A second-generation, point-of-care immunoassay provided improved detection of Anaplasma and Ehrlichia antibodies in PCR-positive dogs naturally infected with Anaplasma or Ehrlichia species.

A validated second-generation SNAP 4Dx Plus (Idexx) incorporates new peptides for improved detection of antibodies against Anaplasma and Ehrlichia tick-borne pathogens in dogs. We compared the first- and second-generation SNAP 4Dx Plus using dogs naturally infected with Anaplasma or Ehrlichia species, or dogs seroreactive by an E. canis indirect fluorescent antibody test (IFAT). The second-generation immunoassay was more sensitive than the first-generation for dogs infected with A. phagocytophilum (51.1% and 29.2%, respectively), A. platys (63.6% and 35.3%, respectively), E. canis (96.2% and 88.3%, respectively), or E. ewingii (73.7% and 70.8%, respectively), and for dogs seroreactive by E. canis IFAT (87.3% and 83.9%, respectively). The second-generation immunoassay detected significantly more Anaplasma- or Ehrlichia-infected dogs that were Anaplasma (p < 0.001) or Ehrlichia (p = 0.031) seroreactive, respectively, than did the first-generation test. When Ehrlichia seroreactivity by E. canis IFAT and both immunoassays was compared, significantly more E. canis-infected dogs were seroreactive by E. canis IFAT than the first-generation (p = 0.006) but not the second-generation (p = 0.125) immunoassay. Significantly more E. ewingii-infected dogs were seroreactive by the first- (p = 0.011) and second-generation (p = 0.049) immunoassays than the E. canis IFAT. Medical records available for 7 dogs that were Anaplasma seroreactive by the second-generation but not the first-generation immunoassay revealed case management decisions that might have been different with an immediate anaplasmosis diagnosis, including earlier doxycycline therapy and less hospitalization. The second-generation SNAP 4Dx Plus test offered improved serologic detection of Anaplasma and Ehrlichia in naturally infected dogs.

An Improved Point-of-Care ELISA for the Diagnosis of Anaplasmosis and Ehrlichiosis During the Acute Phase of Tick-Borne Infections in Dogs.

Veterinarians often test for serologic evidence of vector-borne infections in sick dogs presenting with clinical signs or to screen for subclinical chronic infections. Additional peptide targets for the detection of antibodies to Anaplasma phagocytophilum, Anaplasma platys, and Ehrlichia canis were added to an existing point-of-care (POC) ELISA test (SNAP 4Dx Plus Test, IDEXX Laboratories, Westbrook, ME). This second-generation, multi-analyte test detects Dirofilaria immitis antigen and antibodies to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. The second-generation test is expected to better meet the needs of practicing veterinarians and their patients. To assess this expectation, the second-generation POC test was evaluated with serum samples from experimentally infected dogs and a broader field population of dogs. Compared to the first-generation test, most dogs experimentally infected with A phagocytophilum (n = 7/8), A platys (n = 4/6), or E canis (n = 4/6) had detectable antibody responses 3-22 days earlier post-infection; these results demonstrated better alignment with polymerase chain reaction (PCR) amplification results and the onset of clinical signs. Using a convenience sample set of 510 sera from both academic and commercial veterinary diagnostic laboratories, the second-generation test had sensitivities greater than 90% for Anaplasma spp. (94.1%), B burgdorferi (95.5%), Ehrlichia spp. (93.4%) and D immitis (98.0%). Specificity ranged from 96.8% - 100% across the four assays. Results from this study demonstrate that the second-generation POC ELISA had an improved ability to detect serologic responses during the acute phase of A phagocytophilum, A platys, and E canis experimental infections. The results from the broader field samples support overall high sensitivity and specificity, consistent with the historical performance of the first-generation POC ELISA test.

Biologic variability of N-terminal pro-brain natriuretic peptide in adult healthy cats.

Objectives The biologic variability of N-terminal pro-brain natriuretic peptide (NT-proBNP) and its impact on diagnostic utility is unknown in healthy cats and those with cardiac disease. The purpose of this study was to determine the biologic variation of NT-proBNP within-day and week-to-week in healthy adult cats. Methods Adult cats were prospectively evaluated by complete blood count (CBC), biochemistry, total thyroxine, echocardiography, electrocardiography and blood pressure, to exclude underlying systemic or cardiac disease. Adult healthy cats were enrolled and blood samples were obtained at 11 time points over a 6 week period (0, 2 h, 4 h, 6 h, 8 h, 10 h and at weeks 2, 3, 4, 5 and 6). The intra-individual (coefficient of variation [CVI]) biologic variation along with index of individuality and reference change values (RCVs) were calculated. Univariate models were analyzed and included comparison of the six different time points for both daily and weekly samples. This was followed by a Tukey's post-hoc adjustment, with a P value of <0.05 being significant. Results The median daily and weekly CVI for the population were 13.1% (range 0-28.7%) and 21.2% (range 3.9-68.1%), respectively. The index of individuality was 0.99 and 1 for daily and weekly samples, respectively. The median daily and weekly RCVs for the population were 39.8% (range 17.0-80.5%) and 60.5% (range 20.1-187.8%), respectively. Conclusions and relevance This study demonstrates high individual variability for NT-proBNP concentrations in a population of adult healthy cats. Further research is warranted to evaluate NT-proBNP variability, particularly how serial measurements of NT-proBNP may be used in the diagnosis and management of cats with cardiac disease.

Analytical validation of an immunoassay for the quantification of N-terminal pro-B-type natriuretic peptide in feline blood.

The measurement of N-terminal pro-B-type natriuretic peptide (NT-proBNP), a biomarker for heart stress detectable in blood, has been shown to have clinical utility in cats with heart disease. A second-generation feline enzyme-linked immunosorbent assay (Cardiopet® proBNP, IDEXX Laboratories Inc., Westbrook, Maine) was developed to measure NT-proBNP in routine feline plasma or serum samples with improved analyte stability. Results of the analytical validation for the second-generation assay are presented. Analytic sensitivity was 10 pmol/l. Accuracy of 103.5% was determined via serial dilutions of 6 plasma samples. Coefficients of variation for intra-assay, interassay, and total precision were in the ranges of 1.6-6.3%, 4.3-8.8%, and 10.1-15.1%, respectively. Repeatability across 2 lots for both serum and plasma had an average coefficient of determination (r(2)) of 0.99 and slope of 1.11. Stability of the analyte was found to be high. In serum samples held at 4°C for 24-72 hr, the mean percent recovery from time zero was ≥99%. In serum samples held at 25°C for 24 hr, the mean percent recovery from time zero was 91.9%, and for 48 hr, 85.6%. A method comparison of the first- and second-generation assays with a clinically characterized population of cats revealed no difference in the tests' ability to differentiate levels of NT-proBNP between normal cats and cats with occult cardiomyopathy (P < 0.001). Results from our study validate that the second-generation feline Cardiopet proBNP assay can measure NT-proBNP in routine feline plasma and serum samples with accuracy and precision.

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.

OBJECTIVE: To evaluate the sensitivity and specificity of a commercially available in-clinic ELISA for detection of heartworm infection and tick-borne diseases in dogs. SAMPLE POPULATION: 846 serum, plasma, or blood samples obtained from dogs. PROCEDURES: Samples were evaluated via the in-clinic ELISA to detect antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis (heartworm) antigen. True infection or immunologic status of samples was assessed by use of results of necropsy, an antigen assay for D immitis, and immunofluorescence assay or western blot analysis for antibodies against B burgdorferi, E canis, and A phagocytophilum. RESULTS: Sensitivity and specificity of the in-clinic ELISA for detection of heartworm antigen (99.2% and 100%, respectively), antibodies against B burgdorferi (98.8% and 100%, respectively), and antibodies against E canis (96.2% and 100%, respectively) were similar to results for a similar commercial ELISA. In samples obtained from dogs in the northeast and upper Midwest of the United States, sensitivity and specificity of the in-clinic ELISA for antibodies against Anaplasma spp were 99.1% and 100%, respectively, compared with results for an immunofluorescence assay. Samples from 2 dogs experimentally infected with the NY18 strain of A phagocytophilum were tested by use of the in-clinic ELISA, and antibodies against A phagocytophilum were detected by 8 days after inoculation. Antibodies against Anaplasma platys in experimentally infected dogs cross-reacted with the A phagocytophilum analyte. Coinfections were identified in several of the canine serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The commercially available in-clinic ELISA could be used by veterinarians to screen dogs for heartworm infection and for exposure to tick-borne pathogens.

Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota.

A population of 731 naturally exposed pet dogs examined at a private practice in Baxter, Minnesota, an area endemic for Lyme disease and anaplasmosis, was tested by serological and molecular methods for evidence of exposure to or infection with selected vector-borne pathogens. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and for Dirofilaria immitis antigen. Blood samples from 273 dogs were also analyzed by polymerase chain reaction (PCR) for Anaplasma and Ehrlichia species DNA. Based on the owner history and the attending veterinarian's physical examination findings, dogs exhibiting illness compatible with anaplasmosis or borreliosis were considered clinical cases, and their results were compared to the healthy dog population. Antibodies to only A. phagocytophilum were detected in 217 (29%) dogs; to only B. burgdorferi, in 80 (11%) dogs; and seroreactivity to both organisms, in 188 (25%) dogs. Of 89 suspected cases of canine anaplasmosis or borreliosis, A. phagocytophilum or B. burgdorferi antibodies were detected in 22 dogs (25%) and 8 dogs (9%) respectively, whereas antibodies to both organisms were found in 38 dogs (43%). Ehrlichia canis antibodies and D. immitis antigen were each detected in 11 (1.5%) dogs. Anaplasma phagocytophilum DNA was amplified from 7 of 222 (3%) healthy dogs and 19 of 51 (37%) clinical cases. Seroreactivity to both A. phagocytophilum and B. burgdorferi was detected more frequently in suspected cases of anaplasmosis and/or borreliosis than seroreactivity to either organism alone. Based on PCR testing, A. phagocytophilum DNA was more prevalent in suspected cases of anaplasmosis or borreliosis than in healthy dogs from the same region.

The extreme carboxyl terminus of v-Abl is required for lymphoid cell transformation by Abelson virus.

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells in vivo and in vitro and can transform immortalized fibroblast cell lines in vitro. Although the kinase activity of the protein is required for these events, most previously studied mutants encoding truncated v-Abl proteins that lack the extreme carboxyl terminus retain high transforming capacity in NIH 3T3 cells but transform lymphocytes poorly. To understand the mechanisms responsible for poor lymphoid transformation, mutants expressing a v-Abl protein lacking portions of the COOH terminus were compared for their ability to transform pre-B cells. Although all mutants lacking sequences within the COOH terminus were compromised for lymphoid transformation, loss of amino acids in the central region of the COOH terminus, including those implicated in JAK interaction and DNA binding, decreased transformation twofold or less. In contrast, loss of the extreme COOH terminus rendered the protein unstable and led to rapid proteosome-mediated degradation, a feature that was more prominent when the protein was expressed in Ab-MLV-transformed lymphoid cells. These data indicate that the central portion of the COOH terminus is not essential for lymphoid transformation and reveal that one important function of the COOH terminus is to stabilize the v-Abl protein in lymphoid cells.