Name: zebrafish as a model to assess the teratogenic potential of nitrite
Uploaded: 2016-02-16T14:00:00
Description: Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

VK was funded by grants from the IUP Department of Biology and School of Graduate Studies and Research (Graduate Student Professional Development). CQD and TWS were supported by the IUP School of Graduate Studies and Research (Faculty Publication Costs/Incidental Research Expenses). We also thank members of the Diep laboratory for maintaining the zebrafish facility.

The procedures described in this protocol were approved by the Institutional Animal Care and Use Committee at the Indiana University of Pennsylvania.
1. Harvest Embryos
<ol>
	<li>Maintain zebrafish at 28.5 &#176;C, pH 7, conductivity between 500-1,500 &#181;S, and a light/dark cycle of 14 hr light and 10 hr dark24. Use wild-type strains such as T&#252;, AB or T&#252;/AB hybrid. Different strains may respond differently to chemical treatment25.</li>
	<li>Set up the fish ...

<ol>
	<li>Gilbert-Barness, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogenic+causes+of+malformations.">Teratogenic causes of malformations.</a> Ann Clin Lab Sci. 40 (2), 99-114 (2010).</li><li>Brent, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cause+and+prevention+of+human+birth+defects:+What+have+we+learned+in+the+past+50+years.">The cause and prevention of human birth defects: What have we learned in the past 50 years.</a> Con Anom. 41 (1), 3-21 (2001).</li><li>Lin, S., Zhao, Y., Nel, A. E. <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+in+vivo+model+for+nano+EHS+studies.">Zebrafish: an in vivo model for nano EHS studies.</a> Small. 9 (9-10), 1608-1618 (2013).</li><li>Pamanji, R., 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=Toxicity+effects+of+profenofos+on+embryonic+and+larval+development+of+zebrafish+(Danio+rerio).">Toxicity effects of profenofos on embryonic and larval development of zebrafish (Danio rerio).</a> Environ Toxicol Pharmacol. 39 (2), 887-897 (2015).</li><li>Simmons, A. E., Karimi, I., Talwar, M., Simmons, T. W. <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+nitrite+on+development+of+embryos+and+early+larval+stages+of+the+zebrafish+(Danio+rerio).">Effects of nitrite on development of embryos and early larval stages of the zebrafish (Danio rerio).</a> Zebrafish. 9 (4), 200-206 (2012).</li><li>Mantecca, 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=Toxicity+Evaluation+of+a+New+Zn-Doped+CuO+Nanocomposite+With+Highly+Effective+Antibacterial+Properties.">Toxicity Evaluation of a New Zn-Doped CuO Nanocomposite With Highly Effective Antibacterial Properties.</a> Toxicol Sci. , (2015).</li><li>Jensen, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+formation+from+nitrite+in+zebrafish.">Nitric oxide formation from nitrite in zebrafish.</a> J Exp Biol. 210, 3387-3394 (2007).</li><li>Scholz, S., 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=The+zebrafish+embryo+model+in+environmental+risk+assessment--applications+beyond+acute+toxicity+testing).">The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing).</a> Environ Sci Pollut Res Int. 15 (5), 394-404 (2008).</li><li>Howe, K., 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=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>CDC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+abortions+possibly+related+to+ingestion+of+nitrate-contaminated+well+water--LaGrange+County,+Indiana,+1991-1994.">Spontaneous abortions possibly related to ingestion of nitrate-contaminated well water--LaGrange County, Indiana, 1991-1994.</a> MMWR Morb Mortal Wkly Rep. 45 (26), 569-572 (1996).</li><li>Brender, J. D., 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=Prenatal+nitrate+intake+from+drinking+water+and+selected+birth+defects+in+offspring+of+participants+in+the+national+birth+defects+prevention+study.">Prenatal nitrate intake from drinking water and selected birth defects in offspring of participants in the national birth defects prevention study.</a> Environ Health Perspect. 121 (9), 1083-1089 (2013).</li><li>Huber, J. C., 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=Maternal+dietary+intake+of+nitrates,+nitrites+and+nitrosamines+and+selected+birth+defects+in+offspring:+a+case-control+study.">Maternal dietary intake of nitrates, nitrites and nitrosamines and selected birth defects in offspring: a case-control study.</a> Nutr J. 12, 34 (2013).</li><li>Phillips, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+nitrate+and+nitrite+in+foods+in+relation+to+infant+methaemoglobinaemia.">Naturally occurring nitrate and nitrite in foods in relation to infant methaemoglobinaemia.</a> Food Cosmet Toxicol. 9 (2), 219-228 (1971).</li><li>Moncada, S., Palmer, R. M., Higgs, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+physiology,+pathophysiology,+and+pharmacology.">Nitric oxide: physiology, pathophysiology, and pharmacology.</a> Pharmacol Rev. 43 (2), 109-142 (1991).</li><li>Gladwin, M. T., Crawford, J. H., Patel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+nitric+oxide,+nitrite,+and+hemoglobin:+role+in+blood+flow+regulation.">The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation.</a> Free Radic Biol Med. 36 (6), 707-717 (2004).</li><li>Gupta, S. K., 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=Recurrent+acute+respiratory+tract+infections+in+areas+with+high+nitrate+concentrations+in+drinking+water.">Recurrent acute respiratory tract infections in areas with high nitrate concentrations in drinking water.</a> Environ Health Perspect. 108 (4), 363-366 (2000).</li><li>Kross, B. C., Ayebo, A. D., Fuortes, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methemoglobinemia:+nitrate+toxicity+in+rural+America.">Methemoglobinemia: nitrate toxicity in rural America.</a> Am Fam Physician. 46 (1), 183-188 (1992).</li><li>Greer, F. R., Shannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infant+methemoglobinemia:+the+role+of+dietary+nitrate+in+food+and+water.">Infant methemoglobinemia: the role of dietary nitrate in food and water.</a> Pediatrics. 116 (3), 784-786 (2005).</li><li>Sanchez-Echaniz, J., Benito-Fernandez, J., Mintegui-Raso, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methemoglobinemia+and+consumption+of+vegetables+in+infants.">Methemoglobinemia and consumption of vegetables in infants.</a> Pediatrics. 107 (5), 1024-1028 (2001).</li><li>Virtanen, S. M., 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=Nitrate+and+nitrite+intake+and+the+risk+for+type+1+diabetes+in+Finnish+children.+Childhood+Diabetes+in+Finland+Study+Group.">Nitrate and nitrite intake and the risk for type 1 diabetes in Finnish children. Childhood Diabetes in Finland Study Group.</a> Diabet Med. 11 (7), 656-662 (1994).</li><li>Reimers, M. J., Flockton, A. R., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol-+and+acetaldehyde-mediated+developmental+toxicity+in+zebrafish.">Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish.</a> Neurotoxicol Teratol. 26 (6), 769-781 (2004).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Loucks, E., Carvan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+effects+of+developmental+ethanol+exposure+in+zebrafish.">Strain-dependent effects of developmental ethanol exposure in zebrafish.</a> Neurotoxicol Teratol. 26 (6), 745-755 (2004).</li><li>Bilotta, J., Barnett, J. A., Hancock, L., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+exposure+alters+zebrafish+development:+a+novel+model+of+fetal+alcohol+syndrome.">Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome.</a> Neurotoxicol Teratol. 26 (2), 737-743 (2004).</li><li>Li, J., Jia, W., Zhao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+nitrite+affects+zebrafish+valvulogenesis+through+yielding+too+much+NO+signaling.">Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.</a> PLoS One. 9 (3), e92728 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for chemical analysis of water and wastes. , (1983).</li><li>Loucks, E., Ahlgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+teratogenic+changes+in+a+zebrafish+model+of+fetal+alcohol+exposure.">Assessing teratogenic changes in a zebrafish model of fetal alcohol exposure.</a> J Vis Exp. (61), (2012).</li><li>Addiscott, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilizers+and+nitrate+leaching.">Fertilizers and nitrate leaching.</a> Agricultural Chemicals and the Environment, Issues in Environmental Science and Technology. , 1-26 (1996).</li><li>Su, Y. F., Lu, L. H., Hsu, T. H., Chang, S. L., Lin, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+treatment+of+methemoglobinemia+in+an+elderly+couple+with+severe+cyanosis:+two+case+reports.">Successful treatment of methemoglobinemia in an elderly couple with severe cyanosis: two case reports.</a> Journal of Medical Case Reports. 6 (290), (2012).</li><li>Harvey, M., Cave, G., Chanwai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatal+methaemoglobinaemia+induced+by+self-poisoning+with+sodium+nitrite.">Fatal methaemoglobinaemia induced by self-poisoning with sodium nitrite.</a> Emergency Medicine Australasia. 22, 463-465 (2010).</li><li>Nishiguchi, M., Nushida, H., Okudaira, N., Nishio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autopsy+case+of+fatal+methemoglobinemia+due+to+ingestion+of+sodium.">An autopsy case of fatal methemoglobinemia due to ingestion of sodium.</a> Forensic Research. 6, (2015).</li><li>Avdesh, A., Chen, M., Martin-Iverson, M. T., Mondal, A., Ong, D., Rainey-Smith, S., 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=Regular+Care+and+Maintenance+of+a+Zebrafish+(Danio+rerio)+Laboratory:+An+Introduction.">Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction.</a> J. Vis. Exp. (69), (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>White, R. M., 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+tool+for+chemical+screens.">Zebrafish: A tool for chemical screens.</a> Birth Defects Res C Embryo Today. 90 (3), 185-192 (2010).</li></ol>

The method described here demonstrates the utility of zebrafish in assessing the teratogenic potential of nitrite and nitrate. Compared to other vertebrates, zebrafish have advantages that include high fecundity, external fertilization, optical transparency, and rapid development. Available mutants that lack pigmentation (such as the casper zebrafish36) also help to enhance visibility of internal organs. It is also easy to generate transgenic zebrafish with reporter genes to facilitate analysis in live fish<su...

Teratogenesis is a process that disrupts the normal development of an embryo or fetus by causing permanent structural and functional abnormalities, growth retardation, or miscarriage in severe cases1. It can be caused by certain natural agents (teratogens), which interfere with embryonic development in multiple ways2. During human fetal development, common teratogens such as radiation, infectious agents, toxic metals, and organic chemicals have been reported to cause defects in epicanthic folds (the skin fold in the upper eye lid) and clinodactyly (curved finger or toe) through morphogenetic errors1.
Understanding the molecular mechanism of teratogenesis is the first step towards developing treatment and prevention. Several vertebrate models such as the African clawed frog (Xenopus laevis) and zebrafish (Danio rerio) have been used to determine the molecular pathways affected by teratogens. Previous studies have used zebrafish as a model for epidemiology, toxicology and teratogenesis3-7. Scholz et al. considered zebrafish as a &#34;gold standard&#34; for environmental toxicity assessment. This is due, in part, to the transparency of the zebrafish embryo, which allows researchers to visualize the developmental defect as it occurs8. Approximately 70% of human genes have orthologues in zebrafish, making zebrafish a desirable vertebrate model for studying human defects9.
Some epidemiological studies have suggested that nitrate and nitrite, commonly present in farm foods and water, are associated with birth defects or spontaneous abortions10,11, while other studies do not support this association12. Nitrate (NO3-) and nitrite (NO2-) are naturally present in soil and water. They are a source of nitrogen for plants and are a part of the nitrogen cycle13. Foods such as green beans, carrots, squash, spinach, and beets from farms that use fertilizers high in nitrate have significantly augmented levels of nitrate and nitrite7. Milk from cows fed with high nitrate foods and fish in high nitrate water (mainly from soil runoff30) can lead to humans consuming large amounts of nitrate and nitrite14. Nitrate and nitrite are also commonly used in food preservation, which dramatically increases the amount ingested by humans12.
Optimal levels of nitrate and nitrite play fundamental roles in physiological processes like vascular homeostasis and function, neurotransmission and immunological host defense mechanisms13-15. However, exposure to high levels of nitrate and nitrite may lead to adverse effects, especially in infants and children16. Ingested nitrate is further converted to nitrite in the oral cavity by microflora and in the gastrointestinal tract by intestinal microflora17.
Nitrate puts infants at a high risk for blue baby syndrome by oxidation of hemoglobin to methemoglobin, impairing hemoglobin from its oxygen carrying ability18. This results in the blue color of skin that extends to peripheral tissues in more severe cases. Inhibited oxygenation of tissues results in other symptoms, most severely leading to coma and death19,20. Similar symptoms are observed in babies and adults at higher concentrations of nitrate21. Elevated levels of methemoglobin in adults due to nitrite poisoning results in cyanosis, headache, breathing disorders31, and death if not treated due to complications related to vital tissue hypoxia32,33.
Nitrate ingested at higher levels can also result in various health complications. Childhood diabetes, recurrent diarrhea, and recurrent respiratory tract infections in children have been linked with high nitrate intake11,17,22. Chronic exposure to a high level of nitrate is associated with urination and spleen hemorrhaging. Acute high dose exposure to nitrates can lead to a wide spectrum of medical conditions like abdominal pain, muscle weakness, blood in stools and urine, fainting, and death11. Prenatal exposure to nitrate at high levels has been associated with neural tube and musculoskeletal defect11.
A recent report showed that treating zebrafish embryos with nitrite led to yolk sac edema, craniofacial and axial malformations, and swim bladder noninflation5. In this study, we demonstrate a method for treating zebrafish embryos with nitrate and nitrite to determine their teratogenic potential. Embryos were exposed to nitrite at different concentrations and different lengths of time. Ethanol was used as a positive control, since it is an established teratogen23. Our method showed that both high concentrations and long exposure times to nitrite were detrimental to survival and resulted in various phenotypes, ranging from mild (edema) to severe (gross developmental defects). Therefore, the zebrafish is a useful model for directly exploring the potential teratogenic effects of nitrate and nitrite on embryos to complement epidemiological studies.

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			<td height="20" style="height:20px;width:191px;">DREL/2010 instrument</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">26700-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Ethanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">KIMAX glass Petri Dish</td>
			<td style="width:185px;">VWR</td>
			<td style="width:104px;">89001-244</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">MS-222</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">E10521</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">NitraVer 5 Nitrate Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14034-46</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">NitriVer 3 Nitrite Reagent</td>
			<td style="width:185px;">Hach</td>
			<td style="width:104px;">14065-99</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Parafilm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">3-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Paraformaldehyde</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:104px;">158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">S6E stereomicroscope</td>
			<td style="width:185px;">Leica</td>
			<td style="width:104px;">10446294</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrate</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Sodium nitrite</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">S347</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:191px;">Transfer pipets</td>
			<td style="width:185px;">Laboratory Products Sales</td>
			<td style="width:104px;">L320072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Glass vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:104px;">03-338B</td>
			<td></td>
		</tr>
	</tbody>
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zebrafish as a model to assess the teratogenic potential of nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to 300 mM ethanol for 22 hr had no effect on survival (data not shown), consistent with previous reports5,23,26. This is expected, as ethanol is a known teratogen and served as a positive control. Observed phenotypes included pericardial edema, swim bladder noninflation (Figure 1), craniofacial defects, and developmental delay (data not shown).
Treatment with nitrite resulted in mild to sever...

Watch this Scientific Journal Video about Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite at JoVE.com

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The overall goal of this procedure is to treat zebrafish embryos with nitrite and determine its teratogenic effect. This method can help answer some of the key questions in the field of developmental toxicology such as assessing the teratogenic effects of chemicals on embryonic development. The main advantage of this procedure is that it uses zebrafish embryo which develop externally and can be observed under a microscope.For the experiments demonstrated here maintain wild-type zebrafish strains, such as the Tuebingen strain, at 28.5 degrees Celsius and a light-dark cycle of 14 hours and 10 hours, respectively. The night before harvesting eggs, add fish water and dividers into mating tanks and place male and female fish on separate sides of the divider. To ensure that enough eggs will be produced set up 30 pairs of fish.The next morning, after the lights turn on, remove the dividers to initiate mating. Check the mating tanks for eggs every 15 minutes. Once the fish lay eggs, use a tea strainer to harvest the embryos and combine them all into a large container with E3 buffer.Incubate the eggs at 28.5 degrees Celsius. At 1.5 hours post fertilization, or HPF, under a dissecting microscope use a plastic transfer pipette to discard unfertilized eggs that will appear opaque. Transfer 50 embryos each into 100 by 15 millimeter glass petri dishes containing 50 milliliters of E3 buffer in triplicate for each treatment condition.For example for 11 treatment conditions, prepare 33 dishes of 50 embryos each. To three petri dishes of embryos at 2 HPF remove the E3 buffer and add 50 milliliters of 300 millimolar ethanol diluted in E3 buffer. Cover the dishes with parafilm to minimize evaporation of ethanol.Incubate the embryos in the ethanol buffer for 22 hours at 28.5 degrees Celsius, then remove the solution and use E3 buffer to wash out the ethanol, swirling the dish several times. Repeat the wash two more times. To 3 petri dishes of embryos at 2 HPF remove the E3 and replace with 50 milliliters of fresh E3.For nine petri dishes at 2 HPF remove the E3 and add 50 milliliters of 1, 000 milligrams per milliliter of sodium nitrite dissolved in E3 and incubate at 28.5 degrees Celsius, replacing the sodium nitrite solution daily.After 46 hours, remove three of the nine sodium nitrite plates, remove the solution, and use E3 buffer to wash the plates three times. 24 hours later remove three different sodium nitrite plates from the incubater, remove the solution, and use fresh E3 buffer to wash the embryos three times. 24 hours later, perform similar wash steps for the last three plates.Next, for another set of three petri dishes, replace the E3 buffer with 200 milligrams per milliliter of sodium nitrite, then set up groups of three dishes of 50 embryos each with 400, 600, 800, and 1, 000 milligrams per milliliter of sodium nitrite. Incubate the embryos for 70 hours, replacing the sodium nitrite solution daily. After the incubation, remove the solution from the plates, and use E3 buffer to wash the plates three times.For another three petri dishes of embryos, replace the E3 buffer with 50 milliliters of 1, 000 milligrams per milliliter of sodium nitrate in E3 buffer. After exposing the embryos for 70 hours, use E3 to wash the embryos as previously demonstrated. During each day of exposure, under a stereo microscope count the number of dead embryos, looking for a lack of heartbeat and blood circulation, or a lack of mobility after one minute of observation.Remove the dead embryos. At the end of the experiments, to euthanize the larvae, use a transfer pipette to remove the E3 buffer, then add 50 milliliters of 0.2%MS-222 and incubate for 10 minutes. After one E3 wash to remove the MS-222, leaving some volume to properly pipette the embryo, add the larvae to 4%PFA, swirl the dish several times, and use a transfer pipette to place the larvae into a glass vial with enough PFA to fill the vial.Then fix the larvae in the refrigerator overnight. After fixation, use a stereoscope and a digital camera at 30X magnification to take pictures of the larvae. Orient the larvae so that the anterior is to the left and the dorsal surface is at the top of the field.Exposure to 300 millimolar ethanol for 22 hours had no effect on survival, consistent with previous results. As shown here, observed phenotypes included pericardial edema and swim bladder non-inflation. Cranial facial defects, and developmental delay were also observed and are not shown here.Treatment with sodium nitrite resulted in mild to severe effects on survival, depending on the concentration, with higher concentrations of sodium nitrite resulting in lower survival rates. This figure illustrates that at higher concentrations, such as 1, 000 milligrams per milliliter, longer incubation times also have a more severe impact on survival. Finally, as shown here, unlike nitrate treated fish, nitrite treated larvae have developmental defects.Once mastered, this technique can be done in 3 hours and it's important to remember to remove the embryos daily to minimize the contamination. Following this procedure, other methods, like in situ hybridization, can be performed to assess gene expression. This technique is communion for researchers to explore teratogenic potentials of chemicals on development in zebrafish.

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Immunology and Infection

Unit 1

Adverse Drug Effects and Chemical Toxicity

SCIENTISTS IN ACTION

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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). ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

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 ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.