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DETECTION METHOD FOR WHITE TAIL DISEASE
(WTD) OF Macrobrachium
rosenbergii
Macrobrachium rosenbergii is the most favoured species for farming purposes and being cultured in different parts of the world
Important freshwater prawn producing countries are China, Thailand, India, Taiwan, Bangladesh and Vietnam
One of the major constraints limiting the prawn production all over the world is diseases
Generally, M.rosenbergii is considered to be a moderately disease-resistant species when compared to penaeid shrimp
No serious viral diseases have been reported so far in M.rosenbergii except White Tail Disease (WTD)
INTRODUCTION
The following viruses have been reported in prawn:
1.Macrobrachium hepatopancreatic parvo-like virus (MHPV)
2.Macrobrachium muscle virus (MMV)
3.Infectious hypodermal and haematopoietic necrosis virus (IHHNV)
4.White spot syndrome virus(WSSV)
5.Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus-like particle (XSV)
VIRAL DISEASES OF Macrobrachium
White Tail Disease (WTD) is responsible for high mortalities in hatchery and nursery-reared post larvae of fresh water prawn and subsequent economic loses in prawn culture industry
DISTRIBUTION : This disease was first reported in the French West Indies, later in China, India and very recently in Taiwan, Thailand and Queensland, Australia
CLINICAL SIGNS : The clinical signs of WTD include lethargy and opaqueness of abdominal muscle. Opaqueness gradually extend towards anterior and posterior sides, and degeneration of telson and uropods is also observed in severe cases
WHITE TAIL DISEASE
Hatchery-reared post-larvae of M. rosenbergii having white tail disease(opaqueness of abdominal muscle)
Two viruses namely Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus are responsible for WTD
MrNV is a small icosahedral non-enveloped particle, 26-27nm in diameter
It contains two single-stranded RNAs, RNA1 (RdRP) and RNA2 (Capsid) of 2.9 and 1.26 kb, respectively
Its capsid contains a single polypeptide of 43 kDa
With these characteristics, it is closely related to the Nodaviridae family
CAUSATIVE ORGANISMS
MrNV
Genome Capsid protein
XSV is also non-enveloped icosahedral virus
15nm in diameter
Possesses a linear ssRNA genome of 0.9kb encoding two overlapping structural proteins of 16 and 17kDa
Because of small size and absence of gene-encoding enzymes required for replication, It has been suggested that XSV may be a satellite virus and MrNV is a helper virus
CAUSATIVE ORGANISMS
XSV
Genome Capsid protein
Virus borne diseases are extremely difficult to control and it is one of the major problem of crustacean farming
Up to now preventive measures like early diagnosis is the only way of controlling further disease out break
One of the method of detection of this MrNV viral pathogen is electron microscopy, but it is too cumbersome to be used for routine screening and it requires high level of technical skill
It can also be diagnosed by observing clinical signs, however it can be misinterpreted some times
DETECTION OF WHITE TAIL DISEASE
mAb has been developed against the whole virus and triple antibody enzyme-linked immunosorbent assay (TAS-ELISA) diagnostic method has been developed
Recently Sandwich-Enzyme Linked Immunosorbent Assay (S-ELISA) has been developed for the detection of the MrNV - However ELISA based diagnosis is not as sensitive as genome based methods
Three genome-based diagnostic methods {dot-blot hybridization, in-situ hybridization and reverse transcription-polymerase chain reaction(RT-PCR)} have been developed
RT-PCR is considered as the best method for detecting the MrNV owing to its sensitivity, repeatability in the result and easier way of detecting when compare to other methods
DETECTION OF WHITE TAIL DISEASE
Collection and transportation of WTD suspected prawn
↓
RNA extraction ↓
cDNA synthesis using Reverse transcription (RT) PCR using gene specific reverse primer
↓
PCR amplification
↓
Gel electrophoresis
GENERAL STEPS IN DETECTION OF WTD USING REVERSE-TRANSCRIPTASE PCR
Infected animals will be collected from hatcheries by looking in to the gross clinical signs and transported using RNA fixative solution
Sample transported using RNA fixative will be transferred to 70% alcohol with in 3 days of time
Samples in alcohol will be used for RNA extraction with in 13-15 days of time
Total RNA will be extracted using trizol reagent
COLLECTION OF WTD INFECTED ANIMALS
The muscle tissues will be homogenized by adding trizol reagent
Then the homogenate will be left for incubation for 10’ at room temperature in trizol after mixing
200 µl of chloroform will be added to the homogenate, shaken vigorously for 15 seconds and will be kept for incubation at room temperature for 2-3 minutes
This homogenate will be centrifuged at 10000 g for 15 min at 4ºC
RNA will be precipitated from the aqueous phase with isoproponol and finally washed with 75% alcohol and will be dissolved in DEPC treated water
RNA extracted will be tested for MrNV and XSV by RT-PCR using the primers designed by our lab
RNA EXTRACTION
RT-PCR is done to obtain cDNA of CP-43 gene of MrNV and CP-17 gene of XSV from extracted RNA by using gene-specific reverse primer
PRIMERS USED FOR THE DETECTON OF MrNV AND XSV
Product Primer Sequence PCR Product Size
MrNV Capsid Protein
F : 5’ATG GCT AGA GGT AAA CAA AAT TC 3’R : 5’CTA ATT ATT GCC GAC GAT AGC 3’
1116
XSV Capsid protein
F : 5’ATG AAT AAG CGC ATT AAT CAT 3’R : 5’TTA CTG TTC GGA GTC CCA ATA 3’
525
PRIMER SEQUENCES
3 µl of RNA +4 µl DEPC
4 µl buffer for RT + 200 µM dNTPs+ 2 µl 0.1 M DTT + 20 pmole reverse primer+20 U RNAase inhibitor +100 U Revert Aid made up with DEPC water
synthesis reaction will becarried out at 42 ºC for 1hr+final denaturation at 99 ºC
Initial denaturation at 72 ºC
cDNA SYNTHESIS
Temperature (°C) Time (Minutes)
Initial denaturation 94 5
Denaturation 94 1
Annealing 45 (46.8 for MrNV) 1
Extension 72 1
Final extension 72 10 (20 for MrNV)
Number of cycles:30
PCR CONDITIONS
Agarose gel electrophoresis showing PCR amplification of MrNV and XSV Capsid protein
Lane 1 : 100 bp DNA markerLane 2 : MrNV Capid protein gene (1116 bp)Lane 3 : XSV Capsid protein gene (525 bp)
CONFIRMATION OF WTD INFECTION BY RT-PCR
To avoid the necessity of carrying out two separate RT-PCR reactions
Detection of MrNV and XSV in a single tube can be done by one-step multiplex RT-PCR assay by minor alteration in annealing temperature extension time
MULTIPLEX RT-PCR FOR MrNV AND XSV
Agarose gel picture showing results of multiplex RT-PCR
THANK YOU