The Shiga toxin “A chain” possesses enzymatic activity (N-glycosidase). The mode of action of this toxin is that its enzymatic activity is able to depurinate a specific region of the eukaryotic ribosomal RNA. Such a modified ribosome is no longer able to bind the elongation factor that is needed to create the next peptide link on the polypeptide chain the ribosome is in the process of making. The synthesis of that protein stops where it is because the affected ribosome is now no longer in the protein manufacturing business. Because Shiga toxin is an enzyme, it simply goes looking for new substrate, i.e., other ribosomes to inactivate. Before too long, there is no protein synthesis going on in the cell at all (because all of the ribosomes have been inactivated) and the cell dies. If the appropriate receptors are present on the cells of the organism, the B-subunit of the Shiga toxin can facilitate entry into a new cell containing more healthy ribosomes for the Shiga toxin to inactivate and so on. In cattle, such cell to cell transmission is relatively ineffective, meaning that Shiga toxin cannot hop from one cell to the next, so the effects on protein synthesis are localized. In humans, it tends to go like gang-busters.
Cattle are subject to infection by bovine leukemia virus. Unchecked, this can be a devastating disease ending in death. However, it has been known for some years now that, in bovines, Shiga toxin is able to protect the animal against bovine leukemia virus because the N-glycosidase activity is able to inactivate the RNA-based genetic information that this retrovirus (RNA virus) needs for its own replication. This situation leads to an intriguing speculation. Most of the bovines we humans regularly feast upon spend their last days before slaughter in feedlots where they are quite stressed in consequence of being so crowded together, a condition known to favor infections of all kinds. It is quite conceivable [as has been suggested in the literature – Infection and Immunity (2003) 71(1):327] that under these conditions, evolution is hard at work spreading (with the help of the ever helpful E. coli) the Shiga toxin that helps keep bovines alive under such conditions. In other words, in the presence of Shiga toxin the bovine is able to live and grow with bovine leukemia virus as a chronic disease rather than as an acutely lethal infection. This situation may spare operating efficiencies for feedlot operators, but it creates problems for slaughterhouse customers and, ultimately, for meat consumers.
Microbiological testing is slow. A number of immunology-based assay developers have already come up with specific antibody-based kits able to detect Shiga toxin producing strains after suitable enrichment. Unfortunately, most of these still seem to require an overnight enrichment period before submitting the enrichment culture for testing. This adds considerable time to the assay, although it is still faster than straight microbiology. Nevertheless, if you think that this capability would be a useful addition to your operational capability, (and part of your HACCP plan) please contact Food Safety Analysis, LLC. The remote management of a selected employee (or group of employees) in the use of these kits may allow even a small operation to "borrow" the expertise it needs to perform such a testing program inhouse.
Far more exciting that immunological testing for Shiga toxin is the new method CDC has already come up with, a Shiga-toxin protocol using a synthetic DNA substrate, which allows quantitation of minute amounts of Shiga toxin. This protocol is said to take some two hours to a final result. Since it is a functional assay (measuring N-glycosidase activity itself) it will work equally well with any Shiga toxin. The only real problem with this assay is that it requires not just a mass spectrometer but a fancy one at that (MALDI MS/MS). These instruments are not cheap, neither to buy nor to keep. Nevertheless, Food Safety Analysis, LLC would welcome comments (see contact page) on this scheme from those in the meat or diary industries who might be interested in a rapid and extremely sensitive method that might make for a far more realistic and effective (i.e., no enrichment needed) CCP than the other tests out there right now (microbial isolation or antibody). In the event that there is sufficient interest, Food Safety Analysis, LLC will be able to put together a testing program using this new CDC protocol.
This methodology is expected to be of most interest to late-stage intermediate users of meat and diary products as an integral part of the HACCP program designed to validate the ingredients they purchase in order to ensure that their own end-users are kept safe. For example, the literature contains an instance of a hemolytic uraemic syndrome (HUS) outbreak involving a lot of Pasteurized milk. The milk was tested microbiologically and found to contain no Shiga toxin carrying microorganisms. Indeed, the milk was quite free of microorganisms at all, as expected of Pasteurized milk. Still, people were getting sick. Upon further investigation, this lot of milk was found to contain Shiga toxin. The conclusion was that this milk had been contaminated at one point with E. coli O157:H7, which was then subjected to a Pasteurization step that quite efficiently eliminated the contaminating organism but which was ineffective at inactivating the relatively heat resistant Shiga toxin that had been deposited in the milk prior to Pasteurization. The lesson to be gleaned from this example is that microbiologically clean does not necessarily mean safe (i.e., free of intoxicants). This point may become of greatly increased importance as advances in alternate pasteurization schema are brought out of the laboratory and onto the production floor.
Copyright © 2012 by M. Mychajlonka, Ph. D.