engleski

Establishment of DNA-based test for quick diagnostics of canine babesiosis

Canine babesiosis is a tick-borne disease caused by the parasite B. canis canis (most prevalent in Croatia) and B. canis vogeli. Blood smear observation is the routine procedure for diagnostics but could lead to false negative results and is unable to discriminate subspecies. A new diagnostic test based on qPCR has been set up. Two genes were used to set up PCR: 18SrRNA and HSP70, 18SrRNA being partially available on NCBI. For both B. canis canis and B. canis vogeli 18SrRNA genes, all sequences (96 and 253 respectively) have been assembled to generate the most complete gene sequence. Sequence of HSP70 for both subspecies was already available. Primers were designed and tested with Primer3. 18SrRNA was used to discriminate the 2 subspecies (92.9% homology), with 2 couples of primers (amplicon lengths of 153 and 163 bp). HSP70 was used to design one couple of primer to detect each Babesia genus species (82.7% homology among Babesia genus, amplicon length of 164 bp). A patent is still in negotiation for this original work. First, PCRs specificity have been checked on gel electrophoresis, confirmed on high resolution (0.2°C) qPCRs, which demonstrated that amplicons melting curves (depending on sequences) are dynamically in accordance with in silico simulations (uMelt). Then for routine diagnostic qPCR was used to give an estimation of Babesia per erythrocyte. Each sample was characterized by presence/absence of Babesia genus (HSP70), B. canis canis (18SrRNA), and B. canis vogeli (18SrRNA) with dedicated qPCRs. So far, 29 samples diagnosed for B. canis canis by blood smear have been analysed, 28 were confirmed, and 1 was diagnosed for B. canis vogeli. From 8 samples with negative blood smear, 1 was diagnosed for B. canis canis, confirmed by a second blood smear. This diagnostic tool is able to answer to veterinarian needs (fast, specific, cheap). The next step aims to transfer the technique and invites veterinarians to use it through education and presentations.

canine babesiosis, diagnostics, qPCR

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