Masters Research

Biofilms are surface-attached microbial communities encased by extracellular matrix. This mode of growth enables microbial survival during adverse environmental conditions. During my MS study, I investigated whether bacterial biofilms could serve as potential reservoirs for largemouth bass virus (LMBV), a fish virus that does not infect biofilm microorganisms. LMBV, a member of family Iridoviridae is one of the naturally occurring fish viruses, causing fatal disease of largemouth bass (Micropterus salmoides). The reservoir of LMBV is currently unknown. Laboratory investigations of this phenomenon consisted of mixing various concentrations of LMBV with lab-grown biofilms of Pseudomonas fluorescens ATCC 13525, a bacterium commonly found in aquatic environments. Control experiments consisted of mixing LMBV with the biofilm substratum in the absence of bacteria. LMBV was detected using standard and quantitative (real-time) PCR techniques. Virus infectivity was measured using the tissue culture infective dose (TCID50) technique. Artificially introduced LMBV was detected in lab grown biofilms by PCR and infectivity assays. Real Time PCR was able to quantify a maximum 6.22% of total LMBV in the adjacent bulk environment incorporated within biofilms. The results also indicated that LMBV associated with lab grown biofilms is directly correlated to the LMBV concentration in the adjacent liquid environment. Real Time PCR detected 100 fold less LMBV copy number than conventional PCR. Epifluorescent microscopy of biofilms grown in presence and absence of LMBV did not reveal any structural differences in bacterial community structure caused by virus introduction. LMBV recovered from biofilms were further observed under transmission electron microscope (TEM) and tested for infectivity using the TCID50 method. Biofilmassociation did not change the tissue culture infectivity of LMBV. In a separate experiment, natural biofilm samples were collected from 4 different ponds of A. E. Wood Fish Hatchery, San Marcos, Texas; all of which were screened negative for LMBV by PCR. However, lab studies showed that natural biofilm samples could acquire the virus when placed in LMBV-spiked pond water. This study demonstrates the importance of examining microbial and ecological niches as potential reservoirs in the control of viral diseases. The work was published in Journal of Aquatic Animal Health. The thesis could be found here