How to detect encephalitis in a tick? - briefly
Detect encephalitis in a tick by extracting nucleic acids from the specimen and performing PCR or RT‑PCR assays targeting the relevant viral genome, optionally confirmed with immunofluorescence staining for viral antigens. Positive findings should be validated through sequencing or virus isolation.
How to detect encephalitis in a tick? - in detail
Detecting encephalitis‑causing agents in a tick requires a systematic laboratory workflow. First, remove the arthropod from the host using sterile forceps, place it in a labeled tube, and keep it refrigerated (4 °C) if processing occurs within 24 hours; otherwise freeze at –80 °C. Record species, life stage, collection site, and date, because vector competence varies among taxa.
Sample preparation
- Surface‑sterilize the specimen with 70 % ethanol for 30 seconds, then rinse with sterile water.
- Homogenize the whole tick or dissect salivary glands and midgut in a biosafety cabinet using a bead‑beater or mortar with sterile pestle.
- Transfer the homogenate to a microcentrifuge tube containing lysis buffer appropriate for the downstream assay.
Molecular detection
- Extract nucleic acids with a validated kit (e.g., silica‑column or magnetic‑bead system). Include an internal control to verify extraction efficiency.
- Perform real‑time PCR targeting conserved regions of the flavivirus genome (e.g., NS5 or E gene). Use primers and probes validated for tick‑borne encephalitis virus (TBEV).
- Run a quantitative standard curve to determine viral load; a cycle threshold (Ct) < 35 generally indicates presence of viral RNA.
- Confirm positive results by sequencing the amplicon and comparing it with reference sequences in GenBank.
Serological approaches
- Apply enzyme‑linked immunosorbent assay (ELISA) to homogenate supernatants to detect viral antigens.
- Use indirect immunofluorescence assay (IFA) on tick tissue sections with monoclonal antibodies specific for TBEV antigens. Positive fluorescence under a microscope confirms viral protein presence.
Microscopic examination
- Prepare thin sections of tick tissues, stain with hematoxylin‑eosin, and inspect for cytopathic changes typical of flaviviral infection.
- Electron microscopy can visualize virions within salivary gland cells, providing morphological confirmation.
Quality control
- Include negative extraction controls, no‑template PCR controls, and positive reference material in each batch.
- Perform duplicate assays for each sample to ensure reproducibility.
Interpretation
- Detectable viral RNA or antigen in a tick indicates infection, but does not alone prove transmission capability. Correlate findings with epidemiological data (e.g., prevalence in the area) and host infection status.
- High viral loads (low Ct values) and presence in salivary glands suggest a higher risk of onward transmission to vertebrate hosts.
Following this protocol yields reliable identification of encephalitis‑associated pathogens within ticks, supporting surveillance and risk assessment programs.