Field applications of the second-generation Environmental Sample Processor (ESP) for remote detection of harmful algae: 2006-2007

We assess the application of the second-generation Environmental Sample Processor (ESP) for the detection of harmful algal bloom (HAB) species in field and laboratory settings using two molecular probe techniques: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). During spring 2006, the first time this new instrument was deployed, the ESP successfully automated application of DNA probe arrays for various HAB species and other planktonic taxa, but non-specific background binding on the SHA probe array support made results interpretation problematic. Following 2006, the DNA array support membrane that we were using was replaced with a different membrane, and the SHA chemistry was adjusted. The sensitivity and dynamic range of these modifications were assessed using 96-well plate and ESP array SHA formats for several HAB species found commonly in Monterey Bay over a range of concentrations; responses were significantly correlated (p < 0.01). Modified arrays were deployed in 2007. Compared to 2006, probe arrays showed improved signal:noise, and remote detection of various HAB species was demonstrated. We confirmed that the ESP and affiliated assays can detect HAB populations at levels below those posing human health concerns, and results can be related to prevailing environmental conditions in near real-time.

PCR-based assay for detection of four coral pathogens

Several microorganisms have been identified as pathogenic agents responsible for various outbreaks of coral disease. Little has been learned about the exclusivity of a pathogen to given disease signs. Most pathogens have only been implicated within a subset of corals, leaving gaps in our knowledge of the host range and geographic extent of a given pathogen. PCR-based assays provide a rapid and inexpensive route for detection of pathogens. Pathogen-specific 16S rDNA primer sets were designed to target four identified coral pathogens: Aurantimonas coralicida, Serratia marcescens, Vibrio shilonii, and Vibrio coralliilyticus. Assays detected the presence of targets at concentrations of less than one cell per microliter. The assay was applied to 142 coral samples from the Florida Keys, Puerto Rico, and U.S. Virgin Islands as an in situ specificity test. Assays displayed a high-level of specificity, seemingly limited only by the resolution of the 16S rDNA.

Rapid detection of white spot syndrome virus (WSSV) of Penaeus monodon by latex agglutination test using monoclonal antibodies

Latex beads were sensitized with monoclonal antibodies (MAb) rose against VP28 of WSSV. The optimum concentration of MAb required to sensitize the latex beads was 125 µg/ml. The sensitized latex beads were used to detect WSSV from PCR-positive stomach tissue homogenates obtained from infected shrimp. Stomach tissue homogenates from WSSV-infected shrimp agglutinated the sensitized latex beads within 10 minutes, while uninfected samples did not produce any agglutination, although non-specific agglutinations were observed in some samples. The analytical sensitivity, analytical specificity, diagnostic sensitivity and diagnostic specificity of the (LAT) agglutination test were assessed. The analytical sensitivity of the test was 40 ng of purified WSSV (2 µg/ml). The sensitized latex beads did not agglutinate with normal shrimp tissue or MBV-infected tissue homogenate. The test has a diagnostic sensitivity of 70 and 45%, respectively, compared to single-step and nested PCR. The diagnostic specificity of the test was 82%. This test is a simple and rapid on-farm test which can be used to corroborate clinical signs for the detection of WSSV in grow-out ponds.

Chemical changes in skin mucin as an index of early stages of spoilage in fish

Results of a preliminary investigation on the overall chemical nature of fish skin mucin in lung fish, Clarias batrachus, with special reference to water soluble low molecular weight compounds, are presented. Changes observed during room temperature spoilage have been studied with a view to present a new approach towards the assessment of freshness in fish inspection. pH of the mucin was distinctly alkaline (8.2) and remained unchanged during spoilage. Much of the nitrogen was found to be present in the glycoprotein fraction. Free amino acids and purine bases were present in appreciable quantities in the aqueous extracts which registered a significant increase after 10 hrs. Post-mortem increase in total solids was accompanied by a slight rise in protein nitrogen which may indicate tissue breakdown. Increase in TVN was also observed to occur earlier in the outside mucin as compared to the inside muscle. Presence of free sugars or sialic acid could not be confirmed nor was there any indication of cholesterol and lipoid material as stated in earlier literature.