Theses and Dissertations


Title: DEVELOPMENT OF IMPROVED TECHNIQUES FOR THE DETECTION OF LARGEMOUTH BASS VIRUS

Name: McClenahan, Shasta D.

Degree: MS

Chair: Grizzle, J.

Resides: SFAA

University: Auburn University

Location: Auburn

Date: 2004

Pages: 57

Keywords: fish disease, largemouth bass, virus, detection

Abstract:

Largemouth bass virus (LMBV) is an iridovirus discovered during 1991 in Lake
Weir, Florida. This virus primarily infects largemouth bass Micropterus salmoides;
however, it has also been found in several other species of fish. The disease caused by
this virus typically occurs during the summer in adult largemouth bass. The disease
causes fish to lose equilibrium, swim near the water surface, and sometimes have
enlarged or reddened swim bladders. Largemouth bass virus is typically detected in cell
culture or by the polymerase chain reaction.
Experiments were performed with LMBV to determine the optimal conditions for
the detection of this virus. For isolation in cell culture, we tested 180 largemouth bass (2
types of samples per fish) on bluegill fry (BF-2) and fathead minnow (FHM) cells. The numbers of positive samples detected by each cell line were not significantly different;
however, the use of two cell lines increased the total number of positive samples
detected. We also determined that blind passage was important for detecting virus in low
quantities, and subcultivation was important to eliminate false-positive results.
Other experiments tested incubation temperature, adsorption time, and inoculation
method. The optimal method for the detection of LMBV was to remove the cell culture
medium prior to the addition of virus or homogenized organs, and allow the sample to
adsorb on the cell culture for 40 min with agitation on a shaker table. Following
inoculation, the optimal incubation temperature for the detection of LMBV was 30°C.
We developed a simplified polymerase chain reaction (PCR) method for the
identification of LMBV. For this PCR, cell culture supernatant was used in the PCR
reaction without any additional purification. For supernatants from cell cultures
inoculated with homogenized organs, only 70% of the LMBV positive samples were
positive when unpurified supernatant was used in PCR; however, when these
supernatants were diluted 1:500 in TE buffer, 85% of the failed supernatants became
-
PCR positive. For supernatants taken from blind passage or subcultivation, 93.8% and
97.2%, respectively, of LMBV positive samples were PCR positive with unpurified
supernatant. This simplified PCR method saves both time and money, and is accurate for
the detection of LMBV.

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