Membrane Filtration – Fluorescent Antibody Staining Procedure for Detecting and Quantifying Renibacterium salmoninarum in Coelomic Fluid of Chinook Salmon (oncorhynchus tshawytscha)

Abstract

We developed a rapid method for detecting and quantifying the pathogen Renibacterium salmoninarum in coelomic fluid of spring chinook salmon (Oncorhynchus tshawytscha) by concentrating the bacteria on 0.2-μm polycarbonate filters and staining them with specific fluorescein-labeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphate-buffered saline containing Triton X-100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringe-mounted filter holders, reducing handling of the filters and possible loss of bacteria. The number of bacteria detected by the filtration – fluorescent antibody technique in a broth culture of R. salmoninarum ranged from 6.7 × 107to7.6 × 107/mL and was slightly higher than that determined by plate count (9.6 × 106/mL). Increasing the sample dilution or decreasing the number of microscope fields examined generally increased the variability of filter counts of R. salmoninarum. Using the filtration – fluorescent antibody technique, we detected the bacterium in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population.

Brood stock segregation of spring Chinook salmon Oncorhynchus tshawytscha by use of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody technique (FAT) affects the prevalence and levels of Renibacterium salmoninarum infection in progeny

“…Concentration of Renibactenum salmoninarum from ovarian fluid by membrane filtration, FAT staining, a14tl enumeration of the bacterium were done as described by Elliott & Barila (1987). We used their trypsin digestion method to facilitate sample filtration, and further modified the procedure as follows: nylon filters (5.0 pm, 13 mm diameter; Micron Separations, Inc., Westborough, MA, USA) were placed under the 0.2 Km polycarbonate filters to provide support during filtration, and the Eriochrome black T concentration in the counterstain was increased to 500 mg 1-l. From each female tested by the MF-FAT, a 0.5 m1 subsample of ovarian fluid was processed and filtered, and a total of 150 microscope fields (1000 X magnification) were examined per filter.…”

“…A direct relationship exists between ELISA absorbance values and the R. salmoninarum soluble antigen concentration , Turaga et al 1987, which generally corresponds to the severity of infection , Turaga et al 1987. The test developed by has been used in combination with quantitative (Elliott & Barila 1987) and semiquantitative versions of the direct fluorescent antibody technique (FAT; Bullock et al 1980) to more accurately determine the prevalence and levels of BKD in salmonid populations .…”

Comparison of Diagnostic Tests for Bacterial Kidney Disease in Juvenile Steelhead Trout (Oncorhynchus Mykiss)

“…Bacterial cultures have been made more effective with the identification of the proper incubation time 4 as well as the development of KDM-2 medial 3,18,35 and has been used routinely for confirmation of this infection. Fluorescent antibody testing has been well established 2,8,9,11,19 and is used commonly as a screening tool to determine if fish are infected with BKD. Polyclona1 23,25,33 immunoenzyme assays, 26,28,37 and western blot analyses.…”

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

“…The pellets were resuspended in 35 ml PBS (0.01 M, pH 7.4) supplemented with 0.1% (w/v) peptone (PBS-peptone). The concentrations of bacteria in the broth cultures were determined by a membrane filtration-fluorescent antibody staining procedure (Elliott & Barila 1987) modified for use with 4', 6-diamidino-2-phenylindole (DAPI) stain (Sigma, St. Louis, MO, USA). The modified procedure used 100 µl of DAPI stain at 1 µg ml -1 with PBS (0.01 M, pH 7.2) in place of the fluorescein-labeled goat-anti-R. salmoninarum antibody.…”