Date of Award

2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

H. Wayne Taylor

Abstract

Streptococcus iniae was first isolated in 1972 from an Amazon River dolphin. It reappeared as a fish pathogen in Japan several years later. Since then it has steadily increased in incidence and host range worldwide, and is a known zoonotic agent. In the United States it is the most significant pathogen affecting growth of tilapia aquaculture. Histopathologic descriptions are incomplete and little is known concerning pathogenesis of the disease or virulence mechanisms of the organism. A composite of lesions was compiled from diseased tilapia evaluated by the Aquatic Animal Disease Diagnostic Laboratory, Louisiana State University from 1995--2001. In common with certain other streptococci, S. iniae causes polyserositis and meningoencephalitis. Previously unreported findings include otitis interna, gastroenteritis, and subcutaneous abscesses. Cocci-laden macrophages traverse olfactory, lateral line, and gastrointestinal epithelia, indicating possible routes of shedding. Gonadal infection suggests possible horizontal and vertical transmission. Streptococcus iniae isolates of fish and human origin were evaluated for virulence factors identified in other streptococci. No M-like protein was identified using PCR primers designed to amplify entire M protein genes or conserved anchor regions of M-like proteins. Findings were supported by electron microscopy, which revealed a capsule, rather than M protein. Lysine, with ruthenium red, proved superior to glutaraldehyde fixation alone, indicating a capsule composed of acid-polysaccharides. Using a hyaluronidase plate assay and DNase test agar, 79% and 52% of isolates showed activity for the two enzymes, respectively. Streptokinase-like activity was evaluated using a casein overlay containing human plasminogen and a broth assay using human and tilapia plasma. Streptococcus iniae showed no activity with human plasminogen. In tilapia plasma, results were equivocal, delaying but not completely inhibiting clot formation. The effectiveness of transposon mutagenesis using Tn917 was demonstrated in the production of non-hemolytic mutants. Transposition frequencies, plasmid loss, and frequency of mutant phenotypes were consistent with findings in other streptococci. Single primer PCR, yielded DNA sequence with a high degree of identity to segments of the S. pyogenes sag operon, encoding streptolysin S, an important cytolytic virulence factor in that organism. Unexpectedly, in a competitive elimination trial, the non-hemolytic mutant showed greater persistence than the wild-type strain.

ISBN

9780493328317

Pages

255

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