Experiment 1 Experimental design to characterize the role of tick salivary proteins in allergic reactions to mammalian meat consumption in the zebrafish model of the α-Gal syndrome (AGS). Saliva from semi-engorged Ixodes ricinus female ticks was collected and used in this experiment. Wild type adult AB strain zebrafish (20 animals/group) were treated with PBS, saliva, saliva non-protein fraction (NPF) alone and combined with recombinant tick proteins metalloprotease (MET), allergen-like p23 (p23) and, as control, Subolesin (SUB). Zebrafish were kept on fish feed during pre-treatment and until Day 2. Zebrafish were injected with each treatment at Days 0 and 3, and from Day 2 and until the end of the experiment at Day 8 fish were fed with dog food containing mammalian meat. Zebrafish local hemorrhagic type allergic reactions, abnormal behavior patterns and feeding and accumulated mortality were examined from Day 1 and followed daily until the end of the experiment at Day 8. After fish euthanasia, serum was collected individually to determine IgM antibody titers against tick salivary glands (SG) protein extract, α-Gal and tick proteins. Intestine samples were collected for expression analysis of selected immune response and allergy gene markers by RT-qPCR.

Allergic reactions to mammalian meat consumption in zebrafish treated with tick salivary proteins. Experiment 1. Zebrafish were treated with recombinant tick salivary proteins, metalloprotease (MET) or allergen-like p23 (p23), in combination with saliva non-protein fraction (NPF) and in comparison, with tick saliva, saliva NPF, Subolesin (SUB)-NPF and PBS. Zebrafish were examined daily and the incidence of hemorrhagic type allergic reactions, abnormal behavior and feeding patterns and cumulative mortality were compared between treatments by one-way ANOVA test with post-hoc Tukey HSD test (p < 0.05; n = 13–20 biological replicates). Significant differences between treatments and PBS control are shown with post-hoc Tukey HSD p-values. Color of the asterisk of significant differences are associated with treatment line.

Characterization of IgM antibody titers in zebrafish AGS model treated with tick salivary proteins. Experiment 1. Zebrafish were treated with recombinant tick salivary proteins, metalloprotease (MET) or allergen-like p23 (p23), in combination with saliva non-protein fraction (NPF) and, in comparison with tick saliva, saliva NPF, Subolesin (SUB)-NPF and PBS. (A) IgM antibody titers against tick salivary gland (SG) protein extract. (B) IgM antibody titers against α-Gal. (C) IgM antibody titers against SUB. (D) IgM antibody titers against tick protein p23. (E) IgM antibody titers against tick protein MET. Experiment 2. Zebrafish were treated with a combination (Mix) that included both recombinant tick salivary proteins, MET and p23, with NPF and in comparison, with tick saliva, and PBS. (F) IgM antibody titers against tick SG protein extract. (G) IgM antibody titers against α-Gal. (H) IgM antibody titers against tick protein p23. (I) IgM antibody titers against tick protein MET. Significant differences between treatments and PBS control are shown with post-hoc Pairwise Mann–Whitney p-values (* p < 0.05, ** p < 0.01, *** p < 0.001).

Experiment 2: Experimental design to evaluate the role of combined treatment with tick salivary proteins and fish tuberculosis. Saliva from semi-engorged I. ricinus female ticks was collected and used in this experiment. Wild type adult AB strain zebrafish (30 animals/group) were treated with PBS, saliva, and combined (Mix) tick salivary proteins metalloprotease (MET) + allergen-like p23 (p23) with saliva non-protein fraction (NPF). Zebrafish were kept on fish feed during pre-treatment and until Day 2. Zebrafish were injected via IM with each treatment at Days 0 and 3, and from Day 2 and until the end of the experiment at Days 7 and 25 fish were fed with dog food containing mammalian meat. At Day 7, 10 animals/group were euthanized by prolonged immersion using an overdose of tricaine methane sulfonate (MS222, 200–300 mg/l). The rest of the animals (n = 20) were challenged with M. marinum and maintained until Day 25 in which they were sacrificed. Zebrafish local hemorrhagic type allergic reactions and accumulated mortality were examined from Day 1 and followed daily until the end of the experiment at Days 7 or 25. After fish euthanasia, serum was collected individually to determine IgM antibody titers against tick salivary glands (SG) protein extract, α-Gal, tick proteins and the immunopurified subcomplex protein P22. Intestine samples were collected for expression analysis of selected immune response and allergy gene markers by RT-qPCR and the analysis of M. marinum DNA levels by qPCR.

Allergic reactions to mammalian meat consumption in zebrafish treated with tick salivary proteins and challenged with M. marinum. Experiment 2. Zebrafish were treated with both recombinant tick salivary proteins, metalloprotease (MET) and allergen-like p23 (p23), combined with saliva non-protein fraction (NPF) (Mix) and in comparison, with tick saliva, and PBS (control). Zebrafish (n = 10) were euthanized at Day 7 and the rest (n = 20) were challenged with M. marinum and euthanized at Day 25. (A) Zebrafish were examined daily, and the incidence of hemorrhagic type allergic reactions and mortality were compared between treatments by one-way ANOVA test with post-hoc Tukey HSD test (p < 0.05; n = 13–20 biological replicates). Significant differences between treatments and PBS control are shown with post-hoc Tukey HSD p-values. (B) Zebrafish weight was evaluated at Day 0 and at Day 25 for each animal to calculate weight gain per fish (grams, Day 25 – Day 0) and the percentage (%) of fish showing weight gain on each group. The results were compared with PBS-treated group by Student’s t-test (p < 0.05; n = 15–17 biological replicates).

Quantification of zebrafish gut infection and antibody levels in response to M. marinum infection. Zebrafish were treated with a combination of MET, p23 and NPF (Mix) tick salivary biomolecules and compared with PBS control group. (A) Infection levels were quantified by qPCR using the 16 S ribosomal RNA (16 S rRNA) gene fragment. (B) IgM antibody titers against M. bovis P22 protein complex were determined by ELISA. Significant differences between treatments and PBS control are shown with post-hoc Pairwise Mann–Whitney p-values (* p < 0.05, ** p < 0.01, *** p < 0.001).

Experiment 1: Expression levels of different zebrafish gut immunity and allergy biomarkers. Expression levels were characterized in zebrafish samples collected from Experiment 1 (Fig. 1, Experimental design to characterize the role of tick salivary proteins in allergic reactions to mammalian meat consumption in the zebrafish model of the α-Gal syndrome). The mRNA levels for (A) Cathepsin S, (B) DNA-dependent protein kinase, (C) Tumor necrosis factor alpha, (D) Interleukin 4, (E) Complement C3a, (F) Toll-like receptor 2, (G) Interleukin 1 beta and (H) Interferon 1 were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Significant differences between groups were analyzed using the Real Statistics extension for Microsoft Excel (v2310) and data was depicted with GraphPad Prism (version 8.0.1) (**p < 0.01).

Experiment 2: Expression levels of different zebrafish gut immunity and allergy biomarkers. The mRNA levels were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Expression levels were characterized in zebrafish samples collected from Experiment 2 (Fig. 3, Experimental design to evaluate the role of combined treatment with tick salivary proteins and fish tuberculosis). The mRNA levels for (A) Cathepsin S, (B) DNA-dependent protein kinase, (C) Tumor necrosis factor alpha, (D) Interleukin 4, (E) Complement C3a, (F) Toll-like receptor 2, (G) Interleukin 1 beta and (H) Interferon 1 were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Significant differences between groups were analyzed using the Real Statistics extension for Microsoft Excel (v2310) and data was depicted with GraphPad Prism (version 8.0.1) ((* p < 0.05, ** p < 0.01, *** p < 0.001).

Summary of results. Allergic type reactions to tick salivary compounds and protective mechanisms in response to mycobacteria. Summarized immune effects and molecular mechanismsin zebrafish gut caused by I. ricinus tick saliva treatment and water-borne exposure to M. marinum. Injection of I. ricinus tick saliva containing the glycan α-Gal and proteins like metalloproteases and p23 activates antigen presenting cells (APCs) controlled by the overexpression of cathepsin S (ctss2.1) and other immune modulators. APCs charged with toll like receptors 2 (TLR2) activate Th2 responsive cells that produce interleukin 4 (IL-4), which leads to basophil and mast cell recruitment. This type of response is common in allergic inflammation and parasitic infection. On the other hand, bacterial infection by M. marinum is responsible for the activation of alternative pathway of complement, measured through the genetic expression of C3a. Furthermore, a Th1 driven response is present, marked by TLR2 signaling, activation of pro-inflammatory mediators like DNA-dependent protein kinase (prkdc) and cytokines such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α).