Seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes in Calcutta.

The seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes was studied in 1982 and 1983. The occurrence of this organism in water and sediments at the three sites studied was very infrequent and was restricted to the summer months, although it was not always isolated during these months. The association of V. parahaemolyticus with plankton was chiefly confined to the summer months and progressively declined with the onset of monsoons, remaining below detectable levels during the postmonsoon and winter months. The incidence and counts of V. parahaemolyticus were consistently higher in association with plankton than with water and sediment samples. V. parahaemolyticus could be recovered throughout the period of investigation from freshly caught and market samples of freshwater fishes. The highest recovery rate of this halophile from fishes was invariably from fecal samples. Most of the strains isolated in this study were untypable, and those which could be typed were predominantly serotypes encountered in the environment. All the isolates were Kanagawa negative. From this study, it could be concluded that the survival of V. parahaemolyticus in freshwater ecosystems is transient and dependent on a biological host.     

Incidence and level of Vibrio parahaemolyticus associated with freshwater plankton.

We isolated Vibrio parahaemolyticus from plankton samples collected from different freshwater areas in the vicinity of Calcutta, India. Levels of this organism in association with freshwater plankton were low (less than 90 organisms per g). All of the 16 strains of V. parahaemolyticus isolated in this study were Kanagawa negative, and all except 1 were untypable. The occurrence of V. parahaemolyticus in association with freshwater plankton suggests that adsorption onto plankton may aid the survival of this moderately halophilic organism in freshwater milieus.     

Ecology ofVibrio cholerae in the freshwater environs of Calcutta,India

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.     

The role of sinking particles in the overwintering process of Vibrio parahaemolyticus in a marine environment

Abstract The behavioral pattern of Vibrio parahaemolytics during the winter season (December 1988 to March 1989) in the water column, sediment, plankton and sinking particles was determined in a eutrophic marine environment. A total of 15 environmental parameters and seven microbial characteristics were examined. This halophile was isolated sporadically from bottom water and plankton materials, whereas it was undetectable in the surface water and sediment samples. However, V. parahaemolyticus was isolated from the sinking particles continuously throughout the sampling period with highest counts during February 1989. Out of 195 strains identified, 10 Vibrio spp. and 3 Listonella spp. were observed of which V. alginolyticus was predominantly isolated irrespective of the samples tested. Simple correlation and multiple regression analyses show that the occurrence of V. parahaemolyticus is not governed by any single biotic or abiotic factor of the environment. Possibly, the cumulative effect of all these environmental parameters on the distribution of V. parahaemolyticus is conceivable.     

Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region

Abstract An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus . The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.     

Ecology of Vibrio parahaemolyticus in Chesapeake Bay

A study of the ecology of Vibrio parahaemolyticus and related vibrios in the Rhode River area of Chesapeake Bay was carried out over the period December 1970 through August 1971. The incidence of V. parahaemolyticus and related vibrios was found to be correlated with water temperature. The vibrios could not be detected in the water column during the winter months, although they were present in sediment. From late spring to early summer, when water temperatures were 14 +/- 1 C, vibrios over-wintering in sediment were released from the bottom communities and attached to zooplankton, proliferating as the temperature rose. The number of vibrios in and on plankton was reflected in the water column bacterial population densities at water temperatures of ca. 19 C. Thus, temperature of the water column in the range of 14 to 19 C was found to be critical in the annual cycle of the vibrios. Interaction between sediment, water, and zooplankton was found to be essential in the natural estuarine ecosystem. Bacterial counts of zooplankton were found to be temperature dependent. The bacterial population associated with zooplankton was found to be predominantly on external surfaces and was specific, differing from that of the sediment. Vibrio spp. and related organisms comprised the total bacterial population associated with zooplankton in summer months. The ecological role of Vibrio spp., including V. parahaemolyticus, was found to be significant, with respect to their property of chitin digestion and in relation to the population dynamics of zooplankton in Chesapeake Bay.     

Incidence of Vibrio parahaemolyticus in Chesapeake Bay

A Bay-wide survey of the distribution of Vibrio parahaemolyticus was carried out in Chesapeake Bay during May 1972, to determine whether the annual cycle of V. parahaemolyticus which was observed to occur in the Rhode River subestuary of Chesapeake Bay took place in other parts of Chesapeake Bay. In an earlier study, April to early June, when the water temperature rises from 14 to 19 C, was found to be a critical period in the annual cycle of the organism in the Rhode River, since this is the time period when the annual cycle is initiated. Results of this study, however, revealed that V. parahaemolyticus could not be found in the water column during May 1972. Nevertheless, several samples of sediment and plankton yielded V. parahaemolyticus isolates. Comparison of data with those for the Rhode River area examined in the earlier studies of the annual cycle of V. parahaemolyticus suggests that the time of initiation of the annual cycle of V. parahaemolyticus in the open Bay proper may be influenced by various factors such as temperature and salinity, i.e., deeper water locations may show initiation of the V. parahaemolyticus annual cycle later than shallow areas. Confirmation of the presence of the organisms in the samples studied was accomplished using numerical taxonomy with 19 reference strains also included in the analyses.     

Distribution of Vibrio parahaemolyticus and Related Organisms in the Atlantic Ocean off South Carolina and Georgia

The distribution of Vibrio parahaemolyticus and related organisms in the Atlantic Ocean was determined during the summer of 1971 from samples collected at stations along four transects on the continental shelf off the South Carolina and Georgia coasts. No V. parahaemolyticus strains were isolated from any of the samples of seawater (surface and bottom), sediment, and plankton which were collected. A numerical taxonomy analysis of data on substrate utilization, including 154 organic compounds serving as single carbon sources, was carried out, and four groups of strains were observed. Each group showed well-separated distribution profiles from shore out to the continental shelf. That is, the groupings were observed to correspond to coastal, off-shore and intermediate distribution patterns for the strains. This study provides a useful example of the kind of ecological distributional analysis of bacteria which can be accomplished with numerical taxonomy.     

Incidence of Vibrio species associated with blue crabs (Callinectes sapidus) collected from Galveston Bay, Texas.

Bacteria were readily isolated from the hemolymph of a majority (88%) of the blue crabs collected from Galveston Bay, Texas. The hemolymph of most crabs contained moderate (greater than 10(3) bacteria/ml) to heavy (greater than (10(5) bacteria/ml) infections. Large variances were observed in the bacterial number associated with individual crabs, but no significant difference was observed between the mean bacterial levels in the hemolymph of crabs collected during different seasons of the sampling year. Vibrio spp. were the predominant bacterial types in the hemolymph of infected crabs and increased in number significantly during the summer season. Warmer water temperatures were thought to be responsible for this increase. Bacterial numbers and the percentage of Vibrio spp. were highest in the interior of the crab bodies, especially in the digestive tract. The exterior of the crabs did not appear to be the source of the hemolymph's bacterial flora. Bacteria taxonomically identical to Vibrio cholerae. V. vulnificus, and V. parahaemolyticus were routinely isolated from the crab hemolymph and external carapace. V. parahaemolyticus was the most prevalent of the pathogenic Vibrio spp. and was isolated from 23% of the hemolymph samples. V. vulnificus (7%) and V. cholerae (2%) occurred less commonly in the hemolymph. The incidences of V. parachaemolyticus and V. vulnificus were related and increased in the summer months. Both organisms were frequently isolated from the same crab.     

Rapid detection and enumeration of trh-carrying Vibrio parahaemolyticus with the alkaline phosphatase-labelled oligonucleotide probe

An alkaline phosphatase (AP)-labelled oligonucleotide probe was developed to detect and enumerate trh(+)Vibrio parahaemolyticus in seafood. The probe was evaluated using 40 isolates of V. parahaemolyticus, 45 isolates of other vibrios and 55 non-vibrio isolates. The probe reacted specifically with V. parahaemolyticus possessing either the trh1 or trh2 variant of the trh gene and was found to be 100% specific for trh(+)V. parahaemolyticus. Using the trh probe, V. parahaemolyticus carrying trh gene was targeted in 34 seafood samples by direct plating and colony hybridization procedure. The trh(+)V. parahaemolyticus could be detected in five of 34 (14.7%) samples and the levels ranged from 5.0 x 10(2) to 3.4 x 10(3) cfu g(-1). Colonies of trh(+)V.parahaemolyticus were isolated from the five positive samples. Forty seafood samples were analysed for trh(+)V. parahaemolyticus by colony hybridization following enrichment in alkaline peptone water. 16 samples (40%) were positive for trh gene and trh(+)V. parahaemolyticus was isolated from 15 samples (37.5%). To assess the sensitivity of the trh probe, seafood homogenates spiked with known concentrations of trh-positive V. parahaemolyticus were plated and hybridized. Counts obtained using the probe were similar to those of inocula. The results suggest that the AP-labelled trh probe is useful for the detection and enumeration of trh(+)V. parahaemolyticus in seafood.     

Occurrence of potentially pathogenic vibrios and related environmental factors in Songkhla Lake, Thailand

Vibrios are halophilic bacteria that are ubiquitous in marine environments. Their occurrence in tropical lakes has rarely been investigated. In this study, the predominance and diversity of Vibrio spp. was investigated over a 12-month period in a coastal lagoon, Songkhla Lake, in southern Thailand. Water samples were collected at 2 stations in the estuary near Yor Island in Songkhla Lake. The predominant vibrios were detected by a culture-based method, using thiosulfate-citrate-bile salt-sucrose agar and CHROMagar Vibrio. The diversity of Vibrio spp. was evaluated using denaturant density gradient electrophoresis (DGGE). The highest numbers of total vibrios and Vibrio parahaemolyticus in both areas were observed during the summer. There was no significant correlation between the numbers of vibrios, including V. parahaemolyticus, and either the water temperature or plankton density. Variations in Vibrio species were observed with changes in salinity. Vibrio parahaemolyticus and V. cholerae non-O1/non-O139 were detected during the rainy season when the salinity dropped to nearly 0 parts per thousand. In both areas, V. alginolyticus was the most prominent species detected by the culture method, whereas Vibrio parahaemolyticus was detected by DGGE, every month. Other Vibrio spp. of potential public health concern were also detected by the culture method; they included V. vulnificus , V. fluvialis , and V. mimicus .     

The use of multiple typing methods allows a more accurate molecular characterization of Vibrio parahaemolyticus strains isolated from the Italian Adriatic Sea

Vibrio parahaemolyticus is a natural inhabitant of marine environments and constitutes part of the autochthonous microbial communities, but is also associated with human gastroenteritis, wound infections and septicemia. Recently, a number of clinical cases of infection due to ingestion of seafood contaminated with V. parahaemolyticus and potentially pandemic marine strains isolated from water and plankton have been reported in Europe. To identify the source of virulent strains and to analyze the possible persistence, in time and space, of particular clones, the molecular typing of Vibrio strains is of high epidemiological interest. In this study, we applied pulsed-field gel electrophoresis and two PCR-based techniques (enterobacterial repetitive intergenic consensus- and repetitive extragenic palindromic-PCR) to establish the DNA fingerprints for the analysis of genetic variability among the environmental V. parahaemolyticus strains isolated in the area of the Venetian Lagoon. A temporal distribution of the environmental strains in the studied geographical area and, in some cases, a strong association between a certain genetic profile and a specific source have been evidenced. A number of genetic clusters/clones seem to persist over time, reappearing in the marine environment for subsequent months and also at a 1-year gap. The use of multiple typing methods allowed a more accurate characterization of the environmental strain genetic profiles and the identification of clones hardly revealed through common techniques.     

Detection of free-living and plankton-bound vibrios in coastal waters of the Adriatic Sea (Italy) and study of their pathogenicity-associated properties

Culturable vibrios were isolated from water and plankton fractions collected during an 18-month sampling study performed along the north-central coast of the Adriatic Sea (Italy). Unculturable Vibrio vulnificus and V. parahaemolyticus were detected in plankton fractions by polymerase chain reaction amplification of DNA sequences for cytotoxin-haemolysin and thermolabile haemolysin respectively. The presence of V. parahaemolyticus, V. vulnificus and V. cholerae virulence genes and the expression of pathogenicity-associated traits were analysed in all isolates. The results showed the spreading of these properties among the environmental isolates and confirm the need of both monitoring the presence of vibrios in coastal areas and studying their pathogenicity potential in order to properly protect human health.     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10(4) organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.     

Vibrio virulence genes in fishes collected from estuarine waters in Italy

Aims: The aim of this study was to investigate the presence of Vibrio vulnificus and potentially pathogenic strains of Vibrio parahaemolyticus in mullets collected from estuarine environment in Italy. Methods and Results: Two hundred and ninety‐five mullets were analysed by culture using the selective medium thiosulfate citrate bile salt sucrose agar, during a monitoring period of 2 years (2008–2009). Presumptive Vibrio colonies were initially identified by using biochemical tests, and strains identified as V. parahaemolyticus and V. vulnificus were subsequently examined by PCR for the presence of species‐specific and virulence genes (toxR, trh, tdh and vvh). V. parahaemolyticus was found in 55% (162/295) of fishes and V. vulnificus in 1% (3/295) with a higher presence in summer months. The trh+/tdh? strains were detected in 16% (47/295) of samples and only one strain resulted trh+/tdh+. One of the V. parahaemolyticus trh+ strains isolated belonged to the O1:KUT (K untypeable), a serotype recently associated to gastroenteritis in Italy. Conclusions: This is the first report demonstrating a high percentage of potential pathogenic V. parahaemolyticus trh+ strains in estuarine fishes of the Mediterranean area. Significance and Impact of the Study: These findings indicate the potential human health risk associated with the presence of pathogenic Vibrio spp. in wild fishes.     

Edwardsiella tarda in freshwater catfish and their environment

Edwardsiella tarda was isolated from 47, 88, and 79% of skin, visceral, and dressed-fish samples, respectively. This species was also isolated from 30% of imported dressed fish, 75% of catfish pond water samples, 64% of catfish pond mud samples, and 100% of frogs, turtles, and crayfish from catfish ponds. The incidence of Edwardsiella increased during the summer months, as water temperatures increased. Of several isolation media evaluated, the most effective was selective enrichment in double-strength Salmonella-Shigella broth and subsequent plating on single-strength Samonella-Shigella agar. The significance of the incidence of Edwardsiella in catfish, catfish disease, and public health could not be substantiated.     

Edwardsiella tarda in freshwater catfish and their environment.

Edwardsiella tarda was isolated from 47, 88, and 79% of skin, visceral, and dressed-fish samples, respectively. This species was also isolated from 30% of imported dressed fish, 75% of catfish pond water samples, 64% of catfish pond mud samples, and 100% of frogs, turtles, and crayfish from catfish ponds. The incidence of Edwardsiella increased during the summer months, as water temperatures increased. Of several isolation media evaluated, the most effective was selective enrichment in double-strength Salmonella-Shigella broth and subsequent plating on single-strength Samonella-Shigella agar. The significance of the incidence of Edwardsiella in catfish, catfish disease, and public health could not be substantiated.     

Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Serological method for identification of Vibrio parahaemolyticus from marine samples.

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.