Figure 3.

Experimental design. Experiments were designed and performed to evaluate the allergic reactions and immune response in zebrafish treated with tick saliva and salivary components and in response to red meat consumption. (A) In Experiment 1, zebrafish were injected with 2.5 μL tick saliva and biogenic substances α-Gal and PGE₂ to evaluate the allergic reactions and immune response in fish feeding on fish feed or dog food. Serum and tissue samples were collected to determine anti–α-Gal IgM response, intestine and kidney for qRT-PCR analysis of immune response markers, and half fish for histochemical characterization of local granulocytes. (B) Experiment 2 was conducted to inject fish with less amount of tick saliva than in Experiment 1 (1 μL instead of 2.5 μL R. sanguineus saliva) to reduce response to toxic and anticoagulant biogenic compounds different from α-Gal and PGE₂ present in tick saliva and to better monitor the incidence of allergic reactions, abnormal behavior patterns, and feeding during the experiment. Zebrafish local allergic reactions and behavior were examined immediately after treatment or feed change and followed daily until the end of the experiment at day 14 (Experiment 1) or day 10 (Experiment 2). Fish and tick representative images are shown.