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National Shellfish Sanitation Program
Guide for the Control of Molluscan Shellfish
2003

Guidance Documents

Chapter IV. Naturally Occurring Pathogens

Table of Contents

( .01 Vibrio Risk Management for Oysters | .02 Vibrio vulnificus Management Plan | .03 Vibrio parahaemolyticus Interim Control Plan | .04 Validation/Verification Interim Guidance )

.01 Vibrio Risk Management for Oysters

Background

Current information concerning Vibrio vulnificus, which is responsible for several shellfish associated illnesses and deaths each year can be found in Watkins and McCarthy (1994).

A small number of shellfish-borne illnesses have also been associated with bacteria of the genus Vibrio (Bonner, 1983; Blake et al.,1979; Morris, 1985; Joseph et al.,1982; Roderick, 1982). The Vibrios are free-living aquatic microorganisms, generally inhabiting marine and estuarine waters (Joseph et al, 1982: Spira, 1984; Colwell 1984; Bachman, 1983 ). Among the marine Vibrios classified as pathogenic are strains of non-01 Vibrio cholerae, V. parahaemolyticus, and V. vulnificus (Bachman, 1983; Desmarchelier, 1984; Blake, 1980). All three species have been recovered from coastal waters in the United States and other parts of the world (Joseph, 1982; Colwell, 1984; Blake, 1980; DePoala, 1981; Madden, 1982; Davey, 1982; Oliver, 1983; Tamplin, 1982; NIH, 1984). These and other Vibrios have been detected in some environmental samples recovered from areas free of overt sewage contamination and coliform (Bonner, 1983; Joseph, 1982; Spira, 1984).

In general, shellfish-borne vibrio infections have tended to occur in coastal areas in the summer and fall when the water was warmer and vibrio counts were higher (Bonner, 1983; Morris, 1985; Joseph, 1982). V. parahaemolyticus and non-01 V. cholerae are commonly reported as causing diarrhea illness associated with the consumption of seafood including shellfish (Bonner, 1983; Blake, 1979; Morris, 1985; Joseph, 1982; Baross and Liston, 1970; Morris, 1981). In contrast, V. vulnificus has been related to two distinct syndromes: wound infections, often with tissue necrosis and bacteremia, and primary septicemia characterized by fulminant illness in individuals with severe chronic illnesses such as liver disease, hemochromatosis, thalassemia major, alcoholism or malignancy (Bonner et al., 1983; Tacket, 1984). Increasing evidence shows that individuals with such chronic diseases are susceptible to septicemia and death from raw seafood, especially raw oysters (Bonner et al., 1983; Blake, 1979; Morris, 1985; Rodrick, 1982; Bachman, 1983; Blake, 1980; Oliver, 1983; NIH, 1984; Tacket, 1984; Oliver 1982; FDA, 1985). Shellfish-borne vibrio infections can be prevented by cooking seafood thoroughly, keeping them from cross contamination after cooking, and eating them promptly or storing them at hot (60°C or higher) or cold (4°C or lower) temperatures. If oysters and other seafood are to be eaten raw, consumers are probably at lower risk to vibrio infection during months when seawater is cold than when it is warm (Blake, 1983 and 1984).

**.02 Vibrio vulnificus Management Plan**

The voting delegates at the 1999 Annual Meeting in New Orleans created the Vibrio Management Committee (VMC). Subsequently, Vibrio vulnificus and Vibrio parahaemolyticus subcommittees have been charged to develop appropriate illness control measures for these two pathogens. The VMC provides guidance and oversight to the subcommittees. Subcommittee recommendations are reviewed by the VMC before submittal to Task Forces. At the 2001 annual meeting, Task Forces reviewed the VMC's recommendation of reducing the rate of etiologically confirmed shellfish-borne Vibrio vulnificus septicemia with the intention to submit the recommendation to the voting delegates. The goal is to reduce the rate of illness reported in California, Florida, Louisiana and Texas due to the consumption of commercially harvested raw or undercooked oysters by 40 percent, for years 2005 and 2006 (average) and by 60 percent for years 2007 and 2008 (average) from the average illness rate for the years 1995 - 1999 of 0.306/million. The list of states may be adjusted if after a thorough review, epidemiological and statistical data demonstrates that it would be appropriate. The rate of illness shall be calculated as the number of illnesses adjusted for population. This adjustment will be performed in consultation with statisticians and epidemiologists from California, Florida, Louisiana and Texas and Federal agencies. The baseline data and all future data for measuring illness reduction shall be the reported illnesses in the California, Florida, Louisiana and Texas for the period 1995 to 1999, inclusive, as compiled by the Southeast Regional Office of the U.S. Food and Drug Administration. The data used for measuring goal attainment shall begin with 2002 data. For the purpose of maintaining an accurate count of the number of illnesses report by each state (California, Florida, Louisiana and Texas), the following will apply:

(a) Illness cases counted are those reported by California, Florida, Louisiana and Texas;

(b) Each illness case is recorded under the state that reports it;

(d) In the event more than one report per case is filed, the case is recorded under the state of diagnosis.

The formula for calculating the rate of illness is as follows:

number of cases
population

The V.v. subcommittee members will include, at a minimum, balanced representation from industry and state shellfish control authorities from Vibrio vulnificus Illness Source States California, Florida, Louisiana and Texas, FDA, NOAA, EPA, CDC, state epidemiologists; as well as industry and shellfish control representatives from other regions. Vibrio vulnificus Illness Source States are those states reporting two (2) or more etiologically confirmed shellfish-borne Vibrio vulnificus illnesses since 1995 traced to the consumption of commercially harvested raw or undercooked oysters that originated from the waters of that state. Etiologically confirmed means those cases in which laboratory evidence of a specific agent is obtained and specified criteria are met.

Recognizing the increasing importance and roles for the Committee, leadership will be expanded and structured in a similar manner as stated in the ISSC By-Laws for Task Forces (reference: ISSC By-Law, Article I Task Forces). The VMC Chair shall alternately be selected from a state shellfish control authority and from industry. The Board Chairman, with approval of the Board, shall appoint a VMC Chair and Vice-Chair. If the VMC Chair represents a state shellfish control authority, the Vice-Chair shall be an industry representative. At the end of the VMC Chair's term of office, the Vice Chair will become Chairman and a new Vice Chair will be appointed who represents the same segment of the Conference as the outgoing VMC Chair. A VMC Chair and Vice Chair should be appointed before October 1, 2001 in order to be consistent with plans for annual VMC meetings and with the effective date of Vibrio vulnificus Risk Management Plans. Likewise, the term of office shall be for (2) years.

The VMC will meet at least annually to develop and approve annual VMC work plans for Vibrio vulnificus illness reduction and review progress. A series of work plans, each covering a one-year period shall be adopted. The first work plan and progress review period will cover a seventeen-month period from August 1, 2001 to December 31, 2003 followed subsequently by annual work plans. Work plans will include goals, tasks, performance measures and assessment methods to track and achieve progress towards the illness reduction goals. The work plans will be developed by the VMC and approved by the VMC membership. The chair of the VMC will deliver a written annual progress report, including a summary of the previous year's progress made in the education program, to the ISSC March executive board meeting. The report shall be made available to the general membership. The annual work plan structure, outlined below, provides adaptive management and assures consistent progress towards the illness reduction goals. If annual assessment of progress towards achieving the illness rate reduction goals show inadequate progress the VMC shall incorporate actions into current and subsequent work plans to assure success in achieving those goals. In addition, if annual review shows inadequate progress the VMC will develop issues for deliberation at the 2005 biennial meeting to consider actions such as:

increased educational efforts,
limited harvest restriction,
reduction in time from harvest to refrigeration,
phased-in post-harvest treatment requirements, or
other equivalent controls.

Work plans developed by the VMC shall include the following elements and shall define the administrative procedures and resources necessary for accomplishment (i.e. establishment and maintenance):

(a) An ISSC Consumer Education Program targeted toward individuals who consume raw oysters and whose health condition(s) increase their risk for Vibrio vulnificus infection. The Education Program's objectives will be 1) to increase the target audience's awareness that eating raw, untreated oysters can be life-threatening to them, and; 2) to change the at-risk group's oyster-eating behavior, i.e., to reduce or stop eating raw, untreated oysters. The ISSC Vibrio Management Committee and the Vibrio vulnificus Education Subcommittee will evaluate Year 2001 survey results and compare them with the Year 2003 or 2004 survey results to determine the effectiveness in meeting the two objectives of the Vv education effort: (1) Show 40% increase in awareness of risk from Vv; and (2) Show 15% increase in at-risk consumers no longer eating raw oysters while minimizing impacts to non-at-risk consumer raw oyster consumption.

(i) The Consumer Education Program will focus educational efforts in California, Florida, Louisiana and Texas. The Education Program will make educational materials available to additionalstates upon request.

(ii) Educational approaches will emphasize partnerships with health and advocacy organizations, and include dissemination of printed materials, posting materials on the Internet, broadcast of television spots, press releases, and other measures deemed effective such as the USDA Physician Notification Program.

(iii) Survey assessments at the state level shall be usedas a means of assessing the baseline knowledge and effectiveness of educational interventions.

(b) Administration of a survey to determine the current Vibrio vulnificus disease reporting and education in each state.

(c) Creation of a working group to work cooperatively with local, state, and federal agencies and programs to assist in the collection of environmental and epidemiological data to further expand on the current information available. A coordinator may be utilized to facilitate the activities of this working group to develop standardized collection of environmental and epidemiological information from harvest to consumer.

(d) Industry-implemented post-harvest controls to reduce Vibrio vulnificus levels in oyster shellstock which may include: time-temperature, post harvest treatment (i.e. hydrostatic pressure, cool pasteurization, IQF, and irradiation--pending approval), rapid chilling and other emerging technologies.

(e) Pursuit of ISSC options such as industry education and communication; FDA label incentives; PHT specific growing area classifications; targeted time/temperature assessment by FDA during annual shellfish program evaluations; assistance, as necessary, for the further study and possible implementation of dockside icing to investigate its effects on shelf life and variations in the effectiveness of the method as a result of seasonal and regional differences and incentives to add refrigeration capacity to harvest vessels. The goal will be to provide incentives necessary to post-harvest treat 25 percent of all oysters intended for the raw, half-shell market during the months of May through September harvested from a Source State by the end of the third year (December 31, 2004). The assessment will include the capacity of all operational plants and the capacity of plants under construction. Should the 25 percent goal not be accomplished, the VMC will investigate and report their findings as to why the goal was not reached.

(f) Development by the VMC of a list of issues relating to public health, various technologies including Post-harvest treatments; marketability; shelf -life and similar matters that lend themselves to investigation. The VMC will work with FDA, NOAA, CDC, EPA, the shellfish industry and other entities as appropriate to obtain or facilitate the investigation of the issues listed and take the results into account as it develops plans or recommended Issues for the ISSC.

(g) Provision for VMC compilation and review of the data on rates of illness, which will be made available to the ISSC at the ISSC Biennial meeting following the year in which the data was gathered. In the event that the data is not available at the time of the meeting, the VMC shall meet and review the data when it becomes available and issue a compilation report, which will be made available to the entire ISSC membership. In the event there is no Biennial meeting scheduled for a certain year, the VMC shall meet and review the data when it becomes available and issue a compilation report which will be made available to the entire membership.

(h) Provision for a VMC evaluation of the effectiveness of reduction efforts, which will be conducted at the end of the fifth year (December 31, 2006). The evaluation will determine whether the 40 percent, 5-year goal to reduce the rate of illnessor education/consumer intervention or post harvest controls performance measures set forth in prior work plans have been achieved. Should the VMC evaluation indicate the 40 percent, 5 year goal has not been accomplished, the committee will identify additional harvest controls in the 2007 - 2008 work plan to assure achievement of the 60 percent reductionin the rate of illnessgoal by the close of the seventh year. In addition, the VMC will evaluate the requirements in Section 04.C. with the possibility of changing the controls to achieve remaining illness reduction goals.

Should a disagreement arise between FDA and the Authority on the equivalency of a control as described in .04(C), the V.v. Subcommittee will be requested to provide guidance.

.03 Vibrio parahaemolyticus Interim Control Plan

A. Contingency Plan.

(1) If the waters of a state have been confirmed as an original source of oysters associated with two or more confirmed V. parahaemolyticus illnesses annually in the most recent three years (excluding years when growing areas were closed at least half of the period from June through September), or with an outbreak in the last three years, the Authority should develop and adopt a V. parahaemolyticus contingency plan.

(2) The plan should define the administrative procedures and resources necessary to accomplish the following:

(a) Identify and define growing areas in the state affected by V. parahaemolyticus based on hydrographic and geological parameters and other considerations relevant to control of a naturally occurring pathogen;

(b) Conduct an oyster meat sampling and assay program in those areas which have been associated with a V. parahaemolyticus illness;

(d) Prevent harvesting of affected oysters;

(e) Provide for oyster recall if an oyster growing area is closed as a result of illness;

(f) Notify the shellfish industry and the local health jurisdictions in the state of the potential for illnesses due to V. parahaemolyticus prior to historical times of onset or at a minimum of once a year;

(g) Issue a health advisory to the public about the potential problem and advise the industry to educate wholesalers, retailers, and consumers about the potential problem, with recommendations that oysters not be consumed raw during periods historically affected by V. parahaemolyticus.

(3) The plan may include agreements or memoranda of understanding between the Authority and individual oyster harvesters and processors to allow harvesting of oysters from growing areas which have been placed in the closed status, as specified in C. for:

(a) Post-harvest treatment by a process which has been demonstrated to reduce V. parahaemolyticus levels in oysters to non-detectable; or,

(b) Shucking and labeling "for cooking only"; or,

(c) Under specific circumstances, as approved by the Authority, where the oyster shellstock will be sold to a retailer or food establishment, food processor, or to a shucker-packer and labeled in accordance with (3)(b); or,

(d) Under specific circumstances, as approved by the Authority, where the oyster shellstock will be cooked and controls exist to ensure cooking.

B. Vibrio parahaemolyticus Monitoring

(1) In all areas where two or more confirmed V. parahaemolyticus illnesses have occurred annually in the most recent three years (excluding years when growing areas were closed at least half of the period fromJune through September), representative samples of oysters should be collected at least monthly during harvest periods historically associated with illnesses and otherwise as determined by the Authority. All samples will be analyzed using the direct plating procedures and gene probe methods or enrichment PCR procedures for total (tlh+ colonies) and pathogenic (tdh+ colonies) V. parahaemolyticus. *

(2) In all areas where a confirmed V .parahaemolyticus outbreak has occurred within the last three years, representative samples of oysters should be collected when environmental conditions are favorable for V. parahaemolyticus growth and/or periods historically associated with illness as determined by the Authority Samples should be collected and analyzed weekly during the year of and the first year after an outbreak, and at least monthly during the second and third years after an outbreak. All samples will beanalyzed using the direct plating procedures and gene probe methods or enrichment PCR procedures for total (tlh+ colonies) and pathogenic (tdh+ colonies) V. parahaemolyticus.

(3) In order to determine the number of samples that would be appropriate for V. parahaemolyticus monitoring, the following factors should be considered:

(a) The size of the growing area;

(b) The amount of oyster shellstock typically harvested from the area;

(4) In the event that emerging technologies and research identify pathogenic strains other than or in addition to tdh+ strains, the Authority may adopt and FDA may approve other or additional monitoring and control methods for preventing V. parahaemolyticus illnesses.

C. Closed Status of Growing Area Based On Monitoring Results.

(1) The growing area as defined in accordance with A.(2)(a) should be placed in the closed status for oyster harvest, except as allowed under A.(3), if a total of 5 or more pathogenic (tdh+) V. parahaemolyticus colony-forming units (CFU) per 0.1 gram, confirmed by at least one pathogenic (tdh+) V. parahaemolyticus CFU per 0.1 gram by replicate analysis, are found for any oyster sample from the harvest area. If any sample shows total (tlh+) V. parahaemolyticus counts above 5,000 CFU per gram, then additional samples (twice the number collected as determined by the Authority) should immediately be collected and analyzed for pathogenic V. parahaemolyticus. Should any of these additional samples show 5 or more pathogenic V. parahaemolyticus CFU per 0.1 gram, confirmed by at least one pathogenic V. parahaemolyticus by replicate analysis, the area will be placed in the closed status for oyster harvest, except as allowed under A.(3).

(2) The closed status should remain in effect until two consecutive representative samples of oyster meats, collected a minimum of four days apart, show fewer than 5 pathogenic (tdh+) V. parahaemolyticus CFU in 0.1 gram, or show no pathogenic V. parahaemolyticus by replicate analysis. If any sample shows total V. parahaemolyticus counts above 5,000 CFU per gram, then additional samples (twice the number collected as determined by the Authority) should immediately be collected and analyzed for pathogenic (tdh+) and total (tlh+) V. parahaemolyticus. Should those samples show fewer than 5 pathogenic (tdh+) V. parahaemolyticus CFU in 0.1 gram, or show no pathogenic V. parahaemolyticus by replicate analysis, the growing area should be opened.

(3) The analysis leading to a decision to return a growing area to the open status should be adequately documented.

D. Illness Outbreak.

(1) When a growing area is implicated in a V. parahaemolyticus illness outbreak, the Authority shall follow the procedures prescribed in Chapter II Section@.01A through E. If a growing area is closed due to an illness outbreak, the closed status should remain in effect until two consecutive representative samples of oyster meats, collected a minimum of four days apart, show no pathogenic (tdh+) V. parahaemolyticus CFU in replicate 0.1 gram portions of oyster meat and less than 5,000 total (tlh+) V. parahaemolyticus CFU per gram.

(2) If additional confirmed V. parahaemolyticus illnesses occur within 2 weeks of re-opening, they should be considered a continuation of the illness outbreak. The growing area should immediately be placed in the closed status, and re-opening may only occur when environmental conditions shift to those unfavorable to the growth of V. parahaemolyticus, or the Authority, in conjunction with the state epidemiologist, develops and implements a sampling plan.

E. Records.

The Authority should maintain a copy of all of the following records:

(1) All information, including monitoring data, relating to the levels of V. parahaemolyticus in the oyster growing areas;

(2) Copies of notices placing growing areas in the closed status;

(3) Evaluation reports; and,

(4) Copies of notices returning growing areas to the open status.

* Direct plating procedure by Cook, D.W. et al, 1999. Procedure for enumeration of Vibrio parahaemolyticus in shellfish meats. A collaborative study by shellfish producing states, FDA, and the ISSC; gene probe methods for total (tlh+ colonies) V. parahaemolyticus (McCarthy, S.A. et al, 1999. TRS. Appl. Microbiol.28:66-70) and virulent (tdh+ colonies) V. parahaemolyticus (McCarthy, S.A. et al, 1999. Abstracts of the 99^th General Meeting of the American Society for Microbiology, p.512).

[References for the direct plating, digoxygenin DNA probe method and the enrichment PCR procedure adapted to the VpICP can be provided.]

References for Vibrio parahaemolyticus Methods

Cook, D.W., A. DePaola, and S.A. McCarthy. 2000. Direct plating procedure for the enumeration of total and pathogenic Vibrio parahaemolyticus in oyster meats. FDA, Office of Seafood, Gulf Coast Seafood Laboratory, Dauphin Island, AL. 8 pp.

Gooch, J.A., A. DePaola, C.A. Kaysner, and D.L. Marshall. 2001. Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters. Appl. Environ. Microbiol. 67(2):721-724.

Kaysner, C.A. and A. DePaola, Jr. 2001. Chapter 40, Vibrio, p. 405-420. In Downes, F.P. and K. Ito (eds.), APHA Compendium of Methods for the Microbiological Examination of Foods, 4^th Edition, 2001, American Public Health Association, Washington. D.C.

McCarthy, S.A., A. DePaola, C.A. Kaysner, W.E. Hill, and D.W. Cook. 2000. Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of Vibrio parahaemolyticus. J. Food Protect. 63(12):1660-1664.

McCarthy, S.A., A. DePaola, D.W. Cook, C.A. Kaysner, and W.E. Hill. 1999. Evaluation of alkaline phosphatase- and digoxigenin-labeled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus. Letters in Applied Microbiology 28(1):66-70.

McCarthy, S.A., A. DePaola, C.A. Kaysner, W.E. Hill, and D.W. Cook. 1999. P1. Comparison of PCR and DNA hybridization methods for detection of the tdh gene of Vibrio parahaemolyticus, p. 512. In American Society for Microbiology (ed), Abstracts of the 99^th General Meeting of the American Society for Microbiology. American Society for Microbiology, Washington, D.C.

References

Bachman, B. et al. 1983. Marine Noncholera Vibrio Infections in Florida. So. Med. Jour. 76:296-303.

Baross, J. and J. Liston. 1970. Occurrence of Vibrio parahaemolyticus and Related Hemolytic Vibrios in Marina Environments of Washington State. Appl. Microbiol. 20:179-186.

Blake, P.A. et al. 1979. Disease Caused by a Marine Vibrio, Clinical Characteristics and Epidemiology. N. Eng. J. Med. 300: 1-5.

Blake, P.A. et al. 1980. Disease of Humans (Other Than Cholera Caused by Vibrios). Ann. Rev. Microbiol. 34:341-367.

Blake, P.A. 1983. Vibrios on The Half Shell: What the Walrus and the Carpenter Didn't Know. Ann. of Int. Med. 99:558-559.

Blake, P.A. 1984. Prevention of Food-Borne Disease Caused by Vibrio Species. In: Colwell, R.R., et al., eds. Vibrios in the Environment. John Wiley and Sons. New York, NY. pp. 579-590.

Bonner, J.R. et al. 1983. Spectrum of Vibrio Infections in a Gulf Coast Community. Ann. Intern. Med. 99:464-469.

Colwell, R.R. 1984. Vibrios In The Environment In: Colwell, R.R.; et al., eds. Vibrios in the Environment. John Wiley & Sons. New York, NY. pp. 1-12.

Davey, G.R. et al. 1982. Detection of Vibrio cholerae In Oysters, Water And Sediment From The Georges River. Food Technol. Aust. 34:334-336.

DePaola, A. 1981. Vibrio cholerae in Marine Foods and Environmental Waters. A literature review. Jour. of Food Sci. 46:66-70.

Desmarchelier, P.M. 1984.. Significance Of Vibrio spp. in Foods. Food Technol. Aust. 36:220-222.

Food and Drug Administration. 1985. Vibrio vulnificus and Patients with Liver Disease. In: FDA Drug Bulletin. April. 15(1):5-6.

Joseph, S.W. et al. 1982. Vibrio parahaemolyticus And Related Halophilic Vibrios. CRC Crit. Rev. in Microbiol. 10:77-124.

Madden, J.M. et al. 1982. Vibrio cholerae. In Shellfish From U.S. Coastal Waters. Food Tech. 36(3):93-96.

Morris, J.G. Jr. et al. 1981. Non-O group 1 Vibrio cholerae Gastroenteritis in the United States. Ann. of Int. Med. 94:656-658.

Morris, J.G., Jr. et al. 1985. Cholera And Other Vibrioses In The United States. N. Engl. J. Med. 312:343-350.

National Institute of Health (NIH). 1984. Highly Invasive New Bacterium Isolated From U.S. East Coast Waters. JAMA. 251:323-325.

Oliver, J.D. 1982. The Pathogenicity and Ecology of Vibrio vulnificus. Marine Tech. Soc. Jour. 15:45-52.

Oliver, J.D. et al. 1983. Distribution of Vibrio vulnificus and Other Lactose-Fermenting Vibrios in The Marine Environment. Appl. Environ. Microbiol. 45:985-998.

Rodrick, G.E. et al. 1982. Human Vibrio Gastroenteritis, Symposium On Intestinal Infections. Med. Clinics of North Amer. 66:665-673.

Spira, W.M. 1984. Tactics For Detecting Pathogenic Vibrios In The Environment. In: Colwell, R.R. et al., eds. Vibrios in the Environment. John Wiley & Sons. New York, NY pp 251-268.

Tacket, C.O., et al. 1984. Clinical Features and an Epidemiological Study of Vibrio vulnificus Infections. Jour. Infect. Dis. 149:558-561.

Tamplin, M., et al. 1982. Isolation and Characterization of Vibrio vulnificus From Two Florida Estuaries. Appl. Environ. Microbiol. 44:1466-1470.

Watkins, W. and S. McCarthy. 1994. Proceedings of the 1994 Vibrio vulnificus Workshop. U.S. Department of Health and Human Services, Public Health Service, Office of Seafood (HFS-400), Shellfish Sanitation Branch, 200 C Street, SW, Washington, D.C. 175 pages.

.04 Validation/Verification Interim Guidance

Process Validation

Used for the initial validation of a process or when there has been a change to a previous validation process.

Data on ten processed samples obtained on each of three processing days (total of 30 samples) are required.
All samples used on a processing day must come from the same lot of shellfish and be determined to have an adjusted geometric mean (AGM) MPN of 100,000 per gram or greater as described below for initial load testing. (If some lower initial levels are used the process will only be validated for those maximum initial levels.)
Samples should be distributed throughout the processing day. A sample will consist of a composite of 10 to 12 oysters processed at one time.
The zero hour level may be achieved through naturally occurring Vibrio levels in shellfish and, where not practical, by time/temperature abuse. (Inoculated pack samples may be used as appropriate.)
Analytical methodology to determine Vibrio levels should be the official methods previously endorsed by the ISSC.
Microbiological testing for processed samples will be by a single dilution five-tube MPN, inoculating with 0.01 g of shellfish per tube.
The numerical value of the endpoint criteria should represent the lowest sensitivity of the MPN method, which is less than 30 per gram.
For the process to be validated, no more than three samples out of 30 may fail. Failure is indicated by more than two out of five MPN tubes in any sample being positive. If any one sample has all five MPN tubes positive, the validation process will fail.

Equipment Validation

Used to ensure that each unit of equipment will deliver the validated process. May be accomplished using either of two methods:

The process described under "Revalidation," below;
A physical test of the equipment (e.g., thermal distribution study) that is designed to ensure that, when properly operated, it will consistently deliver the validated process.

Revalidation

Used when verification sampling indicates a failure in the process.

Data on ten processed samples obtained throughout a processing day are required.
All samples used on a processing day must come form the same lot of shellfish and be determined to have an adjusted geometric mean (AGM) MPN of 100,000 per gram or greater as described below in initial load testing.
A sample will consist of a composite of 10 to 12 oysters processed at one time.
The zero hour level may be achieved through naturally occurring Vibrio levels in shellfish and, where not practical, by time/temperature abuse. (Inoculated pack samples may be used as appropriate).
Microbiological testing for processed samples will be by a single dilution five-tube MPN, inoculating with 0.01 g of shellfish per tube.
The numerical value of the endpoint criteria should represent the lowest sensitivity of the MPN method, which is less than 30 per gram.
For revalidation, no more than one sample out of ten may fail. Failure is indicated by more than two out of five MPN tubes in any sample being positive. If any one sample has all five MPN tubes positive, revalidation will fail.

Initial Load Testing

Initial level of vibrios in shellfish for each lot of shellfish used in validation shall be 100,000 MPN per gram or greater based on the adjusted geometric mean (AGM) of the MPNs/g of four samples where the AGM is given by:

AGM = the geometric mean of the 4 MPNs/g multiplied by an adjustment factor of 1.3

Note: If 4 samples from a lot of shellfish with a true density of 100,000 cells per gram are examined by the MPN procedure, the probability of the geometric mean of the MPNs showing 100,000 or greater is about 50%. In an attempt to improve the probability of samples being accepted when the true density is 100,000/g an adjustment factor of 1.3 was selected based upon statistical analysis provided by Dr. Bob Blodgett.

Verification

Used to verify that a previously validated process is working properly.

Process verification by microbiological testing should be done monthly
The number of samples/sub samples for verification and the pass/fail criteria for the verification process will be determined by the validation/verification workgroup following evaluation of statistical data to be supplied by Dr. Bob Blodgett.
The dealer in conjunction with the SSCA shall annually evaluate the previous 12 months of data and the HACCP plan.
The dealer may elect, with SSCA concurrence, to conduct quarterly sampling if the previous 12 verification samples pass.

Verification Sampling Protocol Decision Tree

References for Vibrio parahaemolyticus Methods

References

Bachman, B. et al. 1983. Marine Noncholera Vibrio Infections in Florida. So. Med. Jour. 76:296-303.

Baross, J. and J. Liston. 1970. Occurrence of Vibrio parahaemolyticus and Related Hemolytic Vibrios in Marina Environments of Washington State. Appl. Microbiol. 20:179-186.

Blake, P.A. et al. 1979. Disease Caused by a Marine Vibrio, Clinical Characteristics and Epidemiology. N. Eng. J. Med. 300: 1-5.