PUBLIC HEALTH BRIEFS
Failure to Detect Hepatitis B Surface Antigen (HBSAg) in Certified Shellfish From New England RAYMOND S. KOFF, MD LORNA J.D. CONNELLY The major known viral hazard associated with ingestion of bivalve mollusks (clams, oysters, and mussels) is hepatitis. Epidemiologic investigation implicated ingestion of raw oysters as a mode of transmission of Hepatitis A in Sweden two decades ago,' and a number of outbreaks of hepatitis in the United States were subsequently traced to uncooked bivalve mollusks.2 In addition to recognized outbreaks, sporadic cases of viral hepatitis following ingestion of raw or inadequately cooked clams and oysters have been recorded in New England3 and Germany.4 Recently, Heptatitis B surface antigen (HBSAg), a marker of the Hepatitis B virus (HBV), was detected in one of four soft-shell clams taken from an estuary known to be bacteriologically polluted.5 Similarly, HBsAg was detected in hard-shell clams harvested from a polluted bed on the coast of Maine.6 This clam bed, closed to commercial harvesting, was directly contaminated by drainage of untreated sewage from a small community hospital. In closed system aquariums hard-shell clams were also shown to be able to filter and concentrate HBsAg, and transmit this antigen to other clams.6 No evidence of replication was obtained. These data provide the only known link between bivalve mollusks and Hepatitis B. In order to determine whether bivalve mollusks grown in approved New England waters and purchased on the open market in Boston were contaminated with HB,Ag, the present study was undertaken.
Materials and Methods Hard-shell clams (Mercenaria mercenaria) and softshell clams (Mya arenaria) harvested from certified regions in Massachusetts (Waquoit and Buzzards Bay) and Maine (Millbridge and Cranberry Isle), respectively, were purchased at three to four month intervals during a one-year period. In addition, both hard- and soft-shell clams harvested from approved beds in Narragansett Bay, Rhode Island were studied. All bivalves were certified by respective State health departments as having been grown, processed, and packaged under strict sanitary controls approved by State health authorities as required by the National Shellfish Sanitation Program of the Food and Drug Administration. Approximately 2,000 clams were shelled in the cold at 4°. Viscera were pooled in groups of ten and homogenized in a Waring blender. Homogenates were centrifuged in a BeckFrom the Hepatology Section, Medical Service, Veterans Administration Hospital, Boston University School of Medicine, Bos-
ton, Ma 02130. Address reprint requests to Dr. Koff at the above address. 174
L3-40 Ultracentrifuge at 20,000 rpm for 30 minutes. Supernatants were tested for HB,Ag with the coated-tube solid phase radioimmunoassay of Ling and Overby (Abbott Ausria I).7 To determine ability to recover HBsAg in these man
materials, serum containing HBSAg was added to clam homogenates previously found to be negative for HBsAg. Both
the diluted
homogenates and supernatants obtained following centrifugation, as described above, were tested for
HBSAg.
Results Approximately 99 per cent of the HBsAg added to clam homogenates was recovered from clam supernatants. HBSAg, however, was not detected in any of the tested pooled clams, taken at various seasons from beds in Maine, Massachusetts, or Rhode Island.
Discussion Bivalve mollusks breed, grow, and are harvested in estuaries and protected bay waters from which they extract food and oxygen by filtration. Transport and uptake rates are dependent on a number of factors including water temperature, salinity, particulate matter, and chemical composition. Because this filtering and transport capability lacks selectivity viruses, as well as other pollutants, when present in the aquatic environment, may be concentrated in edible mollusk viscera. The presence of HBsAg in mollusks harvested from polluted waters indicates the presence of this viral product in the environment and that HBV may also be present. Although there is little reason to believe that mollusks contaminated with Hepatitis B virus would differ from Hepatitis A contaminated bivalves as vehicles of infection, there is at present no epidemiologic evidence linking shellfish ingestion with Hepatitis B. The results of the present study provide no evidence that mollusks from certified beds are contaminated with HBsAg and by inference with HBV, but continued surveillance and investigation of outbreaks or sporadic cases of hepatitis may necessitate revision of this assumption.
Summary In this study, hard- and soft-shell clams harvested dur-
ing a one-year period from state approved (certified) clam beds in Maine, Massachusetts, and Rhode Island were tested for HB,Ag by radioimmunoassay. HBsAg could not be de-
AJPH February, 1976, Vol. 66, No. 2
PUBLIC HEALTH BRIEFS
tected in clam viscera. The role of bivalve mollusk ingestion in transmission of Hepatitis B remains speculative. REFERENCES 1. Roos, B. Hepatitis epidemic transmitted by oysters. Svenska Lak.-Tidning, 53:989-1003, 1956. 2. Koff, R. S. Epidemiology of viral hepatitis, Crit. Rev. Environm. Control 1:383-442, 1970. 3. Koff, R. S., Grady, G. F., Chalmers, T. C. Mosley, J. W., Swartz, B. L., and the Boston Inter-Hospital Liver Group. Viral hepatitis in a group of Boston hospitals. III. Importance of ex-
4. 5. 6. 7.
posure to shellfish in a nonepidemic period. New Eng. J. Med. 276:703-710, 1967. Stille, W., Kunkel, B., and Nerger, K. Oyster-transmitted hepatitis. Dtsch. Med. Wschr. 97:145-147, 1972. Kater, R. M. H., Kim, C. Y., and Davidson, C. S. Australia antigen and viral hepatitis. J. Inf. Dis. 120:391-392, 1969. Mahoney, P., Fleischner, G., Millman, I., London, W. T., Blumberg, B. S., and Arias, I. M. Australia antigen: detection and transmission in shellfish. Science 183:80-81, 1974. Ling, C. M. and Overby, L. R. Prevalence of Hepatitis B virus antigen as revealed by direct radioimmune assay with 1251-antibody. J. Immunol. 109:834-841, 1972.
Serosurvey of Dogs for Brucella canis Infection in Memphis, Tennessee GEORGE S. LOVEJOY, MD, HAROLD D. CARVER, MS, I. K. MOSELEY, RPS, and MILDRED HICKS, BS A rare human case of Brucella canis infection occurred in an 18 year old man living in Memphis, Tennessee in 1973. The patient was hospitalized, treated, and eventually recovered. Dogs tested for B canis in the patient's neighborhood, including the patient's own pet, revealed a high rate of positivity.' As a result of the human case and positivity among neighboring dogs, a more extensive survey of dogs for B canis was conducted by the Memphis and Shelby County Health Department between November 1973 and January 1974. Objectives of the serosurvey were: (1) to define the geographical distribution and frequency of B canis infection in the free-roaming and confined dog populations, and distribution by sex, breed, age, underlying illness, and size; (2) to define frequency and distribution of infection in dogs in a similar, but non-contiguous geographic location; and (3) to identify foci of infection within Memphis and Shelby County which would permit future efforts to be directed toward eradication of the foci.*
Procedure Six geographic areas were established for the survey, four within the city of Memphis, and the fifth including all of
The authors are with the Memphis and Shelby County Health Department, 814 Jefferson Avenue, Memphis, TN 38105. Address reprints to Dr. Lovejoy, Director of the Department. This article was submitted to the Journal on April 29, 1975, revised and accepted for publication October 4, 1975. *A suggested protocol for the serosurvey was prepared by Dr. Robert S. Munford of the Center for Disease Control, Atlanta. AJPH February, 1976, Vol. 66, No. 2
Shelby County outside Memphis city limits. The sixth area was the town of Bolivar, Tennessee, 55 miles from Memphis, chosen to reflect a "non-contiguous" geographic location. The survey was undertaken by the field services division of the Memphis and Shelby County Health Department, which includes rabies control. Ten private veterinarians chosen by the local veterinary society from all areas of the city, and the veterinarian in Bolivar were asked to collect sera from the "confined dog" population. Sera from stray dogs scheduled for euthanasia were collected by the Memphis Humane Shelter which serves both the city and county, and the Hardeman County Animal Shelter, which serves Bolivar. Dr. L. E. Carmichael, DVM, Veterinary Virus Research Institute, Cornell University, kindly agreed to test the sera. Special forms, to record pertinent data about the dogs and to secure written owner consent for bleeding dogs seen by private veterinarians, were prepared for the survey which began in December 1973 and continued into January 1974. At the Memphis Humane Shelter, 201 sera were collected from stray dogs picked up in the city and county and scheduled for euthanasia. The ten Memphis veterinarians were able to collect 51 serum specimens. No effort was made to select sick or "suspect" dogs. Personnel assigned by the Hardeman County Health Department surveyed 34 stray dogs scheduled for euthanasia at the Hardeman County Animal Shelter while a private veterinarian at Bolivar sampled 16 non-stray dogs. In all cases eight cc of blood were drawn from the dog's foreleg and transferred immediately to a vacutainer vial. Centrifugation to separate serum and cells was carried out, usually the same day for Shelby County specimens, and the following day in Hardeman County specimens. Each serum 175
Failure to Detect Hepatitis B Surface Antigen (HBSAg) in Certified Shellfish From New England RAYMOND S. KOFF, MD LORNA J.D. CONNELLY The major known viral hazard associated with ingestion of bivalve mollusks (clams, oysters, and mussels) is hepatitis. Epidemiologic investigation implicated ingestion of raw oysters as a mode of transmission of Hepatitis A in Sweden two decades ago,' and a number of outbreaks of hepatitis in the United States were subsequently traced to uncooked bivalve mollusks.2 In addition to recognized outbreaks, sporadic cases of viral hepatitis following ingestion of raw or inadequately cooked clams and oysters have been recorded in New England3 and Germany.4 Recently, Heptatitis B surface antigen (HBSAg), a marker of the Hepatitis B virus (HBV), was detected in one of four soft-shell clams taken from an estuary known to be bacteriologically polluted.5 Similarly, HBsAg was detected in hard-shell clams harvested from a polluted bed on the coast of Maine.6 This clam bed, closed to commercial harvesting, was directly contaminated by drainage of untreated sewage from a small community hospital. In closed system aquariums hard-shell clams were also shown to be able to filter and concentrate HBsAg, and transmit this antigen to other clams.6 No evidence of replication was obtained. These data provide the only known link between bivalve mollusks and Hepatitis B. In order to determine whether bivalve mollusks grown in approved New England waters and purchased on the open market in Boston were contaminated with HB,Ag, the present study was undertaken.
Materials and Methods Hard-shell clams (Mercenaria mercenaria) and softshell clams (Mya arenaria) harvested from certified regions in Massachusetts (Waquoit and Buzzards Bay) and Maine (Millbridge and Cranberry Isle), respectively, were purchased at three to four month intervals during a one-year period. In addition, both hard- and soft-shell clams harvested from approved beds in Narragansett Bay, Rhode Island were studied. All bivalves were certified by respective State health departments as having been grown, processed, and packaged under strict sanitary controls approved by State health authorities as required by the National Shellfish Sanitation Program of the Food and Drug Administration. Approximately 2,000 clams were shelled in the cold at 4°. Viscera were pooled in groups of ten and homogenized in a Waring blender. Homogenates were centrifuged in a BeckFrom the Hepatology Section, Medical Service, Veterans Administration Hospital, Boston University School of Medicine, Bos-
ton, Ma 02130. Address reprint requests to Dr. Koff at the above address. 174
L3-40 Ultracentrifuge at 20,000 rpm for 30 minutes. Supernatants were tested for HB,Ag with the coated-tube solid phase radioimmunoassay of Ling and Overby (Abbott Ausria I).7 To determine ability to recover HBsAg in these man
materials, serum containing HBSAg was added to clam homogenates previously found to be negative for HBsAg. Both
the diluted
homogenates and supernatants obtained following centrifugation, as described above, were tested for
HBSAg.
Results Approximately 99 per cent of the HBsAg added to clam homogenates was recovered from clam supernatants. HBSAg, however, was not detected in any of the tested pooled clams, taken at various seasons from beds in Maine, Massachusetts, or Rhode Island.
Discussion Bivalve mollusks breed, grow, and are harvested in estuaries and protected bay waters from which they extract food and oxygen by filtration. Transport and uptake rates are dependent on a number of factors including water temperature, salinity, particulate matter, and chemical composition. Because this filtering and transport capability lacks selectivity viruses, as well as other pollutants, when present in the aquatic environment, may be concentrated in edible mollusk viscera. The presence of HBsAg in mollusks harvested from polluted waters indicates the presence of this viral product in the environment and that HBV may also be present. Although there is little reason to believe that mollusks contaminated with Hepatitis B virus would differ from Hepatitis A contaminated bivalves as vehicles of infection, there is at present no epidemiologic evidence linking shellfish ingestion with Hepatitis B. The results of the present study provide no evidence that mollusks from certified beds are contaminated with HBsAg and by inference with HBV, but continued surveillance and investigation of outbreaks or sporadic cases of hepatitis may necessitate revision of this assumption.
Summary In this study, hard- and soft-shell clams harvested dur-
ing a one-year period from state approved (certified) clam beds in Maine, Massachusetts, and Rhode Island were tested for HB,Ag by radioimmunoassay. HBsAg could not be de-
AJPH February, 1976, Vol. 66, No. 2
PUBLIC HEALTH BRIEFS
tected in clam viscera. The role of bivalve mollusk ingestion in transmission of Hepatitis B remains speculative. REFERENCES 1. Roos, B. Hepatitis epidemic transmitted by oysters. Svenska Lak.-Tidning, 53:989-1003, 1956. 2. Koff, R. S. Epidemiology of viral hepatitis, Crit. Rev. Environm. Control 1:383-442, 1970. 3. Koff, R. S., Grady, G. F., Chalmers, T. C. Mosley, J. W., Swartz, B. L., and the Boston Inter-Hospital Liver Group. Viral hepatitis in a group of Boston hospitals. III. Importance of ex-
4. 5. 6. 7.
posure to shellfish in a nonepidemic period. New Eng. J. Med. 276:703-710, 1967. Stille, W., Kunkel, B., and Nerger, K. Oyster-transmitted hepatitis. Dtsch. Med. Wschr. 97:145-147, 1972. Kater, R. M. H., Kim, C. Y., and Davidson, C. S. Australia antigen and viral hepatitis. J. Inf. Dis. 120:391-392, 1969. Mahoney, P., Fleischner, G., Millman, I., London, W. T., Blumberg, B. S., and Arias, I. M. Australia antigen: detection and transmission in shellfish. Science 183:80-81, 1974. Ling, C. M. and Overby, L. R. Prevalence of Hepatitis B virus antigen as revealed by direct radioimmune assay with 1251-antibody. J. Immunol. 109:834-841, 1972.
Serosurvey of Dogs for Brucella canis Infection in Memphis, Tennessee GEORGE S. LOVEJOY, MD, HAROLD D. CARVER, MS, I. K. MOSELEY, RPS, and MILDRED HICKS, BS A rare human case of Brucella canis infection occurred in an 18 year old man living in Memphis, Tennessee in 1973. The patient was hospitalized, treated, and eventually recovered. Dogs tested for B canis in the patient's neighborhood, including the patient's own pet, revealed a high rate of positivity.' As a result of the human case and positivity among neighboring dogs, a more extensive survey of dogs for B canis was conducted by the Memphis and Shelby County Health Department between November 1973 and January 1974. Objectives of the serosurvey were: (1) to define the geographical distribution and frequency of B canis infection in the free-roaming and confined dog populations, and distribution by sex, breed, age, underlying illness, and size; (2) to define frequency and distribution of infection in dogs in a similar, but non-contiguous geographic location; and (3) to identify foci of infection within Memphis and Shelby County which would permit future efforts to be directed toward eradication of the foci.*
Procedure Six geographic areas were established for the survey, four within the city of Memphis, and the fifth including all of
The authors are with the Memphis and Shelby County Health Department, 814 Jefferson Avenue, Memphis, TN 38105. Address reprints to Dr. Lovejoy, Director of the Department. This article was submitted to the Journal on April 29, 1975, revised and accepted for publication October 4, 1975. *A suggested protocol for the serosurvey was prepared by Dr. Robert S. Munford of the Center for Disease Control, Atlanta. AJPH February, 1976, Vol. 66, No. 2
Shelby County outside Memphis city limits. The sixth area was the town of Bolivar, Tennessee, 55 miles from Memphis, chosen to reflect a "non-contiguous" geographic location. The survey was undertaken by the field services division of the Memphis and Shelby County Health Department, which includes rabies control. Ten private veterinarians chosen by the local veterinary society from all areas of the city, and the veterinarian in Bolivar were asked to collect sera from the "confined dog" population. Sera from stray dogs scheduled for euthanasia were collected by the Memphis Humane Shelter which serves both the city and county, and the Hardeman County Animal Shelter, which serves Bolivar. Dr. L. E. Carmichael, DVM, Veterinary Virus Research Institute, Cornell University, kindly agreed to test the sera. Special forms, to record pertinent data about the dogs and to secure written owner consent for bleeding dogs seen by private veterinarians, were prepared for the survey which began in December 1973 and continued into January 1974. At the Memphis Humane Shelter, 201 sera were collected from stray dogs picked up in the city and county and scheduled for euthanasia. The ten Memphis veterinarians were able to collect 51 serum specimens. No effort was made to select sick or "suspect" dogs. Personnel assigned by the Hardeman County Health Department surveyed 34 stray dogs scheduled for euthanasia at the Hardeman County Animal Shelter while a private veterinarian at Bolivar sampled 16 non-stray dogs. In all cases eight cc of blood were drawn from the dog's foreleg and transferred immediately to a vacutainer vial. Centrifugation to separate serum and cells was carried out, usually the same day for Shelby County specimens, and the following day in Hardeman County specimens. Each serum 175
