Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules

Summary

Here, zebrafish (Danio rerio) is used as a model to study allergic reactions and immune responses related to alpha-Gal syndrome (AGS) by evaluating allergic reactions to tick saliva and mammalian meat consumption.

Abstract

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health worldwide. In some individuals, high levels of immunoglobulin E antibodies against the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) have been induced by tick bites. Anaphylactic reactions mediated by glycoproteins and glycolipids containing the glycan α-Gal, present in tick saliva, are related to alpha-Gal syndrome (AGS) or mammalian meat allergy. Zebrafish (Danio rerio) has become a widely used vertebrate model for the study of different pathologies. In this study, zebrafish was used as a model for the study of allergic reactions in response to α-Gal and mammalian meat consumption because, like humans, they do not synthesize this glycan. For this purpose, behavioral patterns and hemorrhagic anaphylactic-type allergic reactions in response to Ixodes ricinus tick saliva and mammalian meat consumption was evaluated. This experimental approach allows the obtention of valid data that support the zebrafish animal model for the study of tick-borne allergies including AGS.

Introduction

Ticks are vectors of pathogens that cause diseases and are also the cause of allergic reactions, affecting the health of humans and animals worldwide^1,2. During tick feeding, biomolecules in tick saliva, especially proteins and lipids, facilitate the feeding of these ectoparasites, avoiding host defenses³. Some saliva biomolecules with glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) modifications lead to the production of high anti-α-Gal IgE antibody levels after the tick bite, only in some individuals, which is known as α-Gal Syndrome (AGS)⁴. This is a disease associated with IgE-mediated allergy that may result in anaphylaxis to tick bites, non-primate mammalian meat consumption, and some drugs such as cetuximab⁵. Reactions to α-Gal are often severe and sometimes could be fatal^{6,7,8,9,10,11,12,13,14,15}.

The α-Gal is found in all mammals except for Old World monkeys, apes, and humans that do not have the ability to synthesize α-Gal¹³. However, pathogens such as bacteria and protozoa express this glycan on their surface, which can induce the production of high amounts of anti-α-Gal IgM/IgG antibodies and may be a protective mechanism against these pathogens^16,17. However, the production of anti-α-Gal antibodies increases the risk of developing IgE-mediated anti-α-Gal allergies^7,13. Natural anti-α-Gal antibodies produced in humans, mainly of the IgM/IgG subtypes, could be associated with this modification present in bacteria from the gut microbiota¹⁶. AGS can be a challenging clinical diagnosis, as the main diagnostic method at the moment is based on a clinical history of delayed allergic reactions, especially associated with food allergies (i.e., pruritus, localized hives, or recurrent angioedema to anaphylaxis, urticaria, and gastrointestinal symptoms) and the measurement of IgE anti-α-Gal antibody levels⁹. Current findings suggest tick bites constitute one of the principal risks in the appearance of AGS^18,19, a 20-fold or greater increase in IgE levels to α-Gal following a tick bite¹⁹, a history of tick bites in patients with AGS^20,21,22, the existence of antibodies reactive to tick antigens in AGS patients¹⁹, and that anti-α-Gal IgE are strongly related to anti-tick IgE levels^19,23 but further studies are needed to assess which biomolecules are actually involved.

In addition, another possible scenario is patients who present strong allergic reactions to tick bites and high levels of anti-α-Gal IgE antibodies but are tolerant to mammalian meat consumption¹². Therefore, mammalian meat allergy could be a particular type of tick bite-related allergy. The principal tick species associated with AGS include Amblyomma americanum (USA), Amblyomma sculptum (Brazil), Amblyomma testudinarium and Haemaphysalis longicornis (Japan), Ixodes holocyclus (Australia), and Ixodes ricinus (the main vector of Lyme borreliosis in Europe)^11,24.

The only model that has been used to evaluate IgE production related with tick bites is the mouse model genetically modified with the gene for α−1,3-galactosyltransferase knocked out (α-Gal KO) mice^25,26 because like other mammals, mice also express α-Gal on proteins and lipids and do not produce IgE to α-Gal. However, zebrafish (Danio rerio) is a useful model for biomedical research applied to mammals because it shares many anatomical similarities with mammals and, like humans, is also unable to synthesize α-Gal. Since α-Gal is not produced naturally in zebrafish, it is an affordable model, easy to manipulate, and allows a high sample size for the study of α-Gal-related allergic reactions.

In this study, zebrafish is used as a model organism to characterize and describe local allergic reactions, behavioral patterns, and the molecular mechanisms associated with response to percutaneous sensitization to tick saliva^26,27 and subsequent mammalian meat consumption. For this purpose, fish are exposed to tick saliva by intradermal injection and then are fed with dog feed, that contains mammalian meat-derived products suitable for animal use which contains α-Gal²⁷, then possible related allergic reactions are evaluated. This method may be applied to the study of other biomolecules related to allergic processes, especially those related to AGS.

Protocol

All methods described here have been approved by the Ethics Committee on Animal Experimentation of the University of Castilla La Mancha under the study "Evaluation of the immune response to inactivated M. bovis vaccine and challenge with M. marinum in the zebrafish model number PR-2017-05-12."

Ticks were obtained from the laboratory colony, where representative samples of ticks in the colony were tested by PCR for common tick pathogensto confirm the absence of pathogens, and maintained at the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences (IP BC CAS), Czech Republic.All animal experiments were performed in accordance with the Animal Protection Law of the Czech Republic No. 246/1992 Sb (ethics approval No. 34/2018).

1. Zebrafish treatment

NOTE: The trial is designed to evaluate allergic reactions and the immune response in zebrafish treated with tick saliva in response to mammalian meat consumption.

1. Treat the fish (as explained in section 4) with tick saliva, commercial Gala1-3Gal-BSA 3 (α-Gal) (see Table of Materials), used as a positive control, with phosphate-buffered saline (PBS) as a negative control. Adult zebrafish is randomly distributed into three gender-balanced groups (Figure 1).
   NOTE: Any other desired compound related with AGS can be evaluated using this model.

2. ​ Ixodes ricinus tick saliva extraction

1. Use semi-engorged pathogen-free female ticks fed for 6-7 days on guinea pigs.
2. Treat the tick with 5 µL of a 2% (wt/vol) solution of pilocarpine hydrochloride in PBS (see Table of Materials) at pH 7.4 into the hemocoel using a 50 µL syringe with a 0.33 mm needle as previously was described²⁸ to induce tick saliva production.
   NOTE: Ticks are handled using forceps; be cautious to not apply too much strength when gripping them.
3. Collect saliva using a 10 µL tip mounted on a micropipette.
   1. Introduce the tip inside the tick hypostome carefully.
   2. Keep the saliva in a 1.5 mL tube on ice, pool it, and store it at -80 °C as previously described²⁷.
4. Determine the saliva protein concentration, to establish the amount of protein to be injected into the fish as in previous studies²⁷ using a BCA Protein Assay Kit (see Table of Materials) following the manufacturer's recommendations.

3. Maintenance of zebrafish

1. Maintain zebrafish in a flow-through water system at 27 °C with a light/dark cycle of 14 h/10 h (Figure 2).
2. Feed the fish twice daily at 9:30 a.m. and 1:30 p.m. with dry fish feed (50-70 μg/fish) until day 2.
3. Feed the fish twice daily at 9:30 a.m. and 1:30 p.m. with dry dog feed (50-70 μg/fish) from day 2 after the treatment injection until the end of the experiment

4. Zebrafish injection

1. Select 10 fish per group with a similar ratio of females/males and similar weight.
   NOTE: Group 1 contains fish injected with PBS, group 2 contains fish injected with tick saliva, and group 3 contains fish injected with α-Gal.
2. Anesthetize the fish briefly by immersion in 0.02% tricaine methanesulphonate (MS-222) (Movie 1).
   NOTE: Properly anesthetized fish show normal breathing and no swimming, while they could be placed at the bottom of the water tank or floating. Each fish must be individually anesthetized to avoid possible physiological damage.
3. Capture the anesthetized fish using a fishing net.
4. Place the 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 in order to control the lesions.
5. Inject groups of fish intradermally, as in previous studies²⁶, in the muscle at 5 mm to the caudal fin and at a 45° angle in relation to the body of the fish (Movie 2). Use the appropriate treatment at days 0, 3, and 8 as previously described²⁷ with a 100 µL syringe fitted with a 1 cm, 29 G needle with 1 µL (with 9 µg/µL of protein) of I. ricinus saliva in 10 µL of PBS (tick saliva), 5 µg of α-Gal in 10 µL PBS (α-Gal)²⁷, and 10 µL of PBS (Figure 3).
   NOTE: Handling must be done quickly and carefully to avoid any physical damage to the animal.
   Other biomolecules in tick saliva can be evaluated following this protocol.
6. Place the treated fish back in a freshwater tank without anesthesia for recovery.
   ​NOTE: All the fish of the same group can be placed in the same water tank for recovery.

5. Zebrafish feeding

1. Mash the dog food with a mortar and pestle.
2. Feed 50-70 μg/fish twice daily at 9:30 a.m. and 1:30 p.m. with dry fish feed until day 2.
3. Feed 50-70 μg/fish twice daily at 9:30 a.m. and 1:30 p.m. with mashed dog feed from day 2 after the treatment injection until the end of the experiment on day 8.
   ​NOTE: If immunity markers or antibody titers to α-Gal or IgE antibody in response to the treatments or the feed throughout the different inoculations are to be evaluated, feeding would be necessary until the end of the experiment.

6. Evaluation of allergic reactions, lesions, and behavior in zebrafish

1. Examine the hemorrhagic type of allergic reactions (skin redness, discoloration, and hemorrhage) using a magnifier or stereomicroscope for accuracy and indicate the location of their appearance on the fish following the categorization included in Table 1 (Figure 4A).
   NOTE: The allergic reactions presented in Figure 4 appeared after the injection of tick saliva and the consumption of feed containing red meat. Therefore, the reactions described are the type of reactions associated with AGS, as similar reactions appear in the clinical context.
   1. Observe if any reaction appears after treatments and while administering food twice a day while the fish are in the water tank.
2. Examine the fish behavior by evaluating the changes²⁷ in swimming patterns (mobility, speed, standing motionless at the bottom of the water tank, and zigzag swimming) following the categorization included in Table 1.
3. Evaluate accumulated mortality, reporting the number of dead fish including the time/day of death (Figure 4B).
   NOTE: All parameters are evaluated right after treatment or after the change of feed and followed daily until the end of the experiment on day 8 categorizing qualitative variables (Table 1). As a recommendation, this evaluation should be conducted by a professional with knowledge on zebrafish to consider behavioral changes based on their background and experience working with this animal model.
4. Calculate the number of zebrafish per day with reported allergic reactions, abnormal behavior, and feeding changes in each group and compare between groups by a one-way ANOVA test.

7. Sample collection

1. Euthanize the fish by immersion in 0.04% MS-222 on day 8.
   NOTE: Also collect the samples from the fish that die from allergic reactions during the trial.
2. Fix the fish on a paraffin plate with pins.
3. Collect serum from the gill-blood vessels²⁹ of the fish immediately after euthanasia, when the gills are still irrigated with blood, using a 0.5 mL syringe fitted with a 1 cm, 29 G needle. Store it in a 1.5 mL tube at -20 °C until use (Movie 3).
4. Cut the fish sagittally with a scalpel blade and evaluate the internal lesions (hemorrhagic lesions or granulomas)^27,30 if they appear.
   NOTE: Lesions do not necessarily appear but must be registered if they do.
5. Collect the intestine (Movie 4) and kidney (Movie 5) from each fish in separate empty 1.5 mL tubes, as previously described³¹, and store them at -80 C (Figure 4C).
6. Extract total RNA from the zebrafish intestine and kidney samples using an RNA purification kit (see Table of Materials).
7. Analyze the expression of genes related to immune response as previously described^30,32 (see Table 2 for primer sequences) in zebrafish, performing a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using a reverse transcription mix for RT-qPCR (see Table of Materials), according to the manufacturer's instructions. Normalize the mRNA cT values against D. rerio GAPDH, and compare between groups (fish treated with saliva, α-Gal, and the PBS-treated groups)using a Student t-test with unequal variance.
8. Determine IgM antibody titers that recognize α-Gal in zebrafish in serum samples by ELISA as described previously^27,30. Record the antibody titers as O.D._{450 nm} values, using a plate reader, and compare between groups (fish treated with saliva, α-Gal, and the PBS-treated groups)using a Student t-test with unequal variance.
   NOTE: Determination of IgM antibody titers and expression gene analysis is optional and conducted only if immunological information is required. RT-qPCR mix is a first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.

Results

The protocol presented here is based on several aspects of previously published experiments^27,30 and results performed in our laboratory where the zebrafish model is established and validated for the study of AGS and the immune response to α-Gal because both humans and zebrafish do not synthesize this molecule¹³. This model allows the characterization and evaluation of a variety of allergic reactions as a result of the host response t...

Discussion

Zebrafish is a cost-effective and easy-to-handle model that also has been a very feasible tool for the study of molecular mechanisms of the immune response, pathogen diseases, novel drug testing, and vaccination and protection against infections^33,34,35. The study on the behavior of zebrafish is useful since previous studies have found that some fish species remain motionless at the bottom of the tank when they are stressed, whi...

Materials

Name	Company	Catalog Number	Comments
1.5 mL tube	VWR	525-0990
All Prep DNA/RNA	Qiagen	80284
Aquatics facilities
BCA Protein Assay Kit	Thermo Fisher Scientific	23225
Disection set	VWR	631-1279
Dog Food - Red Classic	Acana
ELISA plates-96 well	Thermo Fisher Scientific	10547781
Gala1-3Gal-BSA 3 (α-Gal)	Dextra	NGP0203
iScript Reverse Transcription Supermix	Supermix	1708840
Microliter syringes	Hamilton	7638-01
Plate reader	any
Phosphate buffered saline	Sigma	P4417-50TAB
pilocarpine hydrochloride	Sigma	P6503
Pipette tip P10	VWR	613-0364
Pipette tip P1000	VWR	613-0359
Premium food tropical fish	DAPC
Sponge Animal Holder			Made from scrap foam
Stereomicroscope	any
Thermal Cycler Real-Time PCR	any
Tricaine methanesulphonate (MS-222)	Sigma	E10521