How are ticks tested for infections? - briefly
Ticks are screened by extracting their DNA/RNA and applying PCR‑based or multiplex molecular assays to detect bacterial, viral, and protozoan pathogens. Positive results are confirmed through sequencing or, where possible, culture methods.
How are ticks tested for infections? - in detail
Ticks are collected by dragging a cloth over vegetation, flagging, or removing them directly from hosts. After removal, specimens are placed in sterile vials containing ethanol (70 %) for DNA preservation or kept alive on moist paper at 4 °C if live‑cell assays are required. Each tick is identified to species, stage, and sex before testing, because pathogen prevalence varies with these factors.
The diagnostic workflow begins with homogenization. Individual ticks are crushed in a bead‑beater or mortar with lysis buffer, ensuring complete disruption of the exoskeleton. Nucleic acids are extracted using silica‑column kits or magnetic‑bead protocols, which yield purified DNA and RNA suitable for downstream amplification. Extraction controls (negative blanks and known positive samples) are processed in parallel to monitor contamination.
Polymerase chain reaction (PCR) is the primary detection method. Conventional PCR targets conserved gene regions of bacteria (e.g., 16S rRNA for Borrelia, gltA for Rickettsia), protozoa (18S rRNA for Babesia), and viruses (NS5 for flaviviruses). Real‑time quantitative PCR (qPCR) adds fluorescence‑based quantification, allowing estimation of pathogen load. Multiplex qPCR panels combine primers for several agents, reducing assay time and sample consumption.
When pathogen diversity is unknown or when co‑infection rates are high, next‑generation sequencing (NGS) provides unbiased detection. Metagenomic libraries prepared from tick extracts are sequenced on platforms such as Illumina MiSeq. Bioinformatic pipelines filter host reads, assemble contigs, and assign taxonomy using reference databases (e.g., NCBI RefSeq). NGS results are confirmed by targeted PCR to validate novel findings.
Serological assays complement nucleic‑acid methods. Enzyme‑linked immunosorbent assays (ELISA) detect antibodies or antigens of specific agents in tick homogenates, useful for viruses that produce low‑copy RNA. Immunofluorescence assays (IFA) visualize intracellular bacteria or protozoa in fixed tick sections, providing morphological confirmation.
Culture is rarely employed because many tick‑borne pathogens are fastidious. When attempted, tick homogenates are inoculated onto specialized cell lines (e.g., Vero for arboviruses, BHK‑21 for rickettsiae) under biosafety level 2 or 3 conditions, depending on the organism.
Quality assurance includes:
- Inclusion of extraction, amplification, and sequencing controls.
- Replication of positive samples in independent runs.
- Use of reference standards for quantification.
- Documentation of tick metadata (location, date, host) for epidemiological correlation.
Interpretation of results distinguishes true infection from environmental contamination. Positive PCR signals from internal tissues (midgut, salivary glands) are considered indicative of vector competence, whereas detection in the exterior exoskeleton may reflect recent blood meals. Quantitative thresholds established by qPCR cycles‑threshold (Ct) values help differentiate low‑level background from clinically relevant infection.
Collectively, these laboratory techniques provide a comprehensive framework for detecting bacterial, viral, and protozoan agents within ticks, supporting surveillance, risk assessment, and public‑health interventions.