James Giese

The title and subject matter for this month’s column were inspired by a symposium presented at PittCon 2001, the analytical laboratory instruments show that was held in New Orleans, La., March 4-9. “It’s a Mad, Mad, Mad, Mad Cow: Detection and Quantification of Prions” was arranged by Brian K. Nunnally of Eli Lilly and Co. and presented some of the latest information on testing for bovine spongiform encephalopathy (BSE).

LABORATORYBSE is known as “mad cow disease.” The chronic, degenerative disease affects the central nervous system of cattle. Worldwide there have been more than 178,000 cases since the disease was first diagnosed in 1986 in Great Britain. The disease has also been confirmed in native-born cattle in other European countries. However, over 95% of all BSE cases have occurred in the United Kingdom. BSE is not known to exist in the United States, although the similar chronic wasting disease is found in wild elk and deer in the U.S.

BSE belongs to the family of diseases known as the transmissible spongiform encephalopathies. These diseases are caused by a transmissible agent which is yet to be fully characterized. The diseases share the following characteristics: (1) a prolonged incubation period of months or years; (2) a progressive debilitating neurological illness which is always fatal; (3) examination of extracts of affected brain tissue by electron microscopy reveal the presence of scrapie associated fibrils; (4) pathological changes such as vacuolization appear in the central nervous system; and (5) the transmissible agent elicits no detectable specific immune response in the host.

A proteinase-resistant form of the proteins called prions have been proposed by Stanley Prusiner, the Institute for Neurodegenerative Diseases, University of Calif., as the transmissible agent that causes BSE and other similar neurodegenerative diseases. Prusiner, who made a presentation at PittCon 2001, was the recipient of the 1997 Nobel Laureate in Medicine for his discovery of prions (PrP)–a new biological principle of infection.

According to Prusiner, prions are the cause of Creutzfeldt-Jakob disease of humans, scrapie of sheep, and BSE of cattle. He outlined four new concepts that have emerged from studies of prions. First, prions are the only known example of infectious pathogens that are devoid of nucleic acid. All other infectious agents possess genomes composed of either RNA or DNA that direct the synthesis of their progeny. Second, prion diseases may be manifested as infectious, genetic, and sporadic disorders. This means that prion diseases have a wide range of clinical manifestations. Third, prion diseases result from the accumulation of PrP Sc, the conformation of which differs substantially from that of its precursor PrP C. Fourth, PrP Sc can exist in a variety of different conformations, each of which seems to manifest in a specific disease phenotype.

One of the difficulties encountered when testing for BSE is that the prions elicit no detectable specific immune response in the host. This has hindered the development of a live animal diagnostic test. Currently, there is no test to detect the disease in a live animal. The only means of diagnosis in live animals at present is by clinical signs by veterinarians. After death, microscopic examination of brain tissue is the primary laboratory method used to confirm a diagnosis of BSE. In addition to microscopic examination, there are several techniques used to detect the partially proteinase-resistant form of the prion protein, PrP Sc. These techniques are the Western blot test and immunocytochemistry.

--- PAGE BREAK ---

These diagnostic tests are based on the interaction of specific antibodies with the disease-causing protein, PrP Sc. The abnormal form of the protein, PrP Sc is more resistant to degradation by enzymes and can be differentiated from normal form, PrP C.

The Western blot test was developed by a Swiss company, called Prionics, (www.prionics.ch). The test is used to detect PrP Sc protein purified from treated brain material (obtained post mortem) by its molecular weight and reaction with specific antibodies. The company is developing the technique to increase the sensitivity and allow its application to tissues other than brain tissue, which may allow it to be used on live animals. Currently, only brain material testing has been fully validated.

The other technique is immunocytochemistry. It also is based on the detection of PrP Sc using specific antibodies. As in the Western blot test, post-mortem brain material is required. This test is performed directly on the tissue section and does not involve any protein purification steps. Immunocytochemistry can also work on other tissues, such as the third eyelid or tonsil of sheep. Research in Europe is trying to determine exactly how useful it would be to take biopsies of these tissues while the animal is still alive as a means of screening flocks. They are not perfect because it looks as if the genetic makeup of the livestock will influence how soon after infection one of these tissues becomes positive for abnormal PrP Sc.

Besides the tests provided by Prionics, the European Union has evaluated others. These include tests manufactured by CEA, Enfer Scientific, and the Veterinary Laboratories Agency. All the tests have only been evaluated on adult cattle showing clinical signs of BSE and confirmed to have the disease. They have not yet been proved to be effective in detecting infected animals during the long incubation period, except perhaps in the last few months before disease becomes apparent.

CEA, a research group in France, has produced a test based on the sandwich immunoassay technique, which is an adapted ELISA test. It also tests brain tissue collected post-mortem. Because it appears to be more sensitive than other tests, it has been used to see if it can detect abnormal protein in the wall of the small intestine of experimentally infected calves. It has also been used to see if it can detect abnormal protein in the brain and spinal cord of experimentally infected animals at an earlier stage than other tests. Stefan Weiss of CEA related at PittCon that the company has selected an RNA aptamer against purified PrP Sc preparations which interacts specifically with PrP Sc. The aptamer recognizes abnormal prions but not the normal type, PrP C. Weiss suggested that the aptamer could act as a powerful tool in diagnosis of BSE.

Enfer Scientific, Tipperary, Ireland, has developed an enzyme linked immunosorbent assay or ELISA test for BSE. The antibody used by the company was manufactured by another British company called Proteus. The assay, like many ELISAs, is very sensitive, and allows a rapid turnaround time with results usually available within 24 hr. The test can be done in high volumes. The company has been commercially testing spinal cord from cattle slaughtered for human consumption in Ireland for a couple of years, and has also tested cattle from BSE affected herds that have been killed by the Irish Authorities. The labs in Ireland test 2,500 samples per day with overnight turnaround. Abbott Laboratories in the U.S. has entered into an agreement with Enfer Scientific Ltd. for the marketing and distribution of two diagnostic tests used to detect BSE in cattle.

An assay, developed by U.S. Dept. of Agriculture/Agricultural Research Service chemist Mary Jo Schmerr, can detect the presence of abnormal prions in the blood of animals and humans. Schmerr, who works at ARS’ National Animal Disease Center in Ames, Iowa, and Andrew Alpert of PolyLC, Inc. in Columbia, Md., are co-inventors of the assay. ARS and Fort Dodge Animal Health of Fort Dodge, Iowa, have signed a Cooperative Research and Development Agreement (CRADA) to develop a test kit for use in diagnosing TSEs in animals.

--- PAGE BREAK ---

Another area of research interest are urine tests. These tests in live animals involve the detection of disease-specific physiological or metabolic markers. These are secondary substances produced as a result of infection. Such markers, apparently related, have been identified in serum and in urine. Whether or not they are specific to BSE or scrapie remains to be proven. Changes have been detected in the constituents of urine of CJD patients, sheep with scrapie and cattle with BSE. Research is being done to determine when the changes occur during the long incubation period of BSE.

None of the tests currently being developed have the prospect of providing reassurance that an animal or carcass is BSE free, only that evidence of infection can or cannot be detected at the time of sampling. Most of the claims for diagnostic tests for BSE have been made on the basis of checks on cattle that are at the end of their incubation period (clinically affected) or on cattle that may never have been infected. According to the European Union testing commission, it is essential that BSE tests are validated on animals that are known to be infected, but which are only part way through their incubation period, before they can legitimately claim to be of value for testing of cattle while alive on farm or dead after slaughter. Another factor in the use of the tests is which tissue should be tested. Most of the immunological tests that can detect abnormal PrP work best on brain tissue. A test which works on the brain of a clinically affected cow may not work as well when used with other body tissue.

Laboratory Cart provides for the easy transfer of equipment from countertop to cart. The stainless steel cart has two removable polished stainless steel pans that hold a variety of laboratory equipment and miscellaneous containers. The pans are two inches deep on three sides with one open side for easy transfer from benchtop to cart. The durable welded frame is one-inch square steel tubing with corrosion-resistant baked-on epoxy powder coating, charcoal in color. The four-inch rubber casters stabilize movement over rough, uneven floors. The cart withstands loads up to 400 lb. For more information, contact Labconco Corp., 8811 Prospect Ave., Kansas City, MO 64132 (phone 816-333-8811; fax 816-363-0130)—or circle 309.

Capillary Columns with Ez-Grips™, may boost laboratory productivity by providing simple, error-free installation and operation of single and dual GC columns. Depending on the application and operator experience, the patent-pending columns can reduce installation time by as much as 90% compared to standard columns. The columns may be used in most GC applications in research, contract, and environmental laboratories. The columns are fitted with a toolbar that uses a series of tabs to hold column elements during installation. These tabs simplify pre-column/retention gap and dual-column installation and ensure secure, error-free connection of single ferrule connectors. The tabs also help identify column inlet and outlet ends for proper installation. For more information, Varian, Inc., 3120 Hansen Way, Palo Alto, CA 94304-1030 (phone 650-213-8000; www.varian.com)—or circle 310.

Recording Thermo/Hygrometer, the Opus 10, is a compact instrument that provides both current and long-term measurement and data logging of interior air temperature and relative humidity. The instrument’s temperature range is 0–50ºC and the relative humidity range is 0–95%. The unit’s power is supplied for two years from one replaceable battery. The unit can be set to capture and store readings at programmable intervals. Internal capacity is available for up to 120,000 stored records. These stored records may be downloaded to a PC via use of an RS232 interface and supplied software. The unit is available with sensors housed within the console or with external sensors. For more information, contact Palmer Wahl Instrumentation Group, 234 Weaverville Hwy., Asherville, NC 28804-1228 (phone 800-421-2853 or 828-658-3131)—or circle 311.

--- PAGE BREAK ---

Powder Rheometer, the ManUmit, may be used to measure the flow properties of powders, granules, and wet mixes. The instrument will help manufacturers to avoid typical problems such as batch and source variation of ingredients, caking during storage, bridging in hoppers and sticking during production. The powder rheometer measures the flow properties not only of powders and granules such as flour, cocoa powder, and sugar, but also wet mixes such as hydrated stabilizer solutions and dough. According to the manufacturer, its innovative approach to the characterization of flow properties provides users and manufacturers with essential data on the rheology of these materials. This rheometer is said to be a user-friendly instrument that is capable of pre-conditioning the powder sample, and then repeating exactly the same flow test time after time. The operating principle is simple–a specially designed rotary blade, similar to a boat’s propeller, is rotated through the powder sample. By measuring the force, torque, and energy profile necessary to rotate the blade through the sample, the rheometer provides highly sensitive data on the sample’s flow properties. To ensure that the data accurately mimic real-life applications, the rheometer can precondition the sample for testing in an aerated or compacted states, as required, and then cause flow by slicing, shearing, compacting, or lifting. The user controls the test speed and the angle of the blade path so as to ensure the flow caused is complementary to the required sample characteristic, or the process conditions. For more information, contact Texture Technologies Corp., 18 Fairview Rd., Scarsdale, NY 10583 (phone 914-472-0531; fax 914-472-0532; www.texturetechnologies.com) —or circle 312.

Amperometric Detector, the BioScan 817, may be used for the determination of carbohydrates but is also capable of analyzing alcohols, sugar alcohols and other oxidizable substances. It may be operated in three modes: DC, pulse, and scan. In DC mode, a constant potential is applied to the working electrode, the analytes are oxidized or reduced in accordance with their electrochemical properties. Pulse mode works with three different working potentials, which are applied cyclically. This frees the electrode surface from any adhering reaction products after each measuring point and an activated surface is produced for the next measurement. The scan mode allows current/potential curves to be recorded. The unit is controlled by a PC and has a column oven for precise temperature control and a low-maintenance detector cell. For more information, contact Metrohm-Peak, Inc., 12521 Gulf Freeway, Houston, TX 77034 (phone 800-410-7118) —or circle 313.

Hygiene Contact Slides, called Hycheck™, allow for the collection of bioburden samples on surfaces and in liquids or semi-solids. These slides are designed with a hinged media paddle that easily bends to make contact with hard-to-reach surfaces. The surface of the paddle is double-sided, allowing separate samples to be taken on each side. The simple sampling procedure begins by pressing the slide to a surface or dipping it in a liquid. The slide is then incubated in an upright position so that the colonies can be counted on each agar surface. Media choices are available for a range of applications including sampling for molds, yeasts, bacteria, and coliform bacteria. A plastic vial screws into the handle of the slide, enclosing it for transport to the laboratory. For more information, contact BD Diagnostics Systems, 7 Loveton Circle, Sparks, MD 21152 (phone 410-316-4000)—or circle 314.

In-Line Refractometer, the PR-111, may be used to measure concentration of dissolved solids in various liquids. For more information, contact AFAB Enterprises, 2151 W. CR44, Eustis, FL 32726 (phone 352-483-1222) —or circle 315.

Portable Spectrophotometer may be used to perform color measurements using adjusted, included, or excluded ultraviolet and specular (gloss) compensation. The Minolta CM-2600d features d/8o, 52mm diameter integrating sphere, dual-beam geometry; 360 to 740 nm wavelength range with 10 nm measurement intervals; selectable 3 mm and 8 mm measurement areas; illuminated sample viewfinder; and a large results display. By acquiring specular included and specular excluded data simultaneously, the user can discriminate both color and appearance differences from a single scan of the sample. The unit achieves this feature through microprocessor-controlled, sequential flashing of multiple pulsed xenon light sources in place of the more traditional single light source. According to the manufacturer, this eliminates mechanical positioning of UV cut-off filters and multiple measurements, and replaces it with instantaneous calibration and adjustment of the UV energy in the light source. The unit measures 69 by 96 by 193 mm, weighs 670 g without batteries, performs up to 1,000 measurements on a single set of batteries. For more information, contact Minolta Corp., Instrument Systems Division, 101 Williams Dr., Ramsey NJ 07446 (phone 201-529-6060; fax 201-529-6070) —or circle 316.

Anaerobic Environments may be produced with the use of the Anoxomat. The new method for producing anaerobic and micro-aerobic environments in a  jar is based on the evacuation replacement method of McIntosh and Fildes. According to the manufacturer the new method produces a micro-aerobic environment with removal of 70% of the original atmosphere within 20 sec. For more information, contact Spiral Biotech, Inc., Two Technology Way, Norwood, MA 02062 (phone 781-320-9000; fax 781-320-8181; www.spiralbiotech.com) —or circle 317.

Internet Editor