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.
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.
1. Bacterial challenge
2. Sampling
3. Dewaxing sections
4. Buffer preparation
5. Immunhistochemistry
6. Mounting
7. Microscope
8. Representative Results:
Because the auto-fluorescence of fish tissues is so high, these tissues will appear orange under the Tri-filter, conveniently providing a background for the bacteria.
Against this background, the individual FitC stained bacteria will appear as bright green dots and, at 200X their rod shape will be recognizable (Fig 1).
Bad sections can constitute false positive (due to a problem in the rinsing phase), in which case a large number of bacteria will appear to be present uniformly on the slide.
Figure 1.Micrograph of a muscle sections showing two E. ictaluri (arrows a and b) (200x).
Figure 2.Overall scheme of the experiment
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 technique is the auto-fluorescence of the tissue. However, it was found that using the Tri-filter mostly solved that problem as the vast majority of the non-specific fluorescence occur outside of the green fluorescent spectrum. Also, while this technique is quite sensitive, it can fail to detect low bacterial loads.
The authors have nothing to disclose.
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.
Name of the reagent | Company | Catalogue number |
---|---|---|
Brain Infusion broth | Becton Dickinson | 237200 |
MS222 | Argent Labs | |
Whole mouse Serum | Cappel | 55989 |
Goat anti-mouse FitC | Southern Biotech | 1010-04 |
Permafluor | Lab Vision | Ta-030-FM |
Potassium chloride (KCl) | Sigma | P-4504 |
Triton-X | Sigma-Aldrich | X100-6X500ML |
Bovine Serum Albumin | Sigma | 85040C |
Potassium phosphate (KH2PO4) | Sigma | P-5379 |
Sodium Bicarbonate (NaHCO3) | Sigma | S-5761 |
Sodium chloride (NaCal) | Sigma | S-9625 |
Sodium phosphate dibasic(Na2HPO4) | Sigma | S-9390 |