Name: use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples
Uploaded: 2011-05-10T12:00:00
Description: Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

The authors wish to recognize the technical assistance of Michelle Banes as well as Dr. Petrie-Hanson, Dr. Eels and Timothy Brown for their assistance developing and performing the immunohistochemistry. This project was funded by USDA CRIS project #MISV-0801310 and by the Mississippi State University College of Veterinary Medicine.

1. Bacterial challenge
<ol>
<li>Grow bacteria overnight at 30&deg;C in shaking Brain Heart Infusion broth.</li> 
<li>Stop water circulation in the fish tanks and add broth in concentration of 1 in 100 (10 ml per liter, for example).</li> 
<li>After one hour incubation, restart water circulation in the tanks to flush out the remaining bacteria.</li> 
</ol>
2. Sampling
<ol>
<li>Sampling times and organs depends of the objective of the study but in our study, we used the following sampling points...

<ol>
	<li>Plumb, J. A., Woo, P. T. K., Bruno, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Diseases and Disorders. , 479-479 (1998).</li><li>Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Pathology. , (2001).</li><li>Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Louisiana State University. , (1997).</li><li>Nusbaum, K. E., Morrisson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+35S-Labeled+Edwardsiella+ictaluri+into+Channel+Catfish.">Entry of 35S-Labeled Edwardsiella ictaluri into Channel Catfish.</a> Journal of Aquatic Animal Health. 8, 146-146 (1996).</li><li>Karsi, A., Menanteau-Ledouble, S., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioluminescent+Edwardsiella+ictaluri+for+noninvasive+disease+monitoring.">Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring.</a> FEMS Microbiology Letters. 260, 286-286 (2006).</li><li>Miyazaki, T., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+Edwardsiella+ictaluri+in+channel+catfish,+Ictalurus+punctatus+(Rafinesque).">Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque).</a> Journal of Fish Diseases. 8, 839-839 (1985).</li><li>Areechon, N., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Edwardsiella+ictaluri+in+Channel+Catfish,+Ictalurus+punctatus.">Pathogenesis of Edwardsiella ictaluri in Channel Catfish, Ictalurus punctatus.</a> Journal of the World Mariculture Society. 14, 249-249 (1983).</li><li>Dumpala, P. R., Lawrence, M. L., Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Edwardsiella+ictaluri.">Proteome analysis of Edwardsiella ictaluri.</a> Proteomics. 9, 1353-1353 (2009).</li><li>Thune, 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=Signature-Tagged+Mutagenesis+of+Edwardsiella+ictaluri+Identifies+Virulence-Related+Genes,+Including+a+Salmonella+Pathogenicity+Island+2+Class+of+Type+III+Secretion+Systems.">Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems.</a> Applied and Environmental Microbiology. 73, 7934-7934 (2007).</li><li>Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence mutant screening strategy for identification of bacterial virulence genes.</a> Applied and Environmental Microbiology. 75, 2166-2166 (2009).</li><li>Hook, E. W., Mandell, G. L., Douglas, G., Bennett, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Infectious Diseases. , 1700-1700 (1990).</li><li>Ainsworth, J., Capley, G., Waterstreet, P., Munson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+monoclonal+antibodies+in+the+indirect+fluorescent+antibody+technique+(IFA)+for+the+diagnosis+of+Edwardsiella+ictaluri.">Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri.</a> Journal of Fish Diseases. 9, 439-439 (1986).</li></ol>

This protocol details a technique for the in situ visualization of the catfish pathogen Edwardsiella ictaluri in histological sections. To the best of our knowledge, this is the first such protocol described.
The most critical step, in our experience, is the rinsing of the antibodies as described in the step 4.4 of the present protocol as insufficient washing may result in false positive.
The main problem with interpreting the results of this techn...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>Brain Infusion broth</td>
 <td>Becton Dickinson</td>
 <td>237200</td>
 </tr>
 <tr>
 	<td>MS222</td>
 <td>Argent Labs</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Whole mouse Serum</td>
 <td>Cappel</td>
 <td>55989</td>
 </tr>
 <tr>
 	<td>Goat anti-mouse FitC</td>
 <td>Southern Biotech</td>
 <td>1010-04</td>
 </tr>
 <tr>
 	<td>Permafluor</td>
 <td>Lab Vision</td>
 <td>Ta-030-FM </td>
 </tr>
 <tr>
 	<td>Potassium chloride (KCl)</td>
 <td>Sigma</td>
 <td>P-4504</td>
 </tr>
 <tr>
 	<td>Triton-X</td>
 <td>Sigma-Aldrich</td>
 <td>X100-6X500ML</td>
 </tr>
 <tr>
 	<td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>85040C</td>
 </tr>
 <tr>
 	<td>Potassium phosphate (KH2PO4)</td>
 <td>Sigma</td>
 <td>P-5379</td>
 </tr>
 <tr>
 	<td>Sodium Bicarbonate (NaHCO3)</td>
 <td>Sigma</td>
 <td>S-5761</td>
 </tr>
 <tr>
 	<td>Sodium chloride (NaCal)</td>
 <td>Sigma</td>
 <td>S-9625</td>
 </tr>
 <tr>
 	<td>Sodium phosphate dibasic(Na2HPO4)</td>
 <td>Sigma</td>
 <td>S-9390</td>
 </tr>
 
 </tbody>
</table>

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

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