Sequence-Based Analysis Aids in the Identification of the Causative Agent of Epitheliocystis in Elasmobranchs
Epitheliocystis is a bacterial disease known to affect the skin and gills of over 50 freshwater and marine teleosts and an increasing number of elasmobranch species. Although reports in wild fish are becoming increasingly common, clinical disease occurs primarily in aquaculture, particularly in early life stages.1-3 Mortalities are strongly influenced by environmental stressors and culture conditions. Diagnosis has traditionally been based on the presence of characteristic, bacteria-laden, intracellular "inclusions" visualized in wet mounts and histologic sections. Historically, lesions have been associated with members of the phylum Chlamydiae, order Chlamydiales, including several candidate species, but a growing body of genetic data suggests otherwise.2-7 Investigations of mortalities in aquarium quarantined, wild-caught spotted eagle rays (Aetobatus narinari) and bonnethead sharks (Sphyrna tiburo) revealed microscopic gill changes consistent with epitheliocystis disease. Lesions varied from epithelial cells with typical "inclusions" and peripheralized nuclei to massive numbers of unidentified hypertrophic cells with abundant, vacuolated, pale eosinophilic cytoplasm and enlarged, vesicular, centrally located nuclei. To enhance histological descriptions, molecular characterization of the bacteria was performed utilizing PCR, Sanger sequencing and in situ hybridization. Mixed pan-chlamydial primers were used to obtain bacterial 16S rRNA sequence data from both elasmobranch species.8 Results of molecular and phylogenetic analysis of near complete 16S rRNA gene sequences supports the presence of a bacterium belonging to the phylum Proteobacteria, rather than a Chlamydiae, in association with the ray and shark gill lesions. The Proteobacteria represent a large phylum of gram-negative bacteria grouped into five classes: the Alpha-, Beta-, Gamma-, Delta-, and Epsilonproteobacteria. While members of the Betaproteobacteria group have been recently reported from lake trout (Salvelinus namaycush) and farmed Atlantic salmon (Salmo salar) with epitheliocystis, sequences from the two elasmobranch species grouped more closely with the Gammaproteobacteria. This group contains many well-known bacterial families, such as the Enterobacteriaceae, with members capable of intracellular survival. Diagnostic investigations in fish intended to prove causation are frequently hindered by a lack of cross reactivity with commonly used mammalian antibodies and suitable culture media. As a surrogate to Koch's postulates, an in situ hybridization test was developed to demonstrate the presence of the bacterial nucleic acid within gill lesions. PCR primers designed from the proteobacterial 16S rRNA sequence were used to generate a digoxigenin-labeled probe. Positive hybridization signals were visualized in formalin-fixed paraffin-embedded tissue sections localizing the proteobacterial nucleic acid in the tissue and lesion of interest. Results of the ISH in conjunction with histologic and molecular evidence support a novel Gammaproteobacteria as a possible cause for epitheliocystis lesions in elasmobranchs.
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+ Student presenter
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