Date Awarded

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Virginia Institute of Marine Science

Advisor

Jeffrey D. Shields

Abstract

Panulirus argus Virus 1 (PaV1) is an emerging disease in Caribbean spiny lobster Panulirus argus. It is considered a threat to the lobster industry in the Florida Keys. In order to understand the infection dynamics of the PaV1 virus in the lobster host, a sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for diagnosis of PaV1 in lobsters. The lower limit of detection using the 110-bp DNA probe in a dot-blot hybridization for PaV1 was 10 pg of cloned DNA template and 10 ng of genomic DNA extracted from hemolymph of diseased spiny lobster. The probe specifically hybridized to PaV1-infected cells in all the tissues tested. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with other virus samples tested. A primary culture of hemocytes was developed for in vitro study of PaV1. The modified Leibovitz L-15 medium supported the best survival of hemocytes in cultures. Hyalinocytes and semigranulocytes maintained higher viability (∼ 80%) after 18 days when cultured separately. Hyalinocytes and semigranulocytes were susceptible to PaV1 in vitro. Cytopathic effects (CPE) were observed as early as 12 h post-inoculation, followed by cell debris and cellular exudates in inoculated cultures. This assay was further developed to assess viral load in hemolymph of diseased lobsters using a 50 tissue culture infectious dose assay (TCID50) based on CPE. The histopathology and hematology of the spiny lobster infected with PaV1 were studied over time courses of experimental infection. The fixed phagocytes in the hepatopancreas were the primary site of PaV1 infection in spiny lobsters. Infection was subsequently observed in the hepatopancreas, gill, heart, hindgut, glial cells around the ventral nerves, as well as in the cuticular epidermis and foregut. as the disease progressed, the hepatopancreas became significantly altered, with hemal sinuses filled with massive amounts of cellular aggregates, including infected circulating hemocytes and a proliferation of infected spongy connective tissues. The virus caused significant decreases in total hemocyte density in later stages of infection and significantly altered several constituents in the hemolymph serum of diseased lobsters, including: glucose, phosphorus, triglycerides, and lipase.

DOI

https://dx.doi.org/doi:10.25773/v5-m7ca-dj49

Rights

© The Author

Download

Share Feedback

Share

COinS