Enteric bacterial population associated with farmed freshwater prawn and its environment, water quality of prawn farm and the existing association between these parameters were studied. Microbiological parameters were determined following the United States Food and Drug Administration (USFDA) methods and the physico-chemical parameters as per the standard methods of American Public Health Association (APHA). Prawn samples yielded a mean Total Plant Count (TPC) in the range of 4.57 to 6.66 log cfu g-1 and was the highest among all other samples. Prawns followed by water samples had the higher level of enteric indicator organisms. Sediment showed higher count of sulphite reducing clostridia. Emerging pathogen E. coli O157:H7 were absent in all the samples analyzed. Enterobacter (31.5%) followed by Citrobacter (13.2%) and non enteric bacteria Aeromonas (11%) were the dominant flora recovered. Escherichia, Klebsiella, Hafnia, Serratia, Salmonella and Shigella were the other opportunistic enteric bacterial pathogens detected from this system. The rearing practices such as use of cow dung as fertilizer and microbiologically contaminated feed could have influenced the enteric flora. Study on various physico-chemical parameters of pond water revealed that they were within the suitable range for the freshwater prawn culture. Correlation analysis revealed a significant positive correlation between pollution indicator parameters such as Total Organic Carbon (TOC), Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD) with that of Total Plate Count (TPC) and Total Enterobacteriaceae Count (TEC) of the pond water and prawn samples. Presence of bacteria of public health significance in the aquaculture pond envisages strict hygienic handling, processing of prawn from this system and cooking prior to consumption to ensure public health safety.
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World aquaculture industry witnessed a phenomenal growth over the last 10 year period by way of increment in production from 24.38 million tones in 1995 to almost double in the year 2006 (51.70 million tones) (FAO, 2009). India with its aquaculture production of 3.12 million tones is placed second only after China in the year 2006 (FAO, 2009). Freshwater crustaceans including prawns though produced in meager quantities (around 953 198 tones) have gained significance as high value commodity by standing as fifth highest valued species group. Farming of giant freshwater prawn Macrobrachuim rosenbergii popularly known as scampi is spreading fast to all Indian states due to its large size attainment, tolerance to water quality changes, ability to cope with handling stress and ability to feed on unconventional feeds. The farming of scampi received a big boost lately owing to the demand and attractive price fetched by the Indian scampi in the international market and the slump in tiger shrimp production due to disease. The production of scampi in India increased from 7150 tones during the year 1999-2000 to 27, 262 tones in 2007-2008 (MPEDA, 2008).
While realizing the potential of aquaculture as an important food producing enterprises, hand in hand there exist, the health risk associated with the aquaculture produce due to contamination of products by chemical and biological agents (Huss et al., 2000). Reviews on the microbiological evaluation of aqua cultured raw fish/shellfish and their growing environments emphasized the need for the assurance of the quality and safety before sending them to retail and export market (Reilly and Kaferstein, 1999). Shellfishes, the most internationally traded food of aquatic origin, are in the forefront of food safety and quality improvement due to stringent microbial quality and safety regulations enforced by international regulatory authorities. Majority of the bacteria implicated in the disease out break of M. rosenbergii and seafood borne bacteria of human health importance belonged to the mesophilic Enterobacteriaceae group (Brock, 1993; Ogbondeminu, 1993). They included pathogenic E. coli, Salmonella, Shigella, Yersinia, Enterobacter, Citrobacter etc., recovered from different aquaculture systems in India and other parts of the world (Anderson et al., 1989; Ogbondeminu, 1993; Jayasree et al., 1999). Threat due to these organisms is more, when earthen pond fertilized with animal manure, was used as the production systems (Ogbondeminu and Okaeme, 1986, 1989). Reports of the occurrence of pathogenic strains of E. coli and outbreaks of illness due to consumption of a wide range of fish and seafood products are common (Samadpour et al., 1994; Kumar et al., 2001; Teophilo et al., 2002). Among the different groups, E. coli O157, an Enterohemorrhagic E. coli, is an important emerging pathogen with very low infective dose (10-100 cells).
It was well established that rearing site water quality and microbiology play a significant role in overall well being of fish, nutrient status of pond, fish yield potential and most importantly quality and safety of fish raised from them (Reilly and Kaferstein, 1999). Therefore, monitoring of productive areas for microbial quality is a fundamental prerequisite to obtain safe produce from the farms. Different researchers reported a direct or indirect correlation between the microbial parameters and water quality parameters (Ogbondeminu, 1993; Jun et al., 2000; Surendraraj et al., 2009). In this study, we report the microbial and limnological quality of scampi farm, the existing correlation if any between this parameter and the presence of bacteria of public health significance to pay way to develop safe farm management practices.
MATERIALS AND METHODS
Description of the Sampling Site
The samples of prawn, pond water, source water, sediment and feed were obtained from a freshwater prawn farm situated in Kumarakom, Kottayam district of Kerala, India. The earthen pond sampled has an area of 0.90 ha with a stocking density of 40,000 Juveniles ha-1. The pond was surrounded by two rearing ponds in two sides, third and fourth side was covered by feeder canal and a transition pond, respectively. Pond dykes had plantations like coconut, banana trees etc and pond bottom was silty. The pond was fertilized with cow dung initially and continued every 15 days according to the nutrient status. Feeding was by the commercial formulated feed obtained from CP Aquaculture India Pvt. Ltd., Chennai. There was periodic water exchange by opening the inlet cum outlet to a small transition pond which was connected to the feeder canal, passing parallel to the farm. Feeder canal has direct contact to the natural freshwater water body.
Five sampling was carried out on 50th, 71st, 127th, 155th and 227th day of culture. Prawn samples were harvested by cast net and put in sterile polythene bag. Water samples were collected from 4 different location of the pond by inverting the sterile polystyrene bottle to about 30 cm below the surface and a well-mixed homogeneous sample was used for analysis. Feeder Canal Water (FCW) samples were also collected in similar manner. Pond sediment were scooped out from four different locations of the pond and collected in a sterile polythene bag. All the samples were kept in ice box carried to laboratory. Analysis was initiated with in 2 h of sample collection. Sampling was done between 08:00 to 09:00 h Indian standard time.
Water Quality Parameters
Water temperature, pH and salinity were determined at the farm location by using thermometer, digital pH meter (Cyberscan, USA) and refractometer (Atago Co. Ltd., Japan), respectively. Transparency was measured using secchi disc (Trivedy and Goel, 1984). Total Suspended Solids (TSS) and Dissolved Oxygen (DO) were analyzed according to the standard methods for examination of water and wastewater (APHA/AWWA/WEF, 1998). TOC, NO3-N (nitrate nitrogen), BOD, COD and surfactant, were measured by properly calibrated pastel UV spectrophotometer (SECOMAM RS 232, France) with a spectral range of 200 to 320 nm. Turbidity was measured by employing the digital turbidity meter (Merck, USA). Conductivity of the water was measured by electronic conductivity meter (CIFT, India).
Ten grams of the prawn (headless) or sediment samples were homogenized for 1 min with 90 mL of saline (0.85% NaCl) in a stomacher 400 lab blender (Seward, London, UK). Prawn, sediment and water samples were serially diluted and used for analysis. The total plate count was enumerated by pour plating the samples on Tryptone Glucose Agar (TGA), Total Enterobacteriaceae count on Violet Red Bile Glucose Agar (VRBGA, Oxoid, Basingstoke, UK) and faecal Streptococci in Kenner Faecal streptococcus agar (KF) (USFDA, 2001).
Total coliforms, faecal coliforms and E. coli counts were estimated for prawn/sediment and water samples by a three-tube and five tube Most-Probable Number (MPN) procedure, respectively, with the following modification. Aliquots of serially diluted samples were inoculated into MacConkey broth (Oxoid, Basingstoke, UK) and incubated at 37°C for 24 to 48 h. Positive tubes were (1) subjected to an MPN procedure in Brilliant Green Lactose Bile Broth (BGLB) (Oxoid, UK) at 37°C for 24 to 48 h and (2) subjected to Elevated Coliform (EC) broth (Difco, Becton Dickinson, Sparks, Md., USA) and indole (Difco, Becton Dickinson, Sparks, Md., USA) broths at 44.5°C. Positives in BGLB, EC broth and indole were noted down and referred to McCardys MPN table to determine the total coliforms, faecal coliforms and E. coli counts, respectively. A loopful of bacterial culture from indole-positive tubes was streaked on Eosine Methylene Blue agar (EMB agar, BBL, Becton Dickinson, Sparks, Md., USA) and characteristic E. coli colonies were isolated and confirmed by Indole-Methylred-Voges-Proskauer-Citrate (IMViC) tests (APHA/AWWA/WEF, 1998). Sulphite reducing clostridia were analyzed by MPN method using differential reinforced clostridia medium (USFDA, 2001).
Detection of E. coli O157:H7
Twenty five gram of the prawn or sediment and 25 mL of water samples were enriched in 225 mL of modified elevated coliform broth (Difco, USA) containing novobiocin (20 mg mL-1; Sigma Chemical Company, St. Louis, Mo., USA) at 42 oC with shaking (150 rpm). After an overnight incubation, diluted enrichment samples were plated on MacConkey sorbitol agar (Oxoid, UK) supplemented with cefixime (SR 0191, Oxoid, UK) and potassium tellurite (March and Ratman, 1986). Plates were incubated overnight at 37°C and sorbitol-negative colonies were isolated at a rate of 2-3 colonies per sample. After isolate purification, they were streaked on EMB agar and confirmed isolates were checked for MUG (methyl umbelliferyl-β-glucuroride) reaction and IMViC test. MUG and sorbitol-positive E. coli (ATCC 25922) were used as controls for checking sorbitol and MUG reactions. Isolates that were sorbitol and MUG negative were tested for latex agglutination with E. coli serotype O157 specific antisera as per manufacturers instruction (Oxoid, UK).
Characterization of Enteric Bacteria
For identification, 2 to 5 well-separated typical colonies from VRBGA plates were selected using Harrisons disc method (Harrigan and McCance, 1976). These cultures were purified and stored for further study in nutrient agar slants. Altogether, 146 pure culture isolates were obtained and identified up to the genus level with the help of an identification scheme from web http//www.vet.uga.edu/WEBFILES/ in consultation with Edwards and Ewing (1972) and MacFaddin (1980). About, 5% of the isolates were crosschecked for identification using analytical profile index 20 E (API 20 E, bioMerieux).
All the results were produced as mean and mean log±SD values for water quality and microbiological parameters respectively. Statistical analysis between the means was accomplished using Tukeys test and a two-tailed Pearson correlation analysis was carried out. The statistical package used in the study is SPSS, 10.
RESULTS AND DISCUSSION
Physico-Chemical Characteristics of Water
In general, water quality parameters showed a distinct variation among different sampling phases and with few exceptions they were within the optimum range reported for scampi culture (Table 1, 2) (Brock, 1993; Saxena, 2003). The temperature and pH of the Pond Water (PW) and FCW ranged between 28.97±0.06 to 31.08±0.06°C and 6.41±0.01 to 7.20±0.03, respectively. Salinity was increasing throughout the farming phase and reached the highest level (2.53 ppt) towards the end of the culture period in PW, which corresponds to the summer season. The observed values were similar to the one reported by Lalitha and Surendran (2004) study on the scampi culture pond.
Water quality parameters of pond water used in giant freshwater prawn (M. rosenbergii) farm*
*Results are presented as Mean±SD. Means in a column with the same superscript letters are not significantly different (p>0.05). TSS: Total suspended solids; TOC: Total organic carbon; COD: Chemical oxygen demand; BOD: Biological oxygen demand; SDT: Secchi disc transparency; DO: Dissolved oxygen; ppt: Parts per thousand; ppm: Parts per million; NTU: Neplometric turbidity unit; cm: Centimeter
Feeder canal water quality for giant freshwater prawn (M. rosenbergii) farm*
*Results are presented as Mean±SD. Means in a column with the same superscript letters are not significantly different (p>0.05). TSS: Total suspended solids; TOC: Total organic carbon; COD: Chemical oxygen demand; BOD: Biological oxygen demand; DO: Dissolved oxygen; ppt: Parts per thousand; ppm: Parts per million; NTU: Neplometric turbidity unit; cm: Centimetre
The pollution indicator parameters, TSS, TOC, BOD, COD and turbidity values showed an increasing trend for pond water as culture progressed. As reported earlier, in our study also bacterial population increased positively with above parameters and hence, control of pollution parameters to the optimum level is important (Martinez et al., 2002). Decomposition of organic matter from aquatic plants and settling of left over feed and excreta from prawns might have contributed to the higher BOD and COD. However, in the case of FCW, similar trend was missing except for TSS. NO3-N and surfactants were below the detection level in most phases both in PW and FCW and were within the permissible level (Brock, 1993). Lalitha and Surendran (2004) observed a DO level of 5 to 6 ppm, where as the DO in the present study varied between 3.18±0.07 to 5.18±0.17 ppm for PW and still lower in FCW. Chien et al. (1999) reported that high conductivity has a direct bearing on the survival of microbes. Conductivity values showed an increasing trend and were highest in phase IV. Though, the microbial counts were comparatively low in the last two phases, similar effect as reported by earlier author is missing in this study.
Microbiology of freshwater prawn farm during different phase of culture*
*Results are presented as Mean log±SD. a,b,c,dMeans in a column with the same superscript letters are not significantly different (p>0.05). TPC: Total plate count; TEC: Total Enterobacteriaceae count; TCC: Total coliforms count; FCC: Faecal coliforms count; ECC: E. coli count; FSC: Faecal Streptococci count; ND: Not detected; SRC: Sulphite reducing Clostridia count; PW: Pond water; FCW: Feeder canal water; cfu: Colony forming unit; MPN: Most probable number
Microbiology of Freshwater Shellfish Farm
The TPC of prawn, sediment and PW samples were significantly different among the different sampling phases (p<0.05) and ranged between 3.34 cfu mL-1 and 6.66 cfu g-1 (Table 3). Prawn samples always had higher TPC than all other samples and were between 4.5 and 6.7 log cfu g-1 among different faming phases. Sediment TPC was 0.5 to 1.5 log cfu g-1 lower than that of prawn and for pond water it was varying from 3.40 to 5.42 log cfu mL-1. The count observed in this study for prawns is slightly higher than the one reported for different types of fresh and processed samples (Bandekar et al., 2004; Lalitha and Surendran, 2006). Higher count for shellfish than sediment and PW samples were also reported by earlier studies (Lalitha and Surendran, 2006; Surendran et al., 2000).
As in TPC, the bacterial indicator organisms such as TEC, TCC, FCC, ECC and FSC were also found to be high in prawn samples than all other samples. The counts were significantly different among different sampling phases (p<0.05) and increased with progress in culture except in phase IV. Sediment samples recorded the least count for the above parameters and in the last two phases they were absent. The PW and FCW water were found to have been contaminated with all the above indicators but there was not much variation among faming phases (p>0.05). The present findings were comparable with the earlier reports on prawn farms from Kerala (Surendran et al., 2000; Lalitha and Surendran, 2006). The microbial analysis of feed and fertilizer used in the pond showed a higher level of TPC, TEC, TCC and FSC. The higher count of TPC and other indicator organisms in prawn samples might be due to their detritus feeding habits and the use of contaminated feed and fertilizers.
Surendran et al. (2000) reported the recovery of Sulphite Reducing Clostridia (SRC) from all the samples of freshwater prawn, sediment and water. In our study also, SRC was detected in all samples. It was higher in sediment/FCW and was low in the case of prawn samples. Contamination by different pathogens such as Salmonella, Vibrio cholerae and pathogenic E. coli were increasingly reported from sediment samples of prawn culture pond (Surendran et al., 2000; Jeyasekaran and Ayyappan, 2002). However, Screening for the emerging pathogen E. coli O157 in this study revealed that they could not be detected in this culture system.
Composition of Enteric Bacteria in Scampi Farms
Characterisation 146 enteric bacterial isolates from scampi farm revealed that Enterobacter (31.5%) was dominant flora. This was followed by Citrobacter (13.7%) and non enteric bacteria Aeromonas (11%). Other genera recovered include Serratia, Escherichia, Salmonella, Klebsiella, Shigella, Morganella, Plesiomonas and Providencia at varying proportions. Earlier studies also reported the recovery of genera belonging to the family Enterobacteriaceae and Aeromonadaceae as dominant flora in the larval rearing and grow out culture environments of M. rosenbergii (Anderson et al., 1989; Lalitha and Surendran, 2004, 2006). Al-Harbi and Uddin (2004) detected Salmonella, Shigella and other pathogens from Macrobrachium culture ponds from Saudi Arabia region. Higher incidence of Aeromonas is of health and safety concern as there were reports of recovery of enterotoxigenic strains of Aeromonas sp. from M. rosenbergii, M. malcomsonii and from aquatic environment (Rahim and Aziz, 1994).
Feeder canal water harbor diverse group of bacteria and as many as 9 genera were detected. Prawn and water samples carried seven enteric bacterial genera each, whereas in sediment only four genera were detected. Enterobacter was the dominant genera detected in prawn, sediment and feeder canal water. In pond water, Citrobacter was the dominant genera recovered (Table 4). Studies on enteric bacterial composition for prawn are scanty. Reports on composition of enteric bacteria on water samples and meat products revealed a similar dominance as observed in our study and the genera recovered were also similar (Ramteke et al., 1992; Jimenez et al., 2003). In the present study, Salmonella (4.5%) was recovered from prawn samples and pond water had Salmonella along with Shigella. Previous studies could recover Salmonella from sediment samples in addition to prawn (Surendran et al., 2000; Jeyasekaran and Ayyappan, 2002). Table 5 shows the status of enteric bacterial pollution during different phases of culture period. Higher density of enteric bacterial genera was observed in phase III followed by phase II and phase IV. Enterobacter dominated from phase II onwards until the last phase with a percentage contribution ranging from 29 to 50.6. Citrobacter was the predominant genera in phase I of culture.
Correlation between Water Quality and Microbiology of Fish and Shellfish Farms
The relationship between physico-chemical parameters and bacterial count attracted much of attention (Ogbondeminu and Adeniji, 1984; Ferguson et al., 1996). In this study, correlation between freshwater prawn pond microbiological quality and physico-chemical parameters was established using the two tailed Pearsons correlation coefficient (p<0.05 and 0.01).
Composition of enteric bacteria among different samples
Changes in the composition of enteric bacteria during different farming phase
Earlier study observed a positive or negative correlation between temperature, salinity and fish pond microbial quality (Sugita et al., 1985; Markosova and Jezek, 1994; Ferguson et al., 1996). However in this study, no consistent relationship could be observed between pond water temperature, pH and salinity with that of microbial quality of pond water and prawns.
TOC, BOD and COD indicate directly or indirectly the organic pollution status and a change in these parameters in water from pond and feeder canal water of scampi culture systems positively affected TPC and TEC (significant at p<0.05 and 0.01) of prawn and pond water. Occasionally in this system similar findings were observed for TSS and turbidity also. It was reported that organic matter helps with greater survival of aquatic bacteria (Gerba and McLeod, 1976) and increase in the level of above parameters lead to the significant increment in density of microbial load (Sugita et al., 1985; Ferguson et al., 1996; Surendraraj et al., 2009). No consistent relationship could be seen between DO and microbial parameters. Several authors reported a significantly positive correlation between TPC of pond water and fish (Ogbondeminu, 1993; Apun et al., 1999). In the present study, pond water microbial parameters TPC, TEC showed a significant positive correlation with fish TPC, TEC, FSC (p<0.01), TCC and SRC (p<0.05).
The TPC and enteric bacterial counts were detected in higher numbers especially in prawn samples and some of the members recovered in this study like Salmonella, Shigella, Hafnia and Klebsiella were established prawn/human pathogens. Further studies on the pathogenic potential/toxin producing ability need to be studied for Aeromonas and E. coli for establishing actual threat posed by these organisms. However, detection of diverse group of enteric bacteria including potential pathogens in scampi culture pond suggests that strict hygiene procedures and proper cooking prior to consumption is essential to safe guard the consumers. Good correlation between the bacterial population and the water quality variables like TOC, BOD, COD opens up an avenue for research in this line to establish a water quality and microbial quality model useful in assessing the quality and safety status of farm prawn.
The authors express their sincere thanks to Director, Central Institute of Fisheries Technology, Cochin, India for providing necessary facilities, support to carry out this study and for giving permission to publish this work. The financial aid as institutional fellowship from CIFE, Mumbai for Mr. R. Yathavamoorthi is greatly acknowledged.
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