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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

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

1.6K 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

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

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). ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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 ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary to perform reproducible burn research. The present protocol describes a simple and reproducible method to create ~20% total body surface area (TBSA) full-thickness burns in rats. Here, a 22.89 cm2 (5.4 cm diameter) copper rod heated at 97&#160;&#176;C in a water bath was applied to the rat skin surface to induce the burn injury. A copper rod with a high thermal conductivity was able to dissipate the heat deeper in the skin tissue to create a full-thickness burn. Histology analysis shows attenuated epidermis with coagulative damage to the full-thickness extent of the dermis and the subcutaneous tissue. Additionally, this model is representative of the clinical situations observed in hospitalized burn patients following burn injury such as immune dysregulation and bacterial infections. The model can recapitulate the systemic bacterial infection by both Gram-positive and Gram-negative bacteria. In conclusion, this paper presents an easy-to-learn and robust rat burn model that mimics the clinical situations, including immune dysregulation and bacterial infections, which is of considerable utility for the development of new topical antibiotic drugs for burn wound and infections.

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...