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A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

ABSTRACT: BACKGROUND:Ehrlichiosis is an important emerging infectious disease of the canid family and humans worldwide. To date, no extensive evaluation or validation of a molecular diagnostic test for ehrlichiosis has been published. Here, we present data for a newly designed TaqMan assay and compare its performance to a commercial technology (PCRun®). Both of these real-time methods of analysis were evaluated using a comprehensive number of prospective and retrospective samples collected from dogs exhibiting symptoms of ehrlichiosis. RESULTS:Whole blood samples collected from dogs, retrospectively in the United Kingdom and prospectively in Israel, were analysed for the presence of Ehrlichia canis and Ehrlichia minasensis DNA using the TaqMan PCR, developed specifically for this study. The results were compared to those of a real time commercial isothermal amplification method (PCRun® system developed by Biogal Galed Labs ACS, Galed, Israel). The sensitivity and specificity (CI: 95%) of the TaqMan PCR and PCRun® were both determined to be 100% and absolute, for all of the samples tested. Interestingly, both tests were demonstrated to be highly comparable, irrespective of differences in amplification chemistry or sequences targeted. Host differences, incidence of disease and geographical location of the isolates had little impact on the positivity recorded by each of the diagnostic methods. CONCLUSIONS:It was evident that both amplification methods were equally suited for diagnosing canine ehrlichiosis and while the PCRun® clearly amplified all clinically relevant Ehrlichia species known to infect dogs and humans, the TaqMan method was more specific for E. canis and E. minasensis. This work demonstrates that despite good analytical sensitivities and specificities for Ehrlichia spp. neither method could fully account for the clinical diagnosis of thrombocytopenia.

SUBMITTER: Thomson K

PROVIDER: S-EPMC6006785 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

Thomson Kirsty K Yaaran Tal T Belshaw Alex A Curson Lucia L Tisi Laurence L Maurice Sarah S Kiddle Guy G

Parasites & vectors 20180618 1

<h4>Background</h4>Ehrlichiosis is an important emerging infectious disease of the canid family and humans worldwide. To date, no extensive evaluation or validation of a molecular diagnostic test for ehrlichiosis has been published. Here, we present data for a newly designed TaqMan assay and compare its performance to a commercial technology (PCRun®). Both of these real-time methods of analysis were evaluated using a comprehensive number of prospective and retrospective samples collected from do ...[more]

PMID: 29914548

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Project description:BackgroundIn the USA, there are several Ehrlichia spp. of concern including Ehrlichia canis, Ehrlichia ewingii, Ehrlichia chaffeensis, Ehrlichia muris eauclarensis, and "Panola Mountain Ehrlichia". Of these, E. canis is considered the most clinically relevant for domestic dogs, with infection capable of causing acute, subclinical, and chronic stages of disease. Changes in climate, land use, habitats, and wildlife reservoir populations, and increasing contact between both human and dog populations with natural areas have resulted in the increased risk of vector-borne disease throughout the world.MethodsA Bayesian spatio-temporal binomial regression model was applied to serological test results collected from veterinarians throughout the contiguous USA between January 2013 and November 2019. The model was used to quantify both regional and local temporal trends of canine Ehrlichia spp. seroprevalence and identify areas that experienced significant increases in seroprevalence.ResultsRegionally, increasing seroprevalence occurred within several states throughout the central and southeastern states, including Missouri, Arkansas, Mississippi, Alabama, Virginia, North Carolina, Georgia and Texas. The underlying local trends revealed increasing seroprevalence at a finer scale. Clusters of locally increasing seroprevalence were seen from the western Appalachian region into the southern Midwest, along the Atlantic coast in New England, parts of Florida, Illinois, Wisconsin and Minnesota, and in a couple areas of the Mountain region. Clusters of locally decreasing seroprevalence were seen throughout the USA including New York and the mid-Atlantic states, Texas, the Midwest, and California.ConclusionsCanine Ehrlichia spp. seroprevalence is increasing in both endemic and non-endemic areas of the USA. The findings from this study indicate that dogs across a wide area of the USA are at risk of exposure and these results should provide veterinarians and pet owners with the information they need to make informed decisions about prevention of tick exposure.

Project description:Pacific salmonid (Oncorhynchus spp.) remains are routinely recovered from archaeological sites in northwestern North America but typically lack sexually dimorphic features, precluding the sex identification of these remains through morphological approaches. Consequently, little is known about the deep history of the sex-selective salmonid fishing strategies practiced by some of the region's Indigenous peoples. Here, we present a DNA-based method for the sex identification of archaeological Pacific salmonid remains that integrates two PCR assays that each co-amplify fragments of the sexually dimorphic on the Y chromosome (sdY) gene and an internal positive control (Clock1a or D-loop). The first assay co-amplifies a 95 bp fragment of sdY and a 108 bp fragment of the autosomal Clock1a gene, whereas the second assay co-amplifies the same sdY fragment and a 249 bp fragment of the mitochondrial D-loop region. This method's reliability, sensitivity, and efficiency, were evaluated by applying it to 72 modern Pacific salmonids from five species and 75 archaeological remains from six Pacific salmonids. The sex identities assigned to each of the modern samples were concordant with their known phenotypic sex, highlighting the method's reliability. Applications of the method to dilutions of modern DNA samples indicate it can correctly identify the sex of samples with as little as ~39 pg of total genomic DNA. The successful sex identification of 70 of the 75 (93%) archaeological samples further demonstrates the method's sensitivity. The method's reliance on two co-amplifications that preferentially amplify sdY helps validate the sex identities assigned to samples and reduce erroneous identifications caused by allelic dropout and contamination. Furthermore, by sequencing the D-loop fragment used as a positive control, species-level and sex identifications can be simultaneously assigned to samples. Overall, our results indicate the DNA-based method reported in this study is a sensitive and reliable sex identification method for ancient salmonid remains.

Dataset Information

A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

Publications

A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

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