Abstract
BackgroundBabesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp.MethodsUsing 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR.ResultsThe LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA qPCR positive.ConclusionsWe have developed a more sensitive qPCR assay with a more expansive range of Babesia spp. detection by targeting a highly conserved region of mtDNA, when compared to an established 18S qPCR.
Highlights
Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals
Results quantitative real-time polymerase chain reaction (PCR) (qPCR) efficiency, analytical sensitivity and specificity Amplicon size for each primer pair was visualized on an agarose gel and corresponded to the expected size of ~150 bp for most Babesia spp. and ~230 bp for B. microti-like
In summary, we have developed a qPCR assay with increased sensitivity to detect a broader number of Babesia spp
Summary
Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. Ribosomal DNA sequences contain hypervariable regions, which are frequently used for species-specific amplification and are flanked by highly conserved regions used for broad-range genus amplification This effectively allows amplification and discrimination of most Babesia species. Differences in the 18S rRNA gene sequence of the more distantly related clades, which include B. conradae and B. microti-like parasites, make it difficult to design 18S rDNA assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. This goal was achieved by targeting the Babesia mitochondrial genome (mtDNA)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
