How does the horseshoe crab protect the public health?The horseshoe crab plays a vital, if little-known, role in the life of anyone who has received an injectable medication. An extract of the horseshoe crab's blood is used by the pharmaceutical and medical device industries to ensure that their products, e.g., intravenous drugs, vaccines, and medical devices, are free of bacterial contamination. No other test works as easily or reliably for this purpose.
Why are we concerned about bacterial contamination of pharmaceutical products?
Bacteria are everywhere-from our intestinal tract, to soils, rivers, and oceans. For the most part, bacteria are beneficial, acting to degrade organic waste and recycle nutrients back into the food chain. Sometimes, however, bacteria cause disease. We are all familiar with many specific bacterial diseases such as Salmonella food poisoning or more serious ones such as Cholera and Tetanus.
Bacteria that cause these diseases are referred to as pathogens and usually require an animal host for multiplication or transmission even though they may persist in a soil or aquatic environment for long periods of time. Other bacteria, generally considered non-pathogenic, can cause disease if they enter parts of our body that are usually bacteria-free, such as the bloodstream. In this case, even the ordinarily benign gut bacterium E. coli can cause sepsis and death. Therefore, the pharmaceutical industry takes great care in producing drugs, vaccines, and medical devices (items that deliver drugs or are implanted) that are sterile-free of living microorganisms. Unfortunately, certain bacterial components can, in and of themselves, be toxic. Thus, pharmaceutical manufacturers not only need to be sure their products are sterile but also non-toxic, i.e., contain no bacterial components left from pre-sterilization bacterial contamination.
The bacterial toxin of greatest concern is termed endotoxin, and it is able to withstand steam sterilization. Endotoxin occurs as part of the cell structure of a large class of bacteria that includes both pathogens and non-pathogens. This class of bacteria is known as Gram-negative, for their characteristic of being easily decolorized during the Gram staining procedure.
Over fifty years ago it was recognized that some sterile solutions, when injected into humans or rabbits, caused a fever or pyrogenic response. Scientists soon learned that these so-called "injection fevers" were caused by endotoxin (a potent pyrogen) left over from bacterial components that remained in the injected solutions after sterilization. Fortunately, it was also found that solutions could be screened for pyrogens by injecting small amounts of the batch into rabbits. If the rabbit exhibited a fever, the solution was deemed pyrogenic and was rejected. The rabbit or pyrogen test, along with a sterility test, became the two most important tools of the pharmaceutical industry. The Pyrogen Test employing rabbits is still in limited use, although as you will see below, an endotoxin test using an extract from the blood cells of the horseshoe crab is the predominant pyrogen test today.
How does the horseshoe crab protect itself from disease?
One may wonder why the horseshoe crab is sensitive to endotoxin and, furthermore, how does the crab benefit from this phenomenon? As we know, seawater is a virtual "bacterial soup". Typical near-shore areas that form the prime habitat of the horseshoe crab can easily contain over one billion Gram-negative bacteria per milliliter of seawater. Thus, the horseshoe crab is constantly threatened with infection. Unlike mammals, including humans, the horseshoe crab lacks an immune system; it cannot develop antibodies to fight infection. However, the horseshoe crab does contain a number of compounds that will bind to and inactivate bacteria, fungi, and viruses. The components of LAL are part of this primitive "immune" system. The components in LAL, for example, not only bind and inactivate bacterial endotoxin, but the clot formed as a result of activation by endotoxin provides wound control by preventing bleeding and forming a physical barrier against additional bacterial entry and infection. It is one of the marvels of evolution that the horseshoe crab uses endotoxin as a signal for wound occurrence and as an extremely effective defense against infection.
How are the horseshoe crabs collected? Are they harmed?
In shallow water, horseshoe crabs are collected by hand from a small boat using a clam rake, and the animals are not injured during this process. In deeper water, a dredge is used, and in this case, some horseshoe crabs do get injured. Injured crabs are released immediately and most will survive. It is quite common to find crabs with "scars" of old injuries that have healed.
Once the crabs are caught, they are transported to the laboratory from the fishing pier by truck. Sometimes a refrigerated truck is used, but as long as the animals are kept cool and dark during transport, they exhibit no adverse affects. During the bleeding process, up to 30% of the animal's blood is removed. Research has shown that once returned to the water, the horseshoe crab's blood volume rebounds in about a week. It takes longer for the crab's blood cell count to return to normal, about two to three months. Theoretically, crabs can be bled several times a year, but LAL manufacturers bleed them only once per year.
The Associates of Cape Cod and other LAL manufacturers have studied horseshoe crab mortality following the bleeding procedure and have found it to be quite low, less than 3% when compared to controls handled similarly but not bled. There are no records of a horseshoe crab dying during the bleeding process itself. Other studies conducted by government agencies and universities indicate a mortality of 10-15%. However, the horseshoe crabs in these studies were not handled as carefully as those collected by the LAL industry. Studies done by the Associates of Cape Cod show that not only do the crabs survive one bleeding, but that they can be captured year after year to donate their life-saving blood-much like human blood donors. In addition, their studies indicate that crabs, which are bled and returned to their spawning area, will continue their breeding activity without any ill effect.
Are there other compounds in the horseshoe crab that are of biomedical interest?
Besides LAL, a number of reagents and medically useful compounds have been discovered in the blood of the horseshoe crab. These include:
* A new test for fungal infections (G-Test) which is already in use in Japan and is expected to be licensed in the US next year
* An endotoxin-neutralizing protein which has potential as an antibiotic as well as an alternative endotoxin assay. This protein, ENP, can be made synthetically, which would eliminate the use of live horseshoe crabs for the LAL reagent.
* A number of other proteins that show anti-viral and anti-cancer activity.
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