Current laboratory methods for biological threat agent identification.

The authors present an integrated approach for the identification of biological threat agents. The methods used have been used extensively in field exercises and during response to incidents of biological terrorism. A diagnostic system, which integrates the clinical diagnosis or medical intelligence with immunodiagnostic tests, rapid gene amplification assays, and standard culture, provides results of the highest quality and confidence. In the future, selected reagents and technologies will be distributed through a network of civilian and military laboratories.

Novel self-sampling culture method to monitor excretion of live, oral Shigella flexneri 2a vaccine SC602 during a community-based phase 1 trial.

A culture technique for assessing the excretion of live enteric vaccines was developed and verified during an outpatient safety trial of the Shigella flexneri 2a SC602 vaccine. Preliminary studies showed that SC602 could be recovered on Hektoen enteric (HE) agar plates that had been inoculated with seeded stools in one quadrant, held for up to 48 hours, streaked for isolation, and incubated for 24 +/- 6 hours. Recovery results on HE plates held at 4 degrees C and 25 degrees C were comparable; however, 4 degrees C better inhibited overgrowth before streaking. To prepare for a community-based vaccine trial, volunteers were trained to self-sample fresh stool and to swab-inoculate a single quadrant of HE agar. The trial began with 36 volunteers ingesting 2.5 x 10(4) CFU of SC602 in bicarbonate buffer. During the study, volunteers inoculated HE plates with fresh stool, stored the plates at 4 degrees C, and delivered them to the laboratory within 48 hours. A microbiologist then streaked the HE for isolation, incubated the plates at 35 degrees C +/- 2 degrees C for 24 +/- 6 hours, and identified presumptive S. flexneri colonies by slide agglutination with poly-group B antiserum. The attenuating genetic signature of SC602 was confirmed on selected isolates with the polymerase chain reaction with two specific DNA primer sets. Vaccine was detected from 20% of volunteers on day 1, increasing to 86% by day 4, and all but one vaccinee had excreted SC602 at least once by day 7. The latest initial SC602 detection occurred on day 7, the longest excretion occurred in one vaccinee on day 33, and excretion throughout the trial was intermittent. The trial was terminated by ciprofloxacin treatment on day 35. Volunteer compliance with self-sampling and HE plating was excellent because of the convenience of the method, and the advantage of immediate "bedside" plating was evident in the high recovery rate of excreted vaccine. This method can be applied in other trials of live enteric vaccines that require accurate sampling of excreted organisms.

Association of Cytophaga psychrophila with mortality among eyed eggs of Atlantic salmon (Salmo salar).

Although Pseudomonas fluorescens was the predominant bacterium associated with Atlantic salmon (Salmo salar) eggs incubated at the White River National Fish Hatchery (Bethel, Vermont) during January 1992, the fish pathogen Cytophaga psychrophila was isolated only from specific lots of eggs that displayed poor survival (35% eye-up).

In vitro growth of the bacterial kidney disease organism Renibacterium salmoninarum on a nonserum, noncharcoal-based "homospecies-metabolite" medium.

Laboratory and field trials were conducted to evaluate in vitro growth of Renibacterium salmoninarum in media without serum or charcoal. Growth of this bacterium, the cause of bacterial kidney disease (BKD) in salmonids, is accelerated by addition of a growth enhancing "metabolite" of unknown composition to KDM2 medium, the medium commonly used for isolation of R. salmoninarum. KDM2 medium supplemented with greater than 1% (v/v) metabolite enhanced growth even without addition of either serum or charcoal. Medium containing 5% metabolite (denoted Five-M) allowed optimal growth in laboratory studies and was further evaluated as a primary plating medium for recovery of the bacterium isolated from chinook salmon (Oncorhynchus tshawytscha) exhibiting clinical BKD. Recovery rates of R. salmoninarum using Five-M medium were 4% and 36% higher, respectively, than comparable rates using a serum-based medium for the two salmon populations evaluated. Five-M medium is an effective, inexpensive alternative to serum-based or charcoal-based media.

Molecular and genetic characterization of cytochrome oxidase-negative Aeromonas salmonicida isolated from coho salmon (Oncorhynchus kisutch).

Cytochrome oxidase variants of the bacterial fish pathogen, Aeromonas salmonicida, were characterized for genetic and molecular homology with cytochrome oxidase-positive isolates that typically induce furunculosis in salmonids. Protein and lipopolysaccharide moieties of the cytochrome oxidase-negative variants were similar to their typical counterparts, based on sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Pathogenicity of aberrant isolates to brook trout (Salvelinus fontinalis) was similar to typical cytochrome oxidase-positive isolates. Colorimetric deoxyribonucleic acid (DNA) hybridization in 96-well microplates yielded homology values greater than 82.5% for typical aberrant A. salmonicida isolates when photobiotinylated DNA for reference A. salmonicida 3.101 was used as a probe. The only variation of these isolates from typical A. salmonicida was a negative cytochrome oxidase reaction.

Isolation and phenotypic characterization of an oxidase-negative Aeromonas salmonicida causing furunculosis in coho salmon (Oncorhynchus kisutch).

An oxidase-negative Aeromonas salmonicida was isolated from coho salmon (Oncorhynchus) kisutch) suffering from an epizootic of furunculosis at the state hatchery near Belfair, Washington. Typical, oxidase-positive A. salmonicida was isolated concurrently from the same population of fish. Mortality was controlled with medicated feed treatments. Evidence supporting the identification of the two types of A. salmonicida is presented. Methods for the proper identification of oxidase-negative A. salmonicida isolates are evaluated.

Automated biochemical identification of bacterial fish pathogens using the Abbott Quantum II.

The Quantum II, originally designed by Abbott Diagnostics for automated rapid identification of members of Enterobacteriaceae, was adapted for the identification of bacterial fish pathogens. The instrument operates as a spectrophotometer at a wavelength of 492.600 nm. A sample cartridge containing 20 inoculated biochemical chambers is inserted in the path of the analyzing beam. Reactions are converted into a 7-digit octal biocode, relayed via a sensor to the memory module, and compared to biocodes preprogrammed in the memory. An identification is then printed. Presently, the Quantum II is capable of identifying human strains of Aeromonas hydrophila and Edwardsiella tarda. This study was initiated to determine the feasibility of expanding the use of the Quantum II to include identification of bacterial fish pathogens. Ten to 50 isolates of Edwardsiella ictaluri, Serratia liquefaciens, Yersinia ruckeri, Aeromonas hydrophila, typical Aeromonas salmonicida, and atypical Aeromonas salmonicida were utilized to determine optimal incubation conditions, relative stability of the biochemicals, and ability to obtain consistent biocode numbers. After sorting the octal biocodes from the 169 isolates into groupings using a cluster analysis technique, it was shown by a Chi-square goodness of fit test that isolates of a given species were sorted into the same cluster group at a frequency of at least 99%. Results of this study illustrate the usefulness of the Quantum II BID system for the identification of bacterial fish pathogens not contained within the system's memory module.