Edward P. Larkin

Foodborne Viruses: Their Importance and Need for Research1

All viruses known to be normally transmissible through foods and of concern to human health emanate from the human intestine. The outbreaks of hepatitis A and recently of gastroenteritis attributed to Norwalk-like viruses most likely developed from feces-contaminated fingers of infected food handlers or water polluted with feces. With few exceptions no recorded outbreak has depended on the ability of virus to withstand even limited heating in food. New and better methods of detection are needed for hepatitis A and Norwalk viruses in foods. It has been well documented that international trade in food products of animal origin can result in the introduction of animal disease into areas in which the disease does not exist. This fact has given rise to programs of research and development for industrially applicable technology to rid animal products from the agents of animal diseases. The survival of viruses inclusive of etiological agents of foot-and-mouth disease, African swine fever, swine vesicular disease and hog cholera virus is reviewed in this paper and new research approaches are suggested. The general need for additional research of foodborne viruses is discussed.

Persistence of Polioviruses in Shellstock and Shucked Oysters Stored at Refrigeration Temperature

Poliovirus persistence was monitored in shellstock and shucked oysters stored at 5°C. The oysters either bioaccumulated viruses from seawater contaminated with feces-associated or cell-culture-grown viruses or they were innoculated with a needle and syringe. The viruses were detected in the oysters for > 28 d, the longest estimated time period between harvest and consumption of fresh oysters. These results indicate that if oysters are contaminated when harvested, they will probably be contaminated when purchased for processing in the home or food establishment.

ETIOLOGICAL STUDIES ON BOVINE LYMPHOSARCOMA

European workers believe that the incidence of lymphosarcoma, or “leukosis” t of cattle, has increased since World War I1 in the cattle populations of Denmark, Sweden, and Germany. They also believe that “leukosis” is a slowly advancing infectious disease with a long incubation period in which the initial sign is an increase in the absolute lymphocyte An infectious etiology for lymphosarcoma has also been suggested by Goetze et al. 1956’; by the reported isolation of a virus from “leukosis” cattle by Thorell, 1957,’O Montemagno et ul., 1957,11 and Papparella, 195912; by the cattle transmission experiments of Rosenberger, 196lS; and by the recent reports of McKercher, 1962,13-14 Jarrett, 1962,16 and Sorenson, 1962.I6-l7 However, proof that an infectious agent is involved in the etiology of bovine lymphosarcoma is still lacking. Our group has recently reported on attempts to characterize lymphosarcoma with regard to clinical manifestations, pathologic alterations and familial distribution of cases in high incidence herds.18 Analysis of pedigree data from cattle with lymphosarcoma in multiple-case herds indicates that the disease occurs in related animals. Such a familial aggregation of cases is suggestive of genetic susceptibility to lymphosarcoma and/or vertical transmission of an infectious agent. We have been attempting to exploit the clinical, hematological, and epidemiological data in developing criteria for the selection of cattle to be used in etiological studies of lymphosarcoma. Cattle are considered to be “normal” or “noninfected” 1 if they: 1. Show no clinical signs of lymphosarcoma. 2. Are derived from herds with no past history of lymphosarcoma. 3. Are derived from herds not showing generalized lymphocytosis on re-

Evidence in support of a virus etiology for bovine leukemia

Virus‐like particles resembling the C‐type particles of murine leukemia were detected in tissue biopsies and more frequently in thin sections of pellets following density gradient centrifugation of milk from leukemic cows and cows in a high incidence leukemic herd. These particles had a buoyant density in potassium citrate and sucrose gradients of 1.15 to 1.16 Gm/cc. Fractions in which the particles were found contained both DNA and RNA, with RNA in higher concentration. Virus interference assays on tissue cultures derived from leukemic and leukemia‐free cattle yielded equivocal results. Bone marrow cultures were uniformly resistant to challenge with vesicular stomatitis virus. Interferon was identified in the supernatant fluid of two resistant cultures. Immunofluorescence studies with anti‐Rauscher virus globulin and a globulin from a leukemic calf suggested possible antigenic similarity when the fluorescent globulins were used to stain tissue cultures derived from leukemic cattle.

Thermal resistance of three parvoviruses: a possible human isolate, the minute virus of mice, and the latent rat virus

Thermal inactivation of three parvoviruses - a possible human isolate (H-1), the minute virus of mice (MVM), and the latent rat virus (RV) - was investigated over a wide range of time and temperature contacts. The H-1 virus was inactivated at a lower FN50 value than were the RV and HVM. Ten minute inactivating temperatures were 74.5°C for H-1 virus, 86.5°C for MVM and 88.5°C for RV when inocula concentrations were ca. 1 × 103. All three viruses were shown to survive when processed at the milk pasteurization times and temperatures advocated in the U.S. Public Health Service milk ordinance and code.

Evaluation of a Method for Recovering Poliovirus 1 from 100-Gram Oyster Samples

A method is described that uses commonly available laboratory equipment and materials to detect low numbers of poliovirus 1 in oysters. Thirty 100-g oyster samples inoculated with poliovirus 1 were processed by blending at pH 4.8 in water, centrifuging, extracting the pellet at pH 9 in a mixture of Eagles medium, nonfat dry milk, MgCl2·6H2O, and Freon TF, and centrifuging again. The supernatant fluids were diluted in water, precipitated at pH 4.8 and centrifuged. The pellets were resuspended in Na2HPO4 and Cat-Floc, and centrifuged. The final supernatant fluids (~10 ml per sample) were assayed for viral plaque-forming units (PFU) in BGM African monkey green kidney cell monolayers. The average inoculum per sample was 95 PFU, and the average recovery from 30 samples was 53 PFU. The percent recovery with 95 percent confidence intervals was 55.4 ± 2.1.

Glass Wool-Hydroextraction Method for Recovery of Human Enteroviruses from Shellfish

The Glass Wool-Hydroextraction Method was developed to analyze a number of foods for the presence of contaminating human enteroviruses. This method was modified to examine a variety of shellfish, including oysters and hard- and soft-shell clams. The method consistently recovered ca. 50% of viruses inoculated into shellfish at levels of ca. 10 virus units/100 g. In a multilaboratory study, the method successfully detected all but one of the eight test viruses, and the quantitative recoveries compared favorably with the control laboratory data.

A Method for Recovery of Poliovirus 1 from a Variety of Foods

A method was developed for the recovery of low numbers of plaque-forming units (PFU) of inoculated poliovirus type 1 from seven different foods. Viruses were eluted at pH 9.0 from 25-g food test portions, concentrated at pH 4.5, and eluted at pH 7.5. The final eluates were assayed for PFU in African green monkey kidney (BGM) cell monolayers. The average virus input ranged from 55 to 79 PFU per unit portion. The average percent recoveries were as follows: potato salad, 61; radishes, 54; celery, 45; mushrooms, 44; carrots, 42; clams, 38; and oysters, 35. Three control products were negative for each food.

Fragility differences and recovery of lymphocytes from the blood of normal and leukemic cattle.

Lymphocytes were recovered from large volumes of bovine blood by use of the continuous flow gyrotester. A flow rate of 1 liter/min and an operating speed of 2,500 rpm (450 × g) resulted in high yields of viable lymphocytes. The addition of a concentrated sodium chloride solution to re-establish isotonicity after a 10-second distilled water contact with the blood, increased the yield of viable lymphocytes from all samples tested. A possible lymphocyte fragility difference was noted when the viability of lymphocytes recovered from the blood of normal and leukemic cattle was compared after the hypotonic-gyrotester treatment.

Comparative Thermal Resistance of Human and Simian Rotaviruses Assayed on Cells Grown in a Serum-Free Medium

Thermal resistance of the Wa strain of human rotavirus was compared with that of SA-11 simian rotavirus in a plaque assay with MA-104 cells grown in conventional cell culture medium supplemented with filtrate from reconstituted and acid-precipitated nonfat dry milk. Both viruses produced two-component survival curves at 56 and 60°C. Average D-values (min) for the Wa human virus were 306.3 at 56°C, 47.8 at 60°C, 12.3 at 62.8°C and 2.4 at 65°C, with a z-value of 4.33°C. For the SA-11 simian virus, the average D-values were 890.5 at 56°C, with a z-value of 4.35°C. These values indicate that low levels of rotavirus should be inactivated by pasteurization processes.