Survival and Recovery of Enterovirus from Foods

Transcription

1 APPLIED MICROBIOLOGY, Mar., 1966 Copyright 1966 American Society for Microbiology Vol. 14, No. 2 Printed in U.S.A Survival and Recovery of Enterovirus from Foods RICHARD K. LYNT, JR. Division of Microbiology, Food and Drug Administration, U.S. Department of Health, Education, and Welfare, Washington, D.C. Received for publication 15 October 1965 ABSTRACT LYNT, RICHARD K., JR. (Food and Drug Administration, Washington, D.C.). Survival and recovery of enterovirus from foods. Appl. Microbiol. 14: Type 1 poliovirus and type Bi and B6 coxsackieviruses added to eight commercial frozen or convenience foods before storage at room temperature, 10 C, and -20 C were still viable after various intervals of time up to 1 week, 1 month, and 5 months, respectively. Infectivity titers were determined in monkey kidney tissue culture. Decomposition which took place in food stored at room temperature did not affect the survival of virus, and antibiotics controlled bacterial growth during assay. A rapid, significant reduction in type B6 coxsackievirus was observed in cole slaw. Preliminary data indicate that sodium bisulfite could be the responsible ingredient. A less rapid reduction of type 1 poliovirus and type B6 coxsackievirus was found in hashed brown potatoes when stored at 10 C. The rapid growth in recent years of the frozen and convenience food industry has given rise to increased concern about the possible role of such foods in the dissemination of viral infections. This was strongly emphasized at a Conference on Viruses and Rickettsiae in foods (Public Health Service, Division of Environmental Engineering and Food Protection, 1963) at which it was agreed that inadequate reporting makes it impossible to estimate the extent to which foodborne illnesses may be due to these agents. However, because of the number of outbreaks for which a bacterial etiology is not established, their involvement cannot be ruled out. Excellent reviews of the literature by Berg (1) and Mehsen (8) pertaining to current knowledge of this subject and the scope of the problem have recently appeared. Although many excellent epidemiological studies have been made, food has seldom been reported as a vehicle for viral distribution. A few outbreaks of poliomyelitis have been traced to raw milk, and, in one incident, food was shown by Ward, Melnick, and Horstman to have been contaminated with the virus by ffies (12). Personal contact and mechanical distribution by flies and roaches are believed to be the usual routes of infection with the enteric viruses. Recently a number of outbreaks of infectious hepatitis have occurred in which foods appear to be the most probable means of transmission. Epidemiological evidence suggests that ingestion of raw clams or oysters taken from sewage- 218 polluted waters could be one means by which infectious hepatitis is spread (Morbidity and Mortality Weekly Rept. 10, No. 17, Communicable Disease Center, Atlanta, Ga.; Hepatitis Surveillance Rept. No. 6, 11, 14, 18, 23, Communicable Disease Center; 4; 7; 11). Isolation and identification of the virus from shellfish, however, has not been reported. On the other hand, the recovery of several enteroviruses from oysters taken from polluted beds has been reported by Metcalf and Stiles (9). Atwood, Cherry, and Klein (Hepatitis Surveillance Rept. No. 20) and Hedstrom and Lycke (6) previously had shown that clams and oysters could remove enteroviruses from water and retain them even after the clams and oysters were removed to clean water. A number of factors can influence the importance of any food as a vehicle for the spread of a virus. Among these are the opportunity for contamination with infectious virus, the ability of a virus to survive and remain infectious under conditions of handling and storage, the extent of adsorption to the food, the effect of ingredients such as flavorings and preservatives, and competition from other contaminants. However, unlike bacteria, viruses will not multiply in the foods. They will either survive at a constant level or die out. Since some frozen foods are subject to a great deal of handling in manufacture, are kept frozen, and require little or no cooking in the home, they would appear to be likely sources of viral distribution, should they become contaminated during

2 VOL. 14, 1966 SURVIVAL AND RECOVERY OF ENTEROVIRUS 219 preparation. Their actual involvement cannot be definitely demonstrated, however, in the absence of practical methods for the isolation of viruses from them. It is unlikely that clinical methods will be satisfactory, since the concentration of the agents is not likely to be high. Cliver, Gibbs, and Yeatman (2, 3, 5) have studied methods of concentrating viruses which may be applicable to foods. This report is concerned with the survival of several added enteroviruses and their recovery from commercial foods under different storage conditions. Large quantities of virus were used to offset dilution factors inherent in the procedures and to detect any losses that might occur. It is recognized that such concentrations would be unlikely in naturally contaminated foods. MATERIALS AND METHODS Viruses. Type 1 poliovirus (attenuated), strain CHAT, and type B6 coxsackievirus, strain Schmitt, were obtained from the Viral and Rickettsial Registry of ATCC. Type Bi coxsackievirus, originally obtained from the Communicable Disease Center, Atlanta, Ga., was kindly supplied by the Washington Hospital Center through the courtesy of Vernon E. Martens. All viruses were propagated in primary rhesus monkey kidney monolayers (MK) in medium 199 (10) without serum. When cytopathic effects (CPE) were complete, the cultures were frozen and thawed for three cycles and the debris was spun out. The clear supernatant fraction, which served as the virus pool, was frozen and stored at -70 C in a mechanical freezer. The poliovirus and B6 coxsackievirus pools represented third MK passage in this laboratory; the coxsackie B1 was a second passage pool. All virus pools were checked for identity against commercially obtained rabbit antisera. Cell cultures. Cell cultures used in these studies were commercial MK in test tubes. Depending upon when they were received, either they were inoculated directly or the medium was replaced with 1.0 ml of medium 199 containing 1% bovine serum shortly before they were inoculated. In some experiments, to use a large amount of inoculum, 0.1 volume of 10 times concentrated medium 199 containing 10% serum was added to the undiluted slurry, 1.1 ml of which replaced the medium already on the cells. Cultures were fed once a week. Preparation of inoculum. The foods used in these experiments were obtained during inspection of the manufacturing plant. Replicate 50-g portions were weighed into sterile glass screw-cap jars and inoculated by injecting 1.0 ml of a virus pool of known titer into the food with needle and syringe. For control, 50-ml portions of buffer were inoculated in the same way. One portion of each was assayed immediately to establish a base line, while the remainder was held at room temperature, under refrigeration (10 C), or in a food freezer at -20 C. At intervals, portions of inoculated food and control were removed and assayed for virus. They were blended with 450 ml of M phosphate buffer, ph 7.2, for 2 min at room temperature in a hood under ultraviolet light to minimize the hazard from aerosols. A sample was removed and centrifuged at 4,000 rev/min for 30 min. Fat which collected at the surface was removed with a sterile cotton swab, and 10 ml of the supernatant fraction was removed to a fresh tube. Enough antibiotic solution to give a final concentration of 100 units of penicillin, 100 Ag of streptomycin, 25,ug of tetracycline, and 50 units of nystatin per ml were added to each, and they were held at room temperature for 1 hr. With grossly decomposed foods, five times the above concentration of antibiotics was used. Preliminary experiments had shown that when a variety of foods were made into 10% slurries, particulate matter was spun out, and the supernatant fraction was treated with antibiotics at room temperature for 1 hr, they could be used in 0.1-ml quantities in MK tube cultures without harmful effect. By adding 10 ml of a slurry so treated to 1.0 ml of lox concentrated medium 199 containing 10% serum, the inoculum could be increased to 1.1 ml, usually with satisfactory results. If slurries were not centrifuged and treated with antibiotics, they were generally unsatisfactory, regardless of the volume used. Virus assay. Serial 10-fold dilutions of the slurry were prepared in buffer, and 0.1 ml was inoculated into each of two MK tubes per dilution. In some experiments, 1.0-mi volumes replaced the culture medium, and the dilutions were prepared in medium 199. Both methods gave the same infectivity titers per milliliter. Preliminary experiments showed that when serial dilutions of virus were added to food slurries or directly to the food, virus recovery approached the theoretical limit of infectivity, and 1.0 ml of inoculum was proportionately more sensitive than 0.1 ml. RESULTS When foods were contaminated with type 1 poliovirus, it was found that the virus survived under all storage conditions and could be recovered in the presence of gross decomposition. The results of this experiment are given in Table 1. It can be seen that, after 7 days at room temperature, the virus had dropped to a very low level in the pizza, and after a month at 10 C it was not recovered from the hashed brown potatoes. With these two exceptions, the results fall within the range of repeated assays of this virus pool. Even the difference between the virus content of the pizza and shrimp roll at zero time and after 1 month at 10 C are about at the extremes of this range, and there is less difference between them and the blanks. Thus, comparison of the blanks with the contaminated foods gives no indication of adsorption or inactivation of the virus. The results of a similar experiment with type Bi coxsackievirus are shown in Table 2, and are essentially the same as those obtained with poliovirus. All titers are within the range of repeated

3 220 LYNT APPL. MICROBIOL. TABLE 1. Recovery of added type I poliovirus from food preparations after storage* Zero time 3 to Blank Hashed brown potatoes < Potato salad Pizza Shrimp roll * Total TCDbo (X 106). TABLE 2. Recovery of added type BJ coxsackievirus from food preparations after storage* Zero time 3 to Blank Hashed brown potatoes Potato patties Shrimp roll Breaded shrimp Cream pie Pizza * Total TCD5O (X 105). TABLE 3. Recovery of added type B6 coxsackievirus from food preparations after storage time 3 to Blank Hashed brown potatoes < Breaded shrimp Shrimp roll Cole slaw < < * Total TCDfs (X 106). titrations of the same virus pool by this method, and suggest that the virus is not affected by these foods. Table 3 shows the results of experiments with type B6 coxsackievirus. Although it appears that there is a drop in the level of virus recovered in all samples after zero time, most of the results are within the range of titers obtained with this virus pool, and comparison of virus content of the foods with that of the corresponding blank shows them to be essentially the same. Exception to this will be noted in the hashed brown potatoes after 1 month of refrigeration at 10 C and the cole slaw in all samples after zero time; virus was not

4 VOL. 14, 1966 SURVIVAL AND RECOVERY OF ENTEROVIRUS 221 TABLE 4. Bacterial content and physical condition offoods studied Condition after storage Product Composition Avg plate EscAerichia cols 3 to 4 days at 7 days at 1 week at 10 C room temp room temp Iwe t1 Hashed brown Partially cooked shredded 14,000 9 Discolored; foul Discolored; foul Looked normal, potatoes potatoes fermented Potato salad Cooked potatoes, salad 15,300 Negative No change Watery; no No change dressing, relish, spices, change in vinegar Pizza Pastry, tomatoes, cheese 2,600,000 Negative Becoming Moldy; fer- No change moldy mented Shrimp roll Shrimp, cabbage, celery, 132,000 Negative No change Moldy; foul No change spices, pastry, egg Breaded Raw, peeled shrimp, 2,900, Strong seafood Moldy; rancid, No change shrimp batter, bread crumbs fishy Cream pie Shortening, sugar, syrup, 29, Becoming Moldy; fer- No change molasses, pastry, milk moldy, sour mented solids, flavoring Cole slaw Cabbage, vegetables, salad 200 Negative No change Slight "off" No change dressing, vinegar, spices recovered from the potatoes, and had dropped to a barely detectable level in the cole slaw. The potato experiment has been repeated on the same lot of potatoes with the same results, and three brands of cole slaw, produced by different manufacturers, have given identical results. An experiment with several separate ingredients known to be present in the cole slaw seems to indicate that, whereas acetic acid, sodium benzoate, benzoic acid, and cabbage itself have no adverse effect on this virus, sodium bisulfite may. Thus, additional studies are indicated. Storage at room temperature was not extended beyond 1 week in these experiments, because by that time the foods were grossly decomposed except for the salads. They had only a slightly "off" and had not changed in appearance. Other foods usually reveal decomposition by in 4 days without much change in appearance. At 10 C, the changes were more gradual, so that after 1 month at this temperature, spoilage was less pronounced than after 1 week at room temperature. Those in the freezer remained unchanged. The original bacterial count and Escherichia coli MPN are given in Table 4, together with an appraisal of the physical condition of the samples after storage. DISCUSSION It is significant that in spite of the gross decomposition of some of these foods, the contaminating virus was still recovered, indicating that the normal flora does not destroy the virus either directly or through decomposition products. Moreover, the bacteria present can be controlled during assay by antibiotics commonly used in tissue culture media. The quantity of virus surviving usually was sufficient to require dilution for assay, but when it was necessary to use the undiluted slurry, bacterial contamination ordinarily was not a problem. Under these circumstances, some foods in themselves were toxic for the cells. For example, the cole slaw preparation had to be replaced with fresh medium the next day, otherwise the cells degenerated without evidence of viral CPE or bacterial growth. This also has occurred with other food materials and must be taken into account when concentration methods are considered. The evidence obtained from these experiments does not permit an unequivocal statement regarding adsorption or inactivation of the added viruses by most of these foods, since the titration method is not sufficiently precise to be significant for any but very large differences in titer. However, it is evident that some ingredient of the cole slaw caused a marked reduction in the amount of virus recoverable, and that hashed brown potatoes had the same effect on two of these viruses after a month of refrigeration. In all other cases, the data indicate that adsorption and inactivation were not sufficient to preclude the recovery of the contaminating virus. Since these foods represent a fairly wide variety of products containing spices, flavorings, and sauces of rather complex nature, and since they represent fats, starches, carbohydrates, vegetable products, and seafood, it seems likely that if a food becomes contaminated with a sufficient

5 222 LYNTT quantity of an enterovirus, it should be possible to detect it in most cases. AcKNowLEDGMENTS The helpful advice and encouragement of Glenn G. Slocum and M. Thomas Bartram are gratefully acknowledged. Barry A. Wentz provided valuable technical assistance, and Bernard F. Surkiewicz supplied the food products used in these experiments, as well as the results of bacteriological tests on them. LITERATURCErr 1. BERG, G The food vehicle in virus transmission. Health Lab. Sci. 1: CLIVER, D Factors in the membrane filtration of enteroviruses. Appl. Microbiol. 13: CLIVER, D. O., AmN J. YEATMAN Ultracentrifugation in the concentration and detection of enteroviruses. Appl. Microbiol. 13: DOUGHERTY, W. J., AND R. ALTMAN Viral hepatitis in New Jersey J. Am. Med. Assoc. 32: GIBBS, T., AND D. 0. CLIVER Methods for APPL. MICROBIOL. detecting minimal contamination with reovirus. Health Lab. Sci. 2: HEDSTROM, C. E., AND E. LYcKE An experimental study on oysters as virus carriers. Am. J. Hyg. 79: MASON, J. O., AND W. R. MCLEAN Infectious hepatitis traced to the consumption of raw oysters. Am. J. Hyg. 75: MEHSEN, J Virus diseases transmitted through foods. Quarterly Bull. Assoc. Food Drug Officials U.S. 29: METCALF, T. G., AND W. C. STILES The accumulation of enteric viruses by the oyster Crassostrea virginica. J. Infect. Diseases 115: MORGAN, J. F., H. J. MORTON, AND R. C. PARKER Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium. Proc. Soc. Exptl. Biol. Med. 73: Roos, B Hepatitis epidemic transmitted by oysters. Svenska Lakartidn. 53: WARD, R., J. L. MELNICK, AND D. M. HORSTMAN Poliomyelitis virus in fly contaminated food collected at an epidemic. Science 101: Downloaded from on July 3, 2018 by guest