Geology topics

C.L. McKibben

Publications and source records attributed to C.L. McKibben.

4 recordsLinked to original sources

USGS2013

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum , the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Journal of Fish Diseases↗

USGS1999

Shedding of Renibacterium salmoninarum by infected chinook salmon Oncorhynchus tschawytscha

Laboratory studies of the transmission and pathogenesis of Renibacterium salmoninarum may describe more accurately what is occurring in the natural environment if test fish are infected by waterborne R. salmoninarum shed from infected fish. To quantify bacterial shedding by chinook salmon Oncorhynchus tschawytscha at 13??C in freshwater, groups of fish were injected intraperitoneally with R. salmoninarum at either 1.3 x 106 colony forming units (CFU) fish-1 (high-dose injection group) or 1.5 x 103 CFU fish-1 (low-dose injection group). R. salmoninarum infection levels were measured in the exposed fish by the enzyme-linked immunosorbent assay (BKD-ELISA). At regular intervals for 30 d, the numbers of R. salmoninarum shed by the injected fish were calculated on the basis of testing water samples by the membrane filtration-fluorescent antibody test (MF-FAT) and bacteriological culture. Mean BKD-ELISA optical densities (ODs) for fish in the low-dose injection group were not different from those of control fish [p > 0.05), and no R. salmoninarum were detected in water samples taken up to 30 d after injection of fish in the low-dose group. By 12 d after injection a proportion of the fish from the high-dose infection group had high (BKD-ELISA OD ??? 1.000) to severe (BKD-ELISA OD ??? 2.000) R. salmoninarum infection levels, and bacteria were detected in the water by both tests. However, measurable levels of R. salmoninarum were not consistently detected in the water until a proportion of the fish maintained high to severe infection levels for an additional 8 d. The concentrations of R salmoninarum in the water samples ranged from undetectable up to 994 cells ml-1 on the basis of the MF-FAT, and up to 1850 CFU ml-1 on the basis of bacteriological culture. The results suggest that chinook salmon infected with R. salmoninarum by injection of approximately 1 x 106 CFU fish-1 can be used as the source of infection in cohabitation challenges beginning 20 darter injection.

Diseases of Aquatic Organisms↗

USGS1997

Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

Journal of Aquatic Animal Health↗

USGS1997

Comparison of two fluorescent antibody techniques (FATS) for detection and quantification of Renibacterium salmoninarum in coelomic fluid of spawning chinook salmon Oncorhynchus tshawytscha

Two versions of the fluorescent antibody technique (FAT) were compared for detection and quantification of Renibacterium salmoninarum in coelomic fluid samples from naturally infected spawning chinook salmon Oncorhynchus tshawytscha. For the membrane filtration-FAT (MF-FAT), trypsin-treated samples were passed through 0.2 ??m polycarbonate filters to concentrate bacteria for direct enumeration by immunofluorescence microscopy. For the smear-FAT (S-FAT), samples were centrifuged at 8800 x g for 10 min and the pelleted material was smeared on slides for immunofluorescence staining Detected prevalences of Renibacterium salmoninarum were 1.8 to 3.4 times higher by the MF-FAT than by the S-FAT: differences were significant at p ??? 0.0002. The S-FAT consistently detected R. salmoninarum only in samples with calculated bacterial concentrations ??? 2.4 x 103 cells ml-1 by MF-FAT testing. Increasing the area examined on a filter or slide from 50 to 100 microscope fields at 1000x magnification resulted in the detection of a maximum of 4% additional positive samples by the MF-FAT and 7% additional positive samples by the S-FAT. In individual samples for which bacterial counts were obtained by both the MF-FAT and the S-FAT, the counts averaged from 47 times (??30 SD) to 175 times (??165 SD) higher by the MF-FAT. Centrifugation of samples at 10000 x g for 10 min resulted in a 4-fold increase in mean bacterial counts by the S-FAT compared with a 10-min centrifugation at 2000 x g, but the highest calculated bacterial concentration obtained by S-FAT testing was more than 6-fold lower than that obtained for the same sample by MF-FAT testing. Because of its greater sensitivity, the MF-FAT is preferable to the S-FAT for use in critical situations requiring the detection of low numbers of R. salmoninarum.

Diseases of Aquatic Organisms↗