Impact of climate change on the population structure and virulence of viral and bacterial pathogens in
marine environment and host response alterations due to pathogen-abiotic synergy
Environmental stress of high temperature and salinity induced genomic variations in Vibrio Multi abiotic stress caused by high salinity and temperature was found to bring about pathogen-abiotic synergy causing increased cumulative mortality in juvenile shrimps due to WSSV.
Medicinal herbs incorporated feed was found to upregulate immune gene (tubulin and penaeidin)
expression in juvenile tiger shrimps. Herbal feed was found to delay and reduce the mortality
rate in WSSV infected shrimps at low temperature (24 ºC) and at normal temperature (29 ºC) conditions.
Quantitative infectivity potential assessment of WSSV and genotyping of virulent strains
WSSV prevalence was found to be at 41.99 % during the project period 2010-12 and the strains were found to be different genotypes based on ORF 94 and ORF 125, transposase region and ORF14/15
Infectivity of the WSSV isolates was found to vary from 100 % to 45.83 % mortality and passaging of the virus resulted in genotype shift in one of the low virulent isolates (Q13).
At low doses of WSSV inoculation, both high and low pH induced high mortality in shrimps while the mortality stagnated at 50% in shrimps in normal pH seawater. Induction of mortality was lower at 22°C compared to both 34 and 30 ° Litopenaeus vannamei was found to survive with low dose of WSSV with limited mortality
Comparative mortality pattern of the juvenile shrimps administered with two WSSV isolates
InNoVacc: Indo-Norwegian platform for the development of candidate vaccines for invertebrate, piscine and avian
species - Fisheries College and Research Institute, Tuticorin
Nodavirus (LCNNV-In01) was isolated from Asian seabass (Lates calcarifer) juveniles and
characterised through electron microscopy, serum neutralisation, RT-PCR and sequence analysis.
Full length genes of coat protein (1.4 kb - RNA2) and RNA dependent RNA polymerase (3.1 kb RNA1) were cloned and sequenced (Genbank Acc.Nos. HM485328 & JQ073720). Two clones of RNA2 were delivered to Dr. Sunil K. Lal, ICGEB, New Delhi for generation of VLP using the self assembling avian influenza viral coat protein genes.
The cDNA of RNA1 and RNA2 was transcribed invitro and RNA was transfected into SSN-1 cells
at equimolar concentrations using lipofectamine. CPE was obtained after 60 h of incubation and virus was rescued from SSN-1 cells.
Gene expression of five immune genes viz. IFN, Mx, ISG-15, IRF-3 and Vig-1 was investigated
with beta actin as the reference gene. CpG and LCNNV upregulated IFN, Mx, ISG-15 and IRF-3 in
seabass cell line, while in clownfish cell lines LCNNV, CpG and poly I:C upregulated Mx,
ISG-1 and Vig-1 genes. The study revealed that the innate immunity in Asian seabass
could be enhanced by application of PRR ligands like CpG ODN, which has high application potential
for improving the health status of the fish both by non-specific immunostimulation and also by
increasing vaccination efficiency as adjuvants.
TEM image of LCNNV-In01 showing non-enveloped, icosahedral virus particles of 25–30 nm size packed in aggregates and in intercellular spaces in the SSN-1 cells.
Cytoplasmic aggregations of virus particles in special membranous envelopes observed in the cell cytoplasm.
Characterisation and maintenance of cell lines of fish and crab
Cell lines were used for virus isolation and characterisation from infected fishes and two isolates were recovered. One of the isolate has been characterised and reported as koi ranavirus, which is the first ever ranavirus reported from a koi and the first report of a fish ranavirus from India.
TEM image of koi ranvirus KIRV grown in SNKD2a cells showing icosahedral particle of 100-120 nm size. Virus particles are at the end of virus morphogenesis (bar = 200nm).
Four cell lines developed from spleen, fin, caudal peduncle and brain of the clownfish tissues viz. CFSP, CFFN2, CFCP1 & CFBR were deposited at National Repository for Fish Cell lines at NBFGR, Lucknow.
Molecular characterization of pathogens associated with fish diseases in Assam
Infected freshwater fish samples with different stages of ulcer were collected from two North-Eastern States viz. Assam and Manipur and analysed for the presence of viruses by diagnostic PCR for four DNA and three RNA viruses.
Samples were also processed for virus isolation using cell culture systems. The infected fishes collected from Imphal and Silchar carried ranavirus infections indicated by the amplicons of specific sizes obtained by PCR.
Cell culture analysis could not yield a virus isolate as yet. However, the samples are being further processed by bioassay studies to find if the filtered extracts could regenerate the infection in susceptible species for virus isolation.
One of the amplicon generated for EHNV primers having a length of 321 bp sequenced to find out the homogeneity indicated that the amplicon has 99 % homogeneity to the major capsid protein gene of largemouth bass virus (LMBV) (Genbank Acc.No.FR682503)
Surveillance of fish and shellfish diseases in selected districts of Tamil Nadu
Awareness Programmes were attended for 87 and 70 shrimp farmers in and around Vedaranyam and Manamelkudi, respectively. Talks on “Role of surveillance in disease control and health management”, “EMS or AHPND, an emerging disease of very serious concern among the Southeast Asian countries”, “Viral diseases affecting Penaeus vannamei” and “Best management practices to prevent the disease occurrence in shrimp farming” were given.
Three districts covered under active surveillance for marine shrimp farming are Nagapattinam (South of Kariakkal), Thanjavur and Pudukottai. Samplings were conducted across from 14 locations from farms under operation and those farms that reported on set of mortality. Of the 122 individuals collected through 34 sampling from 20 farms, 45 samples were analysed for the viral pathogens viz. WSSV, IHHNV, MBV and BP. Ten out of 45 were positive for WSSV (22%) and 7 for IHHNV (15.5%). MV and BP were negative.
Suspected EMS sample were collected in live condition to test for histopathology, bacterial isolation and identification. Histopathological analysis did not show typical APHNS. Bacterial isolates were tested based on the primer sequences provided by Dr Flegel and both AP1 and AP2 primers did not give typical amplicons. The sequence analysis did not match with the sequence provided by Dr Flegel. Molecular analysis of 16srRNA of 1.5 kbp of two isolates was found to blast to parahaemolyticus and V. alginolyticus. However, the biochemical reactions did not corresponded to the typical V. parahaemolyticus. Sequence analysis of IHHNV showed 98% homogeneity with Taiwan isolate (AY355307). None of the samples indicated the presence of typical AHPND.
