Distribution of vibrio species in shellfish and water samples collected from the atlantic coastline of south-east Nigeria.

Crayfish, lobster, and sea-water samples collected from five fishing islands on the Atlantic coast-Bight of Biafra (Bonny)-belonging to Ibaka Local Government Area of Akwa-Ibom State of Nigeria were bacteriologically evaluated on thiosulphate citrate bile-salt sucrose (TCBS) agar for Vibrio load and pathotypes. Mean log10 Vibrio counts of 7.64+/-2.78 cfu/g (in crayfish), 5.07+/-3.21 cfu/g (in lobster), and 3.06+/-2.27 cfu/mL (in sea-water) were obtained in rainy season (June-July) while counts in the dry season (November-December) were 6.25+/-1.93 cfu/g, 5.99+/-1.54 cfu/g, and 3.84+/-1.78 cfu/mL respectively. The physicochemical measurements (temperature, pH, and total dissolved solutes) of the sea-water did not vary significantly in the two seasons across all five islands. Vibrio species isolated were Vibrio cholerae (both O1 and non-O1 serotypes), V parahaemolyticus, V vulnificus, V mimicus, and V fluvialis. Both Ogawa and Inaba subtypes of V cholerae O1 serotype were found. In addition, the Hikojima subtype, which had not been previously reported in the region, was isolated in two samples. The results show that these Vibrio species are endemic in the area.


INTRODUCTION
The interest in Vibrio species worldwide stems from the history and epidemiology of cholera, a severe diarrhoeal disease thought to have originated from the Far East (Asia) thousands of years ago. The spread of cholera was sea-borne at first, affecting particularly coastal towns and fishing villages. As it gained foothold within the regions, the tribal behaviours of the populations, including funeral ceremonies, movement of people within and between countries, and the use of rivers for transportation, trade, and travel as well as source of domestic water became important factors in the dissemination of the disease on a scale reminiscent of the 19 th century pandemics (1,2).
The long-distance spread of the disease to Europe and the Americas through the Middle East region is thought to have begun in 1817. The Ogawa se-sources of infection, and Vibrio species have largely been isolated from these sources in the southeastern region of Nigeria (7,11,12). It is against this background that shellfish and water samples from five fishing islands in this region, which are the major sources of shellfish sold in inland markets of Nigeria, were screened for Vibrio species and pathotypes.

Description of the project area
The study was carried out around five fishing islands-Asia-Obufa, Obio-Iwang, Ikot-Itie-Idung, Utam-Iyata, Utam-Antai located on the Bight of Biafra (Bonny) and belonging to Ibaka LGA of Akwa-Ibom State, Nigeria. Each island has 1,000-1,500 inhabitants. All inhabitants have homes in villages in the mainland and stay in make-shift shelters with their families to engage in daily out-sea fishing activities and shellfish processing for dispatch to the inland markets. The inhabitants have no toilet facilities and defaecate at the banks or inside the sea.

Collection of shellfish and water samples
Freshly-harvested crayfish and lobsters were purchased from fishermen as they came ashore after the night-fishing activities. These were immediately put in sterile polythene bags to avoid further contamination and conveyed to the laboratory sandwiched between ice-blocks in insulated ice-boxes.
Three sea-water samples (250 mL each) were also collected from each of the five islands as follows: using a sterilized conical flask, the sea-water samples were collected at 3 points in each island-one just at the ocean bank, the second at about 100 m from the shore, and the third at about 500 m from the shore into the sea. The conical flask was held with its mouth down and, in that position, it was submerged into depth of about 100 cm before the mouth was turned upwards under water to fill. All water samples were transported to the laboratory under the prevailing atmospheric conditions.

Crayfish and lobster
Approximately 2.0 g of crayfish or lobster sample was taken and homogenized into smooth slurry, using a sterile pestle and mortar; 1.0 g of the homogenate was suspended first in 1.0 mL of normal saline in a 25-mL Pyrex test tube and the volume made up to 10 mL with more saline to obtain a 1% (w/v) suspension. Subsequently, the suspension of homogenized tissue was serially diluted 10-fold, down to 10 -6 ; then, 1.0 mL of each dilution was spread over a freshly-prepared thiosulphate citrate bile-salt sucrose (TCBS) agar (Oxoid) plate, using a sterile bent glass-rod. The plate was incubated at 37 o C in a humidified incubator for 24 hours. Triplicate plates were prepared in the same way from a separate 2.0 g sample of the same batch of crayfish or lobster. After 24 hours, the incubated plates were observed for characteristic Vibrio colonies. Each colony, distinguished mainly by colour and size, was counted separately. Then, discrete representatives of each colony type were randomly selected for purification and characterization.

Water
Water samples collected from different islands were analyzed for temperature and pH at the point of collection and for total dissolved solids (TDS), using a TDS meter (Korea, HM Digital Inc.), and values were expressed in parts per million (ppm). Subsequently, approximately 200 mL of each seawater sample was filtered through a sterile 0.45-μm membrane, using a vacuum pump to facilitate the process; then, the filter unit was carefully dismantled, the membrane aseptically removed with sterile forceps and placed on TCBS agar. After incubation at 37 o C for 24 hours, the plate was examined for characteristic Vibrio colonies which were then counted and discrete colonies randomly selected for characterization.

Characterization of isolates suspected to be Vibrio
The randomly-selected colonies were first Gramstained to ascertain the curved rod cell morphology of Vibrio species before purification by three successive streaking and re-isolations on TCBS agar. The purified isolates were cultured on nutrient agar slants and stored at 4 o C. From the stock culture, each isolate was assayed for oxidase, urease and indole production, motility, and fermentation of various sugars, using the method described by Cheesbrough (13). All isolates preliminarily identified to be Vibrio cholerae from the above assay results were serologically tested for agglutination with polyvalent O1 antiserum (ANTEC, UK). Isolates which tested positive with polyvalent O1 antiserum were further tested with anti-Ogawa and or anti-Inaba sera to differentiate them into subtypes.
Further characterization of the colony morphology types showed that the yellow colonies (sucrose fer-menters) were predominantly either V. cholerae or V. fluvialis while the non-sucrose fermenters were either V. parahaemolyticus, V. mimicus, or V. vulnificus. All five Vibrio species were isolated from crayfish and lobster samples screened from each of the five islands with the frequencies shown in Table 3 and 4 in both rainy (June-July) and dry (November-December) seasons. The differences in frequency of isolation of species from different islands and in the two seasons were not statistically significant (p>0.05). Table  5 shows proportions of the Vibrio cholerae isolates obtained from the shellfish and water samples that were O1 and non-O1 serotypes from each of the five islands both in the rainy season and in the dry season. With the exception of the crayfish samples of rainy season and the water samples (obtained both in rainy and dry seasons) in which there were preponderance of V. cholerae O1 serotypes over the non-O1 strains, the relative occurrence of each serotype varied with locations. The Inaba and Ogawa subtypes of the V. cholerae O1 serotype were found in all locations in both shellfish and water samples. The Hikojima subtype was isolated only twice.
The physicochemical analyses of sea-water from locations in the five islands showed that the temperatures ranged between 27 o C and 28 o C in the rainy season (June-July) and was 28 o C allover in the dry season (November-December). The pH  (Table 6).

DISCUSSION
The shellfish (crayfish and lobster) harvested from waters of the Atlantic coast were heavily contaminated with Vibrio species; Vibrio counts were estimated to be as high as 10 4 -10 9 cfu/g, much higher than the counts recorded by Chigbu and Iroegbu (7). Samples of the water habitat from where the shellfish was harvested were equally heavily contaminated with Vibrio counts reaching 10 7 -10 9 cfu/ mL in some places. This could be assumed to be the natural source of shellfish contamination. This does not preclude the chances of the crayfish and lobsters concentrating the Vibrio cells as filter feeders (14). Human agencies, including direct daefecation into the sea and faecally-contaminated surface water running into the sea (prevalent in the area) could be the main contributors to contamination of the sea-water itself. Direct defaecation into the sea or river was less apparent in the locations researched by Chigbu and Iroegbu (7) and could account for the wide differences in Vibrio counts obtained in both studies.
All five Vibrio species isolated from shellfish and sea-water of the Atlantic coast of South-Eastern Nigeria are known to be pathogenic to humans. Vibrio cholerae and V. parahaemolyticus certainly, but V. fluvialis and V. mimicus also, have been associated with gastroenteritis or diarrhoeal diseases. Vibrio vulnificus, on the other hand, has been associated with wound infection and has been mentioned in connection with bathing in the sea and handling of fish and other sea-foods. Thus, isolation of all the five pathogenic Vibrio species from sea-water and shellfish harvested from this habitat is of great public and environmental health significance, given that members of the fishing communities have a culture of eating the shellfish raw, handling sea-food harvests, and swimming in the sea. It is interesting to note that Chigbu and Iroegbu (7), working in the same region but in different locations, encountered only V. cholerae, V. parahahaemolyticus (also isolated in this study), and V. alginolyticus (not isolated in this study). The main difference in the two studies is that the earlier work was carried out around the Cross River estuary (around Calabar city) and in Ikom, more than 200 km from the Atlantic coastline where environmental samples were drawn from freshwater sources; the study reported here was carried out in islands located far into the sea and where human activity was constantly within the water environment. It is likely that such human activity may have influenced both Vibrio counts and the variety of Vibrio species.
Among the five Vibrio species isolated, Vibrio cholerae is the recognized benchmark of Vibrio pathogenicity, and it is based on what was earlier known about the epidemiology of this species that dates back to 1883 through the early 1980s. It was as-  TDS=Total dissolved solids sumed that the human intestine was the sole reservoir of V. cholerae and that the organism was incapable of surviving for long in water. It was, therefore, held that infection had to be due to recent faecal contamination of drinking-water and foods. In the same thinking, humans have been thought to be the source of contamination of the sea-water and sea-foods (as mentioned earlier). This is buttressed by the culture of direct defaecation into the sea-water, a culture that prevails among fishing communities in the five islands sampled (Eyisi, personal observation). It was, however, later observed that V. cholerae exhibited a capacity of entering a rest-period during adverse environmental conditions (15)(16)(17). This changed all the earlier hypotheses; V. cholerae can apparently survive in marine waters, frequently associated with phytoor zoo-plankton, for several months. During seasons, when the temperatures and abundance of nutrients favour bloom of planktons, the bacteria multiply greatly (18,19). This does not contradict the fact that faecally-contaminated drinking-water constitutes the main source of infection with V. cholerae; the water then contaminates sea-foods as earlier posited. This sufficiently explains the muchreported relative abundance of the organisms with increase in temperature (20), such as during the dry season in the tropical regions of the world (7) or the warm summer months (21). In this work, Vibrio counts did not vary significantly between the dry and rainy seasons, and this may just be explained by the fairly uniform temperatures, pH, and dissolved solutes observed around all five islands in both the seasons. The uniform physicochemical conditions in the two seasons would be expected at about latitude 4 o N (above the equator) where the investigation was carried out. Besides, the dry season (November-December) sampling included the cool harmattan month of December, very close to the time the region may experience the last rains (early October). There may not have been enough time for a significant change in the water temperatures, pH, and nutrient concentrations (for the bloom of planktons).
Both O1 and non-O1 serotypes of V. cholerae were isolated from water and shellfish samples with the O1 serotype predominant at times. The latter observation was unexpected, given that in environmental samples like the ones analyzed in this work, non-O1 serotype is expected to be more abundant. The possible explanation for this unexpected distribution pattern may be that much of the O1 serotype is constantly seeded into the water environment from human sources; and the shellfish pick the Vibrio contaminants, particularly the O1 serotype, from the water environment. This has a further public-health implication in the area-the risk of disseminating the pathogen(s) to the inland fish markets through the contaminated harvests (processed and unprocessed).
Only the Vibrio O1 serotypes were further tested to determine the subtypes present. The Ogawa and Inaba subtypes were abundantly isolated. These have largely been reported even in epidemic cholera along the West coast of Africa and in South-Eastern Nigeria in particular (7,11,12). It is significant that Hikojima subtype was isolated in two locations in this work. This is probably the first time the Hikojima subtype has been reported in Nigeria. It is not certain whether this subtype got to the West African coastline from Asia as well or it genetically arose in habitat as intermediate between the Ogawa and Inaba subtypes here in the West African or Nigerian coastal waters.