Aquatic Animal Health and Environment Division

Salient Research Achievements
  • Epidemiology

Viral and bacterial diseases 

  • The impact of diseases in brackishwater cultured shrimp on economic loss in the states of Odisha and West Bengal along the East Coast and Goa and Karnataka along the West Coast were studied. Studies conducted during 2005-07 revealed that the national loss of shrimp production was estimated to be 48717 metric tonnes of product and the economic loss due to diseases was estimated to the tune of Rs. 1022.13 crore per year.
  • The disease incidence among the shrimp farming was higher in traditional culture than shrimp monoculture system. Incidence of white spot disease was found to be 11.8% among the zero water exchange shrimp monoculture system ofWest Bengal followed by Vibriosis (3.9%).
  • Lack of biosecurity and use of PCR untested seeds were found to be responsible for more WSD incidence in shrimp culture in West Bengal.
  • An investigation on the mass mortality of L. vannamei in shrimp farms of 45-80 DOC in Nellore district (AP) during May, 2011 found to be positive for WSSV by nested PCR and YHV by OIE Real Time PCR protocol-1 for specific detection of YHV amplifying 135 bp product
  • Protocols for enumeration of viruses in brackishwater ponds were standardized by epifluorescence microscopy. The viral counts in brackishwater samples ranged from 1.6-4.9 x 107ml-1 in shrimp culture ponds, 1.3-1.5 x 105ml-1 in shrimp hatcheries and 1.8-1.3-1.5 x 105ml-1 in creeks and 3.3-4.2 x 109g-1 in sediments.
  • Laboratory experiments proved that WSSV could be transmitted to shrimp from infected crabs by cohabitation and oral feeding. Experiments to prove the hypotheses of vertical transmission of WSSV in female Scylla spp. revealed low level (8.7x101- 5.63x102 copy numbers g -1 tissue) viral detection in ovarian tissues by qRT-PCR.
  • Luminescent bacterial (LB) disease is one of the major bacterial diseases and has been often responsible for shutting down of shrimp hatcheries, resulting in huge economic losses. From 1195 hatchery samples processed, 256 distinct isolates of V. harveyi were obtained. Shrimp brooders, maturation and spawning tanks are the main sources of luminescent bacterial disease.
  • Molecular epidemiology of betanodavirus infection causing viral nervous necrosis (VNN) in seabass and other fish revealed that majority of infection in fish as sub clinical (18.5%); Clinical and sub clinical cases in farms, only sub clinical cases in wild. Three cases of fish has been found infected with mixed infection of viruses ie. betanodavirus and iridovirus with clinical symptoms among seabass and milk fish.
  • Asymptomatic iridovirus infection (42%) has been detected by histopathology, electron microscopy and molecular methods in brackishwater and marine fishes from Kerala, Tamil Nadu, Andhra Pradesh, West Bengal and Puducherry. Majority of the cases could be detected only by nested PCR indicating a very low viral load due to possible sub-clinical infections. However, Iridovirus was not a significant problem in cultured farm fish as against the ubiquitous presence in many wild aquatic organisms.

Parasitic diseases

  • Studies conducted on myxozoan parasites infecting brackishwater and marine fishes in coastal waters revealed a variety of myxozoan species. Prevalence of myxozoan parasites in brackishwater / marine fishes ranged from 6 to 37% in various fish species. Kudoa spp., Myxobolus spp., Myxidium spp. and Ceratomyxa spp. The myxozoans infecting brackishwater fishes exhibited some degree of host- and tissue-specificity, despite the co-habitation of their hosts.
  • Parasite associated mortality continued to be a concern for the maintenance of finfish in confined water body. Monogeneans were found to cause heavy mortality in larval and fingerlings stages of finfishes. The most common monogeneans in cultured fish are Gyrodactylus sp., Dactylogyrus sp., Pseudorhabdosynochus sp. and Diplectanumsp. in seabass, mullet, pearlspot etc. while some capsalid (Benedenia sp.) parasites are large and were recorded as important pathogens of marine fish (Epinephelus tauvina) eliciting large wounds on the skin or eyes.
  • One important consideration in designing an effective treatment plan is to develop strategic therapeutic and management interventions based on the biology of the parasite. Knowledge on whether the monogenean is viviparous or oviparous help as eggs of some monogeneans is not susceptible to treatment and thus several drug applications may be required for control. Fish to fish transmission can be reduced significantly by avoiding overcrowding in case of viviparous monogenean, where offspring remain on the same host as compared to oviparous monogenean, where a free-swimming larva may attach to the host actively. Further many monogenean species cannot survive for more than 2 weeks off a host giving an option for transferring to fresh tanks in case the number of stock is limited (for eg. broodstock).
  • Studies on brackishwater aquaculture system revealed that three major groups of parasitic crustaceans; Branchiura, Copepoda and Isopoda. Most important ectoparasitic species being; Caligus sp., Ergasilus sp., Lernanthropus sp., andCymothoa sp. among seabass, grouper, mullet and pearlspot. Rarely Lernaea and Argulus in fish species cultured in freshwater are also encountered.
  • Neoplastic diseases
  • Neoplasms or tumours often reported in finfish culture system were also monitored for in-depth studies. A case of reddish polyp like tumour-like formation on the peduncle region of a broodstock grey mullet revealed that the tumourincreased in size with time and histologically fleshy growth was characterized as squamous cell carcinoma with unknown etiology. The tumor formations were found to be benign, as evidenced by the absence of metastases to other organs.
  • A case of rhabdomyoma in milk fish in polyculture system was diagnosed. The neoplastic cells are long and spindle shaped with oval or oblong nuclei. They are arranged in whorls. Fibrous tissue septa divided the muscle bundles into lobules with well formed blood vessels with no evidences of metastasis in vital organs.

Miscellaneous

  • Based on bioassay experiments and histopathological investigations, etiological agent for Loose Shell Syndrome (LSS) in shrimp was found to be a filterable viral-like particle.
  • Monodon Slow Growth Syndrome (MSGS) reported in tiger shrimp was studied and the samples were found positive for Laem Singh Virus (LSNV) by nested and Real Time PCR. Retinopathy was observed in histological sections of eyestalk from affected shrimp. Viral metagenomics involving viral purification and shotgun sequencing was deployed to decipher the Laem Singh virus (LSNV) genome, associated with monodon slow growth syndrome (MSGS) in tiger shrimp.
  • A survey was conducted to find out the loss encountered by shrimp farmers due to different diseases in shrimp. Currently, a project has been undertaken to survey the disease occurrence in Pacific white shrimp in culture ponds and hatcheries, particularly to study the effect of existing pathogens on the species and also to monitor the occurrence of transboundary diseases.

Diagnostics

Commercialisation

  • A nested PCR kit for the detection of WSSV in shrimp was developed commercialized in 2002 to M/s Bangalore Genei,Bangalore. This kit is economical and sensitive to detect 10 copies of the virus.

Development of kits

  • rtPCR (nested) diagnostic kit was developed for the early diagnosis of white muscle disease in the postlarvae of scampi, M. rosenbergii. For the first time in India, the total RNA extracted from viral pathogens isolated from M. rosenbergii affected by white tail disease has been sequenced.
  • Developed and validated the nested RT-PCR based diagnostic assay and an IPR enabled prototype kit for screening clinical and sub clinical infection of betanodavirus in fish.
  • An improved diagnostic nested RT-PCR with custom designed primers targeting RdRp gene of Laem-Singh virus (LSNV) was developed.

Therapeutics/Preventive measures

  • dsRNA therapy for WSD and MBV were developed. By the use of dsRNA, it was possible to control WSSV in tiger shrimp by targeting both the structural and non-structural genes of the virus. Partial sequences of WSSV genes viz., vp28, vp281, W230 and W474 were amplified and cloned in pLitmus28i vector. Corresponding dsRNAs were synthesized by in vitro transcription and injected in P. monodon experimentally infected with WSSV and the efficacy of dsRNA as antiviral therapeutic against WSSV was established.
  • A large number of bacteriophages from Vibrio sp as hosts were collected and tested their ability to control vibriosis of shrimp larvae. Laboratory trial provided promising results in controlling the luminescent and other vibrios. Prospects for up-scaling this technique and development of bacteriophage therapy for the control of luminescent bacterial disease in shrimp hatchery is being explored.
  • Use of medicinal plant Withania somnifera through feed was evaluated as an immunomodulation therapy. Experiments to study immunomodulatory effect of W. somnifera in feed @0.5- 0.75g/kg has potential as prophylactic agent againstV. harveyi and WSSV infection in shrimp.
  • The baits developed with different WSSV proteins (VP28 and VP 15) in yeast two hybrid experiment were found to give protective effect against WSSV infection when fed to shrimp larvae.
  • In order to develop bacterial vaccines for Asian seabass against Vibrio anguillarum, outer membrane protein (OMP) has been isolated from pathogenic V. anguillarum and being tested as a potential vaccine candidate against V. anguillarum infection in seabass. The OMP K was found to be the major expressed protein on the surface of V. anguillarum. The OMP K was amplified from pathogenic V. anguillarum, cloned and expressed in vitro.
  • The survival rate of the chloramphenicol, furazolidone and ciprofloxacin treated P. monodon seed during transport were found to be significantly more (P<0.05) than untreated shrimp seed.

Genomic investigations

  • Viral resistant genes of P. monodon and Fenneropenaeus indicus were amplified and characterized.  A cDNA library ofP. monodon has been constructed and probed for identification of disease resistant genes.
  • Differences in occurrence of WSSV genotypes with respect to variable number tandem repeats (VNTRs) across different geographical locations and difference between genotypes of wild and cultured crab and shrimp species has been documented.
  • Major shrimp allergen (tropomyosin) gene was cloned from P. monodon and expressed in pRSET A T7 expression system. Antimicrobial gene (crustin) and caspase gene, which is a key enzyme in apoptosis, were cloned into pGEM T cloning vector (Promega).
  • The following important genes which play key role in shrimp metabolism; Chitin, β- actin, ESSS subunit of NADH, Ubiquinone oxidoreductase (complex I), NADH dehydrogenase subunit 5 C-terminus and C-type lectin 2 were sequenced.
  • Full length/near full length amplification of betanodavirus RNA2 from four strains of virus samples revealed that the (near) full length RNA2 sequence was 1434 bp in length containing a single ORF spanning across nt 27-nt 1043, coding for viral coat protein of 338 aminoacids.
  • The molecular phylogenetic analysis of the genotype of betanodavirus revealed complete homogeneity of the capsid protein gene to that of seven band grouper nervous necrosis virus strain SGWak97 (Acc. No. AY324870). This finding confirms the existence of RGNNV genotypes known to infect warm water finfish in India.

Cell culture system for research on shrimp virus

  • Studies to test the susceptibility of primary hemolymph and ovarian cell culture systems to white spot syndrome virus (WSSV) revealed cytopathic effects (CPE) after 2-3 days post inoculation. CPE became more pronounced by third passage in hemocytes culture and by fifth passage in ovarian cultures with corresponding increase in viral load by qPCR. The study showed primary hemolymph and ovarian cell cultures are useful for in vitro replication of WSSV.

Studies for virus characterization and development of anti-viral therapy

  • Using the Yeast Two-hybrid technology, research is undertaken to screen host interacting proteins against specific WSSV proteins (Vp15 and Vp28). From this research it is expected to understand the virulence mechanism as well as the entry of the virus into the host.

Agriculturally important microbes

  • A total of 620 isolates of microbes were isolated from different geographic environments of brackishwater eco-system, which comprises 329 bacteria, 66 actinomycetes, 55 fungi 21 yeast isolates and 7 Archae bacterium.
  • Agriculturally and commercially important enzyme like xylanase, pectinase, agarase, cellulase, chitinase, protease, and lipase producing bacteria were isolated, identified and characterized.
  • One hundred microbes from different environments have been isolated and the collection has been deposited to National Bureau of Agriculturally Important Microorganisms, Mau.
  • Under the programme on screening of agriculturally important microbes from brackishwater ecosystem, for novel genes, Isochorismate Isomerase gene, responsible for encoding proteins for resistance against a variety of pathogenic bacteria, was amplified from Vibrio alginolyticus and cloned into pET32A vector system and transformed into E. coliDH5α. Over expression by IPTG induction was done by transforming the plasmid into BL21 E.coli expression host and confirmed by SDS PAGE.
  • Gene for Azurin which has antiretroviral and antimalarial activities and also exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells was amplified from Vibrio alginolyticus. Azurin gene was over expressed by IPTG induction which was confirmed using SDS PAGE. 70% of phage M13 inactivation was observed at a dose of 10μg /ml of recombinant azurin.
  • Studies on microbial diversity revealed 18 isolates V. alginolyticus from various brackishwater ecosystems and confirmed using 16s rRNA gene-specific primer. Molecular typing of these isolates was carried out using ERIC PCR which yielded products with the size range of 400bp to 2.5kb. The isolates could be separated into five clusters based on geographic location with three major clusters at 40% hierarchical level on phylogenetic analysis.
  • Twenty three ammonia oxidizing autotrophic bacteria and 63 sulfide oxidizing autotrophic bacteria have been isolated from shrimp farms and are being characterized.
  • Eight aerobic denitrifying bacteria have been isolated, characterized and identified based on their activity and presence of denitrification genes (nirS, nirK and nosZ genes).
  • In order to develop bioremediation techniques to treat discharge water from shrimp farms, eight denitrifying isolates were confirmed to carry out denitrification under both oxygen tolerant and oxygen limited conditions through reverse transcriptase-PCR for nosZ gene.
  • Lab-scale biofilters using bioballs enriched with chemolithotrophic AOB, NOB and denitrifying bacteria, and filamentous sulphur oxidising bacteria were found to be successful in maintaining ammonia, nitrite, and sulfide levels below detectable level.  

Scientific Staff list

Dr.S.V.Alavandi, 
Principal Scientist & Head 
Email: 
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Phone : +91- 044 - 24618817Ext-208

 

Dr.K.P Jithendran, Principal Scientist

Dr.M.Muralidhar, Principal Scientist

Dr.Ms. M.Poornima,Principal Scientist 

Dr.Ms.R.Saraswathy, Principal Scientist 

Dr. P.K.Patil, Principal Scientist  

Dr. S.K. Otta, Senior Scientist

Dr. Sanjoy Das, Principal Scientist

Dr. R.Ananda Raja, Scientist

Dr. Sujeet Kumar, Scientist

Dr.Ms. Ezhil Praveena, Scientist

Dr.Ms. T.Bhuvaneswari, Scientist

Dr.Ms. N. Lalitha, Scientist

Shri P. Kumararaja, Scientist

Dr. Satheesha Avunje, Scientist 

Smt. R. Mary Lini, Scientist

Ms. Vidya Rajendran, Scientist

Shri. T. Sathish Kumar, Scientist

Ms. Suvana Sukumaran, Scientist

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