How to test for an encephalitic tick?

How to test for an encephalitic tick? - briefly

Collect the tick, isolate its nucleic acid, and run a specific PCR assay for encephalitis‑causing viruses; confirm positives by sequencing or serologic testing. This rapid molecular protocol provides definitive identification of an infected tick.

How to test for an encephalitic tick? - in detail

Testing a tick for the presence of tick‑borne encephalitis (TBE) virus requires a systematic approach that combines accurate field collection with validated laboratory techniques.

The first phase involves acquiring specimens. Collect ticks from vegetation using a white‑flocked cloth or a dragging method. Separate each specimen by species, developmental stage, and engorgement level, because infection rates differ among these categories. Place ticks individually in sterile microcentrifuge tubes containing RNA‑preserving solution (e.g., RNAlater) or in dry, cold conditions if nucleic‑acid extraction will follow within 24 hours. Record GPS coordinates, habitat type, and collection date for epidemiological correlation.

The second phase focuses on preparation for analysis. Under a biosafety cabinet, surface‑sterilize each tick with 70 % ethanol, rinse with sterile phosphate‑buffered saline, and dissect to obtain the salivary glands, midgut, or whole‑body homogenate, depending on the chosen assay. Homogenize tissue in a suitable lysis buffer using a bead‑beater or motorized pestle. Clarify the homogenate by centrifugation and retain the supernatant for downstream testing.

The third phase comprises laboratory detection methods. Choose at least one of the following validated techniques:

• Reverse transcription quantitative PCR (RT‑qPCR).

  • Use primers and probes targeting the conserved E gene of TBE virus.
  • Include an internal extraction control (e.g., housekeeping gene) to verify nucleic‑acid integrity.
  • Set a threshold cycle (Ct) cut‑off based on positive controls; Ct ≤ 35 typically indicates a positive result.

• Enzyme‑linked immunosorbent assay (ELISA) for viral antigens.

  • Apply a sandwich ELISA with monoclonal antibodies specific for the TBE virus capsid protein.
  • Run standards to generate a calibration curve; samples exceeding the established optical density (OD) limit are considered positive.

• Immunofluorescence assay (IFA).

  • Incubate tick homogenate on fixed Vero cell monolayers, then stain with fluorescein‑labeled anti‑TBE antibodies.
  • Observe under a fluorescence microscope; specific cytoplasmic fluorescence confirms infection.

• Virus isolation in cell culture.

  • Inoculate clarified homogenate onto susceptible cell lines (e.g., BHK‑21 or Vero).
  • Monitor for cytopathic effect over 7–14 days; confirm identity by RT‑qPCR or immunostaining of harvested supernatant.

The fourth phase addresses quality control and data interpretation. Include negative extraction controls, no‑template controls, and known positive specimens in each assay batch. Confirm positive results with a second, independent method to reduce false‑positive risk. Record Ct values, OD readings, or fluorescence scores alongside metadata for statistical analysis.

The final phase involves reporting. Summarize findings in a structured format: number of ticks examined, species distribution, infection prevalence, geographic hotspots, and assay performance metrics. Submit data to public health authorities and relevant surveillance databases to inform risk assessments and preventive measures.