Myxosoma cerebralis: trypsinization of plankton centrifuge harvests increases optical clarity and spore concentration
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Geology topics
Publications and source records attributed to M.E. Markiw.
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Eleven dye substances were tested for their ability to stain Myxosoma cerebralis and to be retained through either the pepsin-trypsin-dextrose or the plankton centrifuge method of releasing and concentrating spores. Silver nitrate proved to be the best; it produced a distinctive brown color that has been retained by spores through 2.5 yr of storage. The method has applications in histologic studies of whirling disease, in quantifying the efficiency of spore detection procedures, and in teaching.
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Rabbits were immunized with antigens extracted from mature spores or prespore stages of Myxosoma cerebralis , and the resulting antisera and their globulins were used in direct and indirect fluorescent antibody techniques. Both kinds of antisera reacted with homologous spores and with stages of the organism that precede spores. When tested for specificity against spores of 12 other myxosporidans the direct fluorescent antibody technique showed cross-reactivity with only one other and that was a species of Myxosoma . The indirect fluorescent antibody technique showed some reactions across generic lines. The antisera have application in studies of the parasite's life cycle and in diagnostics.
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The morphology of purified, mature Myxosoma cerebralis spores was examined by thin-section electron microscopy. Surface architecture corresponded closely to that described in an earlier scanning electron microscopy study of this organism (Lom and Hoffman, 1971). A uniformly thick spore wall is composed of finely granular, electron-lucent material. Intrawall "valvoplasm" at the sutural border region contains small, light spheroidal profiles with dense centers situated in a background matrix; such structures are absent in the intermediate wall areas. Ovoid polar capsules contain complex filamentous profiles within the peripheral matrix region. Individual filaments appear twisted into S-shaped configurations and contain moderately electron-opaque material within their lumina.
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An effective sequential procedure for recovery of Myxosoma cerebralis spores from infected trout was developed, and quantification of spores was carried out at each step of release and concentration. Methods are described for fresh and frozen material. Effective concentration of from 1100- to 9000-fold and an estimated efficiency of recovery of about 80% has been achieved. Tabular and graphic data are presented with recommendations for diagnostic applications. The immediate applications of these procedures are in implementing more effective detection and in preparing antigen for the immunologic studies that should provide the most sensitive detection.
Physical and biophysical methods of detecting spores of Myxosoma cerebralis were used in sequence on 87 individual fingerling rainbow trout ( Salmo gairdneri ) from two populations with a low incidence of infection. Physical methods of releasing spores from the organs of equilibrium, gill arches, or the axial skeleton gave an estimated rate of infection of 2.3–4.6%. Each succeeding step — pepsin digestion, trypsin digestion, and differential centrifugation through 55% dextrose solution — revealed additional infected fish. The final step of the sequence detected 10.5 times more infected fish than were found by examination of the organs of equilibrium or the axial skeleton with physical means; the true incidence was at least 24.1%. Procedures are described for using biophysical methods on 60-fish pools for hatchery inspection work and also on the more bony skeletons of mature fish.