Evaluation of nested polymerase chain reaction, pepsin/trypsin digest, and histology for the detection of Myxobolus cerebralis in salmonid fishes of Indiana and Michigan.

The objectives of this study were to survey fish from state hatcheries in Indiana and Michigan and to compare the nested polymerase chain reaction (PCR) test with pepsin/trypsin digest (PTD) and histopathology for the diagnosis of whirling disease (WD). One group of 40 and 9 groups of 60 fish heads, for a total of 580 samples, were submitted from hatcheries in Indiana and Michigan. These samples were examined for myxozoan spores using histopathology, PTD, and PCR tests. The heads were hemisectioned, and one half was fixed in 10% neutral-buffered formalin for histopathologic examination. The other half was processed for PTD. Some of the sediment was examined for the presence of myxozoan spores, and the rest was prepared for the nested PCR. Histologic examinations did not reveal Myxobolus cerebralis in any of the 580 samples. One hundred serial step sections, taken at 5-microm intervals, were evaluated for samples with positive spore identification by PTD. Histologic examination of these sections failed to reveal any myxozoan parasites. Myxozoan spores were observed in 16.9% (98/580) of samples in sediment after PTD. Spores morphologically similar to those of M. cerebralis were observed in 1.0% of PTD samples (n = 6). The nested PCR indicated that M. cerebralis spores were present in 0.5% of samples (n = 3). All 3 nested PCR-positive samples came from the same hatchery, however, spores of M. cerebralis were seen in 1 sample, spores of other myxozoan species were seen in the second sample, and spores were not seen in the third sample. When comparing the PTD to the nested PCR test, the PTD diagnosed 1 true positive, 5 false positives, 2 false negatives, and 572 true negatives, for a sensitivity of 33% and a specificity of 99.1%. Screening for M. cerebrallis infection in this study indicated a low prevalence of the disease. Histopathology was a very insensitive indicator of WD. The PCR test was highly specific and was used to differentiate spores of M. cerebralis from similar spores of other species.