| Summary: | 碩士 === 國立屏東科技大學 === 獸醫學系 === 87 === Abstract
Iridovirus infection in Taiwan and Peng-hu Island (Pescadores) resulted in high morbidity and mortality in marine cage-cultured red sea bream. Histopathological examination revealed that a large numbers of plump, ballooning cells of variable size randomly distributed in gills and spleen. Ultrastructurally, hexagonal virions were found in gill and spleen of these ballooning cells. The virus from gills and spleen of diseased fish was propagated in KRE-3、BF-2 and CHSE-214 cells. Cytopathic effect (CPE) was observed in BF-2 and KER-3 cells in the primary isolation. While the subsequent passage of the isolated virus in the same cells was proven to be a failure. Therefore, to further identification of the virus, PCR and nucleotide sequence analysis were used. Using two sets of each first PCR and nested-PCR primer pairs, DNA from iridovirus was amplified, producing sizes of 433, 332 bp, 335 and 195 bp respectively. To ensure accurate PCR amplification, the PCR products were restricted with restriction enzymes, Dra I and Bfa I. The PCR products (433 bp) were cleaved with Dra I with two fragment of expected sizes of 176 and 257 bp. Digestion of PCR products (332 bp) with Bfa I produced, two fragments of 283 and 49 bp, as expected. In the present study ,the sensitivity increased approximately 103 folds when the DNA was reamplified with a set of nested primers.
Intraperitoneal inoculation of the filtrate of the spleen, head kidney and gills homogenate of the infected fish to the normal red sea bream. At 3 days post-inoculation, the inoculated fish were dead. Grossly, spleen was markedly swollened. And use PCR, the result was the same with the natural infection.
In Taiwan and Peng-hu, the red sea bream with high mortality in marine cultured fish in recent years is not due to parasite and bacteria. In this study, we assess the applicability of several specific nested-PCR primers with subsequent enzyme analysis for the rapid and early diagnosis of iridovirus infections in red sea bream.
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