Name: zebrafish animal model for the study of allergic reactions in response to tick saliva biomolecules
Uploaded: 2022-09-16T14:10:00.000+00:00
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Description: Watch this Scientific Journal Video about Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules at JoVE.com

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 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.
<ol>
	<li>Treat the fish (as explained in section 4) with tick saliva, commercial Gala1-3Gal-BSA 3 (&#945;-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.</li>
</ol>
2. &#8203; Ixodes ricinus tick saliva extraction
<ol>
	<li>Use semi-engorged pathogen-free female ticks fed for 6-7 days on guinea pigs.</li>
	<li>Treat the tick with 5 &#181;L 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 &#181;L 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.</li>
	<li>Collect saliva using a 10 &#181;L tip mounted on a micropipette.
	<ol>
		<li>Introduce the tip inside the tick hypostome carefully.</li>
		<li>Keep the saliva in a 1.5 mL tube on ice, pool it, and store it at -80 &#176;C as previously described27.</li>
	</ol>
	</li>
	<li>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&#39;s recommendations.</li>
</ol>
3. Maintenance of zebrafish
<ol>
	<li>Maintain zebrafish in a flow-through water system at 27 &#176;C with a light/dark cycle of 14 h/10 h (Figure 2).</li>
	<li>Feed the fish twice daily at 9:30 a.m. and 1:30 p.m. with dry fish feed (50-70 &#956;g/fish) until day 2.</li>
	<li>Feed the fish twice daily at 9:30 a.m. and 1:30 p.m. with dry dog feed (50-70 &#956;g/fish) from day 2 after the treatment injection until the end of the experiment</li>
</ol>
4. Zebrafish injection
<ol>
	<li>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 &#945;-Gal.</li>
	<li>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.</li>
	<li>Capture the anesthetized fish using a fishing net.</li>
	<li>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.</li>
	<li>Inject groups of fish intradermally, as in previous studies26, in the muscle at 5 mm to the caudal fin and at a 45&#176; angle in relation to the body of the fish (Movie 2). Use the appropriate treatment at days 0, 3, and 8 as previously described27&#160;with a 100 &#181;L syringe fitted with a 1 cm, 29 G needle with 1 &#181;L (with 9 &#181;g/&#181;L of protein) of I. ricinus saliva in 10 &#181;L of PBS (tick saliva), 5 &#181;g of &#945;-Gal in 10 &#181;L PBS (&#945;-Gal)27,&#160;and 10 &#181;L 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.</li>
	<li>Place the treated fish back in a freshwater tank without anesthesia for recovery. 
	&#8203;NOTE: All the fish of the same group can be placed in the same water tank for recovery.</li>
</ol>
5. Zebrafish feeding
<ol>
	<li>Mash the dog food with a mortar and pestle.</li>
	<li>Feed 50-70 &#956;g/fish twice daily at 9:30 a.m. and 1:30 p.m. with dry fish feed until day 2.</li>
	<li>Feed 50-70 &#956;g/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. 
	&#8203;NOTE: If immunity markers or antibody titers to &#945;-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.</li>
</ol>
6. Evaluation of allergic reactions, lesions, and behavior in zebrafish
<ol>
	<li>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.
	<ol>
		<li>Observe if any reaction appears after treatments and while administering food twice a day while the fish are in the water tank.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
7. Sample collection
<ol>
	<li>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.</li>
	<li>Fix the fish on a paraffin plate with pins.</li>
	<li>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 &#176;C until use (Movie 3).</li>
	<li>Cut the fish sagittally with a scalpel blade and evaluate the internal lesions (hemorrhagic lesions or granulomas)27,30&#160;if they appear. 
	NOTE: Lesions do not necessarily appear but must be registered if they do.</li>
	<li>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).</li>
	<li>Extract total RNA from the zebrafish intestine and kidney samples using an RNA purification kit (see Table of Materials).</li>
	<li>Analyze the expression of genes related to immune response as previously described30,32 (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&#39;s instructions. Normalize the mRNA cT values against D. rerio GAPDH, and compare between groups (fish treated with saliva, &#945;-Gal, and the PBS-treated groups)using a Student t-test with unequal variance.</li>
	<li>Determine IgM antibody titers that recognize &#945;-Gal in zebrafish in serum samples by ELISA as described previously27,30. Record the antibody titers as O.D.450 nm values, using a plate reader, and compare between groups (fish treated with saliva, &#945;-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.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish:+A+fintastic+model+for+hematopoietic+development+and+disease.">The zebrafish: A fintastic model for hematopoietic development and disease.</a> Wiley Interdisciplinary Reviews. Developmental Biology. 7 (3), 312 (2018).</li><li>Katoch, S., Patial, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+An+emerging+model+system+to+study+liver+diseases+and+related+drug+discovery.">Zebrafish: An emerging model system to study liver diseases and related drug discovery.</a> Journal of Applied Toxicology. 41 (1), 33-51 (2021).</li><li>Kalueff, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+comprehensive+catalog+of+zebrafish+behavior+1.0+and+beyond.">Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.</a> Zebrafish. 10 (1), 70-86 (2013).</li><li>Xin, N., Jiang, Y., Liu, S., Zhou, Y., Cheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prednisolone+on+behavior+and+hypothalamic-pituitary-interrenal+axis+activity+in+zebrafish.">Effects of prednisolone on behavior and hypothalamic-pituitary-interrenal axis activity in zebrafish.</a> Environmental Toxicology and Pharmacology. 75, 103325 (2020).</li><li>Alestr&#246;m, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+Housing+and+husbandry+recommendations.">Zebrafish: Housing and husbandry recommendations.</a> Laboratory Animals. 54 (3), 213-224 (2020).</li></ol>

The authors have nothing to disclose.

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.

<table><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&amp;nbsp;</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 (&amp;alpha;-Gal)&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>P6503</td><td></td></tr><tr><td>Pipette tip P10&amp;nbsp;</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&amp;nbsp;</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...

Watch this Scientific Journal Video about Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules at JoVE.com

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 ...

zebrafish-animal-model-for-study-allergic-reactions-response-to-tick

Research

JoVE Journal

Immunology and Infection

1.7K Views.  SaBio. Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM). 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.

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

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Cancer Research

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Human Exposure to Larval Ticks for Tickborne Disease Research

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick must remain attached to the skin of the host and complete its multi-day feeding process to acquire its blood meal. Exposing animals to ticks is a common practice for studying host responses to tick bites and tickborne diseases. We developed the procedure, conducted the first human research study, and published the findings on exposing human volunteers to uninfected larval Ixodes scapularis ticks. This article describes the methodology used to construct the containment dressing, how to apply and secure the ticks to the host, how to maintain the dressing, and how to remove the ticks from the host. Exposing volunteers to tick bites is an experimental procedure and must be performed under a clinical research protocol approved by the appropriate regulatory authorities. This method allows for translational research to better understand the human response to tick bites and foster the development of diagnostics, prevention, and therapies for tickborne diseases.

Author Spotlight: Controlled Human Exposure Model for Tick Research and Lyme Disease Studies

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

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

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Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks