Horseshoe crabs are remarkable, ancient animals—’living fossils’ that evolved 450 million years ago and have survived at least five mass extinctions. These sea-dwelling relatives of spiders can lay millions of eggs, they have four pairs of eyes, and their blood contains amoeba-like immune cells.
These cells from horseshoe crabs, called amebocytes, are indispensable for modern medicine: they are the only known source of limulus amebocyte lysate (LAL), a reagent extraordinarily sensitive to the liposaccharide toxins produced by gram-negative bacteria, which are responsible for 80% of cases of life-threatening sepsis in humans. Each year, around 11 million people die from sepsis worldwide. Since FDA approval in 1977, the LAL assay has been the standard test for detecting the contamination of medical devices by gram-negative bacteria.
But harvesting blood from wild populations is problematic. Every year, up to 600,000 Atlantic horseshoe crabs (Limulus polyphemus) are harvested in the United States alone for transport to the laboratory, bleeding to extract amebocytes for LAL production, and subsequent return to the ocean. The procedure has an estimated mortality rate of 30%, which puts considerable pressure on wild populations, already classified as a vulnerable species at risk from habitat fragmentation, global warming, and harvesting as feedstock for eels and whelks. Now, researchers may have solved this problem.1
“We wanted to find a way to keep horseshoe crabs healthy in the laboratory, in such a way that we could regularly and reliably obtain LAL from them for medical tests while safeguarding their well-being as much as possible,” says Lee Robertson, director of scientific communication and operations at Kepley BioSystems, Greensboro, NC. “Now that we have managed this, harvesting them from the ocean won’t be necessary anymore.”
In order to achieve this new method, the team developed a recirculating aquaculture system to house L. polyphemus. To enable regular extraction of amebocytes, the crabs were gently immobilized while a capped intravascular catheter was implanted through the pericardial membranes under sterile conditions. Every effort was made to safeguard the animals’ well-being: the entire procedure had a zero mortality rate, and the cathetered animals displayed the full range of natural behaviors in aquaculture while maintaining their body weight. Regular body checks and biochemical and cell composition analysis of their blood proved that they remained healthy throughout.
Now, amebocytes can be extracted up to 24 times per year from each horseshoe crab kept long-term in aquaculture, abolishing the need to harvest them from the ocean. The authors calculate that a single cohort of 45,000 L. polyphemus in aquaculture would yield enough LAL for all current needs and even allow its use in new clinical uses.
Testing Human Blood
The new method of extracting amebocytes from horseshoe crabs also solved a second problem. LAL is used to detect gram-negative bacteria on medical devices, but every attempt to use it to detect these pathogens in human blood had failed, due to the presence of substances in blood that inhibit the test.
However, says Robertson, “we also show that LAL from healthy and well-fed horseshoe crabs in aquaculture is of a higher quality, which for the first time makes it possible to do quick, affordable, and precise LAL assays on specially treated human blood.”
“This study offered tremendous opportunity to improve conditions of a threatened animal species that is intrinsically linked with human health,” says Rachel Tinker-Kulberg, PhD, the study’s lead author. “With a carefully developed diet and rigorous monitoring, we were excited to see the health parameters and blood quality respond positively. We have a significant advantage over current LAL collection methods in that our aquaculture-derived LAL has less batch-to-batch variability and it is a more sustainable approach that will yield a more reliable and higher quality product. The horseshoe crabs continue to be lively and active in their new environment and even laid eggs.”
Tests showed that LAL produced from L. polyphemus in aquaculture tends to have a higher activity than lyophilized and preserved LAL from commercial kits. This finding suggests that fresh LAL has a greater concentration of clotting factors necessary for defense against disease-causing bacteria, presumably because animals in aquaculture are better fed and healthier.
LAL derived from aquaculture can be reliably used to detect endotoxins in human blood. The new blood assay—to be described in greater detail in an upcoming study by the same authors—was sufficiently sensitive to detect toxins across the clinically relevant range of 1 to 500,000 colony-forming units (CFUs) per mL of blood.
“LAL has never before been used for patient diagnostics due to cross-reactivity and inhibitors in human blood,” says Anthony Dellinger, PhD, president of Kepley BioSystems. “Using high-quality and potent LAL from aquaculture, we have now developed a method that makes blood samples compatible with the LAL assay, allowing it for the first time to be used in early, potentially life-saving detection of bacteria and fungi in blood.”
For more information, visit Kepley BioSystems.
1. Tinker-Kulberg T, Dellinger K, Brady TE, et al. Horseshoe crab aquaculture as a sustainable endotoxin testing source. Front Mar Sci. Epub April 1, 2020; doi: 10.3389/fmars.2020.00153.
Featured image: Horseshoe crab (Limulus polyphemus). Photo © Jiri Vaclavek, courtesy Dreamstime (ID 118319905).