Thermal resistance of Listeria monocytogenes in inoculated and naturally contaminated raw milk.

Raw whole milk inoculated with 10(5) CFU/ml of Listeria monocytogenes was thermally processed at 60-72 degrees C for a minimum holding time of 16.2 s with survival being observed at temperatures up to 67.5 degrees C. In addition, milk naturally contaminated with L. monocytogenes serotype 1 (around 10(4) CFU/ml) was pooled for 2 to 2.5 days and then run through an HTST pasteurizer at temperatures ranging from 60-78 degrees C. Viable L. monocytogenes were detected in the temperature range of 60-66 degrees C. No viable Listeria were detected after treatment at temperatures of 69 degrees C and above in any of five trials. Efficacy of pasteurization and widespread use of processing conditions well above the minimum HTST guidelines ensure the absence of Listeria in pasteurized milk products. However, survival of Listeria at sub-pasteurization temperatures (60-67.5 degrees C) is of concern with regard to heat-treated or raw-milk cheeses.

Antiviral effect of commercial juices and beverages.

Nineteen commercial juices or beverages were tested for inactivation of poliovirus type 1. Grape and apple juices and tea were particularly antiviral. Although antiviral in aqueous solution, ascorbic acid was ineffective after addition to juices.

Antiviral effect of apple beverages.

A variety of apple beverages were tested for antiviral activity against poliovirus 1 or coxsackievirus B5. Freshly prepared apple juice was particularly antiviral, but its activity declined more readily than that of commercial juice in response to heat and storage. The component responsible for activity was located both in the pulp and skin; after ultrafiltration, activity was present in fractions greater and less than molecular weight 10,000. Virus infectivity was not restored from virus-apple juice complexes with gelatin, serum, Tween 80, or polyethylene glycol.

Response of various cell lines to Escherichia coli toxic products.

Seven cell lines were compared with Y-1 and Vero cells for morphological response to E. coli toxic products. FL and WI-38, like Y-1 and Vero, responded to heat-labile enterotoxin. FL, HEp-2, HeLa, and BGM, like Vero but not Y-1, responded to heat-labile cytotoxin. FL was comparable to Vero in sensitivity to these two toxic products.

Potential use of continuous cell lines to distinguish between pathogenic and nonpathogenic Listeria spp.

Continuous cell lines were tested for their potential use in distinguishing pathogenic from nonpathogenic Listeria species. Listeria monocytogenes and Listeria ivanovii strains were lethal for mice, and culture filtrates were cytotoxic for cultured cells and hemolytic for sheep erythrocytes, while Listeria innocua, Listeria grayi, and Listeria murrayi strains were negative in all three tests. The eight cell lines tested were all affected but varied in sensitivity, with the Chinese hamster ovary cell line being the most sensitive. Cytolytic effect was noted within minutes of the addition of undiluted filtrates, with optimum titers obtained by 24 h.

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays.

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.

A cytotoxic mononuclear phagocytic cell line.

A human mononuclear phagocytic cell line (M22) was established from lymph node cells of a cancer patient. Little or no multiplication occurred in the absence of target cells such as primary or continuous cultures of human, monkey, or mouse. In response to a target stimulus, M22 altered from the resting thick-walled stage and in time, target cells were destroyed. M22 resembled a monocyte, possessing Fc and complement receptors, strong nonspecific esterase activity, pinocytosis, and lacking B- and T-cell markers but differed by lacking lysozyme. It possessed acid and alkaline phosphatases but lacked myeloperoxidase. Unlike other described monocyte lines, M22 was unique in its nonspecific cytotoxic activity.

Virus inactivation by grapes and wines.

Infusions and extracts of different grapes inactivated poliovirus; agents responsible for this property resided in the skin of the grape. Commercial grape juice at both natural and neutral pH inactivate various enteric viruses and herpes simplex virus; a 1,000-fold reduction in poliovirus infectivity occurred after incubation with grape juice, pH 7.0, for 24 h at 4 degrees C. A variety of wines were antiviral but to a lesser extent than grape juice; red wines were more antiviral than white. Antiviral activity was demonstrable in fractions of grape juice varying in molecular weight from less than 1,000 to greater than 30,000 as determined by membrane filtration. Some restoration of poliovirus infectivity from virus-grape juice complexes was achieved with 1% gelatin, 0.1% Tween 80, 0.5% polyvinyl pyrrolidone, and 0.5% polyethylene glycol.

Enterovirus recovery with vegetable floc.

A lettuce floc was prepared and used for recovering enterovirus from an aqueous suspension. The method is simple, and the adsorption of coxsackievirus B5, echovirus 7, and poliovirus 1 is quantitative. The virus-floc complex may be removed from aqueous suspension by low-speed centrifugation and dissolved at an alkaline pH in a small volume of water; virus is then available for assay on cultured cells. Flocs from some other green vegetables also possess the property of virus adsorption

Methodology for isolation of Listeria from foods--a Canadian perspective.

A previously described monoclonal antibody-enzyme immunoassay system for dairy products was examined to determine its potential for detecting Listeria in naturally contaminated ground meats. In addition, a microtiter plate system was developed which has potential in the rapid detection of Listeria species in foods. Experience in Canada with the U.S. Food and Drug Administration procedure for dairy products, the cold enrichment procedure, and the U.S. Department of Agriculture procedure for meats is discussed. Also, the status on attempts to devise improved selective media for the isolation of Listeria species from foods is described.

Comparative studies of five heat-labile toxic products of Escherichia coli.

Five heat-labile, partially purified toxic products of Escherichia coli were distinguished by isoelectric focusing, molecular weight, and neutralization with homologous and heterologous antisera. Only two affected the morphology of Y-1 cells, induced fluid accumulation in rabbit ileal loops, and stimulated production of cyclic AMP in Vero cells; these two did not cross-neutralize and only one showed cross-neutralization with cholera antitoxin. The remaining three products were cytotoxic for Vero cells.

Properties of an Escherichia coli cytotoxin.

Isoelectric focusing of a heat-labile cytotoxin of Escherichia coli H30 revealed the presence of two molecular variants, pI 7.2 and a comparatively small quantity of pI 6.8. Predominant component pI 7.2 had a molecular weight of 28,000, induced some fluid accumulation in rabbit ileal loops, and showed no morphological response in Y-1 cells but a strong cytotoxic effect on Vero cells.

Stimulation of cyclic AMP secretion in Vero cells by enterotoxins of Escherichia coli and Vibrio cholerae.

The morphological response of Vero cells to Escherichia coli heat-labile enterotoxin was similar to that of cholera toxin and was accompanied by increases in the intracellular level of cyclic AMP. The effects of both enterotoxins were enhanced by the presence of phosphodiesterase inhibitor and inhibited by heat or specific antisera. Accumulation of cyclic AMP preceded the morphological response.

Methods for recovering poliovirus and rotavirus from oysters.

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.

Assay of Escherichia coli heat-labile enterotoxin with vero cells.

The continuous cell line of African green monkey kidney, Vero, showed characteristic morphological changes in response to culture filtrates from toxigenic strains of Escherichia coli. The response compared favorably with that of Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells. Vero cells were the simplest and most economical to maintain in the laboratory.

Vero response to a cytotoxin of Escherichia coli.

A cytotoxin was found in culture filtrates of a number of Escherichia coli strains that differed from the known heat-stable and heat-labile enterotoxins of E. coli. It was cytotoxic for Vero but not for Y-1 or CHO cells, and its effect on Vero was distinctly different from that of heat-labile enterotoxin. It was labile to heat and antigenically different from heat-labile enterotoxin, and membrane filtration indicated a molecular weight of 10,000 to 30,000.

Purification of an Escherichia coli serogroup O157:H7 verotoxin and its detection in North American hemorrhagic colitis isolates.

Verotoxin (VT), which is immunologically unrelated to VT1 (Shiga-like toxin I), was purified from the culture filtrate of Escherichia coli hemorrhagic colitis serogroup O157:H7 strain 3657 by copper ion chelate affinity chromatography followed by anion-exchange chromatography. The isoelectric point by sucrose density gradient isoelectric focusing was 5.0, the molecular weight by gel filtration on Superose 12 was about 60,000, and the 50% cytopathic dose for Vero cells was about 1 pg. This toxin was found by immunological methods to be the predominant VT in E. coli O157 isolates associated with illness in North America, with 38 of 42 strains tested producing this toxin, 20 in combination with VT1. VT from strain 3657 is immunologically identical to the described Shiga-like toxin II (VT2) of E. coli strains (from the United States) K-12(pEB1) and C600(933W) but only partially related to VT of strain E32511 (from the United Kingdom), the first to be named VT2.

Characteristics of nonpathogenic strains of Listeria monocytogenes.

Five strains of nonpathogenic Listeria monocytogenes were characterized for (i) hemolysin production, (ii) cytolysis of Chinese hamster ovary (CHO) cells, and (iii) ability to attach and enter intestine 407 cells. Four of the five strains produced variable hemolysis and were weakly cytolytic for Chinese hamster ovary cells, whereas the other isolate was consistently hemolytic and strongly cytolytic for CHO cells. None of the strains was able to penetrate intestine 407 cells. In addition, two of the five strains were found to be nonmotile.