Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA

Qurollo, Barbara A.; Archer, Nikole R.; Schreeg, Megan E.; Marr, Henry S.; Birkenheuer, Adam J.; Haney, Kaitlin N.; Thomas, Brittany; Breitschwerdt, Edward B.

doi:10.1186/s13071-017-2064-1

Cited by 61 publications

(43 citation statements)

References 34 publications

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“…As DNA of blood parasites in faecal samples may be degenerated, we applied also the third PCR protocol to amplify the short fragment (150 or 230 bp depending on species) of Babesia spp. mitochondrial DNA, lsu4-lsu5 [22]. For concurrent amplification of large (i.e.…”

Section: Methodsmentioning

confidence: 99%

Comparison of the detection efficiency of haemoparasite DNA in blood and faecal samples – the way to eco-epidemiological studies

Bajer

Dwużnik

Tołkacz

et al. 2019

Ann Agric Environ Med.

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Introduction and objective. It is easier and non-invasive to obtain faecal samples compared with blood samples. Molecular techniques may enable detection of parasites even in tiny amounts of blood-containing faeces. We aimed to compare the sensitivity of detection of three Babesia species and Hepatozoon canis in blood and faecal samples, including samples derived from naturally infected hosts. Materials and method. Three groups were involved: 1) Nine BALB/c mice infected with Babesia microti sampled during acute (n=3), post-acute (n=3) and chronic phases of infection (n=3); 2) Eight dogs with symptoms of babesiosis; 3) Six red foxes infected with B. vulpes, one fox infected with B. canis, four foxes infected with H. canis. Genomic DNA was extracted from blood and faeces by use of commercial kits and amplified with genus-specific primers in one-step or nested PCR reactions. Selected PCR products were sequenced. Results. No positive results for faecal samples were obtained from H. canis-positive foxes in contrast to Babesia spp. infections. Positive results from PCRs were obtained for all BALB/c mice (100%), five dogs (62.5%) and four of seven foxes (57.1%). Successful sequencing was obtained for six selected murine samples (B. microti), four canine samples (B. canis) and for one fox sample (B. vulpes). The success of B. microti detection in murine faecal samples from acute, post-acute and chronic phases was identical (100%). Conclusions. Detectability of Babesia spp. infections was lower in naturally infected dogs and foxes, compared to experimentally infected mice. Detection of DNA in faecal samples can be useful in the detection of Babesia infection in populations from which blood samples are hard to obtain, but due regard must be given to the possibility that prevalence of infection may be severely underestimated.

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Section: Methodsmentioning

confidence: 99%

Comparison of the detection efficiency of haemoparasite DNA in blood and faecal samples – the way to eco-epidemiological studies

Bajer

Dwużnik

Tołkacz

et al. 2019

Ann Agric Environ Med.

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“…DNA using two independent PCR protocols. One targeting 18S rRNA genes, and a second targeting mitochondrial DNA . Assays were run using quantitative PCR machines (CFX96 Real‐Time System C1000 Touch Thermal Cyclers [Bio‐Rad Laboratories, Inc]), and amplicon detection and melting curve analyses utilized SYBR green dye (SsoAdvanced Universal SYBR Green Supermix [Applied Biosystems, Life Technologies]).…”

Section: Methodsmentioning

confidence: 99%

Babesia gibsoni cytochrome b mutations in canine blood samples submitted to a US veterinary diagnostic laboratory

Birkenheuer

Marr

Wilson

et al. 2018

Veterinary Internal Medicne

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Background: Babesiosis caused by Babesia gibsoni is recognized throughout the world and can be difficult to treat. Resistance to atovaquone is associated with mutations in the B. gibsoni mitochondrial genome, specifically the M128 position of cytochrome b (cytb). The prevalence of cytb mutations in North America has not been reported. Hypothesis/Objectives: The objective of our study was to describe the prevalence of cytb M128 mutations in B. gibsoni in canine blood samples submitted to a US veterinary diagnostic laboratory. A secondary objective was to determine whether or not some dogs had wild‐type cytb in our initial samples then had M128 mutations detected in follow‐up samples. Animals: One‐Hundred seventy‐four dogs that tested positive for the presence of B. gibsoni between 2012 and 2017. Methods: Case series of consecutive samples submitted to a veterinary diagnostic laboratory. Partial B. gibsoni cytb genes were amplified by polymerase chain reaction and screened for the presence of mutations at the M128 position. Results: The overall prevalence of M128 mutants was 3.5% (6/173 dogs) in the initial samples. The incidence of new cytb mutants in dogs that tested positive for B. gibsoni, which then had follow‐up testing, was 12.1% (5/41). Conclusions and Clinic Importance: Our study reaffirms that B. gibsoni infection is widespread and most commonly detected in American Staffordshire Terrier/American Pit Bull Terrier dogs (128/174, 74% of the infected dogs in our study). The prevalence of cytb mutations does not warrant pretreatment genotyping.

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“…The volume of qPCR data is increasing, along with the urgent need for qPCR data integration and centralized documentation. In the past decade, qPCR has been utilized as a tool to support numerous biological fields of inquiry, including natural resource management ( Thomas et al, 2019 ; Fritts et al, 2019 ), food safety ( Amaral et al, 2016 ), conservation planning ( Franklin et al, 2019 ), and disease vector/infectious disease monitoring ( Qurollo et al, 2017 ; Ikten et al, 2016 ). Research using qPCR methodologies extends beyond the detection and quantification of target gene expression.…”

Section: Introductionmentioning

confidence: 99%

Molecular Detection Mapping and Analysis Platform for R (MDMAPR) facilitating the standardization, analysis, visualization, and sharing of qPCR data and metadata

Young

Deeth

et al. 2020

PeerJ

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Quantitative polymerase chain reaction (qPCR) has been used as a standard molecular detection tool in many scientific fields. Unfortunately, there is no standard method for managing published qPCR data, and those currently used generally focus on only managing raw fluorescence data. However, associated with qPCR experiments are extensive sample and assay metadata, often under-examined and under-reported. Here, we present the Molecular Detection Mapping and Analysis Platform for R (MDMAPR), an open-source and fully scalable informatics tool for researchers to merge raw qPCR fluorescence data with associated metadata into a standard format, while geospatially visualizing the distribution of the data and relative intensity of the qPCR results. The advance of this approach is in the ability to use MDMAPR to store varied qPCR data. This includes pathogen and environmental qPCR species detection studies ideally suited to geographical visualization. However, it also goes beyond these and can be utilized with other qPCR data including gene expression studies, quantification studies used in identifying health dangers associated with food and water bacteria, and the identification of unknown samples. In addition, MDMAPR’s novel centralized management and geospatial visualization of qPCR data can further enable cross-discipline large-scale qPCR data standardization and accessibility to support research spanning multiple fields of science and qPCR applications.

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Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA

Cited by 61 publications

References 34 publications

Comparison of the detection efficiency of haemoparasite DNA in blood and faecal samples – the way to eco-epidemiological studies

Comparison of the detection efficiency of haemoparasite DNA in blood and faecal samples – the way to eco-epidemiological studies

Babesia gibsoni cytochrome b mutations in canine blood samples submitted to a US veterinary diagnostic laboratory

Molecular Detection Mapping and Analysis Platform for R (MDMAPR) facilitating the standardization, analysis, visualization, and sharing of qPCR data and metadata

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