Bureau of Aquaculture & Laboratory Services
Services provided by the laboratory support the needs of the Shellfish Sanitation Program which maintains compliance with the US Food and Drug Administration's National Shellfish Sanitation Program Model Ordinance (NSSP-MO). The laboratory provides analyses for environmental quality and aquatic animal health. Tests and analyses performed by the laboratory include fecal coliform bacterial levels in seawater and shellfish, various contaminants, marine biotoxin analysis and shellfish and fish pathology. The laboratory also functions as a research facility for other institutions on a collaborative basis.
Overview
DA/BA maintains an on-site laboratory that processes all of the samples necessary to ensure that Connecticut shellfish are safe.
To maintain compliance with the sampling requirements outlined in the National Shellfish Sanitation Program Model Ordinance (NSSP-MO), the DA/BA laboratory annually processes around 6,000 water samples, 200 shellfish meat samples, 150 viral samples, 200 phytoplankton samples, 12 biotoxin samples, and Vibrio parahaemolyticus samples as necessary. Since shellfish are filter feeders, they can concentrate potentially pathogenic bacteria and viruses, biotoxins, and other harmful substances in their tissues; therefore, the DA/BA lab uses microbiological techniques and indicator organisms to ensure shellfish are safe for human consumption. Tests include fecal coliforms in water and meat samples, male-specific coliphage (the indicator for potentially pathogenic viruses like Norovirus) in meat samples, harmful algal bloom organisms in phytoplankton (water) samples, toxins (produced by phytoplankton) in meat samples, and Vibrio parahaemolyticus in meat samples.
Fecal Coliform Testing in Seawater and Shellfish Meat
In the microbiology laboratory, seawater samples are analyzed for fecal coliform bacteria. Fecal coliforms have a high association with fecal matter from warm-blooded animals. Human sewage contains potentially pathogenic bacteria, viruses, and parasites, which can accumulate in the gut of filter-feeding shellfish and cause illness in people who eat them. Fecal coliforms are a sub-set of the larger coliform group of bacteria, which are characterized as enteric, Gram-negative, facultative anaerobic, rod-shaped bacteria that ferment lactose to produce acid and gas. Although coliforms are easily detected, their association with fecal contamination is questionable because some coliforms are found naturally in environmental samples (Caplenas and Kanarek 1984). Fecal coliforms are readily and inexpensively analyzed in the lab, and have been selected by the U.S. Food and Drug Administration (FDA) as an indicator organism suitable for the evaluation of the sanitary condition of seawater.
Shellfish Meat Samples
Meat samples provide valuable information about fecal coliform levels inside the shellfish tissue to supplement water samples. Meat samples are critical for Connecticut’s advanced relay system and for establishing the criteria for re-opening growing areas faster without compromising public safety. Meat samples are also used for viral, biotoxin, and Vibrio parahaemolyticus testing.
Viral Testing
Bacteriophages are viruses that infect and replicate in bacteria. Coliphages are a sub-group of bacteriophages that target Escherichia coli (E. coli), and are consequently present in sewage. Male specific coliphage (MSC) is a viral indicator used by the DA/BA laboratory to monitor for the presence of viruses in oyster and hard clam meat. While MSC is not harmful to humans, its presence indicates the possibility for potentially pathogenic viruses, like Norovirus and Hepatitis, to also be present. MSC has been shown to be more reflective of infectious virus levels than fecal coliform bacteria (Borrego et al. 1987; Havelaar et al. 1993; Mandilara et al. 2006; McMinn et al. 2017) and have a similar or greater resistance to degradation relative to infectious viruses (Havelaar 1987; reviewed in Grabow 2001). MSC testing is particularly useful following sewage treatment bypasses or failures when large quantities of viruses may have been released and transported to shellfish growing waters. Virus survival increases with cooler temperatures; therefore, MSC testing is used more heavily in the late fall, winter, and early spring (Dancer et al. 2010; Lipp et al. 2001; reviewed in Rzezutka and Cook 2004). MSC can be used in conjunction with fecal coliform testing to reopen shellfish beds that have potentially been impacted by sewage spills/bypasses.
Phytoplankton and Biotoxin Testing
Phytoplankton are microscopic organisms that are invisible to the naked eye, except during favorable conditions when they can bloom and potentially discolor the water. A subset of phytoplankton is called harmful algal bloom (HAB) species because they are in some way “harmful” to humans (e.g. through toxin production), the environment and/or the economy. Biotoxin testing was initiated in Connecticut in 1985 to monitor for the presence of potentially dangerous toxins produced by HABs. The FDA has established toxin regulatory limits based upon levels that are determined to be unsafe for human consumption. Connecticut has a short biotoxin history, with closures in 1985, 1992, and 2003 due to the presence of saxitoxin above the regulatory limit in Groton, CT. Saxitoxin is a potent neurotoxin produced by the phytoplankton genus Alexandrium. Saxitoxin can cause a variety of neurologic and gastrointestinal symptoms, and in severe cases can cause respiratory arrest/paralysis, making the toxin potentially lethal. DA/BA uses sentinel cages of blue mussels to accumulate toxin in Mumford Cove and Palmer Cove, Groton, CT in May-June, periods during which past closures have occurred. Blue mussels are the ideal shellfish species for biotoxin analysis because they readily accumulate and depurate toxins from the water column, providing an accurate and “real-time” image of toxin levels (Bricelj and Shumway 1998; Mafra Jr et al. 2010a&b). While biotoxin testing is limited to the areas of historical concern (Mumford Cove and Palmer Cove, Groton, CT, which have both periodically been closed due to biotoxin levels) and can be expanded as necessary, phytoplankton testing is conducted along the entire coast and provides long-term information about the phytoplankton community. With trained eyes, DA/BA staff can identify potential HAB species before a biotoxin issue arises. See the “Harmful Algal Bloom” section of this website for more information about the monitoring program and types of shellfish poisoning syndromes.
Vibrio parahaemolyticus Testing
Vibrio are bacteria that are naturally-occurring and are more abundant during the summer as the water warms. Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis in the United States and the world (FDA 2005), and has a short outbreak history in Darien, Norwalk, and Westport, CT waters, causing closures in 2012 and closures and recalls in 2013. Connecticut invested in an advanced molecular biology technique, real-time polymerized chain reaction (qPCR), which can directly detect the genetic material of Vibrio parahaemolyticus to determine if it is present in shellfish meat samples. The DA/BA lab uses the FDA-developed qPCR method that analyzes the genes thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and TDH-related hemolysin (trh). tlh is considered a universal marker for Vibrio parahaemolyticus (pathogenic and non-pathogenic) (McCarthy et al. 1999; Taniguchi et al. 1986), while tdh and trh are accepted as pathogenic markers (Honda and Iida 1993; Miyamoto et al. 1969; Shirai et al. 1990). Therefore, the DA/BA lab can determine if Vibrio parahaemolyticus is present and if it is potentially pathogenic when detected. More information about Vibrio parahaemolyticus and other Vibrio species can be found on the Center for Disease Control (CDC) website.
References
Borrego, J.J., Morinigo, M.A., de Vicente, A., Cornax, R., Romero, P. 1987. Coliphages as an indicator of fecal pollution in water. Its relationship with indicator and pathogenic microorganisms. Water Research. 12: 1473-1480.
Bricelj, V.M and Shumway, S.E. 1998. Paralytic Shellfish Toxins in Bivalve Molluscs: Occurrence, Transfer Kinetics, and Biotransformation. Reviews in Fisheries Science. 6: 315-383.
Caplenas, N.R. and Kanarek, M.S. 1984. Thermotolerant non-fecal source Klebsiella pneumonia: validity of the fecal coliform test in recreational waters. American Journal of Public Health. 74: 1273-1275.
Dancer, D., Rangdale, R.E., Lowther, J.A., Lees, D.N. 2010. Human Norovirus RNA Persists in Seawater under Simulated Winter Conditions but Does Not Bioaccumulate Efficiently in Pacific Oysters (Crassostrea gigas). Journal of Food Protection. 73: 2123-2127.
Food and Drug Administration (FDA). 2005. Vibrio parahaemolyticus risk assessment: quantitative risk assessment on the public health impact of pathogenic Vibrio parahaemolyticus in raw oysters. FDA, Washington, DC. http://www.fda.gov.pallas2.tcl.sc.edu/Food/ScienceResearch/ResearchAreas/RiskAssessmentSafetyAssessment /ucm050421.htm.
Grabow, W. 2001. Bacteriophages: Update on application as models for viruses in water. Water South Africa (SA). 27: 251-268.
Havelaar, A.H. 1987. Virus, Bacteriophages and Water purification. Veterinary Quarterly. 9: 356-360
Havelaar, A.H., van Olphen, M., Drost, Y.C. 1993. F-Specific RNA Bacteriophages Are Adequate Model Organisms for Enteric Viruses in Fresh Water. Applied and Environmental Microbiology. 59: 2956-2962.
Honda, Y. and Iida, T. 1993. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct hemolysin and related hemolysins. Reviews in Medical Microbiology. 4: 106-113.
Lipp, E.K, Kurz, R., Vincent, R., Rodriguez-Palacios, C., Farrah, S.R., Rose, J.B. 2001. The Effects of Seasonal Variability and Weather on Microbial Fecal Pollution and Enteric Pathogens in a Subtropical Estuary. Estuaries. 24: 266-276.
Mafra Jr., L.L., Bricelj, V.M., Ouellette, C., Bates, S.S. 2010a. Feeding mechanics as the basis for differential uptake of the neurotoxin domoic acid by oysters, Crassostrea virginica, and mussels, Mytilus edulis. Aquatic Toxicology. 97: 160-171.
Mafra Jr., L.L., Bricelj, V.M., Fennel, K. 2010b. Domoic acid uptake and elimination kinetics in oysters and mussels in relation to body size and anatomical distribution of toxin. Aquatic Toxicology. 100: 17-29.
Mandilara, G.D., Smeti, E.L., Mavridou, A.T., Lambiri, M.P., Vatopoulos, A.C., Rigas, F.P. 2006. Correlation between bacterial indicators and bacteriophages in sewage and sludge. FEMS Microbiology Letters. 263: 119-126.
McCarthy, S.A., DePaola, A., Cook, D.W., Kaysner, A., Hill, W.E. 1999. Evaluation of alkaline phosphatase and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus. Letters in Applied Microbiology. 28: 66-70.
McMinn, B.R., Ashbolt, N.J., Korajkic, A. 2017. Bacteriophages as indicators of fecal pollution and enteric virus removal. Letters in Applied Microbiology. 65: 11-26.
Miyamoto, Y., Kato, T., Obara, Y., Akiyama, S. 1969. In vitro hemolytic characteristics of Vibrio parahaemolyticus: its close correlation with human pathogenicity. Journal of Bacteriology. 100: 1147-1149.
Rzezutka, A. and Cook, N. 2004. Survival of human enteric viruses in the environment and food. FEMS Microbiology Reviews. 28: 441-453.
Shirai, H. Ito, H., Hirayama, T., Nakabayashi, Y., Humagai, K., Takeda, Y., Nishibuchi, M. 1990. Molecular epidemiological evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis. Infection and Immunity. 58: 3568-3573
Taniguchi, H., Hirano, H., Kubomura, S., Higashi, K., Mizuguchi, Y. 1986. Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus. Microbial Pathogenesis. 5: 425-432.