6. HAZARD IDENTIFICATION, HAZARD CHARACTERIZATION AND EXPOSURE ASSESSMENT OF VIBRIO SPP. IN SEAFOOD

6.1. EXECUTIVE SUMMARY

The objective of the work was to undertake the first steps of a risk assessment of Vibrio spp. in seafood products that would have the most impact on public health and/or international trade. These first steps involve taking a risk assessment developed by a member country, generalizing it, and testing its ability to provide predictions that are useful for other member countries. Furthermore, it is desirable to explore the capability of a risk assessment model to be adapted to different commodities and/or related organisms of international and national interest. The approach used by the drafting group was to quantify those illnesses caused by Vibrio spp. in different countries following the consumption of a range of seafoods. Three species, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae were considered as the species responsible for most illnesses caused by Vibrio spp. This report describes the approach suggested to undertake a risk assessment of these three species in specific seafood products.

With regard to pathogen-food commodity combinations, the expert drafting group proposed to undertake a detailed risk assessment of V. parahaemolyticus in oysters as a model was already available. The proposed work on V. vulnificus would demonstrate the application of the previous model to a different organism with appropriate modifications. Non-oyster associated V. parahaemolyticus is important in Japan and some other countries, and consideration of the organism with respect to finfish would give a different viewpoint of the same organism used in the first model, but with cross-contamination as an important factor. V. cholerae is an important pathogen in developing countries and the development of a model for the organism in shrimp would provide a tool to investigate a number of other scenarios. It would also enable an investigation of the risk associated with international trade of this product and of the problems caused with respect to the export market for potentially contaminated shrimp. Since shrimp are usually eaten cooked it would provide an additional example of the use of a cross-contamination model for another Vibrio spp..

6.1.1 Hazard identification of Vibrio spp. in seafood

Vibrio spp. are Gram-negative, facultatively anaerobic rod-shaped bacteria. The genus contains twelve species that can cause food-borne illness (Table 1), most of which is caused by V. cholerae, V. parahaemolyticus or V. vulnificus (Oliver and Kaper, 1997, Dalsgaard, 1998). Some species are primarily associated with gastrointestinal illness (V. cholerae and V. parahaemolyticus) while others can cause non-intestinal illness, such as septicaemia (V. vulnificus).

In tropical and temperate regions, disease-causing species of Vibrio occur naturally in marine, coastal and estuarine (brackish) environments and are most abundant in estuaries. Pathogenic vibrios can also be recovered from freshwater reaches of estuaries (Desmarchelier, 1997). The occurrence of these bacteria does not correlate with numbers of faecal coliforms and depuration of shellfish may not reduce their numbers. Based on data from the United States, there is a positive correlation between water temperature and both the number of human pathogenic vibrios isolated and the number of reported infections, a correlation particularly marked for V. parahaemolyticus and V. vulnificus.

TABLE 1: Vibrio spp. which cause, or are associated with, human infections (after Dalsgaard, 1998)

* The symbol (+) refers to the relative frequency of each organism in clinical specimens and (-) indicated that the organism was not found

In Japan (Twedt, 1989; Japanese Ministry of Health, 2000) and eastern Asian countries V. parahaemolyticus has been recognised as a major cause of foodborne gastroenteritis. By contrast, in most countries outside of Asia, the reported incidence appears to be low, perhaps reflecting a different mode of seafood consumption. Gastroenteritis caused by this organism is almost exclusively associated with seafood consumed raw or inadequately cooked, or contaminated after cooking. In the United States prior to 1997 illness was most commonly associated with crabs, oysters, shrimp and lobster (Twedt, 1989; Oliver and Kaper, 1997). Four V. parahaemolyticus outbreaks associated with the consumption of raw oysters were reported in the United States in 1997 and 1998 (DePaola et al., 2000). A new V. parahaemolyticus clone of O3:K6 serotype emerged in Calcutta in 1996. It has spread throughout Asia and to the United States elevating the status of V. parahaemolyticus to pandemic (Matsumoto et al., 2000). In Australia, in 1990 and 1992, there were two outbreaks of gastroenteritis caused by V. parahaemolyticus in chilled, cooked shrimps imported from Indonesia (Kraa, 1995) and there was also a death in 1992 associated with the consumption of oysters.

V. vulnificus has been associated with primary septicaemia in individuals with chronic pre-existing conditions, following consumption of raw bivalves. This is a serious, often fatal, disease. To date, V. vulnificus disease has almost exclusively been associated with oysters (Oliver, 1989; Oliver and Kaper, 1997). Recently, V. vulnificus infections have been associated with a variety of raw seafood products in Korea and Japan (Personal Communication, Dr. Yamamoto, Japan).

Toxigenic V. cholerae O1 and O139 are the causative agents of cholera, a water- and food-borne disease with epidemic and pandemic potential. Non-O1/non-O139 strains may also be pathogenic but are not associated with epidemic disease. Non-O1 strains are generally nontoxigenic, usually cause a milder form of gastroenteritis than O1 and O139 strains, and are usually associated with sporadic cases and small outbreaks rather than epidemics (Desmarchelier, 1997).

Outbreaks of cholera have been associated with consumption of seafood including oysters, crabs and shrimp (Oliver and Kaper, 1997). The largest outbreak was a pandemic in South America in the early 1990s when V. cholerae O1 caused more than 400,000 cases and 4,000 deaths, in Peru (Wolfe, 1992). Contaminated water used to prepare food, including the popular, lightly marinated fish ceviche, was the cause of the outbreak.

Given the foregoing, the drafting group concluded that four pathogen-product risk assessments should be progressed:

• Vibrio parahaemolyticus in raw oysters

• Vibrio vulnificus in raw oysters

• Vibrio parahaemolyticus in finfish consumed raw

• Vibrio cholerae in shrimp from developing countries for domestic and export consumption.

Accordingly, the drafting group prepared an exposure assessment and hazard characterization for each these pathogen-commodity combinations. The justification for each pathogen-commodity combination is contained in a "Statement of Purpose" included at the head of each assessment.

References^[2]

Dalsgaard, A. 1998. The occurrence of human pathogenic Vibrio spp. and Salmonella in aquaculture. International Journal of Food Science and Technology, 33: 127-138.

DePaola, A., C.A. Kaysner, J.C. Bowers, and D.W. Cook. 2000. Environmental investigations of Vibrio parahaemolyticus in oysters following outbreaks in Washington, Texas, and New York (1997, 1998). Applied and Environmental Microbiology, 66: 4649-4654.

Desmarchelier, P.M. 1997. Pathogenic Vibrios. In A.D. Hocking, G. Arnold, I. Jenson, K. Newton and P. Sutherland, eds. Foodborne Microorganisms of Public Health Significance 5^th Edition, p 285 -312. North Sydney, Australian Institute of Food Science and Technology Inc..

Kraa, E. 1995. Surveillance and epidemiology of foodborne illness in NSW, Australia. Food Australia, 47(9): 418-423.

Matsumoto, C., J. Okuda, M. Ishibashi, M. Iwanaga, P. Garg, T. Rammamurthy, H. Wong, A. DePaola, Y.B. Kim, M.J. Albert, and M. Nishibuchi. 2000. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analysis. Journal of Clinical Microbiology, 38: 578-585.

Ministry of Health, Labour and Welfare, Japan 2000. Statistics of Food Poisoning Japan in 2000.

Oliver, J. D. 1989. Vibrio vulnificus. In M. P. Doyle, ed.. Foodborne Bacterial Pathogens, p569-600. New York, Marcel Decker, Inc..

Oliver, J. D., and Kaper, J.B. 1997. Vibrio Species. In M. P. Doyle, L. R. Beuchat, and T. J. Montville, eds. Food Microbiology: Fundamentals and Frontiers, p228-264. Washington, D.C., ASM Press.

Twedt, R. M. 1989. Vibrio parahaemolyticus. In M. P. Doyle, ed. Foodborne Bacterial Pathogens, p543-568. New York, Marcel Decker, Inc..

Wolfe, M. 1992. The effects of cholera on the importation of foods: Peru- a case study. PHLS Microbiology Digest, 9: 42-44.

Yamamot, S. 2001. Personal communication Vibrio vulnificus in Japan.

6.1.2 Hazard characterization of Vibrio spp. in seafood

Introduction

This section focuses on evaluating the nature of adverse health effects associated with Vibrio spp. in seafood and how to quantitatively assess the relationship between the magnitude of the foodborne exposure and the likelihood of adverse effects occurring. The hazard characterization presents dose-response curves for three important species of Vibrio: V. parahaemolyticus, V. vulnificus and V. cholerae. Infection by V. parahaemolyticus, and V. cholerae is characterized by an acute gastroenteritis. Therefore, the end-point of the dose-response curve is defined as gastroenteritis. V. vulnificus can occasionally cause mild gastroenteritis in healthy individuals, but for specific subpopulations V. vulnificus can cause a serious septicaemia that frequently leads to death in susceptible people. Therefore, the endpoint for the dose-response curve is defined as septicaemia.

Objective

The objective of the hazard characterization is to provide sufficient information to allow for a quantitative measurement of the public health risk from Vibrio spp. associated with the consumption of seafood and foods potentially contaminated by seafood. The quantitative measurement of public health risk is accomplished by the determination of dose-response relationships for each Vibrio spp. based upon the best available data. These data are often sparse and the resulting dose-response relationship is uncertain. This uncertainty of the dose-response curve is accounted for by representing the dose-response relationship in the form of a family of plausible data-derived dose-response curves.

Approach

Human volunteer studies are available for the construction of dose-response curves for V. parahaemolyticus and V. cholerae. These data were analysed using curve-fitting routines to find a best fit for the Beta-Poisson dose-response curve. Because of the sparse data for the human volunteer studies, multiple curve-fits are determined using resampling techniques. The resulting multiple Beta-Poisson dose-response curves may be used in the risk characterization. These multiple dose-response curves represent a key uncertainty in the risk assessment. For V. vulnificus, no human volunteer data were available and an alternate approach was attempted. The dose-response relationship was estimated by fitting a Beta-Poisson model using monthly V. vulnificus levels in the United States Gulf of Mexico oysters and estimated consumption of raw oysters with monthly reported cases of V. vulnificus-associated septicaemia in the United States. With further research, this risk relationship may be applied in a V. vulnificus risk assessment and validated with data on the distribution of V. vulnificus in raw oysters at the point of retail.

Key findings

A review of the literature was undertaken to identify and characterize the infectivity and genetic factors of Vibrio spp. to be modelled. V. parahaemolyticus and V. cholerae have both pathogenic and non-pathogenic forms based on the presence of specific virulence factors: tdh (thermostable direct hemolysin) and trh (tdh-related hemolysin) for V. parahaemolyticus and cholera toxin for V. cholerae. There is not adequate information to differentiate between virulent and avirulent strains of V. vulnificus. Therefore, all V. vulnificus strains were considered to be equally pathogenic. Relevant factors with respect to the host and food matrix have been identified and where data are available may be incorporated into the model.

Reasonable Beta-Poisson dose-response parameters were obtained from data sets for all three organisms examined; however, the human volunteer studies characterize dose-response relationships for pathogens administered with a pH-neutralizing buffer rather than for pathogens administered with a food matrix.

Figure 6.1 shows the most probable dose-response curve for V. parahaemolyticus; however, the family of curves representing uncertainty that surrounds the curve is not shown. These data are from healthy human volunteer studies where gastrointestinal illness was used as the endpoint response.

FIGURE 6.1 Beta-Poisson dose-response curve for V. parahaemolyticus (endpoint modelled is gastrointestinal illness).

Figure 6.2 shows the most probable fits of human volunteer data for several biotypes and serotypes of V. cholerae. Once again, uncertainty is represented by a family of curves surrounding each of the most probable curves, but this is not shown. The endpoint modelled is gastrointestinal illness. The data indicate that the consumption of V. cholerae with foods may significantly shift the dose-response curve to the right, indicating that a higher dose of V. cholerae is needed to cause illness in a comparable number of volunteers when the V. cholerae are consumed with food. It is unknown if specific food matrices have greater or lesser effects on the shift of the dose-response relation.

Figure 6.3 shows the dose-response relationship for V. vulnificus as estimated based upon exposure of the United States susceptible oyster-consuming population and the United States V. vulnificus epidemiology reports. As with the other Vibrio spp., a family of dose-response curves was generated using resampling techniques (bootstrap) but this was not shown. The derived curve is different compared to the other Vibrio spp. because a septicaemia endpoint is modelled instead of a gastrointestinal illness endpoint.

FIGURE 6.2. Beta-Poisson dose-response curves for V. cholerae (endpoint modelled is gastrointestinal illness)

FIGURE 6.3. Beta-Poisson dose-response curve for V. vulnificus (endpoint is septicaemia)

Gaps in the data

• Few human volunteer studies have been carried out - more data are needed and these could be obtained through the collection of quantitative data from outbreak investigations.

• As there is only a small amount of available data on the effect of the food matrix on dose-response, additional data is required in this area.

• There is a need for ongoing V. vulnificus environmental and epidemiological surveillance data to test and to refine the V. vulnificus dose-response relationship.

• There is a lack of information for the characterization of human susceptibility and pathogen virulence variability.

References

Aiso K, Fujiwara K. 1963. Feeding tests of the pathogenic halophilic bacteria. Annual Research Report Institute of Food Microbiology Chiba University, 15:34-38.

Cash R.A., Music, S.I., Libonati, J.P., Snyder M.J., Wenzel, R.P. and Hornick, R.B. 1974. Response of man to infection with Vibrio cholerae. I. Clinical, serologic, and bacteriologic responses to a known inoculum. Journal of Infectious Disease, 129: 45-52.

Levine, M.M., Kaper, J.B., Herrington, D., Losonsky, G., Morris, J.G., Clements, M.L., Black, R.E., Tall, B.D. and, Hall, R. 1988 Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques. Infection and Immunity, 56: 161-167.

Sanyal, S.C., and Sen P.C. 1974. Human volunteer study on the pathogenicity of Vibrio parahaemolyticus. In T. Fujino, G. Sakaguchi, R. Sakazaki, and Y. Takeda. eds. International Symposium on Vibrio parahaemolyticus. p. 227-230. Tokyo, Saikon Publishing Company.

Takikawa I. 1958. Studies on pathogenic halophilic bacteria. Yokohama Medical Bulletin, 9:313-322.

6.1.3. Exposure assessment of Vibrio spp. in seafood

6.1.3.1 Exposure assessment of Vibrio parahaemolyticus in raw oyster

Introduction

In the United States during 1997 and 1998 there were more than 700 cases of illness due to V. parahaemolyticus, the majority of which were associated with the consumption of raw oysters. In two of the 1998 outbreaks a serotype of V. parahaemolyticus previously reported only in Asia, O3:K6, emerged as a principal cause of illness for the first time. It was suggested that warmer than usual water temperatures were responsible for the outbreaks.

In 1999, the United States Food and Drug Administration (FDA) initiated a risk assessment to characterize the public health impact of consuming raw oysters contaminated with V. parahaemolyticus. The FDA Draft Risk Assessment on the Public Health Impacts of V. parahaemolyticus in Raw Molluscan Shellfish (FDA-VPRA) was released for public comment in 2001. The FDA-VPRA contains several key linkages between prevalence of V. parahaemolyticus in oysters and temperature, most notably temperature of harvest waters and of oysters throughout the post-harvest-retail-consumption continuum.

Temperature profiles in the oyster industry of other countries e.g. New Zealand, Australia and Japan indicated the opportunity for growth of pathogenic V. parahaemolyticus to potentially dangerous levels. However, the public health statistics of these countries do not reflect any impact due to this organism in oysters.

Accordingly, an exposure assessment will be undertaken on V. parahaemolyticus in oysters using data from Australia, Canada, Japan, New Zealand and the United States.

Objectives

The objectives are to:

• Quantify the exposure of consumers to pathogenic V. parahaemolyticus from consumption of raw oysters.

• Extend this exposure assessment to consumers in other countries that have oyster industries.

Approach

The approach being taken is to use the FDA-VPRA model as the base and further develop it to accommodate data inputs from other countries. This model incorporates all phases in the harvest - post-harvest - consumption continuum in three modules (Figs 6.4-6.6).

Data for the exposure assessment were obtained via a call for data issues by to FAO and WHO. The data were then analysed for incorporation into the risk assessment model.

FIGURE 6.4: Harvest module for exposure assessment of V. parahaemolyticus in oysters

FIGURE 6.5: Post-harvest module for exposure assessment of V. parahaemolyticus in oysters

FIGURE 6.6: Consumption module for exposure assessment of V. parahaemolyticus in oysters

Key findings

Analysis of the data has not been completed but the key findings at the present stage are as follows:

• In Japan, while V. parahaemolyticus is the cause of significant seafood-based illness, oysters appear not to be an important vehicle. It may be that regulatory practices of controlling the total coliform number in harvesting seawater result in the curtailment of harvesting in the summer months.

• In Australia and New Zealand, some oyster-producing areas have seawater temperature and salinity combinations, together with post-harvest temperatures that are potentially favourable for proliferation of V. parahaemolyticus. This situation should also be considered in the model.

• It may be that not all oyster species are similarly vulnerable to proliferation of pathogenic V. parahaemolyticus in the post-harvest chain. For example, the Sydney Rock Oyster (Saccostrea commercialis) is particularly resistant to stress during post-harvest handling.

Gaps in the data

For each country the following gaps exist:

• Incidence/frequency of pathogenic V. parahaemolyticus in water and shellfish.

  - Factors that affect incidence of pathogenic V. parahaemolyticus in the environment.
  - Growth rate of V. parahaemolyticus within oysters at temperatures other than 26°C, including data on the potential differences in the growth rate of pathogenic strains versus total V. parahaemolyticus populations.

• Potential virulence factors of pathogenic strains other than tdh and trh, e.g. urease, enterotoxins.

• Consumer and industry handling practices for oysters.

• Consumption patterns of oysters in each country.

6.1.3.2 Exposure assessment of Vibrio vulnificus in raw oyster

Introduction

This document outlines the objectives and approach for modelling the risk of V. vulnificus from consumption of raw oysters. This pathogen-commodity pair was proposed by the European Community in the 33^rd session of the CCFH.

The general approach and many of the parameters may be adopted from the FDA-VPRA, which is the only available risk assessment for a Vibrio spp. in raw oysters. Thus the approach taken by the drafting group was to elaborate on the FDA-VPRA. Due to the lack of appropriate data outside of the United States for many of the model inputs this assessment relies almost totally on data from this country. The approach for determining dose-response uses exposure and illness frequency. Because of this approach there are some elements of hazard characterization included in the exposure assessment.

The choice of the United States data is intended only to provide an example on how to apply the exposure model to a different national situation. This model will be further tested when appropriate data from other countries or situations become available.

Objectives

• To adapt the FDA-VPRA model to assess the risk from V. vulnificus associated with the consumption of raw oysters.

• To identify the most appropriate data as well as the data gaps and limitations for modelling V. vulnificus in oysters.

Approach

• Examine V. vulnificus ecology and the epidemiology of V. vulnificus illness.

• Test the appropriateness of transferring inputs for harvest, postharvest and public health modules of FDA-VPRA to the risk assessment of V. vulnificus (Figure 6.7).

• Select data inputs and develop alternate approaches from FDA-VPRA to fit V. vulnificus.

• Validate the predicted exposure with a survey of V. vulnificus levels at retail.

• Determine the dose-response by establishing a mathematical relationship between exposure and illness for each month of the year within a defined geographical region.

• Develop a conceptual model (schematic diagram) of the V. vulnificus risk assessment model showing integration of all the modules.

FIGURE 6.7 Schematic diagram of the V. vulnificus conceptual risk assessment model showing integration of all the modules. Inputs that are not shaded can be transferred directly from the FDA-VPRA and inputs that are shaded require additional data

Key findings

• The FDA-VPRA provides many of the needed inputs and is a useful framework to model the risk of V. vulnificus septicaemia from consumption of raw oysters.

• Reliable data are available for determining V. vulnificus exposure from consumption of raw United States Gulf Coast oysters during each month of the year (levels at harvest and consumption, growth and survival in raw oysters).

• Reliable monthly illness rates are available due to intensive epidemiological surveillance, the severity of primary septicaemia caused by this organism and the nearly exclusive association of illness in the United States to consumption raw Gulf Coast oysters.

• Aggregate population dose-response can be approximated from available data on differences in exposure from consumption of the United States Gulf Coast oysters and reported illness frequency during warm and cold months.

• The approach for determining dose-response circumvents the lack of data on frequency of virulent strains in raw oysters and uncertainty concerning the susceptible population by assuming that these do not vary from month to month.

• This modelling approach is appropriate for determining the potential effectiveness of specific mitigations.

• The dose-response relationship developed using data for raw oyster consumption in the United States may be used for:

  - Other foods for which the distribution of V. vulnificus at the point of consumption is known assuming there is no matrix effect

  - Other countries, but adjustments would need to be made for differences in the susceptible population compared to the United States.

Gaps in the data

• There is insufficient exposure data available for modelling risk of V. vulnificus illness from consumption of raw Gulf Coast oysters in the United States using the proposed approach.

• There is probably a wide variation in the susceptibilities within and among the various risk groups and this is not well understood. The major obstacle for expanding risk assessments to other foods is the lack of data on the distribution of V. vulnificus levels in these foods at the time of consumption and potential matrix effects.

• The incidence of specific risk factors in the population consuming a seafood of interest and exposure associated with this seafood are the primary data needed for applying this model to other countries.

• Validation of the model in a given region or country would require epidemiological data on monthly V. vulnificus primary septicaemia rates.

6.1.3.2 Exposure assessment of Vibrio parahaemolyticus in finfish consumed raw

Introduction

V. parahaemolyticus is a leading cause of seafood-based illness in Japan and other Asian countries. With the globalization of Japanese cuisine and the increased practice of eating raw fish and shellfish, there is increased possibility of V. parahaemolyticus infection.

Outbreaks due to V. parahaemolyticus associated with fish and shellfish other than oysters have been reported in some countries including the United States, Thailand, China (Taiwan) and Spain. Several reports exist on the high prevalence of the organism in a variety of seafoods, especially in finfish, lobster and shrimp. Therefore, eating raw fish and shellfish has potential risks for V. parahaemolyticus infection and it is important to assess the exposure of consumers to V. parahaemolyticus in finfish.

Objectives

The objective of this exposure assessment is to model and quantify the exposure of consumers to V. parahaemolyticus from consumption of raw finfish.

Approach

The approach being taken is to modify the FDA-VPRA model for V. parahaemolyticus in oysters to accommodate data inputs on other seafood from Japan and other countries.

The model has four modules (Figs. 6.8 - 6.11) accommodating the entire continuum from harvest and post-harvest and ending with consumption in the home or in the food service sector.

FIGURE 6.8: Schematic representation of the pre-harvest module for the exposure assessment of Vibrio parahaemolyticus in finfish consumed raw.

FIGURE 6.9: Schematic representation of the harvest module for the exposure assessment of Vibrio parahaemolyticus in finfish consumed raw.

FIGURE 6.10: Schematic representation of the post-harvest module for the exposure assessment of Vibrio parahaemolyticus in finfish consumed raw.

FIGURE 6.11: Schematic representation of the preparation and consumption module for the exposure assessment of Vibrio parahaemolyticus in finfish consumed raw.

Key findings

• V. parahaemolyticus density and prevalence in the seawater are influenced by seawater temperature, salinity of water, existence of plankton, tide and others.

• Many species of finfish could be contaminated with V. parahaemolyticus though prevalence and number of V. parahaemolyticus vary with species. Differences in prevalence and density seemed to be associated with the species and habitat (e.g. coastal or deep-sea).

• Coastal seawater used at landing and at market was shown to be highly contaminated with V. parahaemolyticus and this phase can be an important risk factor for contamination.

• This conceptual modelling approach would be appropriate for determining the potential effectiveness of mitigation strategies such as chlorinated water and thermal processing.

• The effect of time and temperature during transportation and storage may be less important than with raw oysters as V. parahaemolyticus was shown not to proliferate significantly until four hours at 25^oC on finfish samples.

• Washing the external surface of the fish and the visceral cavity with potable water reduced V. parahaemolyticus levels.

Gaps in the data

• Prevalence, number and proportion of pathogenic V. parahaemolyticus cells in various species of finfish

• Frequency of consumption of raw fish

• Transportation practices (time and temperature)

6.1.3.4 Exposure assessment of Vibrio cholerae in shrimps from developing countries for domestic and export consumption

Introduction

Seafood has been incriminated in cholera outbreaks. Shrimp is one of the most important seafood commodities in international trade and most shrimp come from developing countries. While high value shrimp is mostly exported by the developing countries to earn valuable foreign exchange, low value shrimp are consumed domestically. There have been outbreaks of cholera in many shrimp producing countries and such episodes have often adversely affected the international shrimp markets. In this context, it was felt that performing an exposure assessment for V. cholerae in shrimp intended for international trade as well as domestic markets was desirable since there are differences in the way shrimp are handled for these two markets.

Objectives

To perform an exposure assessment of V. cholerae in shrimp for domestic and export markets.

Approach

Shrimp may be contaminated with toxigenic V. cholerae during handling due to poor personal hygiene and washing with contaminated water. Occasionally V. cholerae O1 may be detected in brackish water aquaculture ponds where shrimp are grown. In the case of shrimp intended for export markets, generally specific hygienic practices are implemented to prevent contamination. Toxigenic V. cholerae is rarely isolated from shrimp imported from developing countries and there have only been one or two reported cases associated with shrimp products in developed countries (Infectious Agents Surveillance Report, National Institute of Infectious Disease, Japan, 1998) even though the total world shrimp production is about four million tons, of which 1,3 million are traded internationally with three quarters of this originating in developing countries (FAO. 1999). But in the domestic markets, contamination of seafood with toxigenic V. cholerae has been reported in a number of developing countries. Thus, the consultation suggested that the exposure assessments for shrimp intended for these two types of markets be performed separately as outlined below (Figures 6.12 and 6.13)

Shrimp intended for domestic markets in developing countries are often poorly iced, washed in landing centres where potable water is generally not available, sorted by hand and transported to local markets. Also, it may be prudent to consider the microbiological quality of the water from which shrimp are harvested. Contamination with toxigenic V. cholerae could occur through water used in processing establishments or through handling by asymptomatic carriers (Figure 6.12). In many developing countries, the domestic water supply is often not of potable quality and contamination with toxigenic V. cholerae could occur in kitchens of households and street vendors and also through raw-cook transfer. If such cooked contaminated shrimp are stored at ambient temperature, V. cholerae could multiply to infective doses.

Shrimp intended for export are generally iced immediately after harvest and transported in ice to well equipped processing establishments with hygienic controls, potable washing water, processors with clean gloves, clean handling tables, and HACCP plans etc.. In this setting, chances of contamination with toxigenic V. cholerae are very low. Shrimp frozen in such establishments are exported and thawed and cooked in the receiving country. Since V. cholerae are killed by cooking, the exposure in the importing country is likely to be negligible (Figure 6.13).

Key findings

Among V. cholerae, only serotype O1 and O139 are known to cause epidemic cholera. The major virulence factor of this organism is cholera toxin encoded by the ctx gene. Many environmental strains may be negative for this gene. V. cholerae is not known to colonise shrimp in its natural habitat. The primary source of V. cholerae is the faeces of persons infected with the organism. Asymptomatic carriers are also known to excrete the organism. V. cholerae reaches water through sewage and survives for long periods of time. However, the levels found in shrimp harvesting waters are generally low.

Contamination of shrimp with toxigenic V. cholerae could occur in the environment, through water used during processing or handling by asymptomatic carriers. V. cholerae is highly sensitive to gastric acid and therefore neutralization of gastric acid is found necessary to cause illness in human volunteers. V. cholerae is not known to multiply in raw shrimp, and it is sensitive to heat and eliminated during normal cooking of shrimp. If cooked shrimp is contaminated, the organism can multiply and reach infective doses if not adequately refrigerated.

Gaps in the data

• Data on the levels of toxigenic V. cholerae in natural waters and aquaculture environments.

• Levels of this organism or chlorine levels in the water supply in rural fish markets and in that used to process shrimp in developing countries.

• Data on the extent of cross-contamination and multiplication in cooked shrimp.

• Frequency and amount of shrimp consumption in both developing and developed countries.

FIGURE 6.12: Model for exposure assessment of V. cholerae in shrimp for domestic consumption in developing countries.

FIGURE 6.13. Model for exposure assessment for V. cholerae in shrimp for international markets.

6.2. SUMMARY OF THE DISCUSSIONS

6.2.1. Hazard identification of Vibrio spp. in seafood

The drafting group had not prepared a hazard identification document prior to the expert consultation. During the consultation the need to undertake this was identified and a document was subsequently prepared.

6.2.2 Hazard characterization of Vibrio spp. in seafood

The expert consultation noted that further information might become available which would modify the dose-response parameters for V. parahaemolyticus. This will be addressed when the data is forthcoming. With regard to V. vulnificus and V. cholerae, there was a need to identify the uncertainty in the dose-response relationships.

The consultation recommended that the information on the effect of age, sex and ethnic group, where available, should be presented in a consistent manner for the three Vibrio species. More recent consumption pattern data for these groups in the United States than that presented should be available and could therefore be included in the model.

Further information on dose-response relationships with respect to ingested food could be obtained by appropriate investigation of outbreaks. This has rarely been undertaken in a manner that contributed useful data. Ways should be considered to ensure that more useful information can be extracted from such events.

Consideration of the virulence factors of V. parahaemolyticus needs to include tdh-related haemolysin (trh). Cholera toxin needs to be clearly identified as the pathogenic entity for V. cholerae O1 and O139. A distinction between the biologically active proteins and the associated genetic factors needs to be made for each of these organisms.

6.2.3 Exposure assessment of Vibrio spp. in seafood

The consultation endorsed the approach used in selecting the pathogen-commodity combinations for detailed study but emphasized that the documents should clearly state the purpose for their selection.

6.2.3.1 Exposure assessment of Vibrio parahaemolyticus in raw oysters

A schematic representation of the process pathway used for the model should be included at the beginning of the risk assessment document before discussion of the model.

The expert consultation welcomed the approach to modelling presented in the exposure assessment document. It noted, however, that specific components of the model were developed based on data from the United States and that they may not therefore be suitable for application in different geographical areas. To facilitate a wider application of the model there should be instructions on the type and structure of data that would be needed in order to apply it in other geographical areas. The United States data would then be presented as an example data set and a basis for validation of the model under the United States conditions. Guidance could be given on the critical points in the model and the use of the United States data suggested where appropriate local data were not available, with appropriate riders on the constraints arising from this. It was noted that the United States data sets used in the exposure assessment were available in electronic format. As further data sets become available they should also be made available in this way.

The status of the availability of other data identified in the draft document, or sent in response to the FAO/WHO call for data was questioned. The identified data from Australia and Canada were available but data are still required from New Zealand on industry practices (e.g. time and temperature on boats). It was suggested that all the data received be placed in a table and identified as to whether it was inappropriate, of limited usefulness or good for inclusion in the risk assessment model. The expert drafting group agreed to undertake this for each pathogen-commodity combination individually.

The model used to predict the density of V. parahaemolyticus at harvest is an empirical model derived from data collected in the United States. There were a number of potential differences identified between the situation in the United States and other countries - cultivation and harvesting techniques, temperature and salinity of harvesting areas, consumption practices and frequency. Differences between water temperatures at surface and depth could occur in some situations. Exposure to direct solar heating at low tide could alter the predicted effect of water temperature for intertidal fisheries. The proportion of pathogenic V. parahaemolyticus strains could conceivably vary in different geographical areas (it was noted that pathogenic strains may have been introduced into a United Kingdom estuary by discharge of waste from a plant processing imported seafood). The consultation recommended that ideally all of these factors be considered in the model.

At this stage the FDA-VPRA model incorporates only one potential explanatory variable, temperature. Salinity does not appear to be an influential variable in the United States so was not included in the final model. However, this may not be the case elsewhere and so this element may need to be retained. Other data elements should be investigated and incorporated into the model if they are found to be relevant. Local data should be used where possible to develop an empirical model to predict the density of V. parahaemolyticus at time of harvest. This will reduce the uncertainty that may arise from applying the FDA-VPRA model to environments outside that country.

The introductory section should be expanded to include greater consideration of the pathogenic strains. The consultation proposed the inclusion of two further paragraphs, one on the toxins and associated genes linked to pathogenicity and the other on the pandemic spread of a single clone and these have been provided by Prof. Nishibuchi from Japan. Pathogenic strains should be defined as those carrying the tdh and/or trh gene. The tdh and trh genes encode thermostable direct haemolysin (tdh) and tdh-related haemolysin (trh) respectively. Molecular epidemiologic studies have demonstrated that clinical strains usually carry the tdh gene, the trh gene, or both, whereas distribution of these genes in environmental strains is rare. The incidence (expressed in % of the total V. parahaemolyticus population) can be used in the proposed risk assessment model. The trh-positive strains are more frequently distributed in seafood than are tdh-positive strains (Personal Communication, Dr. Nishibuchi, Japan). Studies on the O3:K6 strains revealed that pandemic spread of infection by a new clone of V. parahaemolyticus is currently spreading around the world. The strains belonging to this clone have been isolated from clinical specimens in Asian countries and the United States and emergence of serovariants of the clone has been reported. Additional research and surveillance programmes are needed to identify and trace the spread of new epidemic strains as they emerge.

With regard to the proportion of pathogenic strains, it was emphasized that tdh-/trh+ isolates occur and it was noted that these strains occur with a greater frequency in Asia than in the United States. There were differences between studies in performance of the Kanagawa test - some gave good correspondence with molecular methods whereas others showed that a high proportion of false positives were obtained with the conventional test. It was thought that the molecular tests were preferable but there was some concern about recommending these for use in developing countries where laboratory facilities might be limited.

In order to improve the degree of confidence in the regressions of V. parahaemolyticus concentration on temperature and salinity, it will be necessary to include the appropriate model statistics. The drafting group indicated that the model's predictions were in good agreement with the measured concentrations from a retail survey. While it is possible that a number of compensating errors may have produced these results it provides good evidence that the model is valid. The model predictions, survey results and associated statistics should be included in the exposure assessment.

The exposure assessment for V. parahaemolyticus in raw finfish quoted models for the effect of temperature and salinity on the concentration of V. parahaemolyticus in raw oysters. The output from the present model should be compared to that of the Japanese models quoted in the finfish section. It should be determined whether the Japanese models have been subjected to validation. The latter models included a concentration factor relating the concentration of V. parahaemolyticus in seawater and oysters - caution was urged regarding this as such concentration factors were known to vary greatly for other bacteria with regard to both shellfish species and temperature.

Further explanation should be sought regarding the model assumptions relating to the growth of V. parahaemolyticus in oysters at various temperatures. It was confirmed that actual growth in oysters had only been experimentally observed at 26°C. These data were compared to a published study that had looked at the growth in broth at a number of temperatures. These temperatures included 26°C, at which temperature the growth rate in oysters was a quarter of that in broth. A triangular distribution of a factor between 3 and 5 had therefore been used for the relationship at other temperatures. Some concern was expressed that the growth rate in oysters could be even lower at lower temperatures because of greater competitive pressures - this would have the effect of making the present model conservative. It was also questioned as to whether the distribution of ambient air temperature during the day had been determined. In the current model, the temperature at noon had been used rather than a distribution, which would be more complicated. The need for growth studies in oysters at other temperatures has been emphasized.

A difference in V. parahaemolyticus growth rate may exist between whole and shucked oysters. Growth may be slower in shucked oysters, partly because the pH drops after shucking, because they are usually kept on ice and because of the release of degradative enzymes. Shucked oysters had not been included in the FDA study because they were normally cooked in the United States - it was noted that this was not necessarily the case in other countries.

Uncertainty had been allowed for in the proportion of V. parahaemolyticus pathogenic strains included in the model. At present, an arbitrary symmetrical triangular distribution around the observed values had been incorporated to make some allowance for the uncertainty. In this regard, a question has been raised as to whether the proportion of pathogenic strains detected in retail samples from an area would be the same as that obtained from harvesting area samples. This might be due to the pathogenic strains in the former samples having grown above a detectable threshold. No conclusions were reached on this matter. It was queried as to whether an uncertainty had been incorporated into the model for V. parahaemolyticus die-off. This was not the case and a point estimate had been used.

There was discussion as to the role of antacids in V. parahaemolyticus infection. It was not known whether data on the use of antacids was available in many countries (in Australia, approximately 10% of the population are thought to be on prescribed acid lowering treatments). This may need to be taken into account, however, the exposure assessment currently considers the effect of susceptible populations with regard to serious sequelae after infection has taken place but not with respect to initiation of infection.

At this stage of the risk assessment, mitigation strategies were used in the model as examples of interventions and this should be clearly stated. Defining other mitigation strategies should be left to risk managers. A more comprehensive discussion on mitigation strategies, together with an appraisal of their effectiveness, may be undertaken at a later stage.

The water activity component in the equation in Section 4.1 of the background document could be replaced by a constant. It should be noted that the T_min and T_max values in the equation were theoretical and not actual, although they would be close to these.

6.2.3.2 Exposure assessment of Vibrio vulnificus in raw oyster

The consultation welcomed the proposal to extend the V. parahaemolyticus model to this organism and noted the clear identification of the work and data requirements. The present draft needs to be put into the format of the assessments on the other pathogen-commodity combinations. It was noted that V. vulnificus infections and deaths had been associated with other seafoods and reference to this needed to be included in the hazard identification (the information was provided to the expert drafting group by Dr. Yamamoto). Information from the draft European Commission report on Vibrios in seafood could also be incorporated when the report is publicly available. The progress of this assessment will depend on priorities identified by CCFH and the subsequent identification of appropriate resources. If a numerical model is produced (based initially on United States FDA data) then the United States Environmental Protection Agency (EPA) data would be provided by Dr Tamplin and this could be used to test the performance of the model using data collected from different sources.

6.2.3.3 Exposure assessment of Vibrio parahaemolyticus in finfish consumed raw

The consultation identified the need for the inclusion of further data on the prevalence of V. parahaemolyticus in seafood. Data from a study of seafood imported into Japan would be made available to the drafting group by Dr. Nishibuchi. These data can complement the V. parahaemolyticus data of the Japanese fish market. The imported seafood study included data on the proportion of tdh- and/or trh-positive strains. The data showed that for fresh imports, V. parahaemolyticus was most commonly found in tuna. The rate for tuna from that study and the data presented in the current draft document has implications for the progress of the model as there was currently an assumption that contamination rates would be highest for seafood from coastal and estuarine locations rather than deep sea.

In order to progress development of a model, it was proposed that a single appropriate finfish species, eaten raw, be identified. Expansion of such a model to include other species and the additional complications of undercooked seafood could then be undertaken later if the need and appropriate resources were identified.

In order to improve the degree of confidence in the regressions of V. parahaemolyticus concentration on temperature and salinity, it will be necessary to include the appropriate model statistics. Presentation of the data used in the development of the model would be useful. Data could be presented graphically as was done in the exposure assessment for V. parahaemolyticus in oysters. For the further development of models in this area it was necessary to compare the data already presented on contamination of fish surface and intestines in order to better identify the relative contribution of these sources to the contamination of the final product. Information was presented in the current draft on the effect of disinfecting process water: the data to be presented to the drafting group on the Japanese imported seafood included information on the chlorination of seafood in the producing countries. It should therefore be possible to include these as example mitigation steps as the model is developed further.

The variation of performance of V. parahaemolyticus isolation and enumeration methods in publications referenced in the assessment should be explicitly acknowledged even if the variation cannot be explicitly allowed for. This had been avoided in the FDA-VPRA as the study group had gathered all of its own data specifically to overcome this problem. The consultation agreed that standard methods should be adopted to facilitate the inclusion of data into risk assessment models.

Data on the frequency of consumption of raw fish was needed for the progression of this work. Further data would be sought but there is a need to gather additional reliable and detailed consumption data for the development of such risk assessments.

6.2.3.4 Exposure assessment of Vibrio cholerae in shrimp from developing countries for domestic and export consumption

The consultation emphasized that the predominant route of spread of V. cholerae O1 is via faecally contaminated water or by food contaminated by such water. The latter may apply to seafood. However, an increasing number of epidemiological studies have shown that food is an equally important route of transmission. In Latin America, undercooked seafood has been associated with a cholera outbreak. Although the latter may be significant in individual cases or outbreaks the risk should not be seen out of context with the other, more significant, routes of spread. The consultation noted the contention in the exposure assessment that the infectious dose of cholera is high and this certainly seems to be the case from the volunteer experiments that have been done. However, other evidence in the working paper suggests that the infectious dose may be as low as 10² organisms. It will be important for the development of a model to be clear as to the infective dose that applies in the case of food-associated infection. There is the possibility that seafood containing low concentrations of V. cholerae could contaminate other foods in which greater multiplication could occur.

The schematic process models for both domestic consumption in cholera-endemic countries (Figure 12) and for international trade (Figure 6.13) need to include additional steps in order to reflect differences in production, processing, transport and storage around the world. It was noted that while the approach proposed for modelling V. cholerae in exported shrimps was valid, there are differences in trade that are not captured. One example is that of cooked peeled shrimp which is a major import into countries such as Australia and the United Kingdom.

As with the consideration of V. parahaemolyticus in finfish, the information on seafood consumption should be expanded to include international data sets and this emphasizes the need to take steps to ensure that data appropriate to risk assessments is gathered and maintained in the future.

6.2.4 Conclusions and recommendations

• The format of the Vibrio risk assessment draft document should be revised to reflect the nature of the data and the models for the pathogen commodity combinations as indicated in Annex 4.

• The V. parahaemolyticus temperature model for oysters should be validated at temperatures other than 26°C.

• Relevant research should be undertaken to determine the proportion of pathogenic strains of V. parahaemolyticus in any region to be subjected to the model.

• The methodology used for the determination of pathogenicity of V. parahaemolyticus should be molecular identification of the presence of tdh and/or trh. The potential lack of availability of these techniques in developing countries needs to be recognized.

• With regards to the V. parahaemolyticus model, data from the five other identified countries should continue to be accessed by the expert drafting group and incorporated where appropriate.

• Local data should be used where possible to develop an empirical model to predict the density of Vibrio spp. in seafood at harvest.

• It is important to consider local conditions when applying a model based on the United States conditions, e.g. in New Zealand higher salinity in oyster leases may be a significant controlling factor.

• The drafting group should suggest ways in which the identified "Gaps in the data" should be addressed in order that any data obtained is appropriate to use in the model. The key data requirements for risk assessment of Vibrio spp. in seafood are outlined in Annex 5.

• The drafting group should identify the form of data needed for input to the model.

• In general terms data suppliers should provide information in an easily usable (spreadsheet) format.

• Risk of V. cholerae O1 infection through consumption of imported shrimp in developed countries is negligible.

• In developing countries, there is a risk of infection due to cross-contamination of shrimp through water or handling by asymptomatic carriers.

• More research is needed to understand the levels of toxigenic V. cholerae in waters and shrimp culture environments.

• Provision should be made for dialogue between risk assessors and risk managers to provide feedback on model creation and model documentation to better serve risk managers.

6.3. ISSUES TO BE BROUGHT TO THE ATTENTION OF FAO AND WHO

• Development of risk assessments requires information on food consumption patterns with the following particular details: demographics, preparation methods (where relevant), frequency of consumption and portion size.

• Applying Vibrio risk assessments to particular regions or areas will require the collection of data on the quantitative prevalence and numbers of the pathogenic vibrios at harvest and retail, with a knowledge of associated storage and transport times and temperatures.

  - Such data will only be of the greatest use in such assessments if there is harmonization and appropriate quality assurance of methodologies used to gather the data. It is important to facilitate co-operation between institutes in developing and developed countries.

• In order to enable international use of models based on the temperature and salinity of coastal and estuarine waters, there is a need to collect data on these variables, combined with estimations of V. parahaemolyticus and V. vulnificus concentrations in seawater.

• Further information is needed on the public health significance of pathogenic vibrios in all regions. This should be obtained by the reporting of human infections and monitoring of food contamination. Illness surveillance systems should be sufficiently thorough to provide useful data and collation of data and surveillance and monitoring reports should be undertaken at both national and regional levels.

• Data on sporadic cases as well as outbreaks would be important in assessing the full public health significance of these organisms.

• Further information is needed on dose-response relationships with respect to ingested food. This may be obtained by intensive investigation of outbreaks combined with analysis of incriminated foods.

• There is an uncertainty associated with the effect of different food matrices on the infective dose of different pathogenic vibrios. Research ought to be undertaken to determine the shift in dose-response curves when vibrios are ingested in different food matrices.

7. CONCLUSIONS OF THE EXPERT CONSULTATION

In addition to their specific conclusions on the risk assessments of Campylobacter in broiler chickens and Vibrio spp. in seafood (sections 5.2 and 6.2), the expert consultation concluded that considering the inherent difficulties and limitations, the draft risk assessments were comprehensive, of high quality and potentially useful for decision-making. Appreciation was expressed for the magnitude of work carried out by the expert drafting groups. The work represented a substantial advance in the application of scientific knowledge to improve the objective basis for managing microbiological hazards relating to Campylobacter in broiler chickens and Vibrio spp. in seafood.

The expert consultation agreed to the need to develop risk assessments for Campylobacter spp. in broiler chickens and Vibrio spp. in seafood and endorsed the approach taken by the drafting groups. The expert consultation furthermore recognized that the frameworks elaborated for risk assessments of Campylobacter and Vibrio spp. may provide the basis for the development of tools that could be customized and applied in different countries throughout the world. However it constitutes a big challenge, to ensure the flexibility necessary to account for regional and national differences. It was the opinion of the expert consultation that the risk assessments, when finalized, will help to guide and support risk management decisions.

The expert consultation concluded that validation of results was an essential part of any modelling exercise. However, there are currently only very limited data available that allow validation of important elements in the models proposed, which makes the validation of the final public health estimates difficult. There is an urgent need for epidemiological research to fill these data gaps, in particular research that can validate dose-response models.

The consultation concluded that the work in progress by the expert drafting groups should be seen as an exercise aimed at demonstrating:

• The applicability and usefulness of the available methodology.

• The need for the collection of data based on surveys and experimental studies specifically designed to provide information for microbiological risk assessment and the initial priorities in this regard.

• The opportunity for performing sensitivity or importance analysis to advise risk managers on where the risk management options can be implemented with the best use of resources.

• The potential of this work to answer specific risk management questions.

The consultation supported the recommendation of the previous expert consultation on risk assessments of Salmonella spp. in eggs and broiler chickens and Listeria monocytogenes in ready-to-eat foods^[3] to develop guidelines for judging the quality of risk assessments.

The consultation furthermore concluded that frequent interaction between the risk assessors and risk managers in the further preparation of the risk assessments should be undertaken. Presentations by drafting group representatives at meetings of the CCFH would be a productive means to provide better understanding among risk managers of the potential uses and limitations of models and address the specific questions and concerns of the CCFH.

The consultation acknowledged the value of placing the presentation of the expert drafting groups on the FAO and WHO webpage.