How to verify if a tick was encephalitic? - briefly
PCR or immunofluorescence testing for encephalitis‑associated viral RNA or antigens determines whether a tick carried the pathogen; a positive result confirms encephalitic infection.
How to verify if a tick was encephalitic? - in detail
Determining whether a tick harbored an encephalitis‑causing virus requires a combination of field assessment and laboratory analysis.
First, identify the tick species and collection context. Ixodes ricinus, Dermacentor reticulatus, and Haemaphysalis spp. are most frequently associated with tick‑borne encephalitis (TBE). Record geographic location, habitat type, and collection date, because TBE prevalence peaks in spring and early autumn in endemic regions.
Second, preserve the specimen appropriately. Place the tick in a sterile tube with viral transport medium or RNAlater and keep at ‑80 °C until processing. Avoid formalin, which degrades nucleic acids.
Third, conduct molecular testing. Extract RNA from the whole tick or dissected salivary glands. Perform reverse transcription quantitative PCR (RT‑qPCR) targeting the conserved envelope (E) gene of the TBE virus. Include positive and negative controls to validate assay performance. A cycle threshold below 35 indicates the presence of viral RNA.
Fourth, confirm positive PCR results with sequencing. Amplify the E gene fragment, purify the product, and submit for Sanger sequencing. Compare the obtained sequence to reference strains in GenBank using BLAST. A ≥98 % identity confirms the specific virus lineage.
Fifth, apply serological methods when viable virus is unavailable. Homogenize the tick in phosphate‑buffered saline, clarify the suspension, and test for viral antigens using a sandwich ELISA with monoclonal antibodies against TBE virus. Positive optical density values above the established cutoff support infection.
Sixth, attempt virus isolation in cell culture. Inoculate Vero or BHK‑21 cells with tick homogenate and monitor for cytopathic effect. Confirm replication by immunofluorescence staining using anti‑TBE virus antibodies. Isolation provides definitive proof but requires biosafety level 3 facilities.
Finally, integrate all data. A positive RT‑qPCR result, corroborated by sequencing, serology, or successful isolation, constitutes definitive evidence that the tick was encephalitic. Absence of detectable viral markers, despite appropriate sample handling, suggests the tick was not infected at the time of collection.