Abstract

In the summers of 1992, 1993, and 1994, epizootics of encephalitis and mass mortality occurred in Atlantic salmon (Salmo salar) smolts at a sea-farm in Ireland.⁵ In 1993 neurologic signs were observed within 6 to 8 weeks of transfer to the sea, and mortalities exceeded 90 percent of the total smolt stocks within 4 weeks of the onset.⁴

Seasonally variable mortalities of smolts at this sea-farm have been associated with variations in the prevalence and density of encephalitis and infection by a neurotropic parasite.^4,2 This parasite was detected in histologic sections of the central nervous system of affected and unaffected smolts.^4,2 In smolts from 1995, the distribution of the parasite in the brain was non-random, and parasites were detected with significantly greater frequency in the optic tectum of the mesencephalon than in other neuroanatomic subdivisions.² Encephalitis was non-suppurative, multifocal, and also non-randomly distributed, with significantly greater frequency of encephalitic foci in the myelencephalon. Based on ultrastructural studies, this neurotropic parasite has been identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, displaying distinct cortical zones of pinocytotic channels, cytoplasmic partitioning into ectoplasm and endoplasm, and large numbers of generative cells arranged singly or in cell doublets. Such morphologic features are characteristic of developmental stages of members of Family Myxobolidae, which includes the genera

Myxobolus, Henneguya, Thelohanellus, and Neothelohanellus.³ No mature spores were detected. Of these genera, neurotropic species of Myxobolus have been identified in salmonid hosts in other European locations. A few species of Thelohanellus and Neothelohanellus are neurotropic, although they have been identified in non-salmonid hosts. On the basis of ultrastructure and the detection of the parasite only in histologic sections of the central nervous system, it has been proposed that this parasite is a proliferative stage of a neurotropic myxosporean, possibly a Myxobolus species.² In order to substantiate the morphological classification of this myxosporean in the absence of detectable sporogony and to generate genomic sequence data for phylogenetic comparisons, polymerase chain reaction (PCR), Southern blot hybridization, and dideoxynucleotide-chain termination DNA sequencing were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of Myxobolus cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of infected brains. Five separate fragments of the SSU rRNA gene from the myxosporean were amplified. Amplicons were separated by agarose gel electrophoresis and detected by Southern blot hybridization using digoxigenin-labeled DNA probes generated from specific sequences of the SSU rRNA gene of M. cerebralis. Fragments ranged in size from 228 bases to 306 bases. Alignments to sequences of the SSU rRNA genes of M. cerebralis, M. squamalis, and M. insidiosus¹ demonstrated 99% homology to that of M. cerebralis. These results are indicative of SSU rRNA sequence homologies which support the morphologic assertion that the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon smolts is a neurotropic myxobolid. The amplification and sequencing of fragments of the SSU rRNA gene from a proliferative stage of this myxosporean provides a reliable and reproducible means of identification of some myxosporean developmental stages in the absence of detectable sporogony. The genetic information from this and similar studies will be useful in the development of probes for the identification of intermediate hosts and the elucidation of life cycles.

Acknowledgements

The assistance of the technical manager and staff of the fish farm is gratefully acknowledged. Timothy S. Gorton of the Department of Pathobiology, University of Connecticut, is acknowledged for his assistance with the creation of primers and the alignments of sequences. Karl B. Andree and Delores B. Antonio of the Fish Health Laboratory, University of California Davis, are acknowledged for their assistance with the initial experiments. This research was partially funded by grants from the Storrs Agricultural Experiment Station, College of Agriculture and Natural Resources, University of Connecticut, and from a private aquaculture company in Ireland.

References

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2.  Frasca Jr. S, SL Poynton, AB West, HJ Van Kruiningen. 1998. Epizootiology, pathology, and ultrastructure of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon (Salmo salar) in Ireland. Diseases of Aquatic Organisms In press.

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