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.

Evaluation of Anaplasma spp. seroprevalence in dogs and association with incidence of human anaplasmosis.

Point-of-care (POC) ELISA tests are routinely used in US veterinary practices to screen canine patients for antibodies to tick-transmitted pathogens. Results are also used to monitor spatial and temporal trends in canine seroprevalence, and these data can build awareness of the risk to humans of tick-transmitted diseases such as Lyme disease and anaplasmosis. This study utilized a second-generation test that has incorporated additional Anaplasma-specific peptides into a commercial POC ELISA test to allow detection of Anaplasma spp. antibodies earlier post-infection. A convenience population consisting of 19,894 canine samples from a US commercial diagnostic laboratory were tested using the second-generation POC ELISA test to describe regional Anaplasma spp. canine seroprevalence and assess correlation to anaplasmosis cases reported to Centers for Disease Control and Prevention by state. Antibodies to Anaplasma spp. were detected in 1646 samples (8.3%) with the Northeast and Midwest US census regions having the highest proportion of positive samples. At the state level, a significant correlation was found between canine Anaplasma spp. seroprevalence and human anaplasmosis incidence (r2 = 0.64). Although estimates of canine Anaplasma spp. seroprevalence presented here using the second-generation POC ELISA are generally increased, especially in the Northeast and Midwest, the regional distribution of canine samples testing positive for Anaplasma spp. antibodies is consistent with previous reports. The observed correlation with human anaplasmosis incidence indicates that results from the second-generation POC ELISA will continue to add value in epidemiological assessment of human anaplasmosis risk.

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.

Validation of immune complex dissociation methods for use with heartworm antigen tests.

BACKGROUND: Antigen testing is routinely used to diagnose canine Dirofilaria immitis infections. Immune complex dissociation (ICD) methods, which were employed in the original heartworm antigen tests to release antigen that was bound by endogenous canine antibodies, were discontinued with improvements in assay reagents. The purpose of this study was to evaluate different ICD methods for detection of heartworm antigen by microtiter plate ELISA and assess the performance in samples from pet dogs. METHODS: The original PetChek® Heartworm Test (IDEXX Laboratories, Inc.) utilized pepsin at an acidic pH for ICD prior to antigen testing. Performance and characteristics of the pepsin ICD method were compared with those for heat treatment (with and without EDTA) and acid treatment. RESULTS: All four methods released complexed antigen in serum samples when tested using microtiter plate ELISA. Heat treatment required ≥600 µL of serum or plasma, whereas pepsin and acid methods needed only a 50-µL sample. Samples from 1115 dogs submitted to IDEXX Laboratories between 2014 and 2016 for investigation of discrepant heartworm results were evaluated with and without pepsin ICD using the PetChek Heartworm Test. Samples from 10% (n = 112) of the dogs were antigen positive with the ICD protocol only while 90% of the results remained unchanged. In a prospective study, antigen levels with and without ICD were evaluated for 12 dogs receiving pre-adulticide heartworm treatment with a macrocyclic lactone and doxycycline for 28 days. Serial samples revealed that three dogs had a reduction in detectable heartworm antigen within 4 weeks of initiating treatment. In these cases, heartworm antigen levels could be recovered with ICD. CONCLUSIONS: Heartworm antigen testing with ICD can be a valuable diagnostic tool for patients with discrepant results that have had intermittent use of a preventive, or have been treated with a macrocyclic lactone and doxycycline. Heartworm therapies may reduce antigen production and favor immune complexing in some dogs, resulting in false-negative results. Therefore, it is important to confirm positive heartworm antigen test results before initiating therapy.