Early Days of Food and Environmental Virology.

In July 1962, the author joined the Food Research Institute (FRI), then at the University of Chicago, to become its food virologist. There was a limited record of waterborne viral disease outbreaks at the time; recorded data on foodborne outbreaks were fewer still. Laboratory environmental (water and wastewater) virology was in its infancy, and food virology was in gestation. Detection of viruses was most often attempted by inoculation of primary primate cell cultures, with observation for plaque formation or cytopathic effects. Focus was initially on enteroviruses and reoviruses. Environmental and food samples had to be liquefied if not already in liquid form; clarified to remove solids, bacteria, and fungi; and concentrated to a volume that could be tested in cell culture. Cytotoxicity was also a concern. Studies at the FRI and some other laboratories addressed all of these challenges. The FRI group was the World Health Organization's Collaborating Center for Food Virology for many years. Other topics studied were virus inactivation as functions of temperature, time, matrix, disinfectants, and microbial action; peroral and ex-vivo infectivity; and the suitability of various virus surrogates for environmental monitoring and inactivation experiments. Detection of noroviruses and hepatitis A virus required molecular methods, most often RT-PCR. When it was found that inactivated virus often gave the same RT-PCR signal as that of infectious virus, sample treatments were sought, which would prevent false-positive test results. Many laboratories around the world have taken up food and environmental virology since 1962, with the result that a dedicated journal has been launched.

Disinfection of animal manures, food safety and policy.

Manure is a resource, but sometimes also a nuisance. Manure management strategies have traditionally focused on soil nutrients (N, P, K), COD, and more recently biological substances (antibiotics, hormones, etc.), with disinfection being a relative afterthought. Zoonotic pathogens (Salmonella and other bacteria, protozoa, etc.) may be present in manure, but only occasionally cause foodborne disease. In countries where food is relatively safe, requiring heroic manure disinfection measures may be a net detriment to public health. Decisions that a new, elegant disinfection technology can, should, or must be done may result from invoking the "precautionary principle." Additional capital and operating costs must be passed to the consumer. Since such measures are likely to prevent very few human illnesses, policymakers should also consider the effect of increased prices on human nutrition and hunger. In most situations, not eating is more dangerous than eating.

Capsid and Infectivity in Virus Detection.

The spectacular achievements and elegance of viral RNA analyses have somewhat obscured the importance of the capsid in transmission of viruses via food and water. The capsid's essential roles are protection of the RNA when the virion is outside the host cell and initiation of infection when the virion contacts a receptor on an appropriate host cell. Capsids of environmentally transmitted viruses are phenomenally durable. Fortuitous properties of the capsid include antigenicity, isoelectric point(s), sometimes hemagglutination, and perhaps others. These can potentially be used to characterize capsid changes that cause or accompany loss of viral infectivity and may be valuable in distinguishing native from inactivated virus when molecular detection methods are used.

Ammonia disinfection of animal feeds --laboratory study.

Animal feeds may be contaminated, accidentally or maliciously, with a number of zoonotic bacteria. Animal infections with these bacterial agents, whether or not they cause animal disease, may lead to human illnesses. Anhydrous ammonia was introduced on farms in developed countries as a high-nitrogen soil amendment, but later found use in enhancing crude protein in low-quality roughage fed to ruminants and in neutralizing mycotoxins in fungus-infested feed grains. Although ammonia has been known to be effective against bacteria in other contexts (e.g., manure, community sewage sludge, seeds for sprouting, and boneless lean beef trimmings), it appears that the antibacterial effect of ammoniating animal feeds had not been tested. In the present study, samples of roughage (wheat straw, corn silage) and concentrates (corn grain, cottonseed) produced as animal feed were contaminated with dried-on zoonotic bacteria (Salmonella Newport in all; Campylobacte jejuni, E. coli O157:H7, Listeria monocytogenes, Yersinia enterocolitica in corn grain only). Disinfection with anhydrous ammonia gas was conducted for 24 h at room temperature ( 25 degrees C). The treatment was least effective in silage because the silage alone showed strong antibacterial activity, which may have been slightly reduced by ammoniation. In the other three feeds, depending on the initial level of contamination, ammonia destruction of >or= 5 log10 cfu/g (99.999%) of the selected contaminant was usually observed.

Pathogen survival in chorizos: ecological factors.

This study addressed health risks from ethnic sausages produced on a small scale, without inspection, in California and elsewhere. Mexican-style chorizo, a raw pork sausage that is not cured, fermented, or smoked, was contaminated experimentally in the batter with Escherichia coli O157:H7, Listeria monocytogenes, or Salmonella serotypes and stuffed into natural casings. Formulations were based on a market survey in California. Physical parameters that were controlled were pH, water activity (a(w)), and storage temperature. The pH was adjusted with vinegar, stabilizing at 5.0 within 24 h. Initial a(w) levels adjusted with salt were 0.97, 0.95, 0.93, 0.90, and 0.85; levels declined with time because of evaporation. Pathogen numbers declined with storage up to 7 days, with few brief exceptions. Main effects and interactions of constant temperature and pH with declining a(w) on survival of the pathogens were determined. Maximum death rates occurred at higher a(w) for E. coli O157:H7 and Salmonella than for L. monocytogenes. Salt used to adjust a(w) affected palatability. Spices (black pepper, chili pepper, chili powder, cumin, garlic, guajillo pepper, oregano, and paprika) comprised another, potentially significant aspect of the sausage formulation. Some (notably black pepper and cumin) carried an indigenous microflora that contributed significantly to the microbial load of the sausage batter. Only undiluted fresh and powdered garlic exhibited a significant antimicrobial effect on the pathogens. Although each of the tested formulations caused death of the inoculated pathogens, none of the death rates was sufficiently rapid to ensure safety within the probable shelf life of the product.

Cutting boards in Salmonella cross-contamination.

Cutting boards are commonly perceived as important fomites in cross-contamination of foods with agents such as Salmonella spp., despite the lack of supporting epidemiological data. A variety of woods and plastics have been used to make work surfaces for cutting. In general, wood is said to dull knives less than plastic, and plastic is seen as less porous than wood. Research to model the hypothetical cross-contamination has been done in a variety of ways and has yielded a variety of results. At least some of the work with knife-scarred plastic indicates that the surface is very difficult to clean and disinfect, although this may vary among the polymers used. High-density polyethylene, which is most used in commercial applications, has been shown to delaminate in response to knife scarring. Wood is intrinsically porous, which allows food juices and bacteria to enter the body of the wood unless a highly hydrophobic residue covers the surface. The moisture is drawn in by capillary action until there is no more free fluid on the surface, at which point immigration ceases. Bacteria in the wood pores are not killed instantly, but neither do they return to the surface. Destructive sampling reveals infectious bacteria for hours, but resurrection of these bacteria via knife edges has not been demonstrated. Small plastic cutting boards can be cleaned in a dishwasher (as can some specially treated wooden boards), but the dishwasher may distribute the bacteria onto other food-contact surfaces. Most small wooden boards (i.e., those with no metal joiners in them) can be sterilized in a microwave oven, but this should be unnecessary if accumulation of food residues is prevented. However, 2 epidemiological studies seem to show that cutting board cleaning habits have little influence on the incidence of sporadic salmonellosis. Further, one of these studies indicated that use of plastic cutting boards in home kitchens is hazardous, whereas use of wooden cutting boards is not.

Central nervous system tissue detection in meat from advanced meat recovery systems.

Three hundred meat samples, recovered from beef neck- and breast-bones using a conventional advanced meat recovery (AMR) system, the de-sinewed minced meat (DMM10) technology, and hand-boning, were collected and tested for presence of central nervous system tissue (CNST) in meat using an ELISA-based test. Samples were collected at two processing facilities (Est. A and B). Sternum meat was the non-CNST reference (control) - it is distant from brain and spinal cord locations on a carcass, with low likelihood of contamination with CNST. Neckbone meat was recovered from bones obtained from carcasses where the spinal cord was removed manually, Est. B, or using a Jarvis circular hydraulic cord remover saw, Est. A. All samples from AMR, DMM, and hand methods showed lower calculated levels of "risk material" than the stated limit of detection (0.1%) of ELISA kit. There was no apparent difference among these, and use of the Jarvis saw had no perceptible advantage.

Survival of Salmonella and Escherichia coli O157:H7 in chorizos.

Mexican-style raw meat sausages (chorizos) are not regulated in California when they are produced in small ethnic food markets. These sausages are sold uncooked, but their formulation imparts a color that may lead the consumer to assume that they are already cooked, and thus the chorizos may sometimes be eaten without proper cooking. If pathogens are present in such cases, illness may result. Survival of Salmonella and Escherichia coli O157:H7 in chorizos was evaluated under different storage conditions selected based on an initial survey of uninspected chorizos in California. Chorizos were formulated with five different initial water activity (aw) values (0.85, 0.90, 0.93, 0.95, and 0.97), stored under four conditions (refrigeration at 6 to 8 degrees C, room temperature at 24 to 26 degrees C, under a hood at 24 to 26 degrees C with forced air circulation, and incubation at 30 to 31 degrees C with convective air circulation), and sampled after 1, 2, 4, and 7 days. The initial pH was 4.8 and remained near 5.0 from day 1 of the sampling period. Two separate studies of packs inoculated with five-strain cocktails of Salmonella and of E. coli O157:H7 were performed twice for each initial aw. The three lowest aw values (0.85, 0.90, and 0.93) and the incubation and hood storage conditions were more effective (P < or = 0.05) at reducing the target pathogen levels in chorizos than were the two highest aw values (0.95 and 0.97) and the refrigeration storage condition, regardless of storage time. These results provide a scientific basis for guidelines given to producers of uninspected chorizo and should reduce the probability of foodborne illness associated with these products.

Survival of Listeria monocytogenes in experimental chorizos.

Chorizos-Mexican-style raw-meat sausages-are a concern in California because their production in small ethnic food markets is unregulated. Their formulation may cause them to appear cooked to the consumer, who may eat the raw sausage without prior proper cooking. Bacterial pathogens in such products may cause illness or even death. Survivability of Listeria monocytogenes in chorizos was evaluated under different storage conditions selected on the basis of an initial survey of uninspected chorizos in California. Sausages were formulated to five different initial water activity (aw) levels (0.85, 0.90, 0.93, 0.95, 0.97), stored under four conditions (refrigeration, "Ref," 6 to 8 degrees C under convective air circulation; room temperature, "RT," 24 to 26 degrees C under convective air circulation; hood, "Hd," 24 to 26 degrees C under forced air circulation; and incubation, "Inc," 30 to 31 degrees C under convective air circulation), and sampled after 1, 2, 4, and 7 days. The initial pH was 4.8 and remained near 5.0 from day 1 of the sampling period. An inoculated-pack study using a five-strain cocktail of L. monocytogenes was performed twice for each initial aw. Results indicated that the three lowest initial aw levels (0.85, 0.90, 0.93) and the Hd and Inc storage conditions were more effective (P < or = 0.05) at reducing L. monocytogenes levels in chorizos than the two highest initial aw levels (0.95 and 0.97) and the Ref storage condition, irrespective of storage time. These results can provide a scientific basis for guidelines given to uninspected chorizo producers in California and reduce the risk of foodborne illness.

Industry practices and compliance with U.S. Food and Drug Administration guidelines among California sprout firms.

Since 1995, raw vegetable sprouts have been implicated as the vehicle of infection in 15 foodborne outbreaks involving Salmonella and 2 foodborne outbreaks involving Escherichia coli O157:H7. To reduce the numbers of sprout-related outbreaks, the U.S. Food and Drug Administration (FDA) published Guidance for Industry: Reducing Microbial Food Safety Hazards for Sprouting Seeds in 1999. Between October 2000 and April 2001, 61.5% (16 of 26) of the known commercial sprout firms in California were enrolled in a survey to evaluate the industry practices of California sprouting operations and to determine compliance with FDA guidelines. A standardized questionnaire was used to collect data on firm demographics and seed disinfection practices. Additionally, free chlorine levels in seed disinfection solutions were measured, and 48-h spent irrigation water samples were collected from each firm. The irrigation water was screened for Salmonella and E. coli O157:H7 with FDA-recommended test kits. Free chlorine levels in the treatment solutions ranged from 50 to 35,000 mg/liter (ppm), with a median of 14,000 mg/liter (ppm). Free chlorine levels were higher for firms producing alfalfa sprouts than for those producing only mung bean or soybean sprouts (P=0.03). Levels of free chlorine tended to be higher for firms using a calcium hypochlorite treatment solution than for firms using a sodium hypochlorite treatment solution (P=0.067). All 32 irrigation water samples screened for Salmonella tested negative. Of the irrigation water samples tested for E. coil O157:H7, 75% (24 of 32) tested negative, and 25% (8 of 32) tested presumptive positive. The eight presumptive positive samples were found to be negative after further testing. These results indicate that producers of alfalfa sprouts are generally achieving the FDA-recommended calcium hypochlorite level of 20,000 mg/liter (ppm), whereas mung bean sprout producers are not.

Infectivity of RNA from inactivated poliovirus.

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.

Capsid functions of inactivated human picornaviruses and feline calicivirus.

The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72 degrees C), and physiological temperature (37 degrees C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37 degrees C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72 degrees C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37 degrees C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72 degrees C inactivation is the capsid and that the target of thermal inactivation (37 degrees C versus 72 degrees C) is temperature dependent.

Spinal cord tissue detection in comminuted beef: comparison of two immunological methods.

Two commercial immunological kits for detection of central nervous system (CNS) tissue in beef were compared: ScheBo® Brainostic™, based on CNS-specific antigen (neuron specific enolase) detection, and Ridascreen® Risk Material 10/5 test, an enzyme immunoassay for glial fibrillary acidic protein. Spinal cord (SC) was added to batches of choice, select, and utility grades of ground fresh beef shoulder clod to yield 0.0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6% SC in meat. Sensitivity and specificity in detecting SC in fresh and frozen samples were determined. Both Brainostic™ and Ridascreen® kits detected SC at claimed levels: 0.25% and 0.11%, respectively. The Ridascreen® test consistently detected SC at 0.025%, below its claimed sensitivity level, expressed for brain and SC combined. The Ridascreen® test was ∼10× more sensitive, easier, faster to run and less expensive than the Brainostic™. Overall, quality grade had no influence on SC detection in fresh or frozen meat.

Potential Salmonella transmission from ornamental fountains.

Besides the usual food and drinking-water vehicles, there are other routes by which Salmonella can be transmitted, especially at outdoor locations. Public fountains containing Salmonella offer models of exposure routes beyond those usually considered in the context of recreational use. The authors studied the bacteriological quality of water sampled from five ornamental fountains in Guadalajara, Mexico during two periods of six and of 10 months. Coliform bacteria and Escherichia coli were detected in 75 percent and 49 percent of the samples, respectively, and various serovars of Salmonella enterica were found in 12.4 percent of samples. In addition to risks from ingestion of the contaminated waters; ornamental fountains may also pose risks to people in the vicinity from inhalation of mists.

Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages.

Norwalk and Norwalk-like viruses (NLV) are major causes of food- and water-related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque-forming units/mL) of UV-inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight-line curves represent exponential inactivation, so UV inactivation can be said to show "one-hit kinetics." The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and phiX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.

Pretreatment to avoid positive RT-PCR results with inactivated viruses.

Enteric viruses that are important causes of human disease must often be detected by reverse transcription-polymerase chain reaction (RT-PCR), a method that commonly yields positive results with samples that contain only inactivated virus. This study was intended to develop a pretreatment for samples, so that inactivated viruses would not be detected by the RT-PCR procedure. Model viruses were human hepatitis A virus, vaccine poliovirus 1 and feline calicivirus as a surrogate for the Norwalk-like viruses. Each virus was inactivated (from an initial titer of approximately 10(3) PFU/ml) by ultraviolet light, hypochlorite or heating at 72 degrees C. Inactivated viruses, that were treated with proteinase K and ribonuclease for 30 min at 37 degrees C before RT-PCR, gave a negative result, which is to say that no amplicon was detected after the reaction was completed. This antecedent to the RT-PCR method may be applicable to other types of viruses, to viruses inactivated in other ways and to other molecular methods of virus detection.

Disinfection of Household Cutting Boards with a Microwave Oven.

Used cutting boards with numerous knife marks, particularly those made of polymers, are difficult to disinfect manually. Plastic cutting boards have been preferred to wood because they can be washed in dishwashers and used in microwave ovens. Our study tested the microwave oven for disinfection of cutting boards. Surfaces of plastic and wooden cutting boards were inoculated with up to 109 CFU of Escherichia coli or other bacteria in broth culture and later sampled by contact with agar medium for CFU assay or by swabbing for ATP bioluminescence assay. On wood, almost total elimination of vegetative cells occurred with exposure times of the 3 to 4 min at a high setting on typical 450 to 600 g wooden boards, depending on board size, bacterial load, and moisture level. On plastic, microwave energy had almost no lethal effect on bacteria: 12 min of exposure did not reduce the number of bacteria significantly. Increased moisture (wetness) enhanced killing efficiency on wood, but was negligible on plastic. Temperatures near the wood surface reached 95°C within the first 4 min, whereas plastic surfaces reached no more than 40°C. Our study indicates that brief "cooking" of wooden boards at a "high" setting in a microwave oven is an effective way to kill bacteria, and thus a very simple and cheap method to protect food against cross-contaminating pathogens. Because plastic is relatively inert to microwaves, disinfection of plastic boards in a microwave oven is impractical.