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

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RNA extraction ↓

cDNA synthesis using Reverse transcription (RT) PCR using gene specific reverse primer

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PCR amplification

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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

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