Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study.

BACKGROUND: Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study. RESULTS: Of the 47 dogs with ClinL, anti-E. canis/ Ehrlichia ewingii antibodies were detected in 17 (36.2%), anti-Anaplasma phagocytophilum/Anaplasma platys antibodies in 5 (10.6%) and antigen for Dirofilaria immitis in 2 (4.3%). Of the 87 control dogs, anti-E. canis/E. ewingii antibodies were detected in 14 (16.1%) and anti-A. phagocytophilum/A. platys antibodies in 2 (2.3%). No anti-Borrelia burgdorferi antibody tests were positive. No statistical differences between the ClinL dogs and control dogs regarding lifestyle or use of ectoparasitic prevention, were identified. The ClinL was significantly associated with anti-E. canis/E. ewingii antibodies (odds ratio = 2.9, 95% confidence interval: 1.3-6.7, P = 0.010) compared to controls by both multivariable logistic regression and structural equation modelling. CONCLUSIONS: It was demonstrated that an increased risk for E. canis/E. ewingii seropositivity is present in dogs with ClinL compared to clinically healthy control dogs, despite similar ectoparasitic prevention use and lifestyle. Based on these findings it is suggested that dogs with ClinL should not only be tested for E. canis co-infection using PCR but also serologically for E. canis/E. ewingii.

Associations between presence of Bartonella species deoxyribonucleic acid and complete blood cell count and serum biochemical changes in client-owned cats.

BACKGROUND: Infection with Bartonella species is common in cats but reported effects of bacteremia on laboratory variables differ. OBJECTIVES: Evaluate for associations between Bartonella bacteremia and CBC and serum biochemical changes in sick and healthy cats throughout the United States. ANIMALS: A total of 3964 client-owned cats. METHODS: Retrospective cohort study using submissions to a commercial laboratory between 2011 and 2017. Serum biochemistry and CBC abnormalities (categorized as above or below reference intervals), age, and location (high- or low-risk state for Ctenocephalides felis) in presumed healthy and sick cats were evaluated for associations with presence of Bartonella spp. DNA, detected by PCR. Univariate and multivariable logistic regression analyses were performed. RESULTS: Bartonella spp. DNA was amplified from 127 (3.2%) of 3964 cats; 126 (99.2%) of 127 were from high flea risk states and 121 (95.3%) of 127 were presumed sick. Fever of unknown origin was the most common PCR panel requested. In the multivariable analysis, neutrophilia, decreased ALP activity, clinical status (presumed sick), and young age (≤2 years) each were positively associated whereas neutropenia and hyperproteinemia both were negatively associated with Bartonella spp. bacteremia. Presence of Bartonella spp. DNA had no association with test results for other infectious disease agents. CONCLUSIONS AND CLINICAL IMPORTANCE: In both healthy and sick cats, active Bartonella infections had minimal association with clinically relevant laboratory abnormalities. However, based on these results, in areas considered high risk for C. felis, active infection with Bartonella spp. is a reasonable differential diagnosis for cats presented with unexplained fever and neutrophilia, particularly if the cat is young.

Distribution of the feline lungworm Aelurostrongylus abstrusus in the USA based on fecal testing.

OBJECTIVES: The aim of this study was to compile commercial reference laboratory data over a 10-year period to determine the distribution of Aelurostrongylus abstrusus, commonly known as feline lungworm, within the USA based on widespread fecal testing in cats. METHODS: The results of 3,610,455 feline ova and parasite (O&P) zinc sulfate centrifugation fecal flotation tests performed at IDEXX Reference Laboratories in the USA from January 2008 to December 2017 were compiled and sorted for tests positive for A abstrusus larvae. The results of 3625 Baermann tests, currently considered the gold standard diagnostic for feline lungworm, were also retrieved from the same period. RESULTS: Of the tests performed, 4721 (0.13%) feline O&P zinc sulfate centrifugation fecal flotation tests and 75 (2.07%) of the Baermann tests conducted were positive for the presence of A abstrusus larvae. The O&P data revealed a significant association between infection status and sex, while male cats in both the O&P and Baermann data sets had a higher risk of A abstrusus infection than females. Significant variation in positive rates were observed by region and most positive cases were clustered in the Northeast, Midwest and West regions of the USA. CONCLUSIONS AND RELEVANCE: This study highlights the distribution of feline lungworm in the USA and the limitations of using current testing to diagnose this infection. The introduction of higher throughput, less labor-intensive diagnostic methods could help increase awareness of this parasite among veterinary professionals, achieve a greater understanding of epidemiological factors, and improve the care and treatment for clinically ill feline patients.

Prevalence of Borrelia burgdorferi, Anaplasma spp., Ehrlichia spp. and Dirofilaria immitis in Canadian dogs, 2008 to 2015: a repeat cross-sectional study.

BACKGROUND: Vector-borne pathogens are emerging concerns in multiple regions of Canada. Determining regional prevalence of canine vector-borne pathogens and documenting change will improve clinician awareness, enable targeted prevention, enhance diagnosis and ideally reduce the risk of disease. Study objectives were to: (i) estimate the prevalence of positive canine vector-borne test results from samples submitted in Canada; (ii) assess change in prevalence over time, from baseline (2008) to 2015; and (iii) estimate the prevalence of pathogen co-infections. METHODS: This repeat cross-sectional study evaluated 753,468 test results for D. immitis antigen and B. burgdorferi, Ehrlichia canis/ewingii/muris serology, and 753,208 test results for Anaplasma phagocytophilum/platys serology using the SNAP® 4Dx®Test and SNAP 4Dx® Plus Test. RESULTS: Based on all submitted samples from Canada (2008-2015), the period seroprevalence of B. burgdorferi, Ehrlichia spp., Anaplasma spp. and D. immitis antigen were 2.0%, 0.5%, 0.4% and 0.2%, respectively. Over the 7 years (2008 compared to 2015) we observed a significant increase in seroprevalence for B. burgdorferi (144.4%) and Ehrlichia spp. (150%). Co-infections (positive for two or more pathogens on a single 4 pathogen test kit) were estimated at 5.4% (1162/21,612) of total positive tests. CONCLUSIONS: The temporal rise and geographical differences in prevalence detected for these pathogens (notably B. burgdorferi) are consistent with anecdotal information on canine illness related to tick-borne pathogen exposure in multiple regions of Canada, particularly canine Lyme disease.

Biologic variability of N-terminal pro-brain natriuretic peptide in healthy dogs and dogs with myxomatous mitral valve disease.

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 (CVA), within-subject (CVI), and between-subject (CVG) 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: CVA = 4.2%; CVI = 25.2%; CVG = 49.3%; IoI = 0.52, and CCV = 70.8%. For dogs with MMVD, calculated NTproBNP values were: CVA = 6.2%; CVI = 20.0%; CVG = 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.

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.

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network.

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.

Analytical validation of a second-generation immunoassay for the quantification of N-terminal pro-B-type natriuretic peptide in canine blood.

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 (r (2)) 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, r (2) = 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.

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network.

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.

DNA mutation detection in a polymer microfluidic network using temperature gradient gel electrophoresis.

A miniaturized system for DNA mutation analysis, utilizing temperature gradient gel electrophoresis (TGGE) in a polycarbonate (PC) microfluidic device, is reported. TGGE reveals the presence of sequence heterogeneity in a given heteroduplex sample by introducing a thermal denaturing gradient that results in differences between the average electrophoretic mobilities of DNA sequence variants. Bulk heater assemblies are designed and employed to externally generate temperature gradients in spatial and temporal formats along the separation channels. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, are achieved using both single- and 10-channel parallel measurements in a microfluidic platform. Additionally, a comprehensive polymer microfluidic device containing an integrated microheater and sensor array is developed and demonstrated for performing spatial TGGE for DNA mutation analysis. The device consists of two PC modular substrates mechanically bonded together. One substrate is embossed with microchannels, and the other contains a tapered microheater, lithographically patterned along with an array of temperature sensors. Compared with the external heating approaches, the integrated platform provides significant reduction in power requirement and thermal response time while establishing more accurate and highly effective control of the temperature gradient for achieving improved separation resolution.