The question is why only some individuals exposed to tick bites develop allergic reactions. For us, elucidating the biomolecules in tick saliva and the immune mechanisms involving this process is key to address this question. The zebrafish animal model of the alpha-Gal syndrome reproduces human behavioral patterns and allergic reactions in response to tick saliva.
This experimental approach advances understanding of the tick-host molecular interaction in which we have conflict associated with the alpha-Gal syndrome and cooperation associated with the antibody response to alpha-Gal, which are protective against pathogen infection. To treat the fish with tick saliva, distribute adult zebrafish into three gender-balanced groups. Use commercial Gala1-3Gal-BSA 3 or alpha-Gal as a positive control and PBS as a negative control.
For saliva extraction, use semi-engorged, pathogen-free, female ticks fed for six to seven days on Guinea pigs. Treat the tick with five microliters of a 2%solution of pilocarpine hydrochloride in PBS at pH 7.4 into the hemocoel using a 50-microliter syringe with a 0.33-millimeter needle. Collect saliva from tick hypostome using a 10-microliter tip mounted on a micropipette.
Place the saliva in a 1.5-milliliter tube on ice, and store it at minus 80 degrees Celsius. Determine the saliva protein concentration to establish the amount of protein to be injected into the fish using a BCA protein assay kit, following the manufacturer's recommendations. Maintain zebrafish in a flow-through water system at 27 degrees Celsius with a light-dark cycle of 14 to 10 hours.
Next, select 10 fish per group with a similar ratio of females to males and similar weight for injection. Anesthetize the fish briefly by immersion in 0.02%tricaine methanesulphonate. Place the anesthetized fish on its half side using forceps or hands carefully, on a wet sponge, with the caudal fin on the right side to inject the compounds in the same direction to control the lesions.
Inject groups of fish intradermally in the muscle with a 100-microliter syringe fitted with a one-centimeter, 29-gauge needle at five millimeters to the caudal fin and a 45-degree angle to the body of the fish. After injection, place the treated fish back in a freshwater tank without anesthesia for recovery. Feed the fish twice daily at 9:30 a.m.
and 1:30 p.m. with 50 to 70 micrograms of dry fish feed per fish until day two. Then mash the dog food with a mortar and pestle to feed the fish from day two after the treatment injection until the end of the experiment on day eight.
For sample collection, fix the euthanized fish on a paraffin plate with pins. Collect the serum from the gill blood vessels of the fish when the gills are still irrigated with blood using a 0.5-milliliter syringe fitted with a one-centimeter, 29-gauge needle. Store the collected serum in a 1.5-milliliter tube at 20 degrees Celsius until use.
Cut the fish sagittally with a scalpel blade, and evaluate the internal lesions. Then collect each fish's intestine and kidney in separate 1.5-milliliter tubes. Extract total RNA from the intestine and kidney using an RNA purification kit.
Analyze the expression of genes related to immune response, performing quantitative reverse transcription-polymerase chain reaction, or RT-qPCR using a reverse transcription mix for RT-qPCR, according to the manufacturer's instructions. Normalize the mRNA cT values against zebrafish GAPDH, and compare between groups using a Student t-test with unequal variance. Determine IgM antibody titers that recognize alpha-Gal in serum samples by ELISA.
Record the antibody titers as optical density 450-nanometer values using a plate reader, and compare between groups using a Student t-test with unequal variance. This zebrafish model allows the characterization and evaluation of various allergic reactions due to the host response to tick saliva and their implication in alpha-Gal syndrome, or AGS. In addition, compared to the control fish, alterations in behaviors such as slow swimming, lying on the bottom of the tank, and not eating, vibrating or zigzagging motion was observed in tick saliva-treated fish.
A significant incidence of allergic reactions was observed in fish treated with tick saliva, showing rapid desensitization and tolerance. The behavioral change was more pronounced in the fish treated with tick saliva than with just alpha-Gal. Additional analysis of the expression of the representative immune markers was performed by RT-PCR.
The results showed differences between zebrafish groups in the kidney, where the immune response markers were downregulated in fish treated with saliva and alpha-Gal compared to the control group. Further, the zebrafish treated with saliva and alpha-Gal developed IgM antibodies against alpha-Gal that showed higher levels than in fish treated with PBS. Injection of the treatment, evaluation of allergic reactions and behavior, and reporting the number of dead fish from allergic reactions are the most important aspect of the protocol.
Using omics technologies at the mRNA, protein, and also microbiome levels to characterize the response to tick saliva allows us to identify the key biomolecules involved in this process.
