Development of a risk-ranking framework to evaluate potential high-threat microorganisms, toxins, and chemicals in food.

Through a cooperative agreement with the U.S. Food and Drug Administration, the Institute of Food Technologists developed a risk-ranking framework prototype to enable comparison of microbiological and chemical hazards in foods and to assist policy makers, risk managers, risk analysts, and others in determining the relative public health impact of specific hazard-food combinations. The prototype is a bottom-up system based on assumptions that incorporate expert opinion/insight with a number of exposure and hazard-related risk criteria variables, which are propagated forward with food intake data to produce risk-ranking determinations. The prototype produces a semi-quantitative comparative assessment of food safety hazards and the impacts of hazard control measures. For a specific hazard-food combination the prototype can produce a single metric: a final risk value expressed as annual pseudo-disability adjusted life years (pDALY). The pDALY is a harmonization of the very different dose-response relationships observed for chemicals and microbes. The prototype was developed on 2 platforms, a web-based user interface and an Analytica(R) model (Lumina Decision Systems, Los Gatos, Calif., U.S.A.). Comprising visual basic language, the web-based platform facilitates data input and allows use concurrently from multiple locations. The Analytica model facilitates visualization of the logic flow, interrelationship of input and output variables, and calculations/algorithms comprising the prototype. A variety of sortable risk-ranking reports and summary information can be generated for hazard-food pairs, showing hazard and dose-response assumptions and data, per capita consumption by population group, and annual p-DALY.

Role of epidemiology in microbial risk assessment.

Microbial risk assessment (MRA) is a systematic tool to evaluate the likelihood of exposure to food-borne pathogens and the resulting impact of exposure on consumer health. In addition, MRA can be used to evaluate the public health impact of intervention or control measures designed to prevent or reduce pathogens at any or all of the steps in our complex food production system. Epidemiological studies provide useful information and data for MRA. This paper discusses the use and limitations of epidemiological data in the development and validation of MRA using examples from published microbial risk assessments.

Determining the microbiological criteria for lot rejection from the performance objective or food safety objective.

The Microbiological Criteria (MC) is a set of parameters used to determine whether a specific lot of food is acceptable or not. These parameters are the microbial test protocol and its sensitivity, the confidence level that an unacceptable lot will be detected, the number of samples to be taken and the number of positive samples that are allowed before rejecting the lot. Determining the microbiological criteria begins with knowledge of the distribution of contamination from samples within a lot, particularly within a lot that is just at the unacceptable level of the microbial hazard. The just unacceptable lot can be defined by the Food Safety Objective (FSO) or Performance Objectives (PO), the small fraction of samples that can exceed these values and the standard deviation of the samples from the lot. With this information, a microbial test protocol is chosen to have a sensitivity level that would detect between approximately 15% and 45% of the samples. A confidence level for the MC and the number of positive samples that would be acceptable (c value which is usually zero) are also chosen. With this information the number of samples (n) required can be calculated. A critical factor in setting the microbiological criteria is the sensitivity of the microbiological test (m value). The sample size (weight) and sampling procedure can affect the standard deviation of the samples, particularly foods with non-homogeneous distribution and low numbers of microorganisms. Sampling, sample preparation and analytical procedures that reduce the variation between the samples will affect the choice of m value and maximum lot mean that meets the MC.

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

Perkinsus marinus extracellular protease modulates survival of Vibrio vulnificus in Eastern oyster (Crassostrea virginica) hemocytes.

The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues.

Adherence to and invasion of tissue culture cells by Vibrio hollisae.

The adherence to and invasion of cultured epithelial cells by Vibrio hollisae were examined by quantitative studies and by light, fluorescent, and electron microscopy. Condensed actin was observed around clustered adherent and intracellular bacteria. Bacteria multiplied intracellularly. Inhibitor studies indicated that internalization occurred by an integrated pleiotropic process involving eukaryotic and prokaryotic protein syntheses, microfilaments, microtubules, and receptor-mediated endocytosis.

Purification and characterization of a Chinese hamster ovary cell elongation factor of Vibrio hollisae.

The halophilic bacterium Vibrio hollisae, isolated from patients with diarrhea, produces an extracellular toxin which elongates Chinese hamster ovary (CHO) cells. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, gel filtration with Sephacryl S-200, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, ion-exchange chromatography with DEAE-Sephadex A-50, and affinity chromatography. The toxin is heat labile and sensitive to proteases, with an isoelectric point of about 6.5 and molecular weights of about 83,000 and 80,000, as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The toxin did not react with immunoaffinity-purified antibodies to cholera toxin in a plate enzyme-linked immunosorbent assay and in a Western blot, and its activity could not be neutralized by anti-cholrea toxin serum. Mixed gangliosides and gangliosides GM1, GD1a, GD1b, Gq1b, GT1b, GD2, GD3, GM2, and GM3 failed to block its activity. Elongation of CHO cells induced by the toxin was not accompanied by an increase in the levels of cyclic AMP. The toxin induced intestinal fluid accumulation in suckling mice. These results and the lack of homology between V. hollisae DNA and DNA coding for cholera toxin or the heat-labile toxin of Escherichia coli suggest that the V. hollisae toxin is structurally and functionally different from other CHO cell-elongating toxins.

DNA probe for detecting Salmonella enteritidis in food.

Salmonellosis is the most frequently reported foodborne illness in the United States, with Salmonella enteritidis being the leading cause of these outbreaks. Nucleotide sequence comparisons of the Salmonella plasmid virulence (spv) genes of S. enteritidis with those of S. typhimurium and S. dublin have revealed that a single base-pair change unique to S. enteritidis is present in the spvA gene. An 18-base synthetic oligonucleotide probe (SE-probe) that is completely homologous to the spvA gene of S. enteritidis but which has one base pair mismatch with other salmonellae was shown to be specific for S. enteritidis. In colony hybridization blots, 129 isolates of S. enteritidis, 29 other species of Salmonella, and 17 non-Salmonella spp. were tested with the SE-probe. The SE-probe hybridized with 96% of the S. enteritidis strains tested but did not react with the other Salmonella or non-Salmonella strains. These data suggest that the SE-probe can be used in a specific and rapid detection assay for S. enteritidis.

Acridine orange stain for determining intracellular enteropathogens in HeLa cells.

Green-fluorescent intracellular enteropathogenic bacteria were observed after infected HeLa cell monolayers were stained with acridine orange and counterstained with crystal violet at least 3 h after infection.

Identification of a conjunctivitis-associated gene locus from the virulence plasmid of Yersinia enterocolitica.

The virulence plasmid (pYV) of Yersinia enterocolitica is necessary for production of conjunctivitis in guinea pigs and for mouse lethality. To identify the genes responsible for production of conjunctivitis in guinea pigs, we subcloned the BamHI and SalI restriction fragments of the virulence plasmid of Y. enterocolitica A2635 (serotype O:8) into derivatives of the broad-host-range plasmid pRK290 and introduced the constructions into plasmid-negative Y. enterocolitica strains. A mild, transient conjunctivitis was evident 24 h after inoculation with strains containing a 2.8-kilobase (kb) BamHI fragment of pYV. These strains were cytotoxic to HEp-2 cells but did not cause death in iron-loaded adult mice. When the 2.8- and adjacent 0.5-kb BamHI fragments were deleted from the virulence plasmid of a fully virulent Y. enterocolitica isolate, the resultant strain did not cause conjunctivitis in guinea pigs and was not cytotoxic to HEp-2 cells. However, the strain with the deletion appeared to be more virulent for mice, with more rapid dissemination after orogastric inoculation, compared with that of the parent strain. When the deletion was complemented by introduction of a plasmid containing the 2.8-kb BamHI fragment, the strain again caused conjunctivitis but had decreased virulence for mice.

Development and testing of a synthetic oligonucleotide probe for the detection of pathogenic Yersinia strains.

A 24-base oligonucleotide probe specific for a region of the Yersinia enterocolitica virulence plasmid (pYV) associated with HEp-2 cell cytotoxicity and the Sereny reaction was constructed by using sequences flanking critical TnphoA insertions in a subcloned fragment of pYV. This probe, highly specific and sensitive for virulent yersiniae, detected pathogenic Y. enterocolitica isolates in artificially inoculated foods.

Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model.

The invasive capacity of 13 enteropathogenic Escherichia coli strains was assessed in vitro in Henle 407 cell culture. Both fluorescent microscopy of infected monolayers stained with acridine orange and electron microscopy revealed the presence of intracellular bacteria. As shown by acridine orange-stained infected monolayers, the number of internalized bacteria increased with time. Monolayers infected for 3 h were treated with antibiotics and either [14C]glutamine or [3H]leucine and incubated for various time intervals, after which the amount of radioactivity present in the washed monolayers was measured. A significant (P less than 0.005) increase in uptake was evident for up to 4 h after the addition of radiolabeled amino acid. This finding was confirmed by an increase in bacterial number in cultured cells and in protein concentration of infected cells with time. None of the South African enteropathogenic E. coli isolates used in these studies produced Vero cytotoxin. These findings demonstrate that, in addition to adherence, cell penetration and intracellular multiplication take place in epithelial cell-derived tissue culture cells infected by enteropathogenic E. coli.

Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species.

The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67- to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup O:8, biotype 1B isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.

The risk of infection from Giardia lamblia due to drinking water supply, use of water, and latrines among preschool children in rural Lesotho.

Stool samples were collected from 267 rural, preschool children in four districts in Lesotho during October-November, 1984. Sixty-three children (23.6%) were tested positive for Giardia lamblia, the most commonly recovered parasite from stool samples. The use of low amounts of water for personal hygiene was associated significantly with having G. lamblia (OR = 2.42), but the use of traditional, non-improved drinking water sources (OR = 1.38) or lack of latrines (OR = 0.94) was not. Although G. lamblia may be primarily waterborne in developed countries, the amount of water that is used for personal and domestic hygiene may be more important than the quality of drinking water in developing countries. Other risk factors that were identified to be associated significantly with having or not having Giardia were child older than 24 months (OR = 6.79), mother less than 20 years of age (OR = 5.18), residing in Mohales Hoek district (OR = 2.33), and possessing several agricultural tools (OR = 0.70).

Comparison of preservation media and freezing conditions for storage of specimens of faeces.

To evaluate the long-term recoverability of bacterial enteropathogens, two freezing conditions (deep-freezing at -70 degrees C and liquid nitrogen) and three preservation media (Cary-Blair, Amies, and buffered glycerol-saline) were tested. These were compared with storage in containers with no preservation medium and refrigeration at 4 degrees C. At 4 degrees C, viability of organisms could not be consistently maintained beyond one month; Cary-Blair medium generally gave the best results and storage without preservation medium was the least efficient. Storage in liquid nitrogen and deep-freezing effectively preserved all organisms except Campylobacter jejuni for the entire period of study (12 months). There was no difference between the various preservation media, or between storage with or without medium. Storage in preservation medium was superior to storage without such supplement for C. jejuni. We conclude that most enteropathogens survive in faecal specimens for as long as 12 months when stored at very low temperatures (-70 degrees C) whether or not preservation media are used.

Human Yersinia enterocolitica infection in the eastern Cape. A report on 18 cases and a review of yersiniosis in the RSA.

Yersinia enterocolitica infections with a wide spectrum of clinical presentations have been reported in South Africa. An additional 18 cases were encountered in the eastern Cape region during the period May 1982 - December 1984. Y. enterocolitica isolates comprised 1% of 1,634 faecal examinations performed by the Microbiology Department of the South African Institute for Medical Research in Port Elizabeth during this period, compared with isolation rates of 5.1% for shigellae, 3.7% for salmonellae and 1.3% for Campylobacter jejuni. The majority of Y. enterocolitica infections were in children who presented with diarrhoea and only 2 systemic cases were documented. Although more cases were seen in summer there was no distinct seasonal incidence and this may be attributed to the even climatic conditions prevailing in the region.

In vitro and in vivo cholera toxin production by classical and El Tor isolates of Vibrio cholerae.

A comparative study was carried out on the in vitro production of cholera toxin by 19 Vibrio cholerae El Tor isolates from patients with cholera in South Africa, one El Tor isolate from a patient in Malawi (a country approximately 1000 km north-northeast of South Africa), 6 El Tor and 12 classical type isolates from patients in Bangladesh, and 5 culture collection classical strains. Identical phage types and indistinguishable toxigenicities among the South African and Malawi V. cholerae, representing isolations obtained over a 10-year period, indicated that essentially a single strain was involved in the cholera of these regions. Similarly, phage typing and toxin profiles indicated that the 12 classical and 6 El Tor V. cholerae cultures in Bangladesh, all isolated in November 1983, represented just two strains. As assessed by titrations in Y-1 mouse adrenal and Chinese hamster ovary cell lines, the general order of toxigenicities was Bangladesh and culture collection classical greater than Bangladesh El Tor greater than southern African El Tor. The African isolates consistently gave rise to very low titers. Their relative reluctance to produce the toxin in vitro compared with the culture collection classical strains, particularly strain 569B, was confirmed by rocket electrophoresis. In somewhat of a contrast, maximum in vivo titers in rice water stools from cholera patients in South Africa and from both classical and El Tor type cholera patients in Bangladesh were essentially equal. It is postulated that under the continuous culture conditions that occur in vivo, cholera toxin concentrations can accumulate to a maximum level, depending on the rate of purging by the diarrheal fluid rather than the toxigenicity of the infecting stain. The relevance of these findings to the relative severities of classical and El Tor types of cholera is discussed.