Evaluation of enrichment media for improved PCR-based detection of V. cholerae and V. vulnificus from estuarine water and plankton.

AIMS: Pathogenic Vibrio spp., including V. cholerae and V. vulnificus, are commonly found along the estuaries of the south-east United States; however, it is often difficult to recover these species directly from environmental samples. Pre-enrichment assays are commonly used to improve the detection of pathogenic vibrios from environmental sources. Here, we evaluated a novel enrichment procedure using freshly collected and autoclaved natural estuarine water amended with 1% peptone (designated as estuarine peptone water, EPW) and compared it to traditional alkaline peptone water (APW) for detection by PCR of V. cholerae and V. vulnificus. METHODS AND RESULTS: Of the 50 samples collected in total, V. cholerae DNA was detected in APW 10% of the time and in EPW 40% of the time. Likewise, the cholera toxin gene (ctxA) was detected in 4 vs 18% of the samples using APW and EPW, respectively. Conversely, APW showed improved recovery for V. vulnificus relative to EPW with respective detection frequencies of 46 and 20%. Results showed similar patterns across different sample types (water and plankton). CONCLUSIONS: While enrichment in traditional APW was adequate for the recovery of Vibrio vulnificius, use of sterile estuarine water amended with peptone significantly improved the detection of V. cholerae and the virulence gene ctxA from estuarine sources.

Emerging marine diseases--climate links and anthropogenic factors.

Mass mortalities due to disease outbreaks have recently affected major taxa in the oceans. For closely monitored groups like corals and marine mammals, reports of the frequency of epidemics and the number of new diseases have increased recently. A dramatic global increase in the severity of coral bleaching in 1997-98 is coincident with high El Niño temperatures. Such climate-mediated, physiological stresses may compromise host resistance and increase frequency of opportunistic diseases. Where documented, new diseases typically have emerged through host or range shifts of known pathogens. Both climate and human activities may have also accelerated global transport of species, bringing together pathogens and previously unexposed host populations.

Climate variability and change in the United States: potential impacts on water- and foodborne diseases caused by microbiologic agents.

Exposure to waterborne and foodborne pathogens can occur via drinking water (associated with fecal contamination), seafood (due to natural microbial hazards, toxins, or wastewater disposal) or fresh produce (irrigated or processed with contaminated water). Weather influences the transport and dissemination of these microbial agents via rainfall and runoff and the survival and/or growth through such factors as temperature. Federal and state laws and regulatory programs protect much of the U.S. population from waterborne disease; however, if climate variability increases, current and future deficiencies in areas such as watershed protection, infrastructure, and storm drainage systems will probably increase the risk of contamination events. Knowledge about transport processes and the fate of microbial pollutants associated with rainfall and snowmelt is key to predicting risks from a change in weather variability. Although recent studies identified links between climate variability and occurrence of microbial agents in water, the relationships need further quantification in the context of other stresses. In the marine environment as well, there are few studies that adequately address the potential health effects of climate variability in combination with other stresses such as overfishing, introduced species, and rise in sea level. Advances in monitoring are necessary to enhance early-warning and prevention capabilities. Application of existing technologies, such as molecular fingerprinting to track contaminant sources or satellite remote sensing to detect coastal algal blooms, could be expanded. This assessment recommends incorporating a range of future scenarios of improvement plans for current deficiencies in the public health infrastructure to achieve more realistic risk assessments.

The role of seafood in foodborne diseases in the United States of America.

In the United States of America, seafood ranked third on the list of products which caused foodborne disease between 1983 and 1992. Outbreaks connected with fish vectors were caused by scombroid, ciguatoxin, bacteria and unknown agents; in shellfish, unknown agents, paralytic shellfish poisoning, Vibrio spp. and other bacteria, followed by hepatitis A virus, were responsible for the outbreaks. At least ten genera of bacterial pathogens have been implicated in seafood-borne diseases. Over the past twenty-five years, bacterial pathogens associated with faecal contamination have represented only 4% of the shellfish-associated outbreaks, while naturally-occurring bacteria accounted for 20% of shellfish-related illnesses and 99% of the deaths. Most of these indigenous bacteria fall into the family Vibrionaceae which includes the genera Vibrio, Aeromonas and Plesiomonas. In general, Vibrio spp. are not associated with faecal contamination and therefore faecal indicators do not correlate with the presence of Vibrio. Viruses are the most significant cause of shellfish-associated disease: in New York State, for example, 33% and 62% of 196 outbreaks between 1981 and 1992 were caused by Norwalk virus and gastrointestinal viruses (small round structured viruses), respectively. In addition, several illnesses are a result of toxic algal blooms, the growth of naturally occurring bacteria and diatoms causing neurotoxic shellfish poisoning, paralytic shellfish poisoning, diarrhoetic shellfish poisoning, amnesic shellfish poisoning and ciguatera. Current estimates place the annual number of ciguatera cases at 20,000 world-wide. Scombroid poisoning is the most significant cause of illness associated with seafood. Scombrotoxin is of bacterial origin and halophilic Vibrio spp. causing high histamine levels are implicated as the source. Scombroid poisoning is geographically diverse and many species have been implicated, namely: tuna, mahi-mahi, bluefish, sardines, mackerel, amberjack and abalone. Temperature abuse has been cited as a major cause of scombroid poisoning. For routine work, the use of faecal indicators to predict the relative level of faecal contamination should not be disposed of. However, the main source of seafood illness is due to species which are not predicted by these organisms. In order to protect public health, routine surveillance using new pathogen-specific techniques such as polymerase chain reaction should be used. This, in combination with risk assessment methods and hazard analysis and critical control points, will begin to address the need for improvement in the safety of seafood.

Assessment and impact of microbial fecal pollution and human enteric pathogens in a coastal community.

The goals of this study were to assess watersheds impacted by high densities of OSDS (onsite sewage disposal systems) for evidence of fecal contamination and evaluate the occurrence of human pathogens in coastal waters off west Florida. Eleven stations (representing six watersheds) were intensively sampled for microbial indicators of fecal pollution (fecal coliform bacteria, enterococci, Clostridium perfringens and coliphage) and the human enteric pathogens, Cryptosporidium, Giardia, and enteroviruses during the summer rainy season (May-September 1996). Levels of all indicators ranged between < 5 and > 4000 CFU/100 ml. Cryptosporidium and Giardia were detected infrequently (6.8% and 2.3% of samples tested positive, respectively). Conversely, infectious enteroviruses were detected at low levels in 5 of the 6 watersheds sampled. Using cluster analysis, sites were grouped into two categories, high and low risks, based on combined levels of indicators. These results suggest that stations of highest pollution risk were located within areas of high OSDS densities. Furthermore, data indicate a subsurface transport of contaminated water to surface waters. The high prevalence of enteroviruses throughout the study area suggests a chronic pollution problem and potential risk to recreational swimmers in and around Sarasota Bay.

Detection of viral pathogens by reverse transcriptase PCR and of microbial indicators by standard methods in the canals of the Florida Keys.

In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, enterococci, coliphages, F-specific (F(+)) RNA coliphages, Giardia lamblia, Cryptosporidium parvum, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1, 410, E. coli organisms from <1 to 130, Clostridium spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F(+) phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks.