and 2.8% of samples, respectively). In addition, a significant correlation was detected between the mean number of reported cases of monocytic ehrlichiosis in humans (by state, per million people)44 and the seroprevalence of E ewingii and E chaffeensis in dogs,35 indicating that dogs tested with the in-clinic ELISA could potentially serve as regional sentinels to gauge potential risk for human infections with these tick-transmitted pathogens. In the context of one health, veterinarians play an increasingly important public health role as it relates to zoonotic and vector-borne pathogens such as this.

Anaplasma platys is currently diagnosed through identification of platelets containing morulae in blood smears, detection of cross-reactive antibodies by IFA for A phagocytophilum,16 or PCR assay.10 Bacteremia with this organism is cyclic, and infection in a dog may be overlooked or may not be promptly diagnosed if a PCR assay or blood film evaluation has negative results owing to low numbers of circulating organisms at the time of evaluation.10 This may be more likely to occur at times when a patient is severely thrombocytopenic. The Anaplasma spp sample spot of the in-clinic ELISA uses the same peptide for detection of antibodies against A phagocytophilum that is used in another in-clinic test.4,5 Prior studies5 have shown that this peptide reacts with antibodies in samples from dogs experimentally infected with A platys. The in-clinic ELISA had sensitivity and specificity of 89.2% and 99.2%, respectively, for detection of antibodies against A platys (presumed on the basis of geographic location) in samples from dogs living in A platys–endemic areas13–15 where Ixodes spp, the vectors for A phagocytophilum, have not been reported to reside.

The in-clinic ELISA was also evaluated for detection of anti–A phagocytophilum, –E canis, and –B burgdorferi antibodies and D immitis antigen in dogs. The performance of the in-clinic ELISA for these analytes was similar to that reported for other commercial assays.5,45,46 The lower specificity of the assay for detection of anti–E canis antibodies (92.3%), compared with 100% specificity obtained with another commercial in-clinic test,5 could potentially reflect antibodies against E chaffeensis that were not detected by E canis IFA but by the in-clinic ELISA.8,21,22 Other results of our study confirmed that the E canis peptides in the in-clinic assay detect antibodies against E chaffeensis. Inclusion of the peptide for E ewingii detection did not appear to affect the overall performance of the in-clinic ELISA for detection of other analytes. For example, the in-clinic ELISA had a sensitivity of 98.9% and specificity of 99.3% for D immitis antigen, with only 2 of 271 confirmed-negative samples testing positive.

Detection of antibodies against a tick-borne pathogen with the in-clinic ELISA indicates that the dog has mounted an immune response against the organism, but this does not necessarily indicate active infection. To determine whether a disease or illness is caused by one of these pathogens, in addition to the serologic evidence, a complete medical history, physical examination, and appropriate hematologic, biochemical, and confirmatory PCR-based diagnostic tests may be needed.

Studies21,47 have demonstrated that coinfection occurs in dogs in defined endemic areas and that dogs infected with multiple vector-borne pathogens may have more pronounced clinical signs than those infected with a single pathogen. The ability of the in-clinic ELISA to accurately and rapidly identify exposure to multiple vector-borne organisms may help improve understanding of the clinical signs associated with each type of infection as well as the atypical and complex clinical signs that can develop when coinfections are present. In the present study, 354 of 1,195 samples contained detectable antibodies against > 1 species of vector-borne pathogen. The highest proportion of dogs with evidence of exposure to multiple tick-borne pathogens had antibodies against A phagocytophilum and B burgdorferi simultaneously detected (112/354), followed by those with antibodies against A platys and E canis (85/354), reflecting the tick species involved in the transmission of these organisms.16 Anaplasma phagocytophilum and B burgdorferi are transmitted by Ixodes spp, and A platys and E canis are transmitted by R sanguineus.10 Evidence supporting coexposure of dogs to B burgdorferi and A phagocytophilum or to A platys and E canis has been reported, particularly in areas endemic for both organisms.21,47 Although simultaneous transmission of both pathogens by 1 tick may be a rare event, exposure to multiple ticks may contribute to concurrent or sequential infection with these organisms.

Because the in-clinic ELISA provides a rapid way to confirm exposure to as many as 6 vector-borne pathogens in a single canine blood sample, this test can provide valuable information about relative disease risk.28 Screening patients with tools such as the in-clinic ELISA can inform veterinarians and clients about transmission of vector-borne pathogens in their local environment,28,36 as well as providing evidence of pathogen exposure as part of a diagnostic workup.

ABBREVIATION

IFA

Indirect immunofluorescence assay

a.

SNAP 4Dx Plus Test Kit, IDEXX Laboratories, Westbrook, Me.

b.

Stillman BA, Beall MJ, Monn M, et al. Performance of the new in-clinic SNAP 4Dx Plus test for the detection of Ehrlichia ewingii (granulocytic ehrlichiosis) and Anaplasma platys (thrombocytotropic anaplasmosis) in dogs (abstr). J Vet Intern Med 2012:26:795.

c.

IDEXX Reference Laboratories, IDEXX Laboratories, Westbrook, Me.

d.

Quality Biological Inc, Gaithersburg, Md.

e.

PetChek Heartworm PF Antigen Test, IDEXX Laboratories, Westbrook, Me.

f.

Analyse-it standard edition, Analyse-it Software Ltd, Leeds, West Yorkshire, England.

References

  • 1. O'Connor TP, Saucier JM, Daniluk D, et al. Evaluation of peptide- and recombinant protein-based assays for detection of anti–Ehrlichia ewingii antibodies in experimentally and naturally infected dogs. Am J Vet Res 2010; 71: 11951200.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Gusa AA, Buller RS, Storch GA, et al. Identification of a p28 gene in Ehrlichia ewingii: evaluation of gene for use as a target for a species-specific PCR diagnostic assay. J Clin Microbiol 2001; 39: 38713876.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Zhang C, Xiong Q, Kikuchi T, et al. Identification of 19 polymorphic major outer membrane protein genes and their immunogenic peptides in Ehrlichia ewingii for use in a serodiagnostic assay. Clin Vaccine Immunol 2008; 15: 402411.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Zhi N, Ohashi N, Tajima T, et al. Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks. Infect Immun 2002; 70: 11751184.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Chandrashekar R, Mainville CA, Beall MJ, et al. Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs. Am J Vet Res 2010; 71: 14431450.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Ohashi N, Unver A, Zhi N, et al. Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis. J Clin Microbiol 1998; 36: 26712680.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Levy SA. Use of a C6 ELISA test to evaluate the efficacy of a whole-cell bacterin for the prevention of naturally transmitted canine Borrelia burgdorferi infection. Vet Ther 2002; 3: 420424.

    • Search Google Scholar
    • Export Citation
  • 8. Little S. Ehrlichiosis and anaplasmosis in dogs and cats. Vet Clin North Am Small Anim Pract 2010; 40: 11211140.

  • 9. Martin AR, Brown GK, Dunstran H, et al. Anaplasma platys: an improved PCR for its detection in dogs. Exp Parasitol 2005: 109: 176180.

  • 10. Greene CE. Infectious diseases of the dog and cat. New York: Elsevier Health Sciences, 2006.

  • 11. Simpson RM, Gaunt SD, Hair JA, et al. Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys. Am J Vet Res 1991; 52: 15371541.

    • Search Google Scholar
    • Export Citation
  • 12. Baker DC, Gaunt SD, Babin SS. Anemia of inflammation in dogs infected with Ehrlichia platys. Am J Vet Res 1981; 49: 10141016.

  • 13. Diniz PPVP, Beall MJ, Omark K, et al. High prevalence of tick-borne pathogens in dogs from an Indian reservation in Northeastern Arizona. Vector Borne Zoonotic Dis 2010; 10: 117123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Yabsley MJ, McKibben J, Macpherson CN, et al. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada. Vet Parasitol 2008; 151: 279285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Hoff B, McEven B, Peregrin AS. A survey for infection with Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Babesia canis in feral and client-owned dogs in the Turks and Caicos Islands, British West Indies. Can Vet J 2008; 49: 593594.

    • Search Google Scholar
    • Export Citation
  • 16. Kordick SK, Breitschwerdt EB, Hegarty BC, et al. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol 1999; 37: 26312638.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Gaunt S, Beall M, Stillman B, et al. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasit Vectors 2010; 3: 3342.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Krupka I, Straubinger RK. Lyme borreliosis in dogs and cats: background, diagnosis, treatment and prevention of infections with Borrelia burgdorferi sensu stricto. Vet Clin North Am Small Anim Pract 2010; 40: 11031119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Levy SA, O'Connor TP, Hanscom JL, et al. Quantitative measurement of C6 antibody following antibiotic treatment of Borrelia burgdorferi antibody-positive nonclinical dogs. Clin Vaccine Immunol 2008; 15: 115119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Little SE, Heise SR, Blagburn BL, et al. Lyme borreliosis in dogs and humans in the USA. Trends Parasitol 2010; 26: 213218.

  • 21. Suksawat J, Hegarty BC, Breitschwerdt EB. Seroprevalence of Ehrlichia canis, Ehrlichia equi, and Ehrlichia risticii in sick dogs from North Carolina and Virginia. J Vet Intern Med 2000; 14: 5055.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Procajło A, Skupie EM, Bladowski M, et al. Monocytic ehrlichiosis in dogs. Pol J Vet Sci 2011; 14: 515520.

  • 23. Peleg O, Baneth G, Eyal O, et al. Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli. Vet Parasitol 2010; 173: 292299.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Goldman EE, Breitschwerdt EB, Grindem CB, et al. Granulocytic ehrlichiosis in dogs from North Carolina and Virginia. J Vet Intern Med 1998; 12: 6170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Hegarty BC, Maggi RG, Koskinen P, et al. Ehrlichia muris infection in a dog from Minnesota. J Vet Intern Med 2012; 26: 12171220.

  • 26. Loftis AD, Mixson TR, Stromdahl EY, et al. Geographic distribution and genetic diversity of the Ehrlichia sp. from Panola Mountain in Amblyomma americanum. BMC Infect Dis 2008; 8: 5461.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Yabsley MJ, Adams DS, O'Connor TP, et al. Experimental primary and secondary infections of domestic dogs with Ehrlichia ewingii. Vet Microbiol 2011; 150: 315321.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Neer TM, Breitschwerdt EB, Greene RT, et al. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. J Vet Intern Med 2002; 16: 309315.

    • Search Google Scholar
    • Export Citation
  • 29. McQuiston JH, McCall CL, Nicholson WL. Ehrlichiosis and related infections. J Am Vet Med Assoc 2003; 223: 17501756.

  • 30. Cohn LA. Ehrlichiosis and related infections. Vet Clin North Am Small Anim Pract 2003; 33: 863884.

  • 31. Anziani OS, Ewing SA, Barker RW. Experimental transmission of a granulocytic form of the tribe Ehrlichiae by Dermacentor variabilis and Amblyomma americanum to dogs. Am J Vet Res 1990; 51: 929931.

    • Search Google Scholar
    • Export Citation
  • 32. Anderson BE, Greene CE, Jones DC, et al. Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis. Int J Syst Bacteriol 1992; 42: 299302.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Childs JE, Paddock CD. The ascendancy of Amblyomma americanum as a vector of pathogens affecting humans in the United States. Annu Rev Entomol 2003; 48: 307337.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Liddell AM, Stockham SL, Scott MA, et al. Predominance of Ehrlichia ewingii in Missouri dogs. J Clin Microbiol 2003; 41: 46174622.

  • 35. Beall MJ, Alleman AR, Breitschwerdt EB, et al. Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America. Parasit Vectors 2012; 5: 2941.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Little SE, O'Connor TP, Hempstead J, et al. Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States. Vet Parasitol 2010; 172: 355360.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Bowman DD, Atkins CE. Heartworm biology, treatment, and control. Vet Clin North Am Small Anim Pract 2009; 39: 11271158.

  • 38. Bowman DD. Introduction: heartworm. Top Companion Anim Med 2011; 26:159.

  • 39. McCall JW, Genchi C, Kramer LH, et al. Heartworm disease in animals and humans. Adv Parasitol 2008; 66: 193285.

  • 40. Nelson CT, McCall JW, Rubin SB, et al. 2005 guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in cats. Vet Parasitol 2005; 133: 267275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. Griner DF, Mayewski RJ, Mushlin AI et al. Selection and interpretation of diagnostic tests and procedures. Principles and applications. Ann Intern Med 1981; 94: 557592.

    • Search Google Scholar
    • Export Citation
  • 42. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977; 33: 159174.

  • 43. Rikihisa Y, Ewing SA, Fox JC. Western immunoblot analysis of Ehrlichia chaffeensis, E canis, or E ewingii infections in dogs and humans. J Clin Microbiol 1994; 32: 21072112.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. CDC. Summary of notifiable diseases: United States, 2008. MMWR Morb Mortal Wkly Rep 2010; 57: 194.

  • 45. O'Connor TP, Esty E, Machenry P, et al. Performance evaluation of Ehrlichia canis and Borrelia burgdorferi peptides in a new Dirofilaria immitis combination assay, in Proceedings. Recent Adv Heartworm Dis Symp Am Heartworm Soc 2001; 7784.

    • Search Google Scholar
    • Export Citation
  • 46. Bowman D, Little SE, Lorentzen L, et al. Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic survey. Vet Parasitol 2009; 160: 138148.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47. Beall MJ, Chandrashekar R, Eberts MD, et al. Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis 2008; 8: 455464.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 1. O'Connor TP, Saucier JM, Daniluk D, et al. Evaluation of peptide- and recombinant protein-based assays for detection of anti–Ehrlichia ewingii antibodies in experimentally and naturally infected dogs. Am J Vet Res 2010; 71: 11951200.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Gusa AA, Buller RS, Storch GA, et al. Identification of a p28 gene in Ehrlichia ewingii: evaluation of gene for use as a target for a species-specific PCR diagnostic assay. J Clin Microbiol 2001; 39: 38713876.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Zhang C, Xiong Q, Kikuchi T, et al. Identification of 19 polymorphic major outer membrane protein genes and their immunogenic peptides in Ehrlichia ewingii for use in a serodiagnostic assay. Clin Vaccine Immunol 2008; 15: 402411.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Zhi N, Ohashi N, Tajima T, et al. Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks. Infect Immun 2002; 70: 11751184.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Chandrashekar R, Mainville CA, Beall MJ, et al. Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs. Am J Vet Res 2010; 71: 14431450.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Ohashi N, Unver A, Zhi N, et al. Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis. J Clin Microbiol 1998; 36: 26712680.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Levy SA. Use of a C6 ELISA test to evaluate the efficacy of a whole-cell bacterin for the prevention of naturally transmitted canine Borrelia burgdorferi infection. Vet Ther 2002; 3: 420424.

    • Search Google Scholar
    • Export Citation
  • 8. Little S. Ehrlichiosis and anaplasmosis in dogs and cats. Vet Clin North Am Small Anim Pract 2010; 40: 11211140.

  • 9. Martin AR, Brown GK, Dunstran H, et al. Anaplasma platys: an improved PCR for its detection in dogs. Exp Parasitol 2005: 109: 176180.

  • 10. Greene CE. Infectious diseases of the dog and cat. New York: Elsevier Health Sciences, 2006.

  • 11. Simpson RM, Gaunt SD, Hair JA, et al. Rhipicephalus sanguineus as a potential biologic vector of Ehrlichia platys. Am J Vet Res 1991; 52: 15371541.

    • Search Google Scholar
    • Export Citation
  • 12. Baker DC, Gaunt SD, Babin SS. Anemia of inflammation in dogs infected with Ehrlichia platys. Am J Vet Res 1981; 49: 10141016.

  • 13. Diniz PPVP, Beall MJ, Omark K, et al. High prevalence of tick-borne pathogens in dogs from an Indian reservation in Northeastern Arizona. Vector Borne Zoonotic Dis 2010; 10: 117123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Yabsley MJ, McKibben J, Macpherson CN, et al. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada. Vet Parasitol 2008; 151: 279285.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Hoff B, McEven B, Peregrin AS. A survey for infection with Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Babesia canis in feral and client-owned dogs in the Turks and Caicos Islands, British West Indies. Can Vet J 2008; 49: 593594.

    • Search Google Scholar
    • Export Citation
  • 16. Kordick SK, Breitschwerdt EB, Hegarty BC, et al. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol 1999; 37: 26312638.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Gaunt S, Beall M, Stillman B, et al. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic and molecular findings. Parasit Vectors 2010; 3: 3342.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Krupka I, Straubinger RK. Lyme borreliosis in dogs and cats: background, diagnosis, treatment and prevention of infections with Borrelia burgdorferi sensu stricto. Vet Clin North Am Small Anim Pract 2010; 40: 11031119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Levy SA, O'Connor TP, Hanscom JL, et al. Quantitative measurement of C6 antibody following antibiotic treatment of Borrelia burgdorferi antibody-positive nonclinical dogs. Clin Vaccine Immunol 2008; 15: 115119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Little SE, Heise SR, Blagburn BL, et al. Lyme borreliosis in dogs and humans in the USA. Trends Parasitol 2010; 26: 213218.

  • 21. Suksawat J, Hegarty BC, Breitschwerdt EB. Seroprevalence of Ehrlichia canis, Ehrlichia equi, and Ehrlichia risticii in sick dogs from North Carolina and Virginia. J Vet Intern Med 2000; 14: 5055.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Procajło A, Skupie EM, Bladowski M, et al. Monocytic ehrlichiosis in dogs. Pol J Vet Sci 2011; 14: 515520.

  • 23. Peleg O, Baneth G, Eyal O, et al. Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli. Vet Parasitol 2010; 173: 292299.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Goldman EE, Breitschwerdt EB, Grindem CB, et al. Granulocytic ehrlichiosis in dogs from North Carolina and Virginia. J Vet Intern Med 1998; 12: 6170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Hegarty BC, Maggi RG, Koskinen P, et al. Ehrlichia muris infection in a dog from Minnesota. J Vet Intern Med 2012; 26: 12171220.

  • 26. Loftis AD, Mixson TR, Stromdahl EY, et al. Geographic distribution and genetic diversity of the Ehrlichia sp. from Panola Mountain in Amblyomma americanum. BMC Infect Dis 2008; 8: 5461.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Yabsley MJ, Adams DS, O'Connor TP, et al. Experimental primary and secondary infections of domestic dogs with Ehrlichia ewingii. Vet Microbiol 2011; 150: 315321.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Neer TM, Breitschwerdt EB, Greene RT, et al. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. J Vet Intern Med 2002; 16: 309315.

    • Search Google Scholar
    • Export Citation
  • 29. McQuiston JH, McCall CL, Nicholson WL. Ehrlichiosis and related infections. J Am Vet Med Assoc 2003; 223: 17501756.

  • 30. Cohn LA. Ehrlichiosis and related infections. Vet Clin North Am Small Anim Pract 2003; 33: 863884.

  • 31. Anziani OS, Ewing SA, Barker RW. Experimental transmission of a granulocytic form of the tribe Ehrlichiae by Dermacentor variabilis and Amblyomma americanum to dogs. Am J Vet Res 1990; 51: 929931.

    • Search Google Scholar
    • Export Citation
  • 32. Anderson BE, Greene CE, Jones DC, et al. Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis. Int J Syst Bacteriol 1992; 42: 299302.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Childs JE, Paddock CD. The ascendancy of Amblyomma americanum as a vector of pathogens affecting humans in the United States. Annu Rev Entomol 2003; 48: 307337.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Liddell AM, Stockham SL, Scott MA, et al. Predominance of Ehrlichia ewingii in Missouri dogs. J Clin Microbiol 2003; 41: 46174622.

  • 35. Beall MJ, Alleman AR, Breitschwerdt EB, et al. Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America. Parasit Vectors 2012; 5: 2941.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Little SE, O'Connor TP, Hempstead J, et al. Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States. Vet Parasitol 2010; 172: 355360.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Bowman DD, Atkins CE. Heartworm biology, treatment, and control. Vet Clin North Am Small Anim Pract 2009; 39: 11271158.

  • 38. Bowman DD. Introduction: heartworm. Top Companion Anim Med 2011; 26:159.

  • 39. McCall JW, Genchi C, Kramer LH, et al. Heartworm disease in animals and humans. Adv Parasitol 2008; 66: 193285.

  • 40. Nelson CT, McCall JW, Rubin SB, et al. 2005 guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in cats. Vet Parasitol 2005; 133: 267275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. Griner DF, Mayewski RJ, Mushlin AI et al. Selection and interpretation of diagnostic tests and procedures. Principles and applications. Ann Intern Med 1981; 94: 557592.

    • Search Google Scholar
    • Export Citation
  • 42. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977; 33: 159174.

  • 43. Rikihisa Y, Ewing SA, Fox JC. Western immunoblot analysis of Ehrlichia chaffeensis, E canis, or E ewingii infections in dogs and humans. J Clin Microbiol 1994; 32: 21072112.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. CDC. Summary of notifiable diseases: United States, 2008. MMWR Morb Mortal Wkly Rep 2010; 57: 194.

  • 45. O'Connor TP, Esty E, Machenry P, et al. Performance evaluation of Ehrlichia canis and Borrelia burgdorferi peptides in a new Dirofilaria immitis combination assay, in Proceedings. Recent Adv Heartworm Dis Symp Am Heartworm Soc 2001; 7784.

    • Search Google Scholar
    • Export Citation
  • 46. Bowman D, Little SE, Lorentzen L, et al. Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic survey. Vet Parasitol 2009; 160: 138148.

    • Crossref
    • Search Google Scholar
    • Export Citation
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