843 Journal o f Food Protection, Vol. 77, No. 5, 2014, Pages 843-848 doi: 10.4315/0362-028X. JFP-13-413 Copyright © , International Association for Food Protection
Research Note
Effect of Cooling Rates and Temperatures on Quality and Safety of Quahog Clams (Mercenaria mercenaria) LINDA ANKENMAN GRANATA,1* DIANNE WALL BOURNE, 1 GEORGE J. FLICK, JR . , 1 MICHAEL PEIRSON,2t TARA RILEY,2+ ROBERT E. CROONENBERGHS, 3 a n d JENNIFER KENSLER4 5 1Department o f Food Science and Technology, Virginia Polytechnic Institute and State University, 360 Duck Pond Drive, Blacksburg, Virginia 24061; 2Cherrystone Aqua Farms, P.O. Box 222, 15026 Harbor Lane, Eastville, Virginia 23347;3Division o f Shellfish Sanitation, Virginia Department o f Health, 109 Governor Street, Room 614, Richmond, Virginia 23219; 4LISA (Laboratory for Interdisciplinary Statistical Analysis), Virginia Polytechnic Institute and State University, 405 Hutcheson Hall, Blacksburg, Virginia 24061; and 5Scientific Test and Analysis Techniques Test and Evaluation Center o f Excellence (STAT T&E COE), Air Force Institute o f Technology, 2950 Hobson Way, Building 646, Room 201, Wright-Patterson AFB, Ohio 45433, USA MS 13-413: Received 27 September 2013/Accepted 13 January 2014
ABSTRACT The model ordinance in the National Shellfish Sanitation Program’s Guide fo r the Control ofMolluscan Shellfish was initially established for oysters; however, the clam industry also follows the protocol. Rapid cooling during periods when the growing waters exceed 80°F (26.7°C) results in cold shock, which causes unacceptable mortalities in clams. The clam industry was looking for a procedure to lower the clams to the standard temperature while minimizing shell shock mortalities during the warm summer months. Three tempering treatments were examined, and total aerobic plate counts (APCs) and most-probable-number (MPN) counts of Vibrio, V. parahaemolyticus, and fecal coliforms were enumerated. In treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage. In treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65°F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long-term storage. In treatment 3, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45°F (7.2°C) for long-term storage. Three replicate trials were performed with triplicate analyses during late June through early to midAugust. The current National Shellfish Sanitation Program standard is treatment 1; it contained statistically (P s 0.05) higher total APCs than treatments 2 and 3 throughout the 21-day storage period. APCs ranged from 2.3 x 104 immediately after harvest to 2.7 x 106, 1.6 x 105,and4.8 x 10s for treatments 1,2, and 3, respectively, after 14 days of storage. A statistical analysis showed that treatments 2 and 3 had significantly lower total MPN per gram Vibrio than treatment 1 on day 7 but were equal to treatment 1 on days 1 and 14. MPN per gram for V. parahaemolyticus was statistically lower in treatments 2 and 3 than in treatment 1 on storage days 1 and 7. However, on day 14, treatment 3 was significantly lower than treatments 1 and 2. There was no statistical difference for fecal coliforms. The greatest mortality occurred in treatment 1 (87.4%), followed by treatment 2 (83.3%) and treatment 3 (66.0%). The outcome of this research clearly shows that treatments 2 and 3 can cool clams to a temperature of 45°F (7.2°C) without compromising quality or safety and can reduce the number of dead clams introduced into the marketplace.
In the United States, contaminated seafood is respon sible for 26.5% of all foodbome disease outbreaks, with the majority of these illnesses associated with the consumption of raw bivalve molluscan shellfish (9, 16). The U.S. Food and Drug Administration reported that fishery seafood products ranked second to nuts and edible seeds for the products most commonly recalled for microbiological contamination in fiscal years 2003 through 2011 (8). Vibrio parahaem olyticus is naturally present in estuarine, marine, and coastal environments throughout the world (4 -7 , 1 1 14). This microorganism is frequently isolated from a variety of raw seafood, particularly shellfish. Consumption of raw or partially cooked seafood is the primary cause of infection (11). The U.S. Centers for Disease Control and * Author for correspondence. Tel: 540-231-9570; Fax: 540-231-9293; E-mail: [email protected]. f Retired. { Present address: 2 Bathing Beach Road, Nantucket, MA 02554, USA.
Prevention estimated that 4,500 cases of V. p arahaem oly ticus infections occur each year in the United States (I), and the microorganism is also a common cause of foodbome illness in European and Asian countries (19). To reduce illness from V. parahaem olyticus and other pathogenic microorganisms, the National Shellfish Sanita tion Program (17) published a model ordinance for the control of molluscan shellfish. The model ordinance was established for oysters; however, the regulation has been applied to clams even though there is lack of scientific information or health statistics to support the inclusion of clams. The model ordinance provides guidance on the time required (10 to 36 h) to lower shell stock temperature to 50°F (10°C) or less and maintain it at that temperature. When molluscan growing waters exceed 85°F (29.4°C), rapid cooling to meet National Shellfish Sanitation Program temperature standards results in significant shell stock mortalities due to thermal shock. Adult Asiatic clams suffered 50% mortalities when exposed to a postharvest
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temperature o f 2°C (35.6°F) for 30 min (15). Dead clams must be discarded by the shell stock shipper or by the receiving agent, resulting in significant financial loss. Also, dead shell stock presents a greater safety risk to consumers through the growth o f pathogenic microorganisms. H ow ev er, it is sometimes difficult to distinguish between dead and live shell stock if the dead shell stock shell remains closed. A staged temperature reduction that will maintain shell stock viability while preventing the proliferation o f V. parahaemolyticus w ill ensure product safety and quality. The objectives o f this research were to determine the populations o f V. parahaemolyticus in quahog clams (Mercenaria mercenaria) stored under staged temperatures to reach 50°F (10°C) compared with immediate storage at 45°F (7.2°C) right from harvest.
MATERIALS AND METHODS Tempering methods investigated (staged storage). Three tempering methods were investigated. In treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage. This method was the National Shellfish Sanitation Program standard (17) at the time the research was conducted. In treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65 °F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long term storage. This tempering method is the first choice for an alternative procedure. In treatment 3, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45 °F (7.2°C) for long-term storage. This tempering method is the second choice for an alternative procedure. Three replicate trials were performed with triplicate analyses over a 6-week period during the midsummer months (late June through early to mid-August) to ensure the worst-case scenario for harvesting conditions. A total of 22,832 clams were used in this study: 7,391 clams for the first tempering method, 7,323 clams for the second method, and 8,118 clams for the third method. All clams were harvested randomly from various locations within the growing area. Trials were run using live clams only. Data was kept on the clam death rate throughout each trial on each treatment. Temperature monitoring. Temperatures throughout the project were monitored using SmartButton Temperature Data Loggers (ACR Systems, Surrey, British Columbia, Canada) and were read using the SmartButton Reader software. One SmartBut ton was placed in the clam bed (in Cherrystone Creek, North ampton, VA) 2 weeks prior to the start of the study and was left there until the project was completed to monitor any changes in the bed temperatures due to weather and tide. One SmartButton was placed inside each of the rooms where the clams were stored to monitor changes in room temperature. To monitor the temperature in the clam totes, three clams from each storage treatment were opened and the clam was removed. Button-sized SmartButton Data Loggers that were preprogrammed to record throughout the experiment were placed inside each shell to record how long it took the clams in each treatment to reach the final temperature of 45°F (7.2°C). The shell was then taped shut using waterproof tape and was randomly placed in the totes among the clams. The SmartButton Data Loggers were removed at the end of each experiment. The recorded information was downloaded using the SmartButton Reader software program.
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Location of clam bed. The clam beds were located in Cherrystone Creek at the Reid Farm in Northampton County, VA. The growing waters in this area were designated as “ approved” by the Virginia Department of Health, Division of Shellfish Sanitation. The bed was chosen because it contained microbiolog ical population, water depth, tidal flows, and water temperature that were similar to other commercially managed clam growing areas in the mid-Atlantic region. The depth of the water ranges from 3 to 4 ft (0.91 to 1.22 m) depending on the tidal conditions, with average temperatures ranging from 76.5 to 89.6°F (24.7 to 32°C) during the summer months, when this study was conducted. The clams were harvested between 5:00 and 8:00 a.m. and were placed under cover to prevent direct contact with the environment. All clams were brought to the processing and storage facility within 4 h after harvesting was initiated. The processing facility is located on Cherrystone Creek, and the clam boats unload directly at the facility. The clams were rinsed with fresh potable water, sorted into bins, and then placed into the designated storage areas. Clam storage. After the clams were unloaded from the harvest boat, they were immediately sent through the initial grader to remove all cracked shells, mudders (unopened shells containing a dead clam), and dead clams. The clams were then separated into totes and were placed in the various chilling rooms according to the three tempering methods. At each sampling interval, 50 clams were randomly removed from totes stored at the appropriate temperature and were brought into the laboratory. Shucking procedure, hi clam shucking, gloves are worn and clams are cleaned with alcohol swabs at the area where the two shells meet and the shucking knife is inserted. Before use, the shucking knife is dipped into alcohol and is flamed. The shucking knife is inserted on one side, and one of the adductor muscles is cut in half (with care taken not to cut the entire clam in half). Then the knife is brought to the other side of the clam, and the second adductor muscle is cut in half. The adductor muscles are cut from both the top and bottom of the shells, and then the clam and liquor are placed into a sterile container. A professional clam shucker opened the clams using the technique described above, and approximately 200 g of clam and liquor was placed into a tared sterile Mason jar and then weighed. An equal amount of phosphate-buffered saline (PBS) was added, and the mixture was blended for 90 s using a Classic Osterizer blender (model 4096, Sunbeam Products, Inc., Neosho, MS). This 1:2 dilution homogenate was used for enumeration of total Vibrio, V. parahaemolyticus, aerobic plate counts (APCs), and coliforms, following the U.S. Food and Drug Administration’s Bacteriolog ical Analytical Manual (12) approved methods. The microbiolog ical media used for isolation and enumeration was obtained from Difco (BD, Sparks, MD) unless otherwise stated. Microbiological methods. For isolation and confirmation of V. parahaemolyticus, 20 g of clam homogenate was weighed into a bottle containing 80 ml of PBS. Serial dilutions were made using alkaline peptone saline tubes to determine a three-tube most probable number (MPN) for Vibrio. After 16 to 18 h of incubation at 35°C, a 3-mm loopful from the top 1 cm of the alkaline peptone saline tubes containing the three highest dilutions of sample showing growth was streaked onto thiosulfate citrate bile salts sucrose agar and was incubated for 18 to 24 h at 35°C. Typical blue-green V. parahaemolyticus colonies on the thiosulfate citrate bile salts sucrose agar were recorded for the MPN (12). Representative colonies were picked onto Trypticase soy agar
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QUALITY AND SAFETY OF QUAHOG CLAMS
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TABLE 1. SmartButton data fo r the clam storage roomsa Room temp Avg temp Temp range
90°F (32.2°C) 90.5°F (32.5°C) 68.9-104°F (20.5^-0°C)
65°F (18.3°C) 61.6°F (16.4°C) 59-63.5°F (15-17.5°C)
55°F (12.8°C) 57.7°F (14.3°C) 56.3-58.1°F (13.5-14.5°C)
45°F (7.2°C) 49.8°F (9.9°C) 46.4-59.9°F (8-15.5°C)
“ The buttons were used and monitored from 24 June to 24 August 2007. plus 2% saline slants for confirmation using API 20E diagnostic strips (bioMerieux, Inc., Durham, NC). Other nontypical V. parahaemolyticus colonies were recorded. These resulting Vibrio colony types were then used for MPN counts to determine total Vibrio spp. For APC, from the initial 1:10 dilution and from each subsequent dilution, 1 ml was pipetted into each of two aerobic count Petrifilm plates (3M Laboratories, St. Paul, MN) and was incubated for 48 h at 35°C. For the presence or absence of fecal coliforms, MPN was determined using a five-tube MPN method, with lauryl tryptose broth tubes containing fermentation vials and incubation at 35°C. The tubes were examined for gas and reactions were recorded after 24 h, i.e., displacement of medium in fermentation vial or effervescence when tubes were gently agitated. After an additional 24-h incubation, gas-negative tubes were examined and reactions were recorded (48 h). From each gassing lauryl tryptose broth tube, a loopful of suspension was transferred to a tube of EC broth, incubated for 24 h at 44.5°C in a circulating water bath, and examined for gas production. Any negative EC tubes were incubated and examined again at 48 h. The gas-positive tubes were used to calculate fecal coliform MPNs. From any positive EC broth tubes, a loopful was streaked onto 3M Escherichia coli Petrifilm and incubated for 24 h at 35°C. Any colonies that appeared blue were picked for E. coli confirmation using API 20E diagnostic strips.
SmartButtons. Three SmartButton Data Loggers from ACR Systems were placed in each tote of clams to monitor the change in temperature and to record how long it took the clams in each treatment to reach the final temperature of 45°F (7.2°C). Once reached, the temperatures remained constant. One SmartButton Data Logger was also placed inside each of the rooms where the clams were stored to monitor changes in room temperature throughout the storage. One SmartButton Data Logger was placed in the clam bed located in Cherrystone Creek. Statistics. Analysis of variance (ANOVA) and Tukey tests were performed on all the data, using a significance level of a = 0.05. The statistics were run by the Laboratory for Interdisciplinary
Statistical Analysis group at Virginia Polytechnic Institute and State University (Blacksburg).
RESULTS AND CONCLUSIONS SmartButton data for the storage rooms is condensed in Table 1; the buttons were used and monitored from 24 June to 24 August 2007. The averages were calculated from over 8,000 data points; the temperature was recorded every 5 min. Temperatures were controlled to minimize exposure to outside air, the constant in and out of rooms, and loading and unloading of clams. Treatments. The three tempering conditions used will be labeled as treatments 1, 2, and 3 for ease in tables and discussion. Treatment 1 was 5 h at 90°F (32.2°C) before being moved to storage at 45°F (7.2°C); treatment 2, 5 h at 90°F (32.2°C), 12 h at 65°F (18.3°C), and 12 h at 55°F (12.8°C) before being moved to storage at 45°F (7.2°C); and treatment 3, 5 h at 90°F (32.2°C) and 24 h at 65°F (18.3°C) before being moved to storage at 45°F (7.2°C). APCs. Separate ANOVAs for 1, 7, 14, and 21 days after clam harvesting were performed to compare APCs for the three treatments. Because clams in different repetitions were harvested at different times, the analysis treated repetitions as blocks. The analysis also treated each treatment within each repetition as the experimental unit. The ANOVAs for days 1, 7, 14, and 21 indicate significant differences in the plate counts for the treatments. On days 1, 7, and 21, the Tukey follow-up tests show that, whereas plate counts for treatments 2 and 3 are not significantly different from one another, they are signifi cantly lower than those of treatment 1 (Table 2). The Tukey follow-up test for day 14 indicates that the plate count for treatment 2 was significantly lower than the plate counts for treatments 1 and 3.
TABLE 2. Total aerobic plate count o f clams fo r each storage treatmenta APC (CFU/g)
Treatment* Control ambient temp 1 2 3
Day 0
Day 1
Day 7
Day 14
Day 21
2.2 x 106 a 5.6 x 105 B 8.9 x 105 b
4.4 x 106 a 1.1 x 106 B 1.3 x 106 b
4.1 X 106 A 9.0 X 105 B 1.3 x 106 a
2.7 x 106 a 1 . 6 x 105 b 4.8 x 105 b
2.3 X 104
a The letters indicate statistical significance at P < 0.05 for each column. The values with the same letter are statistically the same; the value with the different letter is statistically different. b Treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage; treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65°F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long-term storage; treatment 3, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45°F (7.2°C) for long-term storage.
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TABLE 3. Total Vibrio counts o f clams for each storage treatmenta Counts (MPN/g) Treatment* Control ambient temp 1 2 3
Day 0 1.4
x
Day 7
Day 1
Day 14
104 2.7 3.6 2.3
x x x
105 a 104 a 105 a
2.8 9.1 5.3
x x x
105 A 104 b 104 b
7.4 7.2 6.2
x x x
104 104 104
a a
A
a Vibrio species include, but are not limited to, parahaemolyticus, alginolyticus, fluvialis, vulnificus, Aeromonas hydrophila (Aeromonas species included in the Vibrionaceae family). The letters indicate statistical significance at P < 0.05 for each column. The values with the same letter are statistically the same; the value with the different letter is statistically different. * Treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage; treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65°F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long-term storage; treatment 3, clams were harvested and held for 5 h at 90=F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45 °F (7.2°C) for long-term storage.
V. cholerae non-01 (10,18,20,21), with none of the samples harboring species-specific virulence factors. Other studies have also found Aeromonas in 66.1% of samples tested (2). Dalsgaard (3) reports that non-01 and non-0139 strains of V. cholerae are normally present in non-fecal-polluted aquaculture environments but have a low pathogenic potential. Based on these surveys, the number of pathogenic Vibrios in this clam tempering study should constitute only a minor percent of the total Vibrio population because the predominant Vibrio spp. is anticipated to be V. alginolyticus and many of the V. parahaemolyticus strains may not contain virulence factors. Therefore, the levels of V. parahaemolyticus contained in the quahog clams in our study should not present a health hazard to healthy individuals. However, consumers with weakened immune systems or chronic liver disease should avoid consuming raw shellfish.
In summary, the data indicate that the clams kept at 90°F (32.2°C) for 5 h before being cooled to 45°F (7.2°C) contained more aerobic bacteria than the clams initially cooled to 65°F (18.3°C) or 55°F (12.8°C) within 5 h of unloading on days 1, 7, and 14 after harvest. ANOVAs were performed on days 1, 7, 14, and 21 to compare Vibrio levels for the treatments. There were no significant differences in Vibrio levels between treatments for days 1 and 14 (Table 3). On day 7, the ANOVA showed significant differences in Vibrio for treatments. The follow up Tukey test indicated that treatment 1 had significantly higher levels of Vibrio than treatments 2 and 3, which were not statistically different. V. parahaemolyticus. The control values on day 0 serve as a baseline to show that the levels of V. parahaemolyticus were low at the beginning of the study. The ANOVAs for days 1 and 7 indicate that treatment 1 had a significantly higher level of V. parahaemolyticus than treatments 2 and 3; treatments 2 and 3 were not significantly different (Table 4). On day 14, the levels of V. parahaemolyticus were significantly higher for treatment 3 than for treatments 1 and 2. In numerous studies of Vibrio species occurrence, the most prevalent species identified is V. alginolyticus, followed by Kanagawa-negative V. parahaemolyticus, V. fluvialis, and
Fecal coliform of clams for each treatment. There were no significant differences in the fecal coliform count for the clams for any treatment. All three treatments were similar in fecal coliform counts. The MPN for fecal coliforms for the control clams was
Research Note
Effect of Cooling Rates and Temperatures on Quality and Safety of Quahog Clams (Mercenaria mercenaria) LINDA ANKENMAN GRANATA,1* DIANNE WALL BOURNE, 1 GEORGE J. FLICK, JR . , 1 MICHAEL PEIRSON,2t TARA RILEY,2+ ROBERT E. CROONENBERGHS, 3 a n d JENNIFER KENSLER4 5 1Department o f Food Science and Technology, Virginia Polytechnic Institute and State University, 360 Duck Pond Drive, Blacksburg, Virginia 24061; 2Cherrystone Aqua Farms, P.O. Box 222, 15026 Harbor Lane, Eastville, Virginia 23347;3Division o f Shellfish Sanitation, Virginia Department o f Health, 109 Governor Street, Room 614, Richmond, Virginia 23219; 4LISA (Laboratory for Interdisciplinary Statistical Analysis), Virginia Polytechnic Institute and State University, 405 Hutcheson Hall, Blacksburg, Virginia 24061; and 5Scientific Test and Analysis Techniques Test and Evaluation Center o f Excellence (STAT T&E COE), Air Force Institute o f Technology, 2950 Hobson Way, Building 646, Room 201, Wright-Patterson AFB, Ohio 45433, USA MS 13-413: Received 27 September 2013/Accepted 13 January 2014
ABSTRACT The model ordinance in the National Shellfish Sanitation Program’s Guide fo r the Control ofMolluscan Shellfish was initially established for oysters; however, the clam industry also follows the protocol. Rapid cooling during periods when the growing waters exceed 80°F (26.7°C) results in cold shock, which causes unacceptable mortalities in clams. The clam industry was looking for a procedure to lower the clams to the standard temperature while minimizing shell shock mortalities during the warm summer months. Three tempering treatments were examined, and total aerobic plate counts (APCs) and most-probable-number (MPN) counts of Vibrio, V. parahaemolyticus, and fecal coliforms were enumerated. In treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage. In treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65°F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long-term storage. In treatment 3, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45°F (7.2°C) for long-term storage. Three replicate trials were performed with triplicate analyses during late June through early to midAugust. The current National Shellfish Sanitation Program standard is treatment 1; it contained statistically (P s 0.05) higher total APCs than treatments 2 and 3 throughout the 21-day storage period. APCs ranged from 2.3 x 104 immediately after harvest to 2.7 x 106, 1.6 x 105,and4.8 x 10s for treatments 1,2, and 3, respectively, after 14 days of storage. A statistical analysis showed that treatments 2 and 3 had significantly lower total MPN per gram Vibrio than treatment 1 on day 7 but were equal to treatment 1 on days 1 and 14. MPN per gram for V. parahaemolyticus was statistically lower in treatments 2 and 3 than in treatment 1 on storage days 1 and 7. However, on day 14, treatment 3 was significantly lower than treatments 1 and 2. There was no statistical difference for fecal coliforms. The greatest mortality occurred in treatment 1 (87.4%), followed by treatment 2 (83.3%) and treatment 3 (66.0%). The outcome of this research clearly shows that treatments 2 and 3 can cool clams to a temperature of 45°F (7.2°C) without compromising quality or safety and can reduce the number of dead clams introduced into the marketplace.
In the United States, contaminated seafood is respon sible for 26.5% of all foodbome disease outbreaks, with the majority of these illnesses associated with the consumption of raw bivalve molluscan shellfish (9, 16). The U.S. Food and Drug Administration reported that fishery seafood products ranked second to nuts and edible seeds for the products most commonly recalled for microbiological contamination in fiscal years 2003 through 2011 (8). Vibrio parahaem olyticus is naturally present in estuarine, marine, and coastal environments throughout the world (4 -7 , 1 1 14). This microorganism is frequently isolated from a variety of raw seafood, particularly shellfish. Consumption of raw or partially cooked seafood is the primary cause of infection (11). The U.S. Centers for Disease Control and * Author for correspondence. Tel: 540-231-9570; Fax: 540-231-9293; E-mail: [email protected]. f Retired. { Present address: 2 Bathing Beach Road, Nantucket, MA 02554, USA.
Prevention estimated that 4,500 cases of V. p arahaem oly ticus infections occur each year in the United States (I), and the microorganism is also a common cause of foodbome illness in European and Asian countries (19). To reduce illness from V. parahaem olyticus and other pathogenic microorganisms, the National Shellfish Sanita tion Program (17) published a model ordinance for the control of molluscan shellfish. The model ordinance was established for oysters; however, the regulation has been applied to clams even though there is lack of scientific information or health statistics to support the inclusion of clams. The model ordinance provides guidance on the time required (10 to 36 h) to lower shell stock temperature to 50°F (10°C) or less and maintain it at that temperature. When molluscan growing waters exceed 85°F (29.4°C), rapid cooling to meet National Shellfish Sanitation Program temperature standards results in significant shell stock mortalities due to thermal shock. Adult Asiatic clams suffered 50% mortalities when exposed to a postharvest
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temperature o f 2°C (35.6°F) for 30 min (15). Dead clams must be discarded by the shell stock shipper or by the receiving agent, resulting in significant financial loss. Also, dead shell stock presents a greater safety risk to consumers through the growth o f pathogenic microorganisms. H ow ev er, it is sometimes difficult to distinguish between dead and live shell stock if the dead shell stock shell remains closed. A staged temperature reduction that will maintain shell stock viability while preventing the proliferation o f V. parahaemolyticus w ill ensure product safety and quality. The objectives o f this research were to determine the populations o f V. parahaemolyticus in quahog clams (Mercenaria mercenaria) stored under staged temperatures to reach 50°F (10°C) compared with immediate storage at 45°F (7.2°C) right from harvest.
MATERIALS AND METHODS Tempering methods investigated (staged storage). Three tempering methods were investigated. In treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage. This method was the National Shellfish Sanitation Program standard (17) at the time the research was conducted. In treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65 °F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long term storage. This tempering method is the first choice for an alternative procedure. In treatment 3, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45 °F (7.2°C) for long-term storage. This tempering method is the second choice for an alternative procedure. Three replicate trials were performed with triplicate analyses over a 6-week period during the midsummer months (late June through early to mid-August) to ensure the worst-case scenario for harvesting conditions. A total of 22,832 clams were used in this study: 7,391 clams for the first tempering method, 7,323 clams for the second method, and 8,118 clams for the third method. All clams were harvested randomly from various locations within the growing area. Trials were run using live clams only. Data was kept on the clam death rate throughout each trial on each treatment. Temperature monitoring. Temperatures throughout the project were monitored using SmartButton Temperature Data Loggers (ACR Systems, Surrey, British Columbia, Canada) and were read using the SmartButton Reader software. One SmartBut ton was placed in the clam bed (in Cherrystone Creek, North ampton, VA) 2 weeks prior to the start of the study and was left there until the project was completed to monitor any changes in the bed temperatures due to weather and tide. One SmartButton was placed inside each of the rooms where the clams were stored to monitor changes in room temperature. To monitor the temperature in the clam totes, three clams from each storage treatment were opened and the clam was removed. Button-sized SmartButton Data Loggers that were preprogrammed to record throughout the experiment were placed inside each shell to record how long it took the clams in each treatment to reach the final temperature of 45°F (7.2°C). The shell was then taped shut using waterproof tape and was randomly placed in the totes among the clams. The SmartButton Data Loggers were removed at the end of each experiment. The recorded information was downloaded using the SmartButton Reader software program.
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Location of clam bed. The clam beds were located in Cherrystone Creek at the Reid Farm in Northampton County, VA. The growing waters in this area were designated as “ approved” by the Virginia Department of Health, Division of Shellfish Sanitation. The bed was chosen because it contained microbiolog ical population, water depth, tidal flows, and water temperature that were similar to other commercially managed clam growing areas in the mid-Atlantic region. The depth of the water ranges from 3 to 4 ft (0.91 to 1.22 m) depending on the tidal conditions, with average temperatures ranging from 76.5 to 89.6°F (24.7 to 32°C) during the summer months, when this study was conducted. The clams were harvested between 5:00 and 8:00 a.m. and were placed under cover to prevent direct contact with the environment. All clams were brought to the processing and storage facility within 4 h after harvesting was initiated. The processing facility is located on Cherrystone Creek, and the clam boats unload directly at the facility. The clams were rinsed with fresh potable water, sorted into bins, and then placed into the designated storage areas. Clam storage. After the clams were unloaded from the harvest boat, they were immediately sent through the initial grader to remove all cracked shells, mudders (unopened shells containing a dead clam), and dead clams. The clams were then separated into totes and were placed in the various chilling rooms according to the three tempering methods. At each sampling interval, 50 clams were randomly removed from totes stored at the appropriate temperature and were brought into the laboratory. Shucking procedure, hi clam shucking, gloves are worn and clams are cleaned with alcohol swabs at the area where the two shells meet and the shucking knife is inserted. Before use, the shucking knife is dipped into alcohol and is flamed. The shucking knife is inserted on one side, and one of the adductor muscles is cut in half (with care taken not to cut the entire clam in half). Then the knife is brought to the other side of the clam, and the second adductor muscle is cut in half. The adductor muscles are cut from both the top and bottom of the shells, and then the clam and liquor are placed into a sterile container. A professional clam shucker opened the clams using the technique described above, and approximately 200 g of clam and liquor was placed into a tared sterile Mason jar and then weighed. An equal amount of phosphate-buffered saline (PBS) was added, and the mixture was blended for 90 s using a Classic Osterizer blender (model 4096, Sunbeam Products, Inc., Neosho, MS). This 1:2 dilution homogenate was used for enumeration of total Vibrio, V. parahaemolyticus, aerobic plate counts (APCs), and coliforms, following the U.S. Food and Drug Administration’s Bacteriolog ical Analytical Manual (12) approved methods. The microbiolog ical media used for isolation and enumeration was obtained from Difco (BD, Sparks, MD) unless otherwise stated. Microbiological methods. For isolation and confirmation of V. parahaemolyticus, 20 g of clam homogenate was weighed into a bottle containing 80 ml of PBS. Serial dilutions were made using alkaline peptone saline tubes to determine a three-tube most probable number (MPN) for Vibrio. After 16 to 18 h of incubation at 35°C, a 3-mm loopful from the top 1 cm of the alkaline peptone saline tubes containing the three highest dilutions of sample showing growth was streaked onto thiosulfate citrate bile salts sucrose agar and was incubated for 18 to 24 h at 35°C. Typical blue-green V. parahaemolyticus colonies on the thiosulfate citrate bile salts sucrose agar were recorded for the MPN (12). Representative colonies were picked onto Trypticase soy agar
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QUALITY AND SAFETY OF QUAHOG CLAMS
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TABLE 1. SmartButton data fo r the clam storage roomsa Room temp Avg temp Temp range
90°F (32.2°C) 90.5°F (32.5°C) 68.9-104°F (20.5^-0°C)
65°F (18.3°C) 61.6°F (16.4°C) 59-63.5°F (15-17.5°C)
55°F (12.8°C) 57.7°F (14.3°C) 56.3-58.1°F (13.5-14.5°C)
45°F (7.2°C) 49.8°F (9.9°C) 46.4-59.9°F (8-15.5°C)
“ The buttons were used and monitored from 24 June to 24 August 2007. plus 2% saline slants for confirmation using API 20E diagnostic strips (bioMerieux, Inc., Durham, NC). Other nontypical V. parahaemolyticus colonies were recorded. These resulting Vibrio colony types were then used for MPN counts to determine total Vibrio spp. For APC, from the initial 1:10 dilution and from each subsequent dilution, 1 ml was pipetted into each of two aerobic count Petrifilm plates (3M Laboratories, St. Paul, MN) and was incubated for 48 h at 35°C. For the presence or absence of fecal coliforms, MPN was determined using a five-tube MPN method, with lauryl tryptose broth tubes containing fermentation vials and incubation at 35°C. The tubes were examined for gas and reactions were recorded after 24 h, i.e., displacement of medium in fermentation vial or effervescence when tubes were gently agitated. After an additional 24-h incubation, gas-negative tubes were examined and reactions were recorded (48 h). From each gassing lauryl tryptose broth tube, a loopful of suspension was transferred to a tube of EC broth, incubated for 24 h at 44.5°C in a circulating water bath, and examined for gas production. Any negative EC tubes were incubated and examined again at 48 h. The gas-positive tubes were used to calculate fecal coliform MPNs. From any positive EC broth tubes, a loopful was streaked onto 3M Escherichia coli Petrifilm and incubated for 24 h at 35°C. Any colonies that appeared blue were picked for E. coli confirmation using API 20E diagnostic strips.
SmartButtons. Three SmartButton Data Loggers from ACR Systems were placed in each tote of clams to monitor the change in temperature and to record how long it took the clams in each treatment to reach the final temperature of 45°F (7.2°C). Once reached, the temperatures remained constant. One SmartButton Data Logger was also placed inside each of the rooms where the clams were stored to monitor changes in room temperature throughout the storage. One SmartButton Data Logger was placed in the clam bed located in Cherrystone Creek. Statistics. Analysis of variance (ANOVA) and Tukey tests were performed on all the data, using a significance level of a = 0.05. The statistics were run by the Laboratory for Interdisciplinary
Statistical Analysis group at Virginia Polytechnic Institute and State University (Blacksburg).
RESULTS AND CONCLUSIONS SmartButton data for the storage rooms is condensed in Table 1; the buttons were used and monitored from 24 June to 24 August 2007. The averages were calculated from over 8,000 data points; the temperature was recorded every 5 min. Temperatures were controlled to minimize exposure to outside air, the constant in and out of rooms, and loading and unloading of clams. Treatments. The three tempering conditions used will be labeled as treatments 1, 2, and 3 for ease in tables and discussion. Treatment 1 was 5 h at 90°F (32.2°C) before being moved to storage at 45°F (7.2°C); treatment 2, 5 h at 90°F (32.2°C), 12 h at 65°F (18.3°C), and 12 h at 55°F (12.8°C) before being moved to storage at 45°F (7.2°C); and treatment 3, 5 h at 90°F (32.2°C) and 24 h at 65°F (18.3°C) before being moved to storage at 45°F (7.2°C). APCs. Separate ANOVAs for 1, 7, 14, and 21 days after clam harvesting were performed to compare APCs for the three treatments. Because clams in different repetitions were harvested at different times, the analysis treated repetitions as blocks. The analysis also treated each treatment within each repetition as the experimental unit. The ANOVAs for days 1, 7, 14, and 21 indicate significant differences in the plate counts for the treatments. On days 1, 7, and 21, the Tukey follow-up tests show that, whereas plate counts for treatments 2 and 3 are not significantly different from one another, they are signifi cantly lower than those of treatment 1 (Table 2). The Tukey follow-up test for day 14 indicates that the plate count for treatment 2 was significantly lower than the plate counts for treatments 1 and 3.
TABLE 2. Total aerobic plate count o f clams fo r each storage treatmenta APC (CFU/g)
Treatment* Control ambient temp 1 2 3
Day 0
Day 1
Day 7
Day 14
Day 21
2.2 x 106 a 5.6 x 105 B 8.9 x 105 b
4.4 x 106 a 1.1 x 106 B 1.3 x 106 b
4.1 X 106 A 9.0 X 105 B 1.3 x 106 a
2.7 x 106 a 1 . 6 x 105 b 4.8 x 105 b
2.3 X 104
a The letters indicate statistical significance at P < 0.05 for each column. The values with the same letter are statistically the same; the value with the different letter is statistically different. b Treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage; treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65°F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long-term storage; treatment 3, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45°F (7.2°C) for long-term storage.
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ANKENMAN GRANATA ET AL.
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TABLE 3. Total Vibrio counts o f clams for each storage treatmenta Counts (MPN/g) Treatment* Control ambient temp 1 2 3
Day 0 1.4
x
Day 7
Day 1
Day 14
104 2.7 3.6 2.3
x x x
105 a 104 a 105 a
2.8 9.1 5.3
x x x
105 A 104 b 104 b
7.4 7.2 6.2
x x x
104 104 104
a a
A
a Vibrio species include, but are not limited to, parahaemolyticus, alginolyticus, fluvialis, vulnificus, Aeromonas hydrophila (Aeromonas species included in the Vibrionaceae family). The letters indicate statistical significance at P < 0.05 for each column. The values with the same letter are statistically the same; the value with the different letter is statistically different. * Treatment 1 (control), clams were harvested, held for 5 h at 90°F (32.2°C), and then moved to 45°F (7.2°C) for storage; treatment 2, clams were harvested and held for 5 h at 90°F (32.2°C), followed by 12 h at 65°F (18.3°C) and 12 h at 55°F (12.8°C), and then were moved to 45°F (7.2°C) for long-term storage; treatment 3, clams were harvested and held for 5 h at 90=F (32.2°C), followed by 24 h at 55°F (12.8°C) before being moved to 45 °F (7.2°C) for long-term storage.
V. cholerae non-01 (10,18,20,21), with none of the samples harboring species-specific virulence factors. Other studies have also found Aeromonas in 66.1% of samples tested (2). Dalsgaard (3) reports that non-01 and non-0139 strains of V. cholerae are normally present in non-fecal-polluted aquaculture environments but have a low pathogenic potential. Based on these surveys, the number of pathogenic Vibrios in this clam tempering study should constitute only a minor percent of the total Vibrio population because the predominant Vibrio spp. is anticipated to be V. alginolyticus and many of the V. parahaemolyticus strains may not contain virulence factors. Therefore, the levels of V. parahaemolyticus contained in the quahog clams in our study should not present a health hazard to healthy individuals. However, consumers with weakened immune systems or chronic liver disease should avoid consuming raw shellfish.
In summary, the data indicate that the clams kept at 90°F (32.2°C) for 5 h before being cooled to 45°F (7.2°C) contained more aerobic bacteria than the clams initially cooled to 65°F (18.3°C) or 55°F (12.8°C) within 5 h of unloading on days 1, 7, and 14 after harvest. ANOVAs were performed on days 1, 7, 14, and 21 to compare Vibrio levels for the treatments. There were no significant differences in Vibrio levels between treatments for days 1 and 14 (Table 3). On day 7, the ANOVA showed significant differences in Vibrio for treatments. The follow up Tukey test indicated that treatment 1 had significantly higher levels of Vibrio than treatments 2 and 3, which were not statistically different. V. parahaemolyticus. The control values on day 0 serve as a baseline to show that the levels of V. parahaemolyticus were low at the beginning of the study. The ANOVAs for days 1 and 7 indicate that treatment 1 had a significantly higher level of V. parahaemolyticus than treatments 2 and 3; treatments 2 and 3 were not significantly different (Table 4). On day 14, the levels of V. parahaemolyticus were significantly higher for treatment 3 than for treatments 1 and 2. In numerous studies of Vibrio species occurrence, the most prevalent species identified is V. alginolyticus, followed by Kanagawa-negative V. parahaemolyticus, V. fluvialis, and
Fecal coliform of clams for each treatment. There were no significant differences in the fecal coliform count for the clams for any treatment. All three treatments were similar in fecal coliform counts. The MPN for fecal coliforms for the control clams was
