Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters.

Male-specific bacteriophage (MSB) densities were determined in animal and human fecal wastes to assess their potential impact on aquatic environments. Fecal samples (1,031) from cattle, chickens, dairy cows, dogs, ducks, geese, goats, hogs, horses, seagulls, sheep, and humans as well as 64 sewerage samples were examined for MSB. All animal species were found to harbor MSB, although the great majority excreted these viruses at very low levels. The results from this study demonstrate that in areas affected by both human and animal wastes, wastewater treatment plants are the principal contributors of MSB to fresh, estuarine, and marine waters.

Infectious diseases associated with molluscan shellfish consumption.

A history of shellfish-vectored illnesses (i.e., those associated with consumption of clams, oysters, mussels, and scallops) occurring in the past nine decades is presented. Typhoid fever was a significant public health problem among consumers of raw molluscan shellfish earlier in this century. The development of more effective sewage treatment procedures and the institution of a national program following these outbreaks led to a series of measures which eventually eliminated shellfish-associated typhoid fever. Present-day problems associated with this food source still involve some wastewaterborne bacterial illnesses. However, the principal public health concerns are with wastewater-derived viral pathogens and with bacterial agents of an environmental origin. The nature, occurrence, and magnitude of these public health problems are described.

Effect of gamma irradiation on shelf life and bacterial and viral loads in hard-shelled clams (Mercenaria mercenaria).

The feasibility of using 60Co gamma irradiation to inactivate total coliforms, fecal coliforms, Escherichia coli, Clostridium perfringens, and F-coliphage in hard-shelled clams, Mercenaria mercenaria, was investigated. The results of three trials indicated average D10 values of 1.32 kGy for total coliforms, 1.39 kGy for fecal coliforms, 1.54 kGy for E. coli, 2.71 kGy for C. perfringens, and 13.50 kGy for F-coliphage. Irradiation doses of > 0.5 kGy were significantly lethal to the shellfish.

Growth kinetics of Aeromonas hydrophila in freshwaters supplemented with various organic and inorganic nutrients.

Freshwaters of varying natural nutrient enrichment were used as growth media for the culture of an autochthonous, heterotrophic, freshwater bacterium, Aeromonas hydrophila. The growth rate of the bacterium in eutrophic waters was increased to the greatest extent by adding carbon, as glucose; generation times decreased by up to 65%. Additions of carbon and phosphorus increased the maximal cell densities by over 25-fold. In oligotrophic waters, bacterial growth was most strongly promoted by the simultaneous additions of carbon (as glucose) and phosphorus (as KH2PO4). In these waters, stationary phase densities were increased as much as 100-fold, with a corresponding 70% increase in growth rate. These data provide at least a partial explanation for the previously observed correlation between A. hydrophila densities and the trophic states of freshwaters.

Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica).

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.

Survival and replication of male-specific bacteriophages in molluscan shellfish.

The survival and replication of male-specific bacteriophages in hard-shelled clams (Mercenaria mercenaria) and their homogenates were examined to further assess their potential utility as indicator organisms. Trials were conducted in the presence and absence of a suitable bacterial host, Escherichia coli HS[pFamp]R. Results of this study demonstrated that male-specific bacteriophages were unable to replicate in hard-shelled clams, with or without added host cells. In addition, the densities of these bacteriophages were stable for up to 7 days in shellfish held at ambient seawater temperatures (less than 25 degrees C). Evidence of replication, although not observed in live shellfish, was found to occur in temperature-abused shellfish homogenates and supernatants, but only when a suitable bacterial host was present.

Seasonal effects on accumulation of microbial indicator organisms by Mercenaria mercenaria.

The ability of hard-shelled clams (Mercenaria mercenaria) to accumulate fecal coliforms and other microorganisms (Escherichia coli, Clostridium perfringens, and male-specific bacteriophages) was determined over a 1-year period. Twenty separate trails were conducted during different seasons to encompass a wide range of water temperatures. The greatest accumulation of microorganisms in hard-shelled clams occurred during certain periods in the spring, at temperatures ranging from 11.5 to 21.5 degrees C. These periods of hyperaccumulation did not always coincide for all organisms; the accumulation of bacteriophages was not predicted by the accumulation of either fecal coliforms or C. perfringens. Bacteriophages and C. perfringens showed significantly higher rates of accumulation than either the fecal coliform group or E. coli, especially during the spring. The higher incidence of human viral gastroenteritis associated with the consumption of shellfish during this period may be a result of the extraordinary concentration of certain microorganisms, including enteric viral pathogens.

Novel compound for identifying Escherichia coli.

A new chromogenic compound, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide, was found to be useful for the rapid, specific, differential identification of Escherichia coli in the sanitary analysis of shellfish and wastewater. Of 1,025 presumptively positive colonies (blue) and 583 presumptively negative colonies (nonblue), only 1% false-negative and 5% false-positive results were found.

Occurrence ofAeromonas hydrophila in limnetic environments: Relationship of the organism to trophic state.

The densities ofAeromonas hydrophila in various natural waters were found to be strongly correlated with a relative index proposed for use in trophic state assessments of fresh waters. No such correlation was found with the recoverable heterotrophic population of whichA. hydrophila is a part.A. hydrophila was found to be seasonally distributed with maximal densities occurring during summer through early fall. It was also found to be spatially distributed within a pond with the most consistent densities occurring from 1 m depth down to that depth in the water column where the temperature reaches 16°C. The densities of the organism correlated most strongly with total phosphorus, chlorophylla, and Secchi depth. Moderate correlations were found with dissolved phosphorus, Kjeldahl nitrogen, and dissolved organic carbon. Little or no correlation was obtained with ammonia, orthophosphate, pH, alkalinity, or dissolved oxygen. The discriminating ability that theA. hydrophila density measurements provide in the oligotrophic through mesotrophic range appears to exceed those of presently available methods. The facility and sensitivity of the enumeration method forA. hydrophila should make it a useful tool for trophic state assessments.

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.