Analytical validation of an ELISA for the measurement of feline pancreas-specific lipase and re-evaluation of the reference interval and decision threshold for diagnosing pancreatitis.

BACKGROUND: The diagnosis of feline pancreatitis can be challenging. The clinical presentation often includes mild, nonspecific clinical signs, such as vomiting, anorexia, and weight loss. Measurement of feline pancreatic lipase immunoreactivity (fPLI) concentration in serum has been reported to be sensitive and specific for a diagnosis of pancreatitis in cats. However, analytical validation for a widely available commercial assay for the measurement of fPLI concentration has not been published. OBJECTIVE: We aimed to analytically validate the Spec fPL assay (IDEXX Laboratories, Westbrook, ME), a commercial ELISA for the measurement of fPLI concentration, and re-evaluate its reference interval and decision threshold for diagnosing pancreatitis in cats. METHODS: Dilutional linearity, accuracy, precision, and the effect of interfering substances were assessed. The upper limit of the reference interval was calculated based on the 95th percentile of results from clinically healthy cats (n = 107), and a decision threshold for diagnosing pancreatitis was calculated with an expected specificity of 99%. RESULTS: Analytical validation demonstrated good linearity, accuracy, and precision, as well as the absence of interference from lipemia, hemolysis, or icterus. The upper limit of the reference interval for Spec fPL was determined to be 4.4 µg/L, and the decision threshold (a theoretical cut-off) for diagnosing pancreatitis was determined to be 8.8 µg/L based on a desired specificity of 99%. CONCLUSIONS: The Spec fPL assay is analytically valid, and results suggest that a decision threshold of 8.8 µg/L would have high diagnostic specificity for excluding clinically healthy cats.

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.

Typical and atypical manifestations of Anaplasma phagocytophilum infection in dogs.

Eighteen clinically ill dogs, naturally infected with Anaplasma phagocytophilum, were examined at a veterinary practice in Baxter, Minnesota. A clinical examination, complete blood cell count, enzyme- linked immunosorbent assay (ELISA) for A phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and Dirofilaria immitis antigen, and a polymerase chain reaction test for A phagocytophilum DNA were obtained for all dogs. Physical examination findings included fever, arthropathy, lymphadenopathy, epistaxis, acute gastritis, cervical hyperpathia, and central nervous system dysfunction. Complete blood cell count abnormalities included thrombocytopenia, morulae in neutrophils, anemia, leukopenia, eosinopenia, lymphopenia, and monocytosis. Seroreactivity to A phagocytophilum was found in 61%, B burgdorferi antibodies in 17%, and D immitis antigen in 5% of the dogs. Fever, arthropathy, neurologic dysfunction, and epistaxis are clinical syndromes that can be associated with A phagocytophilum infection. Treatment with doxycycline resulted in rapid resolution of clinical signs in all dogs.

Comparison of Anaplasma and Ehrlichia species-specific peptide ELISAs with whole organism-based immunofluorescent assays for serologic diagnosis of anaplasmosis and ehrlichiosis in dogs.

OBJECTIVE: To compare the performance of 5 synthetic peptide-based ELISAs with that of 3 commercially available immunofluorescent assays (IFAs) for serologic diagnosis of anaplasmosis and ehrlichiosis in dogs. SAMPLE: A convenience set of 109 serum samples obtained before and at various times after inoculation for 23 dogs that were experimentally infected with Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Ehrlichia chaffeensis, or Ehrlichia ewingii and 1 uninfected control dog in previous studies. PROCEDURES: All serum samples were assessed with 5 synthetic peptide-based ELISAs designed to detect antibodies against A phagocytophilum, A platys, E canis, E chaffeensis, and E ewingii and 3 whole organism-based IFAs designed to detect antibodies against A phagocytophilum, E canis, and E chaffeensis. The species-specific seroreactivity, cross-reactivity with the other tick-borne pathogens (TBPs), and diagnostic sensitivity and specificity were calculated for each assay and compared among assays. RESULTS: All serum samples obtained from dogs experimentally infected with a TBP yielded positive results on a serologic assay specific for that pathogen. In general, sensitivity was comparable between ELISAs and IFAs and tended to increase with duration after inoculation. Compared with the IFAs, the corresponding ELISAs were highly specific and rarely cross-reacted with antibodies against other TBPs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that peptide-based ELISAs had enhanced specificity relative to whole organism-based IFAs for detection of antibodies against Anaplasma and Ehrlichia spp, which should facilitate accurate diagnosis and may help detect dogs coinfected with multiple TBPs.

Measuring the Humoral Immune Response in Cats Exposed to Feline Leukaemia Virus.

Retroviruses belong to an important and diverse family of RNA viruses capable of causing neoplastic disease in their hosts. Feline leukaemia virus (FeLV) is a gammaretrovirus that infects domestic and wild cats, causing immunodeficiency, cytopenia and neoplasia in progressively infected cats. The outcome of FeLV infection is influenced by the host immune response; progressively infected cats demonstrate weaker immune responses compared to regressively infected cats. In this study, humoral immune responses were examined in 180 samples collected from 123 domestic cats that had been naturally exposed to FeLV, using a novel ELISA to measure antibodies recognizing the FeLV surface unit (SU) glycoprotein in plasma samples. A correlation was demonstrated between the strength of the humoral immune response to the SU protein and the outcome of exposure. Cats with regressive infection demonstrated higher antibody responses to the SU protein compared to cats belonging to other outcome groups, and samples from cats with regressive infection contained virus neutralising antibodies. These results demonstrate that an ELISA that assesses the humoral response to FeLV SU complements the use of viral diagnostic tests to define the outcome of exposure to FeLV. Together these tests could allow the rapid identification of regressively infected cats that are unlikely to develop FeLV-related disease.

Feline Leukemia Virus p27 Antigen Concentration and Proviral DNA Load Are Associated with Survival in Naturally Infected Cats.

Longitudinal studies of cats naturally infected with feline leukemia virus (FeLV) are important for understanding disease outcomes. Levels of p27 antigen and copy numbers of proviral DNA have been associated with FeLV-infection courses. The purpose of this prospective study was to establish cutoff values for p27 antigen concentration and proviral DNA load that distinguished high positive from low positive groups of cats and to evaluate an association with survival. At enrollment, 254 cats were tested by point-of-care and microtiter plate enzyme-linked immunosorbent assays (ELISAs) for p27 antigen and real-time polymerase chain reaction (PCR) for proviral DNA. The 127 positive cats were retested monthly for six months and monitored for survival over the four-year study. A receiver operating characteristic-based analysis of samples with concordant or discordant qualitative results for p27 antigen and proviral DNA was used to establish cutoff values, and when applied to test results at enrollment for classifying cats as high positive or low positive, a significant difference in survival was observed. High positive cats had a median survival of 1.37 years (95% CI 0.83-2.02) from time of enrollment, while most low positive cats were still alive (93.1% survival). Quantitative results for p27 antigen concentration and proviral DNA load were highly correlated with survival times in FeLV-infected cats.

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.

Global distribution of canine Babesia species identified by a commercial diagnostic laboratory.

Babesia species are important canine pathogens with a nearly worldwide distribution. Our understanding of the distribution of these parasites is continually improving. This is in large part, due to improved molecular diagnostic capabilities. However, it can be difficult to assimilate and compare previous reports from various regions due to differences in molecular methods. In this report, we characterize the results of over 100,000 canine samples from 52 different countries and territories spanning 4 continents that were submitted to a commercial diagnostic laboratory for Babesia testing by polymerase chain reaction. The same diagnostic algorithm was used for all samples and is designed to identify and differentiate B. gibsoni, B. canis, B. vogeli, B. rossi and B. conradae. Overall 3.4% of the samples submitted tested positive for the presence of Babesia sp. DNA and were differentiated to the species level. Babesia gibsoni was the most commonly identified species (48.8% of the positive results) followed by B. canis (35.2%) then B. vogeli (15.3%). Babesia gibsoni and B. vogeli were more widely distributed than B. canis, which was primarily found in Europe. This is the largest study of its type and these data provide a global overview of which Babesia species veterinarians could expect to find in their practice area.

Comparison of Diagnostic Tools for the Detection of Dirofilaria immitis Infection in Dogs.

In the last two decades, reports of canine heartworm (HW) infection have increased even in non-endemic areas, with a large variability in prevalence data due to the diagnostic strategy employed. This study evaluated the relative performance of two microtiter plate ELISA methods for the detection of HW antigen in determining the occurrence of Dirofilaria immitis in a dog population previously tested by the modified Knott's test and SNAP 4Dx Plus test. The prevalence of this infection in the sheltered dog population (n = 363) from a high-risk area for HW infection was 44.4% according to the modified Knott's test and 58.1% according to a point-of-care antigen ELISA. All serum samples were then evaluated by a microtiter plate ELISA test performed with and without immune complex dissociation (ICD). The prevalence increased from 56.5% to 79.6% following ICD, indicating a high proportion of samples with immune complexing. Comparing these results to that of the modified Knott's test, the samples negative for microfilariae (mfs) and those positive only for D. repens mfs demonstrated the greatest increase in the proportion of positive results for D. immitis by ELISA following ICD. While the ICD method is not recommended for routine screening, it may be a valuable secondary strategy for identifying HW infections in dogs.

Detection of feline panleukopenia virus using a commercial ELISA for canine parvovirus.

Feline panleukopenia virus (FPV) is a significant pathogen of cats. Rapid virus detection is critical for treatment and management, especially in populations in which spread may occur. This study investigated the ability of the SNAP Canine Parvovirus Antigen Test Kit (SNAP Parvo, IDEXX Laboratories) to detect FPV with confirmation of viral identity by polymerase chain reaction (PCR) assay and genetic sequencing on fecal samples (n = 97) from cats with suspected FPV infection. Fifty-five samples were positive by SNAP Parvo; 54 of 55 were also positive by conventional PCR assay and were identified as FPV by genetic sequencing. This study demonstrates that SNAP Parvo can detect FPV in clinical samples.

Borrelia burgdorferi Antibody Test Results in Dogs Administered 4 Different Vaccines.

Vaccines against Borrelia burgdorferi are administered frequently to dogs in areas endemic for the infection. These vaccines produce an antibody response to spirochetal proteins that cross-react in many antibody tests, including immunofluorescence assay, Western blot, and whole cell ELISA used to document exposure to B. burgdorferi. The purpose of this study was to determine whether the in-clinic C6 ELISA assay (SNAP® 4Dx® Plus; IDEXX Laboratories, Inc., Westbrook, Maine) and the quantitative format C6 ELISA (Lyme Quant C6® Antibody Test, IDEXX Laboratories, Inc., Westbrook, ME) react to sera from dogs that have been vaccinated with 1 of 4 different commercially available B. burgdorferi vaccines. Four groups of 3 dogs each were each administered one of the 4 vaccines and sera evaluated over time by indirect fluorescent antibody assay, western blot immunoassay, the in-clinic C6 ELISA assay, and the quantitative format C6 ELISA. While all dogs developed B. burgdorferi antibodies detectable by indirect fluorescent antibody assay and western blot immunoassay after vaccination, none of the samples were positive in either of the C6 peptide-based assays. Based on these results, positive anti-C6 antibody results in client-owned dogs are likely to reflect exposure to B. burgdorferi rather than vaccination.

Evaluation of a quantitative enzyme-linked immunosorbent assay for feline leukemia virus p27 antigen and comparison to proviral DNA loads by real-time polymerase chain reaction.

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. While higher viral RNA and proviral DNA loads have been correlated with progressive infections and disease, a similar correlation has been suggested for p27 antigen concentrations. This analytical study compared the results of a quantitative ELISA for p27 antigen with quantitative real-time PCR results for FeLV proviral DNA in patient samples. A significant positive correlation between copies of proviral DNA and the concentration of p27 antigen was identified (r = 0.761, P < 0.0001). Samples with high proviral DNA loads, at least 1 × 106 copies/mL of whole blood, typically had p27 antigen concentrations greater than 30 ng/mL in plasma. Samples with proviral DNA loads below this level all had concentrations of p27 antigen in plasma that were less than 10 ng/mL. Given this correlation, it is hypothesized that the concentration of p27 antigen at a given point in time may help to indicate the likelihood of a progressive or regressive infection similar to what has been demonstrated for proviral DNA loads.

Clinicopathological findings in 41 dogs (2008-2018) naturally infected with Ehrlichia ewingii.

BACKGROUND: Ehrlichia ewingii is the most seroprevalent Ehrlichia-infecting dogs in the southern and mid-western United States. Fever, lameness, and polyarthritis are commonly reported findings in dogs naturally infected with E. ewingii. OBJECTIVES: To evaluate clinicopathologic findings in a population of dogs naturally infected with E. ewingii. ANIMALS: Forty-one dogs PCR positive for E. ewingii and PCR negative for other targeted vector-borne organisms. METHODS: Retrospective study. Clinical and clinicopathologic data including physical examination findings, CBC, serum biochemistry, urinalysis (UA), symmetric dimethylarginine (SDMA), and vector-borne disease diagnostic results were reviewed. RESULTS: Frequent clinical diagnoses other than ehrlichiosis (28/41; 68.3%) were renal disease (7/41; 17.1%) and immune-mediated hemolytic anemia (IMHA) (6/41; 14.6%). The most frequent physical examination finding was joint pain (14/41; 34.1%). Prominent hematologic and biochemical abnormalities included abnormal lymphocyte counts (22/36; 61.1%); neutrophilia (21/37; 56.8%); increased alkaline phosphatase (20/35; 57.1%) and alanine transaminase (14/35; 40%) activities; and increased SDMA concentration (11/34; 32.4%). Urinalysis abnormalities included proteinuria (20/27; 74%), most with inactive sediments (16/20; 80%). Dogs were seroreactive by Ehrlichia canis immunofluorescence assay (IFA; 17/39; 43.6%) and Ehrlichia ELISA (34/41; 82.9%). Seroreactivity by IFA for other vector-borne pathogens included Bartonella (1/39; 2.6%), Rickettsia rickettsii (spotted-fever group rickettsiae) (12/39; 30.8%), and Borrelia burgdorferi by ELISA (1/41; 2.4%). CONCLUSIONS AND CLINICAL IMPORTANCE: Renal disease, IMHA, proteinuria, neutrophilia, abnormal lymphocytes, and increased liver enzyme activities were common in this group of E. ewingii-infected dogs. Studies are needed to determine if E. ewingii contributes to comorbidities or is a precipitating factor in clinical syndromes in persistently infected dogs.

Coproantigen Detection Augments Diagnosis of Common Nematode Infections in Dogs.

Microscopic methods which employ active or passive flotation have been used to detect parasite diagnostic stages in the feces of companion animals for many years. More recently, coproantigen ELISAs for the detection of excretory/secretory products from intestinal nematodes have been introduced. These assays can identify the presence of parasites when eggs are not recovered by flotation (e.g. prepatent infection or intermittent egg shedding). The study was designed to assess the added benefit of these coproantigen tests in canine fecal diagnostics. The work was performed at 3 separate sites where canine fecal samples were each independently evaluated by both centrifugal flotation with an expert examiner (CFE) and passive flotation with a less experienced examiner. All samples were also tested using coproantigen ELISA to detect ascarid, hookworm, or whipworm antigen (IDEXX Laboratories, Inc, Westbrook, Maine). A total of 1202 samples were collected; 626 were from shelter dogs and 576 were from pet dogs. CFE recovered ascarid eggs in 58 samples, hookworm eggs in 229 samples, and whipworm eggs in 95 samples. Of the positive samples identified by CFE, the PFE and ELISA identified 40 and 51 ascarid samples, 188 and 203 hookworm samples, and 65 and 67 whipworm positive samples, respectively. The coproantigen ELISA identified 8 ascarid, 82 hookworm, and 22 whipworm positive samples that were not detected by CFE. The combined results of passive flotation and the coproantigen ELISA improved the percent agreement with centrifugal flotation, suggesting that greater sensitivity of detection may be achieved through the use of complementary diagnostic methods. However, errors of misidentification and poor recovery apparently introduced by less experienced examiners using an inferior flotation method remained. A diagnostic approach that combines coproantigen assays with centrifugal flotation and examination by an expert allows detection of more ascarid, hookworm, and whipworm infections.

Canine and human infection with Borrelia burgdorferi in the New York City metropolitan area.

BACKGROUND: Autochthonous transmission of Borrelia burgdorferi, the primary agent of Lyme disease in dogs and people in North America, commonly occurs in the northeastern United States, including the New York City metropolitan area, a region with a large human and pet population and broadly diverse demographics and habitats. METHODS: We evaluated results from a specific, C6-based serologic assay performed on 234,633 canine samples to compare evidence of past or current infection with B. burgdorferi (sensu stricto) in dogs to county-wide social and environmental factors, as well as to reported cases of Lyme disease in people. RESULTS: The data revealed a wide range of county level percent positive canine test results (1.2-27.3%) and human case reports (0.5-438.7 case reports/100,000 people). Dogs from highly (> 50%) forested areas and counties with lower population density had the highest percent positive test results, at 21.1% and 17.9%, respectively. Canine percent positive tests correlated with population-adjusted human case reports (R2 = 0.48, P < 0.0001), as well as population density, development intensity, temperature, normalized difference vegetation index, and habitat type. Subsequent multiple regression allowed an accurate prediction of infection risk in dogs (R2 = 0.90) but was less accurate at predicting human case reports (R2 = 0.74). CONCLUSION: In areas where Lyme disease is endemic, canine serology continues to provide insight into risk factors for transmission to both dogs and people although some differences in geographic patterns of canine infection and human disease reports are evident.

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.

Canine infection with Borrelia burgdorferi, Dirofilaria immitis, Anaplasma spp. and Ehrlichia spp. in Canada, 2013-2014.

BACKGROUND: Canine test results generated by veterinarians throughout Canada from 2013-2014 were evaluated to assess the geographical distribution of canine infection with Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia spp., and Anaplasma spp. METHODS: The percent positive test results of 115,636 SNAP® 4Dx® Plus tests from dogs tested were collated by province and municipality to determine the distribution of these vector-borne infections in Canada. RESULTS: A total of 2,844/115,636 (2.5%) dogs tested positive for antibody to B. burgdorferi. In contrast, positive test results for D. immitis antigen and antibodies to Ehrlichia spp. and Anaplasma spp. were low, with less than 0.5% of dogs testing positive for any one of these three agents nationwide. Provincial seroprevalence for antibodies to B. burgdorferi ranged from 0.5% (Saskatchewan)-15.7% (Nova Scotia); the areas of highest percent positive test results were in proximity to regions in the USA considered endemic for Lyme borreliosis, including Nova Scotia (15.7%) and Eastern Ontario (5.1%). These high endemic foci, which had significantly higher percent positive test results than the rest of the nation (P < 0.0001), were surrounded by areas of moderate to low seroprevalence in New Brunswick (3.7%), Quebec (2.8%), and the rest of Ontario (0.9%), as well as northward and westward through Manitoba (2.4%) and Saskatchewan (0.5%). Insufficient results were available from the westernmost provinces, including Alberta and British Columbia, to allow analysis. CONCLUSION: Increased surveillance of these vector-borne disease agents, especially B. burgdorferi, is important as climate, vector range, and habitat continues to change throughout Canada. Using dogs as sentinels for these pathogens can aid in recognition of the public and veterinary health threat that each pose.

Analytical validation of a reference laboratory ELISA for the detection of feline leukemia virus p27 antigen.

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. Immunoassays for the p27 core protein of FeLV aid in the detection of FeLV infections. Commercial microtiter-plate ELISAs have rapid protocols and visual result interpretation, limiting their usefulness in high-throughput situations. The purpose of our study was to validate the PetChek FeLV 15 ELISA, which is designed for the reference laboratory, and incorporates sequential, orthogonal screening and confirmatory protocols. A cutoff for the screening assay was established with 100% accuracy using 309 feline samples (244 negative, 65 positive) defined by the combined results of FeLV PCR and an independent reference p27 antigen ELISA. Precision of the screening assay was measured using a panel of 3 samples (negative, low-positive, and high-positive). The intra-assay coefficient of variation (CV) was 3.9-7.9%; the inter-assay CV was 6.0-8.6%. For the confirmatory assay, the intra-assay CV was 3.0-4.7%, and the inter-assay CV was 7.4-9.7%. The analytical sensitivity for p27 antigen was 3.7 ng/mL for inactivated whole FeLV and 1.2 ng/mL for purified recombinant FeLV p27. Analytical specificity was demonstrated based on the absence of cross-reactivity to related retroviruses. No interference was observed for samples containing added bilirubin, hemoglobin, or lipids. Based on these results, the new high-throughput design of the PetChek FeLV 15 ELISA makes it suitable for use in reference laboratory settings and maintains overall analytical performance.

Factors associated with Anaplasma spp. seroprevalence among dogs in the United States.

BACKGROUND: Dogs in the United States are hosts to a diverse range of ticks and tick-borne pathogens, including A. phagocytophilum, an important emerging canine and human pathogen. Previously, a Companion Animal Parasite Council (CAPC)-sponsored workshop proposed factors purported to be associated with the infection risk for tick-transmitted pathogens in dogs in the United States, including climate conditions, socioeconomic characteristics, local topography, and vector distribution. METHODS: Approximately four million test results from routine veterinary diagnostic tests from 2011-2013, which were collected on a county level across the contiguous United States, are statistically analyzed with the proposed factors via logistic regression and generalized estimating equations. Spatial prevalence maps of baseline Anaplasma spp. prevalence are constructed from Kriging and head-banging smoothing methods. RESULTS: All of the examined factors, with the exception of surface water coverage, were significantly associated with Anaplasma spp. prevalence. Overall, Anaplasma spp. prevalence increases with increasing precipitation and forestation coverage and decreases with increasing temperature, population density, relative humidity, and elevation. Interestingly, socioeconomic status and deer/vehicle collisions were positively and negatively correlated with canine Anaplasma seroprevalence, respectively. A spatial map of the canine Anaplasma hazard is an auxiliary product of the analysis. Anaplasma spp. prevalence is highest in New England and the Upper Midwest. CONCLUSIONS: The results from the two posited statistical models (one that contains an endemic areas assumption and one that does not) are in general agreement, with the major difference being that the endemic areas model estimates a larger prevalence in Western Texas, New Mexico, and Colorado. As A. phagocytophilum is zoonotic, the results of this analysis could also help predict areas of high risk for human exposure to this pathogen.