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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

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

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.

Giardia duodenalis is a species complex comprising at least eight assemblages. Most dogs harbor the host-adapted assemblages C and D and approximately 30 % harbor the zoonotic assemblages. Humans and dogs with giardiosis can exhibit a variety of clinical manifestations ranging from the absence of clinical signs to acute or chronic diarrhea. Human studies report conflicting results concerning associations between clinical signs and assemblage type. The objective of this study was to use results of molecular and phylogenetic analyses to evaluate associations between G. duodenalis assemblages and diarrhea in client-owned dogs from the United States. Fecal samples that were positive for Giardia cysts were classified as normal or diarrheal. Samples were analyzed by PCR assays of the beta-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) genes. Sequences of the three genes were analyzed by BLAST analysis and phylogenetic analysis was performed by Neighbor-Joining analysis. Two hundred and eighty-eight Giardia-positive fecal samples were evaluated by the three PCRs. One or more genes were amplified from 95 normal samples and 93 diarrheal samples, 27 samples were positive for one or more genes but could not be sequenced due to low quality DNA, and 73 samples tested negative. Ninety seven percent of the samples (182/188) in both the diarrheal and normal groups typed as dog-specific assemblages (D or C) by at least one gene. Phylogenetic analysis of the three genes placed the isolates from assemblages A, B, C and D separated from each other with strong bootstrap support. Diarrhea was not associated with the Giardia assemblage or other parasitic co-infection in this sample set. Other factors, such as the role of gut microbiota in giardiosis should be considered in future studies.

Canine vector-borne diseases (CVBDs) are caused by a range of pathogens transmitted to dogs by arthropods. The present study investigates Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi sensu lato, and Dirofilaria immitis seroprevalences in hunting dogs from southern Italy. Dogs (no. 1335) were tested using a commercial in-clinic enzyme-linked immunosorbent assay kit. Odds ratios (ORs) were calculated by logistic regression analysis to identify risk factors. Overall, 138/1335 dogs (10.3%) were seroreactive to at least one CVBD pathogen. E. canis, Anaplasma spp., B. burgdorferi s.l., and D. immitis seroprevalences were 7.6, 4.4, 0.3, and 0.2%, respectively. E. canis and Anaplasma spp. co-exposures were found in 30 dogs (2.2%), compared with Anaplasma spp. and B. burgdorferi s.l. co-exposures in 2 dogs (0.1%). Adult age was a risk factor for E. canis (OR 2.35) seroreactivity whereas hunting fur-bearing animals for E. canis (OR 4.75) and Anaplasma spp. (OR 1.87), respectively. The historical presence of tick infestation was identified as a risk factor for positivity to E. canis (OR 2.08) and Anaplasma spp. (OR 2.15). Finally, larger dog pack size was significantly associated with E. canis (OR 1.85) and Anaplasma spp. (OR 2.42) exposures. The results of the present survey indicated that hunting dog populations are at relative risk of CVBDs in southern Italy. Further studies are needed to evaluate the role of hunting dogs in the epidemiology of vector-borne organisms due to sharing common environments with wild, sympatric animal populations.

Hepatozoon canis is a hemoprotozoan organism that infects domestic and wild carnivores throughout much of Europe. The parasite is mainly transmitted through the ingestion of infected ticks containing mature oocysts. The aims of the present survey were to determine the prevalence of H. canis in hunting dogs living in Southern Italy and to assess potential infection risk factors. DNA extracted from whole blood samples, collected from 1433 apparently healthy dogs living in the Napoli, Avellino, and Salerno provinces of Campania region (Southern Italy), was tested by a quantitative real-time polymerase chain reaction (qPCR) assay to amplify H. canis. Furthermore, the investigated dog population was also screened by qPCR for the presence of Ehrlichia canis, a major tick-borne pathogen in Southern Italy, in order to assess possible co-infections. Two hundred dogs were H. canis PCR-positive, resulting in an overall prevalence of 14.0% (CI 12.2–15.9). Breed category (P P = 0.015), and province of residence (P H. canis infection. The presence of H. canis DNA was also significantly associated with E. canis PCR positivity (P H. canis, and the infection is potentially associated with close contact with wildlife. Further studies are needed to assess the pathogenic potential of H. canis, as well as the epidemiological relationships between hunting dogs and wild animal populations sharing the same habitats in Southern Italy.

Data concerning combined molecular and serological prevalence of emerging canine tick-borne pathogens in Serbia are lacking. A large population of outdoor living dogs in Belgrade, Serbia's' capital, present an excellent population for epidemiology study. Blood samples were collected from 111 dogs, including 46 shelter, 31 free roaming, and 34 hunting dogs. Species-specific real-time polymerase chain reaction (PCR) (IDEXX Laboratories, Inc., Westbrook Maine, USA) was applied for the molecular detection of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, Babesia canis, B. gibsoni and B. vogeli. A research based SNAP assay (SNAP® M-A, IDEXX Laboratories, Inc., Westbrook Maine, USA) that uses genus and species-specific peptides was used to asses Anaplasma spp., A. phagocytophilum, A. platys, Ehrlichia spp., E. canis, E. chaffeensis, E. ewingii and Borrelia burgdorferi antibody status. B. canis, B. gibsoni and B. vogeli antibody status was assessed with an indirect immunofluorescence test (MegaCor Diagnostic, Horbranz, Austria). Anaplasma spp. and Ehrlichia spp. DNA was not amplified. One quarter of the dogs were A. phagocytophilum, one dog was A. platys, one was E. ewingii and two dogs were B. burgdorferi seroreactive with the SNAP® M-A. Babesia canis or B. gibsoni DNA was amplified by PCR from 16.2% of dogs, whereas 67.6% were seroreactive to one or more Babesia spp. Babesia vogeli was not PCR amplified. We conclude that outdoor dogs in this territory are reservoirs for B. canis and B. gibsoni and are frequently co-exposed to combinations of Anaplasma and Babesia spp.

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.

Introduction To determine the biologic variability of N-terminal pro-brain natriuretic peptide (NTproBNP) in healthy dogs and dogs with various stages of myxomatous mitral valve disease (MMVD). Animals Thirty-eight privately owned dogs: 28 with MMVD and 10 healthy controls. Materials and methods Prospective clinical study with comprehensive evaluation used to group dogs as healthy or into three stages of MMVD based on current guidelines. NTproBNP was measured hourly, daily, and weekly. For each group, analytical (CV A ), within-subject (CV I ), and between-subject (CV G ) coefficients of variability were calculated in addition to percent critical change value (CCV) and index of individuality (IoI). Results For healthy dogs, calculated NTproBNP values were: CV A = 4.2%; CV I = 25.2%; CV G = 49.3%; IoI = 0.52, and CCV = 70.8%. For dogs with MMVD, calculated NTproBNP values were: CV A = 6.2%; CV I = 20.0%; CV G = 61.3%; IoI = 0.34, and CCV = 58.2%. Conclusions Biologic variability affects NTproBNP concentrations in healthy dogs and dogs with MMVD. Monitoring serial individual changes in NTproBNP may be clinically relevant in addition to using population-based reference ranges to determine changes in disease status.

Pathophysiological cardiac and renal interactions are termed cardiovascular-renal disorder (CvRD). Cardiovascular disease/dysfunction secondary to kidney disease (CvRDK), is a leading cause of death in human chronic kidney disease (CKD) patients. The presence and clinical impact of CvRDK in dogs with CKD is unknown. We hypothesized that echocardiographic measurements, and cardiac and renal biomarkers, will be altered in dogs with CKD and associated with survival. Eleven dogs with CKD (n=6 IRIS stage 2, n=5 IRIS stage 3) and without primary cardiac disease, plus 12 healthy age-matched control dogs, were recruited to this prospective observational study. Dogs underwent standard echocardiography, glomerular filtration rate (GFR) estimation by iohexol clearance, and measurement of plasma cardiac troponin I and N-terminal pro-B-type natriuretic peptide (NT-proBNP), plasma and urinary cystatin B, and urinary clusterin and neutrophil gelatinase-associated lipocalin (NGAL). Values were compared between groups, and their association with all-cause mortality explored. Dogs with CKD had significantly lower GFR and higher NT-proBNP, urinary cystatin B, clusterin, and NGAL, compared to controls (P

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 × 10

Background Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis. Hypothesis Dogs with B. canis-induced APR develop dyslipidemia with altered lipoprotein concentration and morphology. Animals Twenty-nine client-owned dogs with acute B. canis infection and 10 clinically healthy control dogs. Methods Observational cross-sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA-1 (radioimmunoassay) and SAA was determined. Results Dogs with B. canis infection had a marked APR (median SAA, 168.3 mu g/mL; range, 98.1-716.2 mu g/mL) compared with controls (3.2 mu g/mL, 2.0-4.2 mu g/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89-7.64 mmol/L versus 6.15 mmol/L, 4.2-7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6-6.6 mmol/L versus 5.72 mmol/L, 4.68-7.0 mmol/L) (P = .02), and alpha-lipoproteins (77.5%, 27.7%-93.5% versus 89.2%, 75.1%-93.5%) (P = .04), and higher ApoA-1 (1.36 U, 0.8-2.56 U versus 0.95 U, 0.73-1.54 U) concentrations (P = .02). Serum amyloid A correlated with high-density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA-1 (rho = .63, P < .001). Conclusions and Clinical Importance Major changes associated with B. canis-induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA-1 and SAA concentration increase is a unique still unexplained pathophysiological finding.

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.

Mycoplasma haemocanis (Mhc) and “Candidatus Mycoplasma haematoparvum” (CMhp) are the main haemoplasma species known to infect dogs. The aim of this study was to determine the prevalence of haemoplasma species infections in hunting dogs from southern Italy and assess related risk factors. 1,433 hunting dogs living in Campania region were tested by qPCR assay. The prevalence was 19.9 %; 13.1 % for Mhc and 11.4 % for CMhp; 4.6 % showed a coinfection with both haemoplasma species. Statistical analysis revealed living in Salerno province (Mhc: OR 3.72; CMhp: OR 2.74), hound (Mhc: OR 5.26; CMhp: OR 8.46) and mixed breed (Mhc: OR 3.38; CMhp: OR 2.80), rural environment (Mhc: OR 12.58; CMhp: OR 10.38), wild mammal hunting (Mhc: OR 8.73; CMhp: OR 8.32), cohabitation with other animals (Mhc: OR 2.82; CMhp: OR 2.78) and large pack size (Mhc: OR 2.96; CMhp: OR 1.61) as risk factors for haemoplasmas. Male gender (OR 1.44) and tick infestation history (OR 1.40) represented risk factors only for Mhc, while adult age (2 7 years - OR 2.01; > 7 years - OR 1.84) and large body size (OR 1.48) were associated only to CMhp. Mhc infection was significantly associated to Babesia vogeli (p < 0.05) and Hepatozoon canis (p < 0.001), while CMhp with H. canis (p < 0.001). This study adds information on haemoplasma species distribution in hunting dogs in southern Italy. Outdoor lifestyle and contact with wild fauna, through greater exposure to tick infestation, or possibly wounds acquired during hunting or fighting, could be factors contributing to haemoplasma infections.

Better tools are necessary to eliminate visceral leishmaniasis (VL). Modeling studies for regional Leishmania elimination indicate that an effective vaccine is a critical tool. Dogs are the reservoir host of L. infantum in Brazil and the Mediterranean basin, and therefore are an important target for public health interventions as well as a relevant disease model for human VL. No vaccine has been efficacious as an immunotherapy to prevent progression of already diagnostically positive individuals to symptomatic leishmaniasis. We performed a double-blinded, block-randomized, placebo-controlled, vaccine immunotherapy trial testing the efficacy of a recombinant Leishmania A2 protein, saponin-adjuvanted, vaccine, LeishTec®, in owned hunting dogs infected with L. infantum. The primary outcome was reduction of clinical progression, with reduction of mortality as a secondary outcome. Vaccination as an immunotherapy reduced the risk of progression to clinically overt leishmaniasis by 25% in asymptomatic dogs (RR: 1.33 95% C.I. 1.009–1.786 p-value: 0.0450). Receiving vaccine vs. placebo reduced all-cause mortality in younger asymptomatic dogs by 70% (RR: 3.19 95% C.I.: 1.185–8.502 p-value = 0.0245). Vaccination of infected-healthy animals with an anti-Leishmania vaccine significantly reduced clinical progression and decreased all-cause mortality. Use of vaccination in infected-healthy dogs can be a tool for Leishmania control.

N-terminal pro–B-type natriuretic peptide (NT-proBNP) has been shown to have clinical utility as a biomarker in dogs with heart disease. There were several limitations associated with early diagnostic assay formats including a limited dynamic range and the need for protease inhibitors to maintain sample stability. A second-generation Cardiopet® proBNP enzyme-linked immunosorbent assay (IDEXX Laboratories Inc., Westbrook, Maine) was developed to address these limitations, and the present study reports the results of the analytical method validation for the second-generation assay. Coefficients of variation for intra-assay, interassay, and total precision based on 8 samples ranged from 3.9% to 8.9%, 2.0% to 5.0%, and 5.5% to 10.6%, respectively. Analytical sensitivity was established at 102 pmol/l. Accuracy averaged 102.0% based on the serial dilutions of 5 high-dose canine samples. Bilirubin, lipids, and hemoglobin had no effect on results. Reproducibility across 3 unique assay lots was excellent with an average coefficient of determination ( r2) of 0.99 and slope of 1.03. Both ethylenediamine tetra-acetic acid plasma and serum gave equivalent results at time of blood draw (slope = 1.02, r2 = 0.89; n = 51) but NT-proBNP was more stable in plasma at 25°C with median half-life measured at 244 hr and 136 hr for plasma and serum, respectively. Plasma is the preferred sample type and is considered stable up to 48 hr at room temperature whereas serum should be frozen or refrigerated when submitted for testing. Results of this study validate the second-generation canine Cardiopet proBNP assay for accurate and precise measurement of NT-proBNP in routine sample types from canine patients.

The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody–based enzyme-linked immunosorbent assay (macELISA) for detection of allergen-specific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7–16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9–10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5–12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong ( r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA.