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APPENDIX U

INITIAL SURVEY ON CLOSTRIDIUM BOTULINUM

TOXIN, TYPE E

By

Dr. Arthur F. Novak

Department of Food Science & Technology

Louisiana State University

Baton Rouge, Louisiana

Since our time is very limited and since most of this material will be published in the Proceedings which will be available shortly, I am going to present a very brief summary of our research on Clostridium botulinum type E toxin.

We have collected, under a grant from the United States Atomic Energy Commission, approximately 800 to 1,000 samples, which have been plated out according to recommended procedures that we have obtained from the Department of Health, Education, and Welfare. To date we have not isolated one organism from either shrimp or oysters which could be identified as C. botulinum.

However, we started out to collect many samples from growing areas, and areas near growing areas, and just recently we obtained one rather deep mud sample in the intercoastal waterway from which we have isolated a clostridium. It is not Clostridium botulinum, and we are not sure that it will synthesize a toxin, but we are investigating this possibility at present.

Now I understand that a short time ago, someone in Europe requested that some mud samples from various Gulf areas be forwarded to him, and from these he had isolated Clostridium botulinum from one mud sample obtained around the Galveston Bay

area.

Now you know we are here discussing the possibility of health hazards. We are considering numbers of micro-organisms, and I am concerned not necessarily with numbers, but more so with the types. A few pathogenic organisms are likely to be more dangerous than a million non-pathogens.

In summary, we have not isolated one Clostridium botulinum, and have not found any type E toxin in any Gulf Coast shrimp or oyster. We are going to continue this work, and presently we are organizing a series of studies in which we will test for the presence of salmonella organisms. We are exploring the possibility of determining whether or not any virus is associated with shellfish from the Gulf Coast.

This poses a difficult problem. Some of the scientists at Parke-Davis Pharmaceutical Company have been working with virus, and we have contacted them to obtain some methods which we might be able to apply. Of course, this is a long story, and there are some shortcomings to the procedures as we intend to use it, but we will study other methods also. So, I hope that within two or three years, or the next time we have a meeting here, I will be able to report on possible viral infections and other types of pathogenic micro-organisms.

If any of you have any questions I would be happy to answer them. I don't want to go into our procedures in detail because they will be published.

I might add that we have surveyed and searched most of the health records from the Gulf States, and we have not found one case of botulism on record which has been attributed to Gulf Coast shellfish. I think this is a real compliment to the shellfish industry.

APPENDIX V

FIELD AND LABORATORY STUDIES ON

HEAT-SHOCK METHOD OF PREPARATION OF OYSTERS FOR SHUCKING

By

Somers B. Pringle

Shellfish Section

South Carolina State Board of Health
Beaufort, South Carolina

in cooperation with

Gulf Coast Shellfish Sanitation Research Center
Public Health Service
Dauphin Island, Alabama

A field study, designed to evaluate the commercial heat-shock method of preparation of oysters for shucking in terms of the recommendations of the Cooperative Program, was conducted by the South Carolina State Board of Health in cooperation with the Gulf Coast Shellfish Sanitation Research Center.

In addition to the cooperative field study, a laboratory study was also undertaken by the Research Center. This study was carried out to give a more carefully controlled detailed research evaluation of the heat-shock method of opening oysters.

The objective of the field investigational study as well as the controlled laboratory studies was to evaluate the heat-shock method as practiced in South Carolina and to determine whether the method could be incorporated in the Shellfish Manual on the same basis as is given to the normal cold shucking method or, if this were not indicated, to determine what special requirements might be necessary.

This was to be accomplished by a study of the commercial heat-shock process as measured by accepted bacteriological parameters. Included would be (1) a study of the comparative effect of the heat-shock process and normal cold shucking methods of opening oysters on the bacteriological quality of shucked shellfish meats and (2) an examination of the sanitary aspects of the process. Information about factors which influence the public health aspect of the process would be collected and any sanitary requirements determined and appropriate revisions, if required, inserted in the Manual. The results of the laboratory study would serve to provide information obtained under more carefully controlled conditions.

The field study of the commercial aspects of the process was conducted during January of this year and consisted of the bacteriological examination of traced lots of oysters taken from various stages of the heat-shock process as well as the examination of the immersion water. The oyster samples represented shellstock opened before immersion, immediately after immersion and after remaining on the shucking bench for various lengths of time. The water samples represented the immersion water at the time the oysters were dipped.

Five commercial plants were involved in the cooperative study. The study of the sanitary aspects of the process in each plant was accomplished by recording information on harvesting area, date and time of harvest, length of storage and storage conditions, ambient air temperature, volume and temperature of immersion water, volume of oysters per dip, period of time of heat shock, condition of shellstock and any unusual condition of processing. Forms were developed and used to standardize the recording of this information.

The water and oysters were examined for coliform and fecal coliform MPNs and by standard 35°C. plate counts.

In the laboratory investigations particular attention was given to the bacteriological quality of the oyster, however, investigations as to the effect of the heat-shock method on the chemical and physical quality of the oyster were also initiated.

For these investigations, a specially designed all glass water bath with a constant agitation of distilled water was used for immersion of the oysters. The temperatures were controlled with an electric immersion heater in an outer water bath coupled with an electronic thermoregulator. Both Alabama oysters and South Carolina oysters were used in the laboratory studies.

The heat-shock method of preparing oysters for shucking, as carried out in South Carolina, is based upon the premise that immersing the oyster in hot water at a given temperature for a definite period of time, followed by immediate chill down, will result in a product that is easier to open but one that may be approved by the Cooperative Program. The results of this study indicated that certain changes due to the process may be expected to occur in the bacteriological indices used as a measure of the sanitary quality of shellfish. The effect of the heat-shock process was a reduction in the density of the bacterial indices measured. This reduction was evident both in the over-all mean values for the indices from the before immersion stage of the process to the after immersion and shucking bench stages of the process and also in the mean ratios of the bacterial indices at the various stages in the process on a traced lot basis.

Thirty-two lots of shellfish meats and process waters collected at various stages of the commercial process were examined.

The bacterial content of the immersion waters was found to be consistently low. Viable collform organisms were recovered in only 2 of 31 samples tested. Viable fecal coliforms were recovered in only 1 of the 31 samples. The 35°C. plate count ranged from 5 to 3000 organisms per milliliter with a median value of 120 organisms per milliliter.

Variations in the densities of the selected bacterial indices at three stages of the heat-shock process are summarized in Table 1.

Values listed at the three percentile levels were derived from the probability plots with no attempt at traced lot analysis. The heat-shock process resulted in an over-all reduction in the coliform and fecal coliform MPNs at all percentile levels. The greatest reduction occurred in the samples examined immediately after shocking. Holding on the shucking bench appears to result in a slight increase in these two groups of bacterial indices as compared to oysters examined immediately after shocking, however, these levels remain significantly lower than the levels obtained from samples from the cold shucking process.

The 35°C. plate count showed a slight decrease at the 25 and 50 percentile levels both in oysters immediately after shocking and in shocked oysters held on the shucking bench. At the 90 percentile level the plate count of the oysters immediately after shocking was comparable to the count obtained from cold shucked oysters. At this percentile level counts from shocked oysters held on the shucking bench were slightly higher than counts obtained from cold shucked oysters or oysters examined immediately after shocking.

The average percent change in coliform and fecal coliform densities examined on a traced lot basis is shown in Table 2. In most instances the shocking process resulted in a reduction in the coliform and fecal coliform MPNs of the shocked oyster meats. A comparison of oysters examined immediately after shocking to cold shucked oysters indicates a reduction in coliform and fecal coliforms of 50 percent and 25 percent, respectively. A comparison of shocked oysters held on the shucking bench with oysters examined immediately after shocking indicated a 40 percent increase in coliform MPN and a 10 percent increase in fecal coliform MPN. A comparison of shocked oysters held on the shucking bench with cold shucked oysters shows a 47 percent reduction in coliform MPN and a 17 percent reduction in fecal coliform MPN of the shocked oysters over the cold shucked oysters.

In 87 percent of the lots of shocked oysters tested immediately, the shocked oysters showed a decrease or no increase in coliform density when compared to cold shucked oysters. In 72 percent of the lots tested, the shocked oysters showed a decrease or no increase in fecal coliform densities.

Table 1. Densities of Selected Bacterial Indices at Three Stages of Processing of Oyster Meats

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Field Study

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Table 2. Changes in Coliform and Fecal Coliform Densities During

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Shucking Process

Over-all Process

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In a comparison of the shocked oysters examined immediately with shocked oysters held on the shucking bench, 53 percent of the lots tested showed a decrease or no increase in coliform MPN, however, in the remaining 47 percent of the lots there was an increase in coliform MPN. On the same basis of comparison, 43 percent of the lots showed a decrease or no increase in fecal coliform MPN, and 57 percent of the lots showed an increase in fecal coliform density.

In a comparison of shocked oysters held on the shucking bench with cold shucked oysters, 80 percent of the lots tested showed a decrease or no increase in coliform MPN with 20 percent of the lots showing an increase. With respect to fecal coliform MPN, 71 percent of the lots tested showed a decrease or no increase and 29 percent of the lots showed an increase.

The laboratory investigations conducted to determine the influence or the heat shocking process on the bacteriological parameters consistently resulted in reductions In coliform MPNs, fecal coliform MPNs and 35°C. plate counts on both Alabama and South Carolina oysters.

A comparison of results (Table 3) shows that the percent reduction in the selected bacterial parameters was significantly higher in the South Carolina oysters. This difference in bacterial reduction appears to correlate with the heat penetration differential determined in the oysters from the two sources. The average internal temperature of the South Carolina oysters at the end of three minutes immersion was found to be higher than the Alabama oysters immersed for an identical interval.

Table 3. Comparative Reduction in Selected Bacterial Parameters
in Alabama and South Carolina Oysters Immersed Three Minutes at 150°F.

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The results of the studies conducted by the cooperative field investigation team and by the Research Center at Dauphin Island of the heat-shock method of preparation of oysters for shucking show that a definite trend exists in a quantitative reduction in the parameters which are normally used in assessing the bacterial quality of shellfish. The method does not cause an increase and, therefore, the method should be approved on an interim basis for acceptance in the Cooperative Program.

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