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

摘要

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 μm polycarbonate filters to concentrate bacteria for direct enumeration by immunofluorescence microscopy. For the smear-FAT (S-FAT), samples were centrifuged at 8800 × 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 × 103 cells ml-1 by MF-FAT testing. Increasing the area examined on a filter or slide from 50 to 100 microscope fields at 1000× 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 × 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 × 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.