Name: zebrafish animal model for the study of allergic reactions in response to tick saliva biomolecules
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We would like to thank members of the SaBio group for their collaboration in the experimental design and technical assistance with the fish experimental facility and Juan Galcer&#225;n S&#225;ez (IN-CSIC-UMH, Spain) for providing zebrafish. This work was supported by Ministerio de Ciencia e Innovaci&#243;n/Agencia Estatal de Investigaci&#243;n MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00). Marinela Contreras is funded by the Ministerio de Ciencia, Innovaci&#243;n y Universidades, Spain, grant IJC2020-042710-I.

All methods described here have been approved by the Ethics Committee on Animal Experimentation of the University of Castilla La Mancha under the study &#34;Evaluation of the immune response to inactivated M. bovis vaccine and challenge with M. marinum in the zebrafish model number PR-2017-05-12.&#34;
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 pathoge...

<ol>
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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 infections33,34,35. 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...

Ticks are vectors of pathogens that cause diseases and are also the cause of allergic reactions, affecting the health of humans and animals worldwide1,2. 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 Gal&#945;1-3Gal&#946;1-(3)4GlcNAc-R (&#945;-Gal) modifications lead to the production of high anti-&#945;-Gal IgE antibody levels after the tick bite, only in some individuals, which is known as &#945;-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 &#945;-Gal are often severe and sometimes could be fatal6,7,8,9,10,11,12,13,14,15.
The &#945;-Gal is found in all mammals except for Old World monkeys, apes, and humans that do not have the ability to synthesize &#945;-Gal13. However, pathogens such as bacteria and protozoa express this glycan on their surface, which can induce the production of high amounts of anti-&#945;-Gal IgM/IgG antibodies and may be a protective mechanism against these pathogens16,17. However, the production of anti-&#945;-Gal antibodies increases the risk of developing IgE-mediated anti-&#945;-Gal allergies7,13. Natural anti-&#945;-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-&#945;-Gal antibody levels9. Current findings suggest tick bites constitute one of the principal risks in the appearance of AGS18,19, a 20-fold or greater increase in IgE levels to &#945;-Gal following a tick bite19, a history of tick bites in patients with AGS20,21,22, the existence of antibodies reactive to tick antigens in AGS patients19, and that anti-&#945;-Gal IgE are strongly related to anti-tick IgE levels19,23 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-&#945;-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 &#945;&#8722;1,3-galactosyltransferase knocked out (&#945;-Gal KO) mice25,26 because like other mammals, mice also express &#945;-Gal on proteins and lipids and do not produce IgE to &#945;-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 &#945;-Gal. Since &#945;-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 &#945;-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 saliva26,27 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 &#945;-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.

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	<tbody>
		<tr>
			<td>1.5 mL tube</td>
			<td>VWR</td>
			<td>525-0990</td>
			<td></td>
		</tr>
		<tr>
			<td>All Prep DNA/RNA</td>
			<td>Qiagen</td>
			<td>80284</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquatics facilities</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Disection set</td>
			<td>VWR</td>
			<td>631-1279</td>
			<td></td>
		</tr>
		<tr>
			<td>Dog Food - Red Classic</td>
			<td>Acana</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plates-96 well</td>
			<td>Thermo Fisher Scientific</td>
			<td>10547781</td>
			<td></td>
		</tr>
		<tr>
			<td>Gala1-3Gal-BSA 3 (&#945;-Gal)&#160;</td>
			<td>Dextra</td>
			<td>NGP0203</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript Reverse Transcription Supermix</td>
			<td>Supermix</td>
			<td>1708840</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringes</td>
			<td>Hamilton</td>
			<td>7638-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-50TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>pilocarpine hydrochloride&#160;</td>
			<td>Sigma</td>
			<td>P6503</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip P10&#160;</td>
			<td>VWR</td>
			<td>613-0364</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip P1000</td>
			<td>VWR</td>
			<td>613-0359</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium food tropical fish</td>
			<td>DAPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge Animal Holder&#160;</td>
			<td></td>
			<td></td>
			<td>Made from scrap foam</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler Real-Time PCR</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methanesulphonate (MS-222)</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish animal model for the study of allergic reactions in response to tick saliva biomolecules

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 Gal&#945;1-3Gal&#946;1-(3)4GlcNAc-R (&#945;-Gal) have been induced by tick bites. Anaphylactic reactions mediated by glycoproteins and glycolipids containing the glycan &#945;-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 &#945;-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.

The protocol presented here is based on several aspects of previously published experiments27,30 and results performed in our laboratory where the zebrafish model is established and validated for the study of AGS and the immune response to &#945;-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...

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

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.

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health ...

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.

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