Jiayou Liu

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

OBJECTIVEnTo evaluate the performance of an in-clinic ELISA designed for detection of heartworm antigen and antibodies against 5 tick-borne pathogens.nnnDESIGNnValidation study.nnnSAMPLEn1,601 serum or matched serum, plasma, and blood samples from dogs.nnnPROCEDURESnSamples 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.nnnRESULTSnSensitivity 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.nnnCONCLUSIONS AND CLINICAL RELEVANCEnThe in-clinic ELISA provided a comprehensive in-house serologic screening test for all vector-borne pathogens evaluated.

A serological survey of tick-borne pathogens in dogs in North America and the Caribbean as assessed by Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, E. ewingii , and Borrelia burgdorferi species-specific peptides

Introduction Tick-borne pathogens cause a spectrum of disease manifestations in both dogs and humans. Recognizing regional and temporal shifts in exposure are important as tick distributions change. To better delineate regional exposure to canine tick-borne pathogens, an expanded set of species-specific peptides were used to detect Anaplasma phagocytophilum (Aph), Anaplasma platys (Apl), Ehrlichia canis (Ec), Ehrlichia chaffeensis (Ech), Ehrlichia ewingii (Eew), and Borrelia burgdorferi (Bb) antibodies in canine serum. Methods Archived canine serum samples (n=6,582) collected during 2008–2010 and in 2012 from the US, Canada, and the Caribbean were retrospectively screened for antibodies against Ehrlichia and Anaplasma species-specific peptides. Overall, regional and temporal seroprevalence rates were determined. Results Overall Bb and Eew were the most seroprevalent pathogens. During 2008–2010, seroprevalence rates increased overall for Aph and Ech, and regionally, Bb and Aph seroprevalence rates increased in the South. Canada had unexpectedly high seroprevalence rates for Ec and Apl. The most common co-exposures were Eew+Ech, followed by Aph+Bb and Eew+Bb. Conclusions This study demonstrated significant shifts in canine vector-borne disease seroprevalence rates. The use of specific peptides facilitated improved geographic delineation of tick-borne pathogen distributions among dogs, which may enhance epidemiological surveillance of vector-borne pathogens shared by dogs and humans.

Evidence of Anaplasma phagocytophilum and Borrelia burgdorferi infection in cats after exposure to wild-caught adult Ixodes scapularis.

Cats are infected by Anaplasma phagocytophilum and Borrelia burgdorferi when exposed to infected Ixodes scapularis (black-legged ticks). The purpose of our study was to allow wild-caught I. scapularis to feed on healthy research cats (n = 4) and temporally evaluate for A. phagocytophilum DNA in blood by a polymerase chain reaction (PCR) assay as well as for antibody responses to the B. burgdorferi C6 peptide, to the A. phagocytophilum P44 peptide, and to a novel A. phagocytophilum peptide (P44-4). Prior to I. scapularis infestation, all cats were negative for antibodies against both organisms based on a kit optimized for dog serum, and negative for A. phagocytophilum DNA in blood using a conventional PCR assay. Using the pre-infestation samples, an enzyme-linked immunosorbent assay for detecting antibodies against the P44-4 peptide was optimized. Cats were infested with wild-caught I. scapularis for 7 days. Genomic DNA of A. phagocytophilum was amplified from the blood before antibodies were detected in all 4 cats. Antibodies against the C6 peptide, P44 peptide, and P44-4 peptide were detected in the sera of all 4 cats. Antibodies against P44-4 were detected prior to those against P44 in 3 out of 4 cats. The results suggest that a PCR assay should be considered in acutely ill cats with suspected anaplasmosis that are seronegative.

Sensitivity and specificity levels of two rapid assays for antibodies to Anaplasma spp. in dogs

Canine anaplasmosis is a tick-borne disease of dogs that results following infection with Anaplasma phagocytophilum or Anaplasma platys. The SNAP 4Dx Plus test (IDEXX Laboratories) and the VetScan Canine Anaplasma Rapid test (Abaxis) are commercial in-house rapid tests for the detection of antibody to these 2 antigenically related Anaplasma species. We evaluated 2 tests using serum and whole blood samples obtained from reference laboratories and veterinary hospitals. Samples were obtained from regions of the country known to be habitats of the primary tick vectors. The A. phagocytophilum sample set comprised 236 dog sera from the northeastern and midwestern United States; the A. platys sample set comprised 179 sera from dogs living in the southwestern United States. An indirect immunofluorescent antibody (IFA) test and an A. platys species-specific ELISA were used as reference assays for the A. phagocytophilum and A. platys samples, respectively. The SNAP test demonstrated significantly higher sensitivity (84.7% for A. phagocytophilum and 83.1% for A. platys), compared to the VetScan test (39.0% for A. phagocytophilum and 57.6% for A. platys). The specificity of the SNAP test (95.8% for A. phagocytophilum and 99.2% for A. platys) was significantly greater than the VetScan test (85.6% for A. phagocytophilum and 82.5% for A. platys). In a separate clinic study, conducted within an A. phagocytophilum–endemic state (Minnesota) using 154 whole blood samples from client-owned dogs, the VetScan test was negative for 22 of 39 SNAP and IFA seropositive samples.

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

BackgroundAntigen 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.MethodsThe 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.ResultsAll four methods released complexed antigen in serum samples when tested using microtiter plate ELISA. Heat treatment required ≥600xa0μL 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% (nxa0=xa0112) 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 28xa0days. Serial samples revealed that three dogs had a reduction in detectable heartworm antigen within 4xa0weeks of initiating treatment. In these cases, heartworm antigen levels could be recovered with ICD.ConclusionsHeartworm 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.

Comparative Evaluation of Two In-clinic Assays for Vector-Borne Disease Testing in Dogs

ABSTRACT Vector‐borne agents comprise medically important infections affecting dogs throughout much of the world. Sensitive detection of antibodies directed at tick‐borne disease‐causing organisms in dogs is diagnostically important for veterinarians, pets and their owners, and epidemiologically important for public health surveillance. The SNAP 4Dx Plus Test (IDEXX Laboratories, Inc., Westbrook, ME) identifies antibodies to or infection with multiple tick‐borne pathogens and canine heartworm antigen in a single assay. Recently, VetScan FLEX4 Rapid Test (Abaxis, Inc., Union City, CA) was launched as a new assay to detect tick‐borne pathogen antibodies and heartworm antigen. In the present study, we evaluated the comparative performance of SNAP 4Dx Plus (SNAP) and FLEX4 Rapid Test (FLEX4) using samples selected based on geographic distributions for canine vector borne diseases, including Borrelia burgdorferi (n = 105), Anaplasma phagocytophilum (160), Anaplasma platys (115), Ehrlichia canis (154), Ehrlichia ewingii (163), Ehrlichia chaffeensis (151) and Dirofilaria immitis (105). Canine vector borne diseases infection status was established for each sample by a combination of reference methods that included necropsy (D. immitis, heartworm disease), Western immunoblotting (B. burgdorferi), immunofluorescence assays (A. phagocytophilum and E. canis) and species‐specific ELISAs (A. platys, E. canis, E. ewingii and E. chaffeensis). For comparisons among the 2 assays, samples were evaluated per the manufacturers’ instructions for each test kit. By testing each same sample set compared to the defined reference results, sensitivities differed substantially between SNAP and FLEX4, at 95.5 vs. 40.9%, respectively for B. burgdorferi, 97.1% vs. 61.4% for E. canis, 98.2% vs. 59.3% for E. ewingii, 64.3% vs. 35.7% for E. chaffeensis, 84.5% vs. 12.7% for A. phagocytophilum, 83.3% vs. 33.3% for A. platys, and 94.1% vs. 88.2% for D. immitis. Specificities for both rapid assay tests ranged from 98% to 100%. Based upon the comparative results derived from this study, the SNAP test was more sensitive than the FLEX4 test for detection of antibodies to all tick‐borne pathogens and heartworm disease (Dirofilaria immitis) antigen in dogs.

Evaluation of rapid diagnostic test kits for feline leukemia virus infection using samples from naturally infected cats

Objectives Feline leukemia virus (FeLV) is a potentially life-threatening oncogenic retrovirus. The p27 viral core protein is produced by the virus in infected feline cells, is found in the cytoplasm in several blood cells and can be free in the serum and plasma. ELISA or particle-based immunoassay are commonly used to detect the presence of the p27 core protein in samples obtained from blood. The objective of this study was to compare the performance of several in-clinic tests: the SNAP Feline Triple Test (IDEXX Laboratories), the WITNESS FeLV-FIV Test (Zoetis) and the VetScan Feline FeLV/FIV Rapid Test (Abaxis). Methods The sample population (100 positive, 105 negative samples) consisted of serum and plasma samples submitted to IDEXX’s worldwide reference laboratory for feline retrovirus testing. Virus isolation and reverse transcriptase PCR results were not available and so samples were judged to be positive or negative based on the results of the ViraCHEK FeLV (Zoetis) microtiter plate assay. Results The percentage of samples positive and negative for FeLV p27 antigen using the three in-clinic tests compared with the ViraCHEK method were as follows: IDEXX Feline Triple (positive 98.0%, negative 100%); Zoetis WITNESS (positive 79.0%, negative 97.1%); Abaxis VetScan (positive 73.0%, negative 97.1%). Conclusions and relevance The SNAP Feline Triple Test demonstrated a high level of agreement for FeLV-positive and FeLV-negative samples when assessed in this model. Results of FeLV assays can vary among tests.