New Tools for Diagnosing Disease

A new generation of molecular techniques promises rapid detection for the first time

Even the mysterious parasite known as MSX has begun to yield its secrets to scientists.

Dermo disease is inescapable on most harvestable grounds in mid-Atlantic estuaries: though oysters may be infected in the first several months, disease can progress slowly and many oysters will survive for at least a couple of years before succumbing, usually in the third year. But oysters may reach harvestable size sooner, depending on the availability of food and other environmental conditions.

The ability to rapidly detect the progress and intensity of Perkinsus marinus (Dermo disease) or Haplosporidium nelsoni (MSX) would give resource managers and commercial growers a chance to take proactive measures. For instance, in an area where disease is especially intense and oysters are not likely to reach harvestable size, it might make economic sense to move them to less salty waters and mitigate the progress of the disease (see "Modeling Around Disease").

Of equal importance is the ability to detect infections in adult oysters or hatchery-reared seed before they are transferred to regions free of disease -- such transfers in the past may have contributed to the spread of Dermo and MSX.

Certifying that hatchery oysters are free of disease must be a cornerstone of some restoration programs. How certain can we be that young oysters first spawned in hatcheries, placed overboard in Bay waters to harden, and then planted for growing to maturity (growout) are free of Dermo disease?

Multiple-stage testing depends on the availability of detection tools that we haven't had in the past -- tools that can serve as a ready alert to managers and growers that infection is present, or that it is spreading.

Molecular Probes

Since the 50s, scientists have depended on what is still the standard method for determining whether oysters are carrying Perkinsus marinus cells. Called Ray's fluid thioglycolate medium (RFTM), this procedure is labor intensive, time-consuming and relatively insensitive; moreover scientists have discovered in the last couple of years that it is not necessarily specific to P. marinus.

"There are several other Perkinsus species," says Gerardo Vasta, "and the RFTM analysis cannot distinguish among them. Moreover," he says, "there have to be numbers of cells in the oyster tissue to be able to detect the presence of Perkinsus." Reliance on such traditional analysis has shifted, largely because of support by the Oyster Disease Research Program for developing sophisticated molecular diagnostic technologies.

Vasta and other researchers -- among them, Muhammad Faisal and Eugene Burreson of the Virginia Institute of Marine Science and Christopher Dungan at the Cooperative Oxford Laboratory -- have been designing a suite of highly sensitive probes that can detect just one or two cells of a parasite in a newly set oyster, no bigger than a pencil point. On the west coast, Arthur Gee and Ralph Elston at Pacific Lutheran University in Tacoma, Washington, have developed a probe for detecting Pacific oyster nocardiosis (PON) -- this widespread disease consists of different Nocardia bacterial species that afflict adult Pacific oysters. PON can be especially lethal at high temperatures.

A molecular probe is a very sensitive way to identify an organism such as a Perkinsus cell. One scientist has characterized the probe as a flag that you plant on a particular cell or organism to distinguish it from a background of similar cells or organisms.

Probes tag the DNA or RNA of an organism and are specific to a species. Vasta and his co-workers, for example, have identified and isolated a segment of DNA that is unique to P. marinus, cloned the segment, and developed probes (short chains of nucleic acids) which will bind with this specific target. These probes are labeled with a more readily detectable molecule such as a radioactive compound.

They have also designed primers based on this DNA sequence for detection of P. marinus through a technique called PCR (polymerase chain reaction) amplification. To analyze for the presence of P. marinus by PCR, DNA is first extracted from the oyster's mantle, rectal tissues or oyster hemocytes, the disease-fighting blood cells in the circulating fluid (hemolymph) -- this initial extract contains a mix of DNA from both oyster and parasite. The PCR amplification codes the P. marinus DNA segment millions of times, thus raising the concentration of the target DNA segment in the sample to detectable levels on agarose gels stained with ethidium bromide. This method is specific for P. marinus and can detect a single parasite cell in 30 milligrams of oyster tissue.

Vasta has compared this PCR probe to the standard RFTM tests and discovered that about 15 percent of oysters deemed "disease-free" by the older method actually harbored the parasite, a serious concern for managers and growers.

The current test is only semi-quantitative, providing a general estimate of the intensity of the infection. The next step is truly quantitative PCR techniques, which Vasta, as well as Eugene Burreson, and others are pursuing with support from the Oyster Disease Research Program.

Antibody Probes

Another promising diagnostic tool is a rapid assay, or quantitative analysis, based on antibodies produced by a mouse or rabbit that has been inoculated with P. marinus cells. Antibodies are proteins -- they are generated in the blood of a host organism, when invaded by a foreign organism, in order to destroy invaders. Chris Dungan has been developing this diagnostic approach in which antibodies are labeled with a fluorescent dye or linked to a specific enzyme.

On the west coast, the oyster industry is largely based on hatchery-reared Pacific oysters (Crassostrea gigas). Though high summer mortalities are thought to be the result of multiple stresses, scientists are developing molecular diagnostic probes for identifying Nocardia bacterial populations which are suspected as a major cause of mortalities.

In the presence of any Perkinsus cell in an oyster tissue sample, the labeled antibodies bind to it. This reaction can produce a color change in the sample or a fluorescent complex detectable by microscopic examination. Dungan has successfully employed immunoassays which disclose the presence of Perkinsus cells in both oyster tissue and in Chesapeake Bay waters.

However, because his initial polyclonal detection antibodies bind to a range of Perkinsus species, he found the test not specific enough for P. marinus. New culture techniques for the oyster parasite will allow the production of pure P. marinus cell extracts, and the promise of rapid, precise and easily-conducted immunoassays for Dermo disease.

Even the mysterious parasite Haplosporidium nelsoni, known as MSX, has begun to yield its secrets to scientists, who have been frustrated for decades by their inability to grow the organism in laboratory cultures, or even to determine how it is transmitted from oyster to oyster. Many researchers feel that this protozoan may have an intermediate host, or "carrier," though this too is unknown (see "Modeling Around Oyster Disease"). Researchers in Burreson's lab have developed a DNA probe and a PCR-based assay specific for MSX that are far more sensitive to the presence of the parasite in oyster tissue or hemolymph than the standard microscopic examination techniques.

In order to identify the hypothetical carrier of MSX, they are using the PCR assay to detect the organism in environmental samples taken from areas where MSX is prevalent. A positive PCR result indicates that H. nelsoni DNA, and thus the parasite, is present somewhere in the sample. Positive samples are then placed on microscope slides and mixed with the MSX DNA probe in a procedure called in situ hybridization. Only the H. nelsoni cells will stain purple-black, showing the location of the organ-ism within the sample.

The ultimate result of this research will be a toolkit of rapid diagnostic tests that scientists, managers and growers can use in the field. These tests should lead to widespread improvements in managing around disease, reliable certification of disease-free spat and improved screening of disease-resistant adult oysters for brood stock.