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Distribution of Vibrio parahaemolyticus in Farmed shrimp Penaeus vannamei, Farm Water and sediment

the halophilic marine bacterium Vibrio parahaemolyticus is a zoonotic pathogen associated with wildcaught and farmed shrimp. The bacterium is an important cause of gastroenteritis associated with the consumption of raw or undercooked seafood. In the present study, the prevalence and human pathogenic potential of Vibrio parahaemolyticus in Penaeus vannamei (tissue and hepatopancreas) and the farm environment (water and sediment) was investigated by conventional culture and molecular techniques. the total Vibrio counts of P. vannamei ranged from <1 CFU/mL in hemolymph to 7.61 log CFU/g in the hepatopancreas. The sediment samples consistently showed the counts of 6-7 log CFU/g, while the pond water had Vibrio counts in the range of 2-3 log CFU/ml. Of 120 Vibrio isolates identified, 87 were confirmed as V. parahaemolyticus based on the toxR and tlh gene-specific PCR. The virulence marker gene tdh was not detected in any of the isolates, while the trh gene was detected in 3 (3.6%) isolates. Although the incidence of pathogenic V. parahaemolyticus in farmed P. vannamei is low, the high numbers of total vibrios and V. parahaemolyticus demand constant monitoring of animals and the farm environment for human pathogenic strains of V. parahaemolyticus.


INTRODUCTION
Among various pathogenic vibrios, Vibrio parahaemolyticus is frequently associated with seafood-borne human gastroenteritis 1 . Consumption of raw or undercooked shellfish is generally responsible for human infections by V. parahaemolyticus. However, all strains of V. parahaemolyticus are not pathogenic. The strains that produce a thermostable direct hemolysin (TDH) or a TDH-related hemolysin (TRH) are considered pathogenic to humans 2 . The TDHproducing V. parahaemolyticus, which comprises less than 1% of the strains of V. parahaemolyticus, is more frequently involved in human infections. Isolates producing TRH alone were first reported to be involved in the outbreak of human gastroenteritis from a patient in the Republic of Maldives 3 . Studies have shown that TRH-positive isolates are more abundant, sometimes as high as 15% of the total V. parahaemolyticus, and usually cause gastrointestinal infections which are less severe in nature 4,5 . Nevertheless, both TDH-and TRH-positive V. parahaemolyticus are considered human pathogens 5 . Infections with V. parahaemolyticus usually occur during warm summer months in temperate countries. However, in tropical coastal waters with a more or less stable temperature throughout the year, V. parahaemolyticus numbers in seafood are temperature independent but are influenced by fluctuations in salinity 6 . Infections generally occur when seafood containing 10 7 -10 8 CFU/ ml V. parahaemolyticus is consumed 7 . Several studies have reported the occurrence of TDHand TRH-positive V. parahaemolyticus from seafood in India 6,8-12 . Apart from gastroenteritis, V. parahaemolyticus can cause wound infections 13 . V. parahaemolyticus is also a pathogen of fish and shellfish, and has been associated with disease in milkfish (Chanos chanos) in the Philippines 14 , farmed sea bass (Dicentrarchus labrax) in Tunisia 15 , red disease in Penaeus monodon in India 16 . The significance of V. parahaemolyticus as a serious pathogen of farmed shrimp was realized with the establishment of its association with the acute hepatopancreatic necrosis disease AHPND in shrimp 17,18 . With the association of V. parahaemolyticus with diseases in farmed fish and shrimp, the bacterium has been recognized as a zoonotic human pathogen. Considering the human health threat this bacterium poses, it is important to routinely monitor the prevalence of V. parahaemolyticus in farmed shrimp as part of risk assessment studies. In this context, the study was designed to understand the prevalence of total and human pathogenic V. parahaemolyticus in shrimp farm environments on the West Coast in India.

Sample collection and preparation
Twenty-one samples comprising of shrimp (9), water (6), and sediment (6) analyzed in this study were collected from three shrimp (P. vannamei) aquaculture farms in Saphale (19.5708° N, 72.8309° E) and Dahanu (19.9903° N, 72.7397° E) in Palghar district of Maharashtra, India. Fresh shrimp samples were collected directly from the ponds in sampling bags and immediately transported to the laboratory in chilled condition. The water and sediment samples of the ponds were collected from three different locations within a pond and pooled. The total Vibrio counts of shrimp hemolymph, tissue, hepatopancreas, as well as water and sediment samples of the respective ponds were analyzed following standard methods 19 , with some modifications. The samples were surface plated directly on selective agar plates for the quantification of presumptive V. parahaemolyticus without the selective enrichment. The salinity and water temperature were measured before the collection of samples. shrimp samples Different parts of the shrimp i.e., hepatopancreas, tissue, and hemolymph were processed for the isolation and quantification of V. parahaemolyticus. The hemolymph was collected from three live animals from a single pond using sterile syringes and pooled. From this, 0.1 mL was directly surface plated on thiosulphate citrate bile salt sucrose (TCBS) agar. Tissue and hepatopancreas were sampled from three different shrimps from the same pond and pooled and one gram of the pooled sample was processed further. Briefly, one gram of the hepatopancreas or the tissue was homogenized in 9 mL of sterile saline solution (2% NaCl w/v), serially 10-fold diluted in saline, and 0.1 mL of the dilutions was spread plated on TCBS agar plates in duplicate.

Water and sediment samples
In the case of water samples, 0.1 mL was spread plated directly on TCBS agar plates. The sediment sample (10 g) was mixed with 90 ml of 2% (w/v) saline, vortexed for 2 min, and allowed to settle for 5 min. The supernatant was 10-fold serially diluted in 2% saline and 0.1 mL from each dilution was spread plated on TCBS agar plates 20 .
The inoculated plates were incubated at 35°C for 18-24 h. Green colonies typical of V. parahaemolyticus, 3-5 from each TCBS plate, were picked and streaked on Luria Bertani agar plates containing 2% NaCl and incubated for 24 h at 35°C. The bacterial isolates were subjected to presumptive identification of Vibrio spp. using biochemical reactions such as the production of cytochrome oxidase, catalase, resistance to 10 µg concentration of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropyl pteridine), growth at different concentrations of salt (0, 3, 6, 8 and 11% w/v NaCl) and glucose fermentation tests.

Molecular characterization
Vibrio isolates from the samples were subjected to V. parahaemolyticus species-specific PCR targeting the toxR and tlh genes. To detect pathogenic strains of V. parahaemolyticus, the isolates were subjected to PCR specific for the thermostable direct hemolysin gene (tdh) and the TDH-related hemolysin gene (trh) ( Table  1). For preparation of DNA for PCR, a single bacterial colony was suspended in 200 µl 1×TE (10 mM Tris pH 8.0; 1mM EDTA) buffer in a 1.5 mL microcentrifuge tube and vortexed. The bacterial suspension was lysed by placing the tube in a dry bath at 98˚C for 10 min, followed by placing it on ice for 5 minutes. The lysate was centrifuged at 10,000 rpm for 2 min and the resultant supernatant was used as the DNA template in PCR.
PCR was performed in 30 µl reaction volumes consisting of 3 µl of 10X buffer (DSS Takara Bio India Pvt. Ltd.), 200 µM concentrations of each of the four dNTPs, 10 pico moles of each of forward and reverse primers, and 1 U of Taq DNA polymerase (DSS Takara Bio India Pvt. Ltd.) and 3 µl of template DNA. The amplifications were performed in SimpliAmp™ thermal cycler (Thermo Fisher Scientific, USA). V. parahaemolyticus AQ 4037 (trh + ) and SY O3:K6 (tdh + ) were used as the positive controls, while sterile distilled water was used as the negative control. The thermocycling conditions for the amplification of target genes (toxR, tlh, trh and tdh) were essentially the same as previously described 21,22 . The PCR amplicons obtained were resolved on 1.5% agarose gels by electrophoresis. The ethidium bromide-stained gels were then visualized and photographed using a gel documentation system (UVP, CA, USA).

RESULTS AND DISCUSSION Isolation and characterization of V. parahaemolyticus
The presence of pathogenic and nonpathogenic strains of V. parahaemolyticus has been reported from wild-caught seafood in India 23-25 . Although V. parahaemolyticus has been considered as an important human pathogen, the bacterium is also an important shrimp pathogen. Studies have reported V. parahaemolyticus infections of P. monodon and P. vannamei at various life stages, from post-larvae to adult shrimps 26,27 . The zoonotic potential of V. parahaemolyticus makes it an important pathogen of public health significance as well. V. parahaemolyticus in shrimp Twenty-one samples collected from three different shrimp farms were analyzed for the presence of V. parahaemolyticus. A total of 120 isolates, identified as Vibrio spp. by biochemical tests, were recovered from the samples. Of these, 31 isolates were from shrimp tissue, 59 were from shrimp hepatopancreas, and 15 each were from water and sediment samples (Table 2). These isolates were subjected to V. parahaemolyticusspecific PCR targeting toxR and tlh genes. Of 120 isolates, 87 (72.5%) isolates were confirmed as V. parahaemolyticus by PCR (Fig. 1A & 1B). The difference in the nucleotide sequences of toxR gene among different Vibrio spp. has been utilized to develop species-specific primers 21 . The toxR specific PCR has been used by several investigators to identify V. parahaemolyticus isolated from seafood 6,31,32 . The prevalence studies of V. parahaemolyticus from India reported 89% in seafood in general 33 and 80% in shrimps 34 .
Studies on farmed shrimp and the   In our study, the Vibrio counts in animal tissues (muscle and hepatopancreas) were in the range of 2-4 log CFU/g, while the counts in the sediment samples were uniformly around 6-7 log CFU/g (Table 3). In shrimp farm water samples, the counts ranged from 2.48-3.59 log CFU/ml. The water temperature and salinity of ponds sampled in this study did not vary significantly throughout the study period of three months. The salinity was in the range of 22-23 ppt, and the temperature was in the range of 27-29°C (Table 3). Therefore, salinity and temperature presumably did not influence Vibrio populations of shrimp ponds investigated in this study. In general, the Vibrio counts of the sediment samples were higher than the counts in tissues or the water samples (Table  3). The higher surface area of sediment particles and the associated nutrients might support higher bacteria populations in pond sediments compared to water. In our study, the counts from the shrimp sample were higher than counts from the farm environment. Higher Vibrio counts in sediments compared to the water have been reported by other investigators 41,42 . In an elegant study mimicking shrimp farm environment, Zheng et al 43 showed significant dissimilarities in microbial composition and diversity of water, sediment, and shrimp intestine. The microbial communities varied during different stages of shrimp culture. However, the changes in shrimp gut microbial communities did not resemble the changes in the surrounding environment 43 . Differences in water, sediment, and shrimp gut Vibrio counts have been reported by several investigators 44-46 . While physicochemical parameters such as temperature, salinity, pH, and nutrients greatly influence Vibrio counts in the shrimp farm water and sediment, additional factors such as the health and immunity of shrimp, diet, pond application of probiotics, etc. determine the gut microbial communities of farmed shrimp 46 .

Molecular characterization of V. parahaemolyticus
The main goal of this study was to understand the prevalence of human pathogenic V. parahaemolyticus in P. vannamei farm environment. The toxR positive isolates were further screened for the virulence genes trh and tdh. None of the isolates harbored the tdh gene, while the trh gene was detected in 2 isolates from shrimp hepatopancreas and 1 isolate from the pond sediment (Fig. 1 C). These isolates corresponded to two separate samples of shrimp and sediment. Thus, the incidence of trh + V. parahaemolyticus in this study was 22.2% of shrimp samples and 16.6% of sediment samples. The tdh gene, which encodes a thermostable