Clarence J. Gibbs, Jr., Ph.D.: Bovine Spongiform Encephalopathies: An Update.
Bovine spongiform encephalopathy (BSE) is classified as belonging to the transmissible cerebral amyloidosis (TCA), the subacute spongiform encephalopathies (SSE) or more commonly prion diseases. BSE was first reported in 1986 in the United Kingdom and subsequently in the Republic of Ireland, Switzerland, France, Denmark, Portugal, Italy, Oman and the Falkland Islands. Although the basic histological features of BSE are similar to that of scrapie, vacuolation of neuronal perikarya is a less prominent feature of BSE. Of greater diagnostic importance in BSE is the occurrence of spongiform change. Neuronal cell loss is most notable in the vestibular nucleus complex. Cerebral amyloid as plaques appear as sparse focal deposits in a small proportion of cases. Diagnosis can be confirmed by examination of a single section of the medulla at the level of the obex where lesions occur predominantly in the nucleus of the spinal tract of cranial nerve V and the nucleus of the solitary tract. Amyloid fibrils consisting of protease-resistant protein, PrP, are demonstrable in extracts of brain and cervical spinal cord. BSE-like SSE has also been identified in five species of wild Bovidae in captivity, two species of wild Felidae and the domestic cat in the United Kingdom. BSE is a common source epidemic that peaked in 1995 and has markedly subsided since that time. Epidemiological studies by Wilesmith et al., strongly support the notion that BSE originated from infected meat and bone meal cattle feed concentrate. In spite of the significant decline in BSE in the United Kingdom, BSE continues to impact as a potential public health hazard and a major veterinary concern, e.g., Germany has banned milk from the United Kingdom, the European Commission has ruled that the United Kingdom must slaughter 340,000 cattle and Switzerland will cull 230,000 cattle. Switzerland has had 223 cases of BSE since 1990 and Portugal has reported 51 cases--of which eight were imports from the United Kingdom, and Ireland has reported 14 new cases within the September-October 1996 time frame.
The recognition during the past two years of 14 atypical cases of Creutzfeldt-Jakob disease and one atypical case in France and the recent report by Collinge et al. on the analysis of prion protein migration as visualized on Western immunoblotting has led to the conjecture that these atypical cases in humans are likely to have been caused by exposure to infected cattle brain or spinal cord before 1989 at which time these specified offals were banned from entering the food chain.
George J. Jackson, Ph.D.: Pathogenic Protozoa in Food and Drink, An Update: 1681-1996.
Parasitic protozoa periodically make the news as causes of food-and-drinkborne human disease. Well publicized were Chicago's waterborne amebiasis in 1933, Minnesota's foodborne giardiasis in 1979, and Milwaukee's waterborne cryptosporidiosis in 1993. So was cyclosporidiosis during spring and summer 1996 at various US and Canadian locations from the Rocky Mountains east; imported raspberries have been associated epidemiologically with this diarrheal illness. Such better known outbreaks are far from being the only incidents of concern. To formulate strategies for preventing outbreaks, the biology of the incriminated parasites must be understood, as must the epidemiology of the infections caused. Despite significant observations dating back to 1681 and the invention of the microscope, practical methods for detecting and inactivating the resistant environmental survival forms (cysts, oocysts) of these parasites are only lately being developed.
Amy Patterson, M.D.: Xenotransplantation: Public Health Perspectives.
The unmet and growing demand for human grafts for transplantation coupled with recent advances in the science of immunology and molecular biology have catalyzed renewed interest in xenotransplantation, or the use of live animal cells, tissues and organs in lieu of human organs to treat a wide variety of human diseases. One of the major public health dilemmas raised by xenotransplantation is how to counterbalance the potential promise of a new technology to treat a wide variety of human disease and to address the shortage of human organs now available for transplantation, with the potential infection of patients and their contacts with both recognized and unrecognized infectious agents transmitted from xenografts? The human allograft experience has demonstrated the transmission of infections from donor to recipient in the transplant setting. Transplantation of animal grafts into humans disrupts the normal physical and immunologic defense mechanisms that usually limit the spectrum of microbes transmissible from animals to humans. The capacity to produce severe disease is unpredictable when a microbe is transmitted from its natural and evolutionarily adapted host into a new host species. The recipient of a xenotransplant is potentially at risk with infectious agents already known to be transmissible from animals to humans as well as infectious agents which may become apparent only in the transplant setting and which may not be readily identified with current diagnostic tools. Of additional concern is the potential transmission of these infectious agents from the transplant recipient to his/her contacts and to the public at large. The United States Public Health Service agencies including the Center for Disease Control, the Food and Drug Administration, and the National Institutes of Health have collaborated in addressing the public health infectious disease risks posed by xenotransplantation The Draft PHS Guideline on Infectious Disease Issues in Xenotransplantation is the set of recommendations developed to minimize the risk to the public of human disease due to known and emerging infectious agents arising from xenotransplantation. A brief summary of the Draft Guideline will be presented.
Linda Tollefson, D.V.M., M.P.H.: Antimicrobial Resistance Monitoring of Zoonotic Enteric Pathogens.
Expert scientific groups such as the Institute of Medicine, the American Society for Microbiology and the Infectious Disease Society of America have expressed concern about the national and global increase in antibiotic resistance and the complex issues surrounding this increase in both the community and institutional settings. A recent joint advisory committee formed from the Veterinary Medicine and the Division of AntiInfective Drugs Advisory Committees recommended that FDA develop an antimicrobial resistance monitoring system as a postmarketing activity to help ensure the continued safety of antimicrobials approved for use in foodproducing animals. In 1995, the FDA, the Centers for Disease Control and Prevention (CDC), and the USDA implemented a national surveillance program to monitor changes in antimicrobial susceptibilities of zoonotic pathogens from human and animal clinical specimens, from healthy farm animals, and from carcasses of foodproducing animals at slaughter plants. The monitoring system is designed to detect emergence of resistance before it becomes widely disseminated. Veterinary testing is conducted at USDA's Agricultural Research Service's National Animal Disease Center in Ames, Iowa. Salmonella was selected as a sentinel organism. CDC's Foodborne Disease Laboratory is conducting the testing of human isolates, both Salmonella and E. coli O157:H7, submitted by 14 State Public Health Laboratories. Testing of human Campylobacter jejuni and C. coli isolates will be added in January, 1997. This presentation will describe the monitoring program and current summary findings.
Dennis Lang, Ph.D.: Helicobacter pylori - Status of Research and Vaccine Development.
Helicobacter pylori has been established as a causative agent of gastritis and gastric and duodenal ulcer disease and has been linked to gastric adenocarcinoma and lymphoma. It infects approximately 40% of the adult population in the U.S. and close to 100% of the population of developing countries in Asia, Africa and South America. It is frequently labeled as an ýemergingÌ disease because it has only recently been identified, isolated, and studied as a human pathogen. Since its isolation in 1983, H. pylori has received much attention from the research community in the U.S. and to an even greater extent in Europe. This attention has resulted in rapid progress in our understanding of the biology and pathogenesis of the organism. This progress was recognized in 1994 by the convening of an NIH Consensus Development Conference that made specific recommendations for the treatment of peptic ulcer disease by combinations of antibiotic and anti-secretory drugs. The FDA has recently approved a non-invasive breath test for the diagnosis of infection, and a combination of clarithromycin and omeprazole for the purpose of eradicating H. pylori as an ulcer treatment. The availability of a relatively easy diagnosis and an effective therapy will, hopefully, have a major impact on reducing ulcer disease in the United States and elsewhere.
Despite significant progress, much research is needed to answer important questions that remain regarding the pathogenesis of H. pylori. How does the organism colonize gastric mucous membranes, interact with gastric epithelial cells to produce changes in the cell's cytoskeleton or influence gastric lymphocytes? How does the organism manage to persist in the stomach despite a vigorous cellular immune response? Does immune tolerance result? What is the basis for its action as a co-factor in the development of adenocarcinoma? It is not known how it is spread, although its age relatedness and greater prevalence in developing countries suggest a fecal-oral route. The reservoir for human infection has also not been established. Does the resultant achlorhydria associated with infection with H. pylori increase susceptibility to other enteric infectious agents? How rapidly does drug resistance develop, and what is the mechanism? Animal model and human studies will be required to answer these and other questions.
The private sector is actively pursuing the development of vaccines. Animal studies have shown that a vaccine consisting of recombinant urease, and using cholera toxin as adjuvant, not only prevented infection with H. pylori, but could cure infected animals. Human trials are underway, but it is unclear if a vaccine would be cost-effective. A cost-benefit analysis has not been done. If it were cost effective in the U.S., it may not be in developing countries that do not have the infrastructure or financial resources to deliver even basic childhood vaccines.
This presentation will focus on recent research findings and the status of commercial efforts to produce a vaccine.
Peter Feng, Ph.D.: Rapid Methods for the Detection of Foodborne Pathogens.
The analysis of foods for microbial pathogens and toxins is a highly challenging task. Foods come in complex matrices and include an infinite array of ingredients, many of which can interfere with analysis. Raw food products, may also contain large populations of normal microflora, which often mask the presence of pathogenic species that usually occur in low numbers in foods. To overcome these problems, conventional microbiology has relied on cultural enrichment of foods in various selective and nonselective media to amplify and select for specific pathogens. Although enrichment is effective, it is too time consuming for real-time assessment of microbial safety of foods. Recent advances in biotechnology have introduced a number of innovative technologies that greatly reduced assay time. Known as "rapid methods" they have had a tremendous impact on food diagnostics. However, since "rapid" is subject to various interpretations, it encompass a large group of technologies, which include: miniaturized biochemical identification; simple modifications of existing microbiological methods; antibody- and nucleic acid-based methods; and automated instrumental systems. Rapid methods are usually more sensitive and specific and faster than conventional assays. However, they still lack efficiency for direct application to food analysis and have to rely on time-consuming cultural enrichment to amplify the target pathogens. Furthermore, rapid methods are equally susceptible to interference by normal flora and by the complexity of food matrices and must be thoroughly evaluated before use. Currently, only rapid methods that have been evaluated by AOAC collaborative studies are approved for use in food analysis. However, approved methods may be used only for preliminary screening, where negative results are regarded as definitive, but all positive results are considered only presumptive and must be confirmed by standard methods.
Walter E. Hill, Ph.D.: Genetic Detection Methods to Identify Foodborne Pathogens.
The detection of microbial hazards in foods requires rapid, sensitive, and specific methods. While traditional microbiological methods rely on enrichment and selection followed by isolation and biochemical or immunological characterization, genetic methods focus on the detection of specific genes that play a role in pathogenesis. Within the past 20 years, genetic methods, based on DNA hybridization have been used to demonstrate the presence or absence of specific genes in foodborne microorganisms. DNA colony hybridization can reveal the presence of specific genetic information in the cells of a bacterial colony by using a DNA molecule (gene probe) to test for the presence of particular nucleotide sequences representing the capacity to produce a pathogenic effect. Just a decade ago, the polymerase chain reaction (PCR) was shown to be convenient tool for the million-fold amplification of specific DNA regions to detectable levels. While this technique requires neither microbial growth nor the isolation of pure cultures, it does require relatively clean DNA to allow the enzymatically catalyzed repeated duplication of a section of DNA. this method can be targetted to the specific genes involved in causing disease. Hence, a few cells of pathogenic strains of bacteria and viruses can be detected among a high background of indigenous microbial flora which may be non-pathogenic. These methods have been used to detect such pathogenic microbes as Escherichia coli O157, Salmonella, Listeria monocytogenes, Vibrio cholerae, Yersinia enterocolitica, and, more recently, the coccidian parasites, Cyclospora, and Eimeria. Furthermore, these methods can be extended to geneticially characterize individual strains to yield important epidemiological data.
Scott Fritschel, Ph.D.: Development and Commercialization of Polymerase Chain Reaction (PCR)based Methods for the Detection of Foodborne Pathogens.
The Polymerase Chain Reaction (PCR) is an extremely valuable tool for producing many copies of a targeted DNA sequence. PCR has been in extensive use in research laboratories since its discovery in 1988. Until recently, however, routine quality assurance and food testing laboratories have not employed PCR. This paper will discuss the development of standardized assays in formats appropriate for the routine food testing laboratory which employ PCR techniques to detect the presence of foodborne pathogens such as Salmonella, E. coli O157:H7, and Listeria monocytogenes. Additional work to extend the methods to the confirmation of presumptive colonies from cultures will also be described.
Myron Sasser, Ph.D.: Detection of Pathogens by Automated Fatty Acid Analysis.
Bacteria make more than 300 different fatty acids, most of which do not occur in plant or animal materials. The fatty acids may be analyzed by automated gaschromatography and the compositions of organisms can be compared to databases through pattern recognition, allowing naming of the organisms. Thus, rapid identification of cultures can be performed after obtaining them in pure culture. There is an approved AOAC method for use of the system in identification of Vibrio. Also, several publications show the system being used for strain ýtypingÌ in clinical settings.
Direct assay from tissues is the objective of current research which separates bacteria from background materials prior to extraction of the fatty acids. The separation is done through filtration and differential centrifugation. Enhanced sensitivity of the gas chromatographic system is through injection of a larger percentage of the extract, using splitless mode operation and through use of an electron capture detector. Mixed populations cause problems in identification, but software algorithms to deal with this are being developed. Since the organisms grow under variable conditions, new databases are being constructed to cover most of the foodborne pathogens from direct assays.
Carol Gravens, M.A.: Automated ELISA for Detection of Foodborne Pathogens.
Enzyme linked immunosorbant assay techniques for many years have been a recognized technology for screening pathogens in foods. Commercial ELISA test kits introduced in the 1980s offered the food microbiologist the advantages of rapidity, accuracy, and convenience; however, the tedious manual requirements of this technology were significant. In the 1990s test kit improvements have evolved, and automated instruments were developed addressing many of the manual demands of ELISA technology. Immunoassay instruments provide computer-controlled testing, analysis of data, and simultaneous processing of multiple assay types. They also afford time savings and ease of use. With on-line reading of results and computer generated reports, automated instruments present more objective analysis than manual methods. For these reasons automation has revolutionized the immunoassay diagnostics industry. Commercial systems range from microtiter formats, such as TekTIME and MR 7000, to multiparametric, self-contained reagent systems including VIDAS and OPUS. The next generation of automated immunoassays is expected to be even more versatile and sensitive as automated formats expand to include immunocapture and amplification techniques.
J. William Costerton, Ph.D.: The Incidence and Control of Microbial Biofilms in Device-Related Infections.
The direct examination of the surfaces of medical devices that have become foci of devicerelated infections show clearly that these infections are caused by bacteria growing in biofilms. Bacteria within biofilms are remarkably resistant to clearance by host defense mechanisms (antibodies and phagocytes) and to killing by antibiotics. We have used direct methods to monitor the development of biofilms on implanted devices and to deduce the routes by which bacteria growing in this mode of growth colonize surfaces and invade organ systems. We will present evidence for the contribution of both reactiondiffusion limitation and physiological factors in the resistance of these sessile populations to antibacterial agents. We will also describe modern methods of controlling bacterial biofilms by the use of modified surface chemistry and of electrical currents that facilitate the action of antimicrobial agents. We will describe other modern methods of infection prevention that use ecological principles to favor the growth of native bacteria and inhibit that of pathogens.
John E. Kvenberg, Ph.D.: Hazard Analysis Critical Control Points (HACCP).
The Hazard Analysis Critical Control Point (HACCP) system is a preventive system for assuring the safe production of food through the systematic approach of identifying, assessing risk, and controlling biological, chemical, and physical hazards associated with a food production process. HACCP is internationally regarded as the most effective system for producing safe food and has been endorsed by the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) and the United Nations' Codex Alimentarius Committee on Food Hygiene. The international harmonization of food production practices also involves the adoption of HACCP-based systems. In December of 1995, the U.S. FDA adopted final regulations mandating the HACCP approach to the production of seafood. In addition to the regulation, the FDA has published a "Fish and Fisheries Products Hazards and Controls Guide" to assist processors of seafood products in the identification of food safety hazards and development of appropriate HACCP plans. In July of 1996, the U.S. Department of Agriculture published final regulations establishing the HACCP approach to all meat and poultry production. The U.S. FDA has also published an advanced notice of proposed rulemaking which requested comments on whether and how the agency should proceed with adopting HACCP regulations for domestically produced and imported foods other than seafood. Also published was an invitation to the food manufacturing industry to volunteer for a pilot HACCP program in cooperation with the FDA. The pilot program encompasses a wide range of foods and manufacturing processes.
Katharine Merritt, Ph.D.: Infections/Medical Devices/Risk Assessment.
Infection remains a major cause of failure of implanted devices to perform their function. It has been demonstrated by many investigators that the presence of an implanted material greatly increases the risk of infection by decreasing the number of organisms required to establish an infection. The use of aseptic technique and the use of sterile devices have greatly reduced the risk of infection over the years. Sterility is a mathematical determination of the probability of a surviving organism remaining after a sterilization procedure. The Sterility Assurance Level (SAL) is the probability that one device in a sterilization lot will not be sterile (have at least one surviving organism). Valid scientific data are needed to determine the appropriate SAL for a device and application. It is important to determine how many organisms are required to cause an infection and to determine the probability of one surviving organism causing an infection. Most infection studies have been done with high doses of organisms and little data are available on infection rates with 20 or fewer organisms on the device. The data obtained with low doses of organisms can be used in a risk assessment approach to predict infection risks and subsequently derive science based SAL values for medical devices.