Detection of the intranuclear microsporidium Nucleospora salmonis in naturally and experimentally exposed Chinook salmon Oncorhynchus tshawytscha by in situ hybridization.

Nucleospora salmonis, an intranuclear microsporidian parasite of salmonid fish, is often difficult to observe in histological sections or wet mount preparations from lightly infected tissues because of its small size and location within the nuclei of lymphoblast-type cells. Diagnosis of infections by conventional light microscopy is directly dependent upon distinguishing different stages of the parasite from host cell nuclear material or vacuoles. To assist detection of stages of the parasite in tissues of its primary host, the Chinook salmon (Oncorhynchus tshawytscha), we developed a nonradioactive in situ hybridization (ISH) method. The new method was then used to detect N. salmonis among Chinook salmon after both natural and experimental exposures to the parasite. Probes derived from the small subunit ribosomal DNA (ssu-rDNA) sequence of the microsporidium were labeled with digoxigenin deoxyuridine triphosphate (DIG-dUTP) and hybridized to parasite DNA present in infected tissues. The ISH procedure effectively identified merogonic and spore stages of N. salmonis in paraffin-embedded tissues of clinically and subclinically infected fish. A Nucleospora-like microsporidium was also detected by ISH in tissues of a nonsalmonid fish, the English sole (Pleuronectes vetulus), using probes designed to a region of the ssu-rDNA of N. salmonis.

Molecular detection and phylogenetic placement of a microsporidian from English sole (Pleuronectes vetulus) affected by X-cell pseudotumors.

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.

Comparison of small subunit ribosomal RNA gene and internal transcribed spacer sequences among isolates of the intranuclear microsporidian Nucleospora salmonis.

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.

Relationships among members of the genus Myxobolus (Myxozoa: Bilvalvidae) based on small subunit ribosomal DNA sequences.

Sequences representing approximately 1,700 base pairs of the 18S rRNA gene from 10 different species in the genus Myxobolus were found to group them into 3 clusters that showed little correlation with spore morphology and size or host specificity, criteria currently used for both higher and lower taxonomic placements in the Myxozoa. Of the phenotypic criteria examined, tissue tropism was most correlated with the rRNA groupings observed. Spores of similar size and shape (Myxobolus cerebralis vs. Myxobolus squamalis) were distantly related in some instances, whereas spores with divergent morphology and size were sometimes found to be closely related (M. cerebralis and Myxobolus insidiosus). These initial investigations into the phylogenetic relationships of putative members of the genus Myxobolus clearly indicate the potential limitations of groupings based on size and morphological properties of the spores and host species infected. We propose that 18S rRNA gene sequences, combined with information on tissue tropism, host species infected, and developmental cycles in the fish and alternate host (when and if known) be given greater consideration in taxonomic placements of myxosporeans.

Small subunit ribosomal RNA sequences unite alternate actinosporean and myxosporean stages of Myxobolus cerebralis the causative agent of whirling disease in salmonid fish.

The alternating myxosporean and actinosporean stages of the myxozoan parasite Myxobolus cerebralis (Hofer 1903) from its salmonid fish and aquatic oligochaete hosts, respectively, were compared for sequence homology of the small subunit (18S) ribosomal RNA genes. A 99.8% similarity between the sequences of these two stages was substantially greater than that of M. cerebralis compared to two other Myxobolus sp. from salmonid fish. Our results are the first molecular evidence confirming the alternating stages, initially described by Wolf and Markiw [25] for the life cycle of M. cerebralis but found in two different taxonomic classes (Myxosporea and Actinosporea) are indeed forms of the same organism. Sequencing of rRNA genes of the actinosporean stage followed by development of specific primers for DNA amplification of the myxosporean stage, as in our study, should be applied to solve other myxozoan life cycles. Additionally, these approaches will in the future provide useful diagnostic reagents for the detection and study of this important group of fish pathogens.