Diagnosis of canine intestinal parasites: Improved detection of Dipylidium caninum infection through coproantigen testing.

Intestinal parasites, including cestodes like Dipylidium caninum, are common in dogs in the United States of America (USA), but fecal flotation consistently, and, at times, dramatically, fails to identify many of these infections. To determine the extent to which including coproantigen testing for D. caninum would improve the identification of dogs infected with this cestode, we evaluated fecal samples from 877 dogs (589 pet and 288 from municipal shelters) from six USA states using zinc sulfate (specific gravity 1.24) fecal flotation with centrifugation along with coproantigen detection for Giardia sp., hookworms, ascarids, and Trichuris vulpis. For D. caninum, PCR of perianal swabs was included. Intestinal parasite infections were identified, using centrifugal fecal flotation or coproantigen, in 265 dogs (13.2 % pet, 64.9 % shelter). Dipylidium caninum infection was detected in 5.6 % of dogs with the combination of coproantigen and centrifugal fecal flotation, and 7.3 % of dogs when perianal swab results were included; prevalence varied by diagnostic method, population, and geographic region. In pet dogs, D. caninum infection was identified by fecal flotation (0), coproantigen (2.2 %), or perianal swabs (1.2 %). The same methods revealed infection in 0.3 %, 12.5 %, and 11.1 % of shelter dogs, respectively. Frequent use of praziquantel in shelter dogs (116/288; 40.3 %) may have reduced prevalence. Positive and negative agreement of D. caninum coproantigen with perianal swab PCR in pet dogs was 85.7 % and 98.8 %, respectively. Multiple logistic regression analysis accounting for region, population, and age found D. caninum infection to be more common in shelter dogs relative to pet (adjusted OR 4.91 [2.48, 10.24]) and in the Southcentral and Southeast regions relative to North (adjusted OR 9.59 [1.92, 174.13] and 17.69 [3.67, 318.09] respectively). Coproantigen testing also enhanced the detection of other intestinal parasites over fecal flotation alone, including Giardia sp. (14.7 % vs 3.3 %), hookworms (13.8 % vs 8.4 %), ascarids (2.9 % vs 2.2 %), and T. vulpis (2.9 % vs 1.4 %). Together, these data indicate that the coproantigen assay employed increases detection of D. caninum infections several fold, supporting the use of this test in clinical practice, and add to a growing body of research documenting enhanced diagnosis through implementation of multiple laboratory-based methods.

Urinary cystatin B differentiates progressive versus stable IRIS Stage 1 chronic kidney disease in dogs.

BACKGROUND: Early identification of dogs with progressive vs stable chronic kidney disease (CKD) might afford opportunity for interventions that would slow progression. However, currently no surrogate biomarker reliably predicts CKD progression. HYPOTHESIS/OBJECTIVES: Urinary cystatin B (uCysB), a novel kidney injury biomarker, predicts progressive disease in International Renal Interest Society (IRIS) CKD Stage 1. ANIMALS: Seventy-two dogs, including 20 dogs from 4 university centers with IRIS CKD Stage 1, with IDEXX symmetric dimethylarginine (SDMA) concentration up to 17 µg/dL and no systemic comorbidities, and 52 clinically healthy staff-owned dogs from a fifth university center. METHODS: A multicenter prospective longitudinal study was conducted between 2016 and 2021 to assess uCysB concentration in IRIS CKD Stage 1 and control dogs. Dogs were followed to a maximum of 3 years (control) or 25 months (CKD). Stage 1 IRIS CKD was classified as stable or progressive using the slope of 1/SDMA, calculated from 3 timepoints during the initial 90-day period. Dogs with slope above or below -0.0007 week × dL/µg were classified as stable or progressive, respectively. Mixed effects modeling was used to assess the association between uCysB and progression rate. RESULTS: Estimates of first visit uCysB results predictive of active ongoing kidney injury based on the mixed effects models were 17 ng/mL for control, 24 ng/mL for stable CKD, and 212 ng/mL for progressive CKD (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary cystatin B differentiated stable vs progressive IRIS CKD Stage 1. Identification of dogs with progressive CKD may provide an opportunity for clinicians to intervene early and slow progression rate.

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.

Differentiation of stable kidney function versus progressive dysfunction in dogs.

BACKGROUND: Circulating creatinine and symmetric dimethylarginine (SDMA) are biomarkers of kidney function that have been used variously to define stable vs progressive chronic kidney disease (CKD). Slope monitoring of inverse biomarker values (creatinine-1 or SDMA-1 ) has shown promise, but quantitative criteria to distinguish stable vs progressive CKD using this approach are lacking. OBJECTIVE: Assessment of creatinine-1 and SDMA-1 slope cutoffs to distinguish stable vs progressive CKD. ANIMALS: One hundred ten clinically healthy university staff-owned dogs and 29 male colony dogs with progressive X-linked hereditary nephropathy (XLHN). METHODS: Retrospective analysis combining 2 prospective observational studies, 1 tracking kidney function biomarkers in healthy dogs (HDs) to a maximum of 3 years, and 1 tracking kidney function biomarkers in male colony dogs with progressive XLHN to a maximum of 1 year. The minimum slope of creatinine-1 or SDMA-1 as measured using the IDEXX SDMA test from HD was assigned as the slope cutoff for stable kidney function. RESULTS: The stable vs progressive slope cutoff was -0.0119 week × dL/mg for creatinine-1 and -0.0007 week × dL/µg for SDMA-1 . CONCLUSIONS AND CLINICAL IMPORTANCE: In the studied CKD population, progressive dysfunction can be distinguished from stable kidney function by using the slope of creatinine-1 or SDMA-1 . These criteria may serve to characterize CKD in other cohorts of dogs and to establish guidelines for degrees of progression rate in dogs with naturally occurring CKD.

Immunoassay for detection of Dipylidium caninum coproantigen in dogs and cats.

Dipylidium caninum infections in dogs and cats are underestimated because of a lack of proglottid observations and poor recovery of parasite elements by centrifugal flotation. We developed an immunoassay that employs a pair of monoclonal antibodies to capture D. caninum-specific coproantigen in fecal extracts from dogs and cats. Real-time PCR for D. caninum DNA in perianal swabs and observation of proglottids were used as reference methods. In 6 experimentally infected dogs, parasite DNA, coproantigen, and proglottid segments were first detected at 22, 23, and 26 d post-infection, respectively. Praziquantel treatment of 3 experimentally infected dogs resulted in the elimination of both coproantigen and proglottid shedding within 1-5 d post-treatment; however, parasite DNA persisted for 14 d. Immunohistochemistry on immature and mature tapeworm segments using an antibody against the coproantigen supports the premise that the antigen is produced in mature segments. We assessed the performance of our coproantigen test in natural infections in 78 dogs from a flea-endemic area. Of the 12 antigen-positive samples, 11 were confirmed with a positive PCR test and/or proglottid observation. Finally, we evaluated a convenience sample set of 730 canine and 163 feline fecal samples obtained from a commercial diagnostic laboratory; D. caninum antigen was detected in 4.1% of the canine and 12.9% of the feline samples, whereas parasite elements were observed in only 0.028% of samples. Our coproantigen immunoassay provides a sensitive method for the detection of D. caninum infection in dogs and cats.

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.

Use of In-Clinic Diagnostic Kits for the Detection of Seropositivity to Leishmania infantum and Other Major Vector-Borne Pathogens in Healthy Dogs.

Canine Vector-Borne Diseases (CVBDs) are widespread in Europe and enzootic in many other countries. Though severe illnesses may occur, dogs living in enzootic areas often show vague or no clinical signs of CVBDs. Undiagnosed infections/co-infections in subclinically infected animals favor the spread of CVBDs and increase the risk of transmission to other animals and, in some cases, humans. This study has evaluated the exposure of dogs living in key enzootic countries, i.e., Italy and Greece, to major CVBDs via the use of in-clinic diagnostic kits. Overall, 300 privately owned dogs without/with single mild clinical signs living in different regions of Italy (n. 150) and Greece (n. 150) were included in the study. As part of a clinical examination, a blood sample was collected from each dog and subjected to two serological rapid tests, i.e., the SNAP® 4Dx®Plus (IDEXX Laboratories Inc.) for the detection of antibodies against Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi s.l. and Dirofilaria immitis antigen and the SNAP®Leishmania (IDEXX Laboratories Inc.) for the detection of antibodies against Leishmania infantum. In all, 51 dogs (17%; 95% CI 12.9-21.7) were seropositive to at least 1 pathogen, i.e., 4 in Italy (2.7%; 95% CI 1.4-13.1) and 47 in Greece (31.3%; 95% CI 24-39.4). Dirofilaria immitis antigens were found in 39 dogs (13%; 95% CI 9.4-17.3), while antibodies against Ehrlichia, Anaplasma and Leishmania were detected in 25 (8.3%; 95% CI 5.5-12.1), 8 (2.7%; 95% CI 1.2-5.2) and 5 (1.7%; 95% CI 0.5-3.8) dogs, respectively. None of the dogs tested seropositive for B. burgdorferi s.l. Statistical analyses were performed to evaluate associations between exposure to CVBDs and possible risk factors. The present results indicate that dogs living in enzootic areas may be seropositive for one or more CVBDs in absence of clinical signs. Rapid kits are among first line tools for the detection of CVBDs in clinical settings, as they are cost-effective, straightforward and quick to use. Also, in-clinic tests used herein allowed detection of co-exposure to CVBDs investigated.

Evaluation of barcoded magnetic bead-based immunoassays for the simultaneous detection of feline leukemia virus antigen and antibody against feline immunodeficiency virus.

Testing platforms that leverage automation, require minimal sample volume, and enable various tests to be performed simultaneously on a single sample have the potential to improve workflow and efficiency in veterinary diagnostic laboratories. We evaluated a barcoded magnetic bead (BMB) technology using established immunoassays for detection of feline leukemia virus (FeLV) p27 antigen and antibody against feline immunodeficiency virus (FIV). Analytical sensitivity, limit of blank, and limit of detection were used to establish a functional sensitivity of 1.00 ng/mL of inactivated FeLV antigen and 35.7 ng/mL of anti-FIV monoclonal antibody. Common interferents, such as hemoglobin, lipid, and bilirubin, were not found to interfere with the performance of the assay. Intra- and inter-assay CVs were <13% for both assays using manufactured samples. Using a set of 116 feline samples, the diagnostic accuracy of our multiplex assay was 100% compared to reference assays. Performance in a convenience set of 1,000 feline samples submitted to a commercial diagnostic laboratory revealed a proportion of positive results of 1.3% for FeLV and 3.7% for FIV. BMB technology should enable rapid screening of samples for various markers in a single immunoassay well.

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.

Vector-borne pathogens of zoonotic concern in hunting dogs of southern Italy.

Dogs are commonly exposed to vector-borne pathogens (VBPs), yet few data are available on hunting dogs, which are often at high risk of infection due to their involvement in field activities. To investigate the occurrence of VBPs and evaluate the relative performance of different diagnostic tools, blood and serum samples were collected from hunting dogs (n = 1,433) in rural areas of southern Italy. All samples were tested by Knott's technique for filarioids, serologically (SNAP® 4Dx® Plus) for Anaplasma spp., Borrelia burgdorferi sensu lato, Dirofilaria immitis and Ehrlichia spp. and molecularly (qPCR) for all except B. burgdorferi of the above pathogens plus Babesia spp. and Leishmania infantum. Logistic regression was run to evaluate the statistical associations between the risk of VBP infection and independent variables (such as geographic area of provenience, age class and sex) and K-Cohen formula for assessing the concordance among diagnostic tests. Overall, out of 321 dogs (22.4%) positive to at least one VBP, 28 (1.9%) were infected by filarial species at the Knott's technique. In particular, Acanthocheilonema reconditum was the most prevalent (1.6%), followed by D. immitis (0.2%) and Dirofilaria repens (0.1%). One hundred forty (9.8%) and 231 (16.1%) dogs scored positive to VBPs by serological and molecular methods, respectively. The most prevalent pathogens detected were Ehrlichia spp. (7.3%) with SNAP® 4Dx® Plus, and A. reconditum (7.7%) by qPCR. Statistics revealed a significant association (p < 0.001) between A. reconditum infestation and both Ehrlichia spp. seropositivity and geographical origin of dogs. An agreement of 99.9%, 94.0% and 95.7% for Knott - SNAP® 4Dx® Plus, Knott - qPCR and SNAP® 4Dx® Plus - qPCR for D. immitis was found, respectively. Data demonstrate a high prevalence of VBPs in hunting dogs, indicating that this group of animals is largely exposed to several arthropod vector species and suggesting the transmission risk of pathogens to humans in rural areas of southern Italy. A multi-diagnostic approach and a deeper cooperation among healthcare and stakeholders are required to prevent VBP infections to animals and humans.

A Comparison of Copromicroscopic and Molecular Methods for the Diagnosis of Cat Aelurostrongylosis.

The gold standard method for the diagnosis of cat aelurostrongylosis is the detection of Aelurostrongylus abstrusus first stage larvae with the Baermann's examination. Nevertheless, molecular assays have shown higher diagnostic performances compared to copromicroscopy. This study evaluated the usefulness of an A. abstrusus species-specific PCR on different biological samples collected in clinical settings from 100 privately-owned cats in Italy (n. 60) and Greece (n. 40). A fecal sample was collected from each animal and a pharyngeal swab was also obtained for cats from Italy. All stool samples were subjected to flotation and Baermann's test. The cats were categorized in three groups based on the results of copromicroscopy, i.e., Group A (n. 50 cats with A. abstrusus infection regardless of positivity for other helminths), Group B (n. 25 cats negative for A. abstrusus but positive for at least one of any other helminth), Group C (n. 25 cats negative for any helminth). DNA was extracted from individual aliquots of feces, flotation supernatant, Baermann's sediment and the pharyngeal swab and then subjected to a PCR specific for A. abstrusus. At least one fecal aliquot or the pharyngeal swab scored positive by the A. abstrusus-specific PCR for 48/50 (96%) cats enrolled in Group A; in particular, 38/50 (76%), 35/50 (70%), 41/50 (82%) and 21/25 (84%) DNA extracts from feces, flotation supernatant, Baermann's sediment and pharyngeal swabs were positive by PCR. These results confirm that molecular tools are highly sensitive and specific and indicate that pharyngeal swabs are the most suitable sample for molecular analysis in clinical settings.

Bartonella spp. seroepidemiology and associations with clinicopathologic findings in dogs in the United States.

BACKGROUND: Improved understanding of Bartonella spp. serology in dogs may aid clinical decision making. OBJECTIVE: Describe demographic and geographic patterns of Bartonella spp. seroreactivity in dogs, and describe hematologic and serum biochemical abnormalities in Bartonella spp. seroreactive and nonseroreactive dogs. ANIMALS: Serum samples from 5957 dogs in the United States, previously submitted to IDEXX Reference Laboratories. METHODS: Serum was tested using 3 indirect ELISAs for B. henselae, B. vinsonii subsp. berkhoffii, and B. koehlerae. Complete blood count and serum biochemistry panel results were reviewed retrospectively. RESULTS: Overall, 6.1% of dogs were Bartonella spp. seroreactive. Toy breeds were less likely to be seroreactive (3.9%) than mixed breeds (7.5%; adjusted odds ratio [aOR], 0.48; 95% confidence interval [CI], 0.32-0.72), and dogs <1 year old were less likely to be seroreactive (3.4%) than dogs 1 to 5.5 years of age (7.3%; aOR, 0.42; 95% CI, 0.23-0.72). Dogs in the West South Central (9.8%) and South Atlantic (8.8%) regions were more likely than dogs elsewhere in the United States to be seroreactive (aOR, 2.22; 95% CI, 1.31-3.87; aOR, 2.44; 95% CI, 1.38-4.36). CONCLUSIONS AND CLINICAL IMPORTANCE: Demographic and geographic findings for Bartonella spp. exposure were broadly comparable to previously reported patterns.

Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview.

Acanthocheilonema reconditum is a filarial parasite transmitted by arthropods (fleas, lice, and ticks) that infect dogs. There is minimal published data available to date on potential haematological and biochemical changes associated with this parasitic infection. Study aims were (i) provide an overview of A. reconditum in Europe, (ii) define A. reconditum prevalence and risk factors in a specific dog population (hunting) from southern Italy, and (iii) assess the frequency of haemato-biochemical abnormalities associated with infection. Blood samples collected from 3020 dogs were tested by a modified Knott's technique to count and identify microfilariae. Eighty-four dogs were infected by A. reconditum (2.78%; 95% CI 2.19-3.37%). Microfilariae ranged from 1 to 212/ml. Based on clinical examination, all but six dogs with non-specific symptoms were healthy. Haematological abnormalities included leucocytosis (n = 15), with eosinophilia (n = 14) and monocytosis (n = 13). Serum biochemical abnormalities included increased total serum proteins (n = 19), albumins (n = 7), total globulins (n = 14), ALT (n = 1), and ALP (n = 1); one dog was hypoalbuminemic, and BUN was mildly increased in 2 dogs. Risk factors included the province origin (Napoli, OR=5.4, 95%CI: 2.1-14.0; Caserta, OR=5.1, 95%CI: 2.5-10.6), hunting wild mammals (OR=2.8, 95% 95%CI: 1.6-4.8), and ectoparasite infestation (OR=1.9, 95%CI: 1.1-3.1). There was a negative correlation between microfilaraemic load and decreased albumin level (-0.37; p=0.021). Our results showed that A. reconditum circulates within the hunting dog population of southern Italy, with seemingly low pathogenic potential.

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.

Canine infection with Dirofilaria immitis, Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. in the United States, 2013-2019.

BACKGROUND: Dogs in the US are commonly infected with vector-borne pathogens, including heartworm and tick-borne disease agents. The geographic distribution of both arthropod vectors and the pathogens they transmit continues to expand. METHODS: To describe the current geographic distribution and prevalence of antigen of Dirofilaria immitis and antibody to Borrelia burgdorferi, Ehrlichia spp., and Anaplasma spp. in dogs, we summarized over 144 million test results from 2013 to 2019, inclusive, by county, state, and region. Canine seroprevalence by state was compared to population-adjusted human reports of tick-borne diseases. RESULTS: Results varied regionally, with D. immitis antigen and Ehrlichia spp. antibodies more frequently detected in the Southeast (2.6% and 5.2%, respectively) and antibody to B. burgdorferi and Anaplasma spp. most common in the Northeast (12.1% and 7.3%, respectively). Overall, percent positive test results to D. immitis decreased in the Southeast by 33.3% when compared to earlier summaries using the same strategy (from 3.9 to 2.6%). Geographic expansion of areas where dogs commonly test positive for Ehrlichia spp. was evident, likely because of a change in the test made in 2012 to allow detection of antibodies to E. ewingii concomitant with expansion of vector tick populations. Percent positive test results to Ehrlichia spp. increased in every region; this shift was particularly pronounced in the Southeast, where percent positive test results increased fourfold (from 1.3 to 5.2%). Continued geographic expansion of B. burgdorferi and A. phagocytophilum was apparent in the Northeast, Midwest, and Upper South, although canine seroprevalence of antibody to B. burgdorferi was much lower than prior surveys in many Lyme-endemic areas. Annual reports of human cases of Lyme disease, ehrlichiosis, and anaplasmosis were associated with percent positive canine results by state for the three tick-borne disease agents (R2 = 0.812, 0.521, and 0.546, respectively). Within endemic areas, percent positive test results for all three tick-borne agents demonstrated evidence of geographic expansion. CONCLUSIONS: Large scale analysis of results from screening dogs in practice for evidence of vector-borne infections, including those with zoonotic importance, continues to be a valuable strategy for understanding geographic trends in infection risk over time.

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.

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.

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.