Work on disease problems affecting aquaculture

                          Shrimp Viruses

         Bacterial disease of shrimp

      Development of immunostimulant for shrimp

      Development of molecular diagnostic methods

      Development of  Probiotics and Bioremediators for  aquaculture  

	Work on pathogenic Vibrio spp.
	Work on fish pathogens including Aeromonas hydophila
	Work on fish and shellfish toxicities and phytoplankton bloom
	Work on Rapid tests to detect seafood quality
	Work on Listeria monocytogenes

Southern hybridisation

 RFLP analysis

Dot Blot Hybridisation

     Under the DBT funded project, developed a highly efficient immunostimulant for shrimp. The  technology 

           has been transferred to industry and the product  sold under the brand name "AQUASTIM”. 

     Developed for the first  time in the country, a highly sensitive and specific diagnostic method based on

           Polymerase Chain Reaction (PCR) for rapid detection of  Whitespot Syndrome  Virus ( WSSV ) which is 

           affecting aquaculture industry in Asia. This technology has been  transferred to the industry on an

           commercial basis 

      Played a central role in dissemination of PCR diagnostic techniques in aquaculture sector in India.  Having 

           developed PCR diagnostic, trained officers of Marine Products Export Development Authority

            (MPEDA ) of Ministry of Commerce, Government of India. Helped MPEDA, Government of Andhra

            Pradesh, Government of Orissa to set up PCR laboratories.

      Help set up PCR laboratories in various shrimp hatcheries  

      Developed a highly sensitive rapid PCR based method for detection of pathogenic strains of  Vibrio

           parahaemolyticus in seafoods. Presently, in the process of  training MPEDA and Fish Control Officers in this

           technology. Filing of patent application in process.

      For the first time developed PCR based detection method for toxic dinoflagellates and their cysts, which

            cause fish kills and shellfish poisoning.  

      First in the country to document Paralytic shellfish toxicity and characterize the toxins  by high performance

            liquid chromatography (HPLC)

  The Department has been actively involved in identifying disease problems caused  by whitespot

  syndrome virus (Aquaculture:153, 9-13, 1997) and  Monodon  baculovirus (Indian J.virol.: 14,  27-30,

  1998) in India. A portion of the WSSV genome has been sequenced and based on this sequence, a nested

  polymerase chain reaction ( PCR ) method has been developed for rapid detection of this virus in shrimp.

  The nested PCR method has been evaluated extensively and compared with other methods. The results

  show that the PCR method developed by us is highly sensitive and specific. This method has been found to

  be very useful for detection of WSSV even when it is present in dormant state in larvae, broodstock and

  carrier animals  (Dis. Aquat.Org.: 38:67-70,1999). Presently the department has a facility to detect WSSV

  by PCR and nested PCR. Several hatcheries and shrimp farms are taking advantage of this PCR diagnostic

  facility. Using this PCR method, several species of wild shrimps such as Metapenaeus dobsoni, 

   Parapanaeopsis  stylifera, Solinocera indica, Squilla mantis,crabs such as Charybdis Metapograpsus

   messor  and other crustaceans have been demonstrated to be carriers of WSSV (Dis. Aquat.Org.: 38,

   67-70,1999; Aquaculture:198,1-11,2001; Fish Pathology:36,93-95,2001).

  An outbreak of disease in a shrimp hatchery has been studied and involvement of multiple viruses

  (WSSV, MBV and Hepatopancreatic parovirus ) has been demonstrated (Dis.Aquat.Org.: 48: 233

   -236). simultaneous presence of WSSV and MBV in shrimp larvae and post-larvae has been detected

   by PCR. The relation between crop success and PCR positivity of the animals has been studied. It has

   been demonstrated that animals, which are positive for WSSV in only nested PCR, could go through

   the normal culture if the environmental conditions are maintained favourable to the host.

    Vibrio species are one of the most important pathogens in brackish water and marine culture systems. The

  Department was the first to demonstrate that regular use of chloramphenicol in hatcheries could result in

  emergence of chloramphenicol resistance in Vibrio harveyi, which could cause mass mortality in Panaeus

  monodon larvae (Aquaculture: 128,203-209,1994). Ability of shrimp pathogenic Vibrio spp. to form biofilms

  on various surfaces such as concrete, plastic and steel has been reported (Aquaculture:140:241-245,1996).

  A Vibrio vaccine has been developed which improves survival of shrimp larvae by 20% (Fishing Chimes: 16,

  49 - 50,1996).

  To understand the disease problems in shrimp hatcheries and farms, the bacteriological status of hatcheries

   (J.Appl.Ichthyol:17, 59-63, 2001) and farms (J.Aquacul. Tropics.:14,309-318,1999) have been studied and

   conditions leading to enrichment of Vibrio spp. elucidated.

   SEMBV or whitespot disease virus has been causing major problems to shrimp aquaculture in India. 

   Therefore research has been focused on development on methods to overcome the viral disease problems

   and the strategy being, to increase host disease resistance. The effect of glucans and bacterins on the

   non -specific immune response of shrimp have been studied (Adv. Shrimp Biotechnol.:177-170,1998). Under

   a project funded by Department of Biotechnology, Government of India and Indian Council of Agricultural

   Research (ICAR), an immunostimulant with the brand name “Aquastim” was developed. Laboratory trails with

  this immunostimulant has shown to induce microbicidal activities in hemolymph and hemocytes and stimulate

  generation of reactive oxygen species in hemocytes and activate polyphenoloxidase system in Penaeus

  monodon. Field trails have also shown that  it is possible to manage WSSV using Aquastim. In a 15 ha. Farm

  in Goa, India, regular treatment with Aquastim helped shrimps to survive an attack of WSSV and brought

  about a survival rate of over 85% and a shrimp production of 2.6 tons per hectare.(Fishing Chimes,:17,7,1996).

   Detection of pathogens would be highly essential to diagnose diseases.  Conventional microbiological

  methods take 2-3 days  to confirm pathogens and histopathological methods are unsuitable for  quick

  diagnosis.  In the Department of Fishery  Microbiology  PCR based detection methods are being developed

  for pathogens.  A PCR protocol  for detection of V.parahaemolyticus in fish and  shrimp has been developed 

  ( Int. J.Food Microbiol. 31, 317-323,1995;  Dis. Asian Aquaculture III Flegel, T.W. et al (eds)  Asian  Fisheries

  Soc.,  1997,  119-122 ).  Genome characterisation  of  white  spot baculovirus  (WSBV)  is currently in

  progress to develop new  PCR primers  for this virus, in addition to the primers  described  in the earlier

  section.

    A PCR protocol for rapid detection of V.cholerae has been described (Mol.Biol.Biotechnol.:4,365-368, 1995),

  which would be extremely useful in determining seafood safety and by this method, toxigenic V.cholerae 01

  as well as 0139 can be detected (FAO Rep.:563,11-15,1997). PCR technology has also been extended for

  rapid detection of toxic dinoflagellates which cause problems in shrimp  farms  and areas  under 

  molluscan culture. Rapid detection  of  Gymnodinium mikimotoi in field samples by PCR has been

  demonstrated  (Marine Biotechnol. 3, 152-162, 2001)

   It has been realised that chemotherapeutic agents are not useful for control of pathogens in hatcheries and

  shrimp ponds. Naturally occurring microorganisms which are antagonistic to pathogens for use as biocontrol

  agents or probiotics. In this context, a Pseudomonas strain has been found to have antivibrio activity and

  this organism has potential for use as biocontrol agents or probiotics. In this context, a  strain has been found

  to have antivibrio activity and this organism has potential for use as  a biocontrol agent.   

  Most disease problems in aquaculture are due to environmental deterioration. We have been  on the

  development of microbial consortia for bioremediation of aquaculture ponds ( Aquaculture International, 8,

  463-472, 2000 ). Microcosm studies have shown that the consortia developed in our laboratory can clean up

  the blackened shrimp pond sediment and reduce levels of ammonia and hydrogen sulphide.

  The  incidence of hemolytic strains of V.  parahaemolyticus along  the  coast  of India was documented

  for  the  first  time (Indian  J. Med. Res. 72, 619,1980).  A comparative study  of the methods  for  detection

  of V. parahaemolyticus  in  seafoods  was published (Appl. Environ. Microbiol. 52, 583, 1986) and  it   was 

  shown  that  prawn processors along the coast of Karnataka  are able  to meet specifications of the

  International  Commission  on Microbiological Specifications  for Foods  (Can. J. Microbiol. 30, 713, 1984).  A

  chemically defined medium for the production of hemolysins was developed for the first  time  (Appl. Environ.

  Microbiol. 41, 1274, 1981).  It was shown that V. parahaemolyticus can  multiply  in seafoods stored at

  temperature more  than  10°C (Harvest  &  Post-harvest Technology of Fish,  1982).  At lower temperatures, 

  the protective effect of chitin  was  demonstrated (Can.  J. Microbiol. 32, 889,  1986).  It was shown that 

  survival of  V.parahaemolyticus  in dry fishes is for short time  (J.  Food Sci. Tech.  21, 235,1984) but in

  cold smoked fishes, even multiplication  could occur (Ant. Van Luen. 52, 145,  1986).  The role  of hemolysin

  in virulence was demonstrated to be as a source of iron (Infect.  Immun. 46,141, 1984).  Survival of  

  V.parahaemolyticus in the  environment did not depend on ability to produce  hemolysins (Syst.

  Appl.Microbiol. 9, 316,1987) and presence of plasmids did not correlate  with  hemolytic activity (Indian  J.

  Med. Res. 86, 711,  1987) and spontaneous mutation to 0/129  resistance  was demonstrated  (Curr. Sci.

  56, 773, 1987). Selection of  hemolytic strains by mouse passage and existence of heat stable  hemolysin

  immunologically different from TSDH of Vibrio  parahaemolyticus was demonstrated (Indian J. Med. Res. 89,

  386, 1989).  Effect  of chemical preservatives, chlorination on survival of V. parahaemolyticus  in seafoods

  was reported (First Indian  Fisheries  forum 1988).  

  Incidence of Vibrio vulnificus in Indian waters was  reported for  the first time (Indian J. Mar.Sci.16,136,

  1987) and  presence of  non  01 V. cholerae in sea water and  freshly  harvested  fish demonstrated (Asian

  Fish. Sci. 2, 121, 1988).  Virulence of non 01 V.cholerae   was  demonstrated to be  multifactorial  (In‚  

  Quality Assurance  in  Fish  industry, Elsevier Sci.  Publ.  211,  1992 ). Presence  of V. cholerae  and other

  pathogenic marine vibrios  was demonstrated  in  marine sediments (FAO Fish.  Rep.  416,  1993). Some 

  non-hemolytic  strains of V. vulnificus  were  found to  be virulent  (Proc. Second Asian Fish Forum,

  Manila, 931,1990)  and hemolytic  marine vibrios were demonstrated in seafood  harvested along  the  coast

  of Karnataka (FAO Fish Rep.  401,  53,   1990).  Importance  of phosphates for production of hemolysins

  was  documented (Indian  J.  Microbiol. 30, 327, 1990). A review of the methods  to  detect Vibrio  spp

  hazard in  seafood  was  prepared (Proceedings FAO/DANIDA expert consultation).  

  The  presence of  Vibrio  hollisae  in seafoods  was  demonstrated. Hemolysins produced by a virulent

  strain of V.hollisae  were  purified.   Two protein toxins with molecular weights 64 kD and 49 kD with  lethal

  activity against mice have been  detected.  Antisera have been raised against Vibrio hemolysins to screen

  environmental strains  of  vibrios by western blot reaction for productions of toxins.

    Ulcerative  infection of Indian major carps (Curr.  Sci.  55, 1194,  1986)  and septicaemia in cultured fishes

  (Curr.  Sci.  58, 1044,  1989)  were reported.  Virulence associated  characters  of  pond  associated  and fish

  associated strains  were  compared  (In‚ Recent  Advances in Biotechnology and Applied Biology, Hong  Kong,

  205, 1988).  The incidence of A. hydrophila,  A.sobria and  A.caviae in  tropical  aquaculture  ponds was

  reported  and  the  virulence associated  characters  of  these  natural  strains  investigated. Various factors 

  influencing  the invasion of  fish  by  virulent strains of A.hydrophila have been studied (I. Aquacult. tropics

  10, 29-35, 1995).  The relation between virulence and phenotypic  characters  such as surface characters,

  ability  to  overcome bactericidal action of serum, ability to produce various  enzymes, hemagglutinins  have 

  been investigated.  A vaccine  was  prepared from virulent strains and  immunological protection was

  demonstrated (J. Fish Dis. 14, 413, 1991).  Role of thymus in immunological response  was  demonstrated (J.

  Aquacult. in tropics  11,  79-82, 1996)  A vaccine has been developed (Dev. Biol.  Standardisation, 70, 9-13,

  1997) and evaluated.  Field trials  indicate significant improvements in survival of vaccinated fish. A detailed

  study on the immunomodulatory effects of various chemicals used in aquaculture has  been carried out (In

  Dis. Asian Aqua. III, 187-191,1997).

  Virulence characters of A. hydrophila associated with epizootic ulcerative  syndrome  (EUS) was studied.

  Diversity  in  virulence characters  suggested  secondary nature of A. hydrophila  in EUS (Karunasagar  et  al, 

  Dis Asian Aquacult. 1995,  Karunasagar  and Karunasagar, Proc. ODA Regional  seminar,  Bangkok  158 - 170,

  1994).

  Strains  of  A.hydrophila  have been shown  to  be  important agents of spoilage in fresh water fish like

  Labeo rohita.   These strains  of  A. hydrophila  were found to be  psychrotrophic  and capable of  producing

  hydrogen sulphide from sulphur  containing  amino acids (Fish Res 13, 189 - 197, 1992). Aphanomyces

  strains associated with EUS have been  isolated.   

  Molecular epidemiological studies have been conducted on Aphanomyces  strains isolated from different

  geographical areas using polymerase chain reaction based Random Amplification of Polymorphic DNA (RAPD). 

  Such studies indicate genetic diversity among isolates from different regions.

  Work on fish and shellfish toxicities and phytoplankton blooms

  For the first time in the country, the incidence of Paralytic Shellfish  Poisoning (PSP) was demonstrated

  (Curr. Sci. 53,  247, 1984). Due to prompt analysis of toxins and reporting in newspapers,  several  lives were

  saved.  First record  of  diarrhetic shellfish toxin producing dinoflagellates was made from India (In‚ Red Tides,

   Elsevier Sci. Publ. 59, 1989) and the PSP toxins was analysed by HPLC. It was shown that toxins in clams and

  mussels involved in the outbreak were predominated by Gonyautoxin 2,3,8, epigonyautoxin 8 with small

  amounts of  saxitoxin  and neosaxitoxin (Toxicon 28, 868, 1990).  Diarrhetic  toxins  in shellfishes were

  demonstrated to be well below limits of safety (Proc. First Indian Fish. Forum 389, 1988).  For the first time, 

  Gymnodinium  nagasakienase red tide and fish kills were reported  from the  coast of India (J. Shellfish

  Res. 12, 312, 1992). Presence of Gymnodinium catenatum in waters along west coast of India  has been  

  reported (UNESCO Harmful Algal News 15, 1, 1996). A second outbreak of PSP from India has been recorded

  (UNESCO Harmful Algal News,17, 1, 1998). Rapid  PCR based detection of Gymnodinium mikimotoi from

  waters has been demonstrated  (Marine Biotechnol. 3, 152-162,2001). The role of bacteria in toxin production

  by the dinoflagellate  Amphidinium cartarae has  been  elucidated  ( World J. Microbiol. Biotechnol. 16,

  99-101, 2000 ).

    Limulus lysate test was found to be good for rapid determination  of bacterial numbers in iced fish (Ind. J.

   Microbiol.  29, 377,  1989) and resazurin strip test was found to be  useful  for shrimp (Asian  Fish  Sci.  3,

   263, 1990)  and  fish  (Fish  Res. 9,75,1990).   A review on harvest and post harvest  microbiology  of  fish 

   was  published (Indian J. Microbiol.  31,  211,  1991).  Chilled sea water preservation was found to alter

   microbial flora of  shrimp  (J.Food  Sci. Technol. 23, 148, 1986)  and  protease inhibitors altered

   microbial and biochemical quality  of  shrimp (J. Food Agric.  36, 1113, 1985; Indian J.  Microbiol.  28,

   82, 1988) and the factor active in the fraction was purified (J. Food Sci. Technol. 26, 283, 1989). 

   Presence  of histamine in seafoods and  histidine decarboxylating bacteria in seafoods and 

   environs demonstrated  (Indian J. Microbiol.32, 75, 1992;FAO  Fish  Rep. 401, 112, 1990, Food

   Microbiol, 1, 263, 1984, FAO Fish Rep. 317, 363, 1985).

   Presence of Listeria in seafoods was demonstrated (Asian Fish Sci.  4, 119, 1991 Int. J. Food Microbiol. 31,

   330-340, 1996) and genetics of L. ivanovii virulence elucidated (Infect. Immun. 61, 162, 1993). Mechanism

   of L. monocytogenes entry into  intestinal  epithelial  cells  were demonstrated (Infect. Immun. 62,  3556 -

   3558, 1994).  Application of molecular tests such as polymerase chain reaction (PCR) for identification of

   Listeria spp. associated  with seafoods has been reported  (Int. I. Food Microbiol,  31, 333-340,  1996). By

   gene complementation, it  has  been shown  that L.seeligeri can escape from  phagosomal  compartment of 

   cells  if complemented with plc A - prf A genes from  L.monocytogenes (FEMS Microbiol Letter, 146, 303

   -310, 1997).  

College of Fisheries, Mangalore -575 002, Karnataka, India

Telephone : 2246384 , 2249256 ; Fax : 2246384 , 224836

E-mail : mircen@sancharnet.in

Last update : August,  2006

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