RESULTS

4 RESULTS

Majority of shrimp (Penaeus monodon) samples (healthy and moribund shrimps)

analyzed in this study were collected during the disease outbreaks Total numbers of

samples analyzed by PCR were 135 of which 67 were post larvae and rest were adult or

juvenile shrimp Of the 135 shrimp samples analyzed 49 (67) were positive for WSSV

(37 in single step PCR (Fig-1) and 30 in nested PCR (Fig-2)) MBV was detected in 29

(40) of the samples of which 21 were found positive in single step PCR (Fig-3) and 19

were positive in nested PCR (Fig-4) HPV was detected in 34 (47) of the samples (23

were found positive in first step PCR (Fig-5) and 24 in nested PCR (Fig-6) Table-41)

Presence of multiple viral infections was observed in many samples (Table-42)

Twenty-one samples (15) showed the presence of triple viral infections (WSSV MBV

and HPV) Dual viral infection with WSSV and HPV were seen in 17 (125) samples

Dual viral infection with WSSV and MBV were found in 6 and with MBV and HPV in 2

samples

41 Loose Shell Syndrome (LSS)

The LSS affected shrimps show hard or leathery shell but not soft and shrunken

tail meat with gap between the shell and the muscle tissue (causing the ldquoloose shellrdquo)

They also go off feed and may have swollen hind gut with whitish fluid The moribund

shrimp exhibited bacterial fungal and algal parasite infections on the exterior (Plate-1)

The infected shrimps move to the pond margins and finally die Studies conducted so far

have not conclusively determined the etiological agent for this syndrome Usually LSS

was noticed during the fourth month of the culture period In many of the farms the

water colour was dark-greenish (Plate-2) and in some farms LSS was also noticed in

normal bluish-grey colour ponds In some farms the pond bottom was degraded and

turned to black colour with rotten smell Affected shrimps were very lethargic and

reduced preening activity Some shrimps were affected so much that Zoothamnium sp

was densely covered the whole of the carapace and shrimp even lost its normal reflexive

activity when disturbed Some shrimps were discoloured to slight yellowish or greenish

due to algal growth

M 1 2 3 4 5

Fig 1 Agarose gel electrophoresis of WSSV one step PCR products Lane M 100bp

DNA ladder plus Lane 1 Positive control Lane 2-3 Samples positive for WSSV Lane

4 Sample negative for WSSV and Lane 5 Negative control

M 1 2 3 4 5

Fig 2 Agarose gel electrophoresis of WSSV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for WSSV Lane 4

Sample negative for WSSV and Lane 5 Negative control

M 1 2 3 4 5

Fig 3 Agarose gel electrophoresis of MBV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 4 Agarose gel electrophoresis of MBV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 5 Agarose gel electrophoresis of HPV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4

Sample negative for HPV and Lane 5 Negative control

1 2 3 4 5 6 M

Fig 6 Agarose gel electrophoresis of HPV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4-5

Sample negative for HPV and Lane 6 Negative control

Table-41 Results of shrimp samples from farms of South West and South East coast analyzed by PCR for WSSV MBV and HPV

WSSV MBV HPV No of Samples analyzed

I step II step I step II step I step II step

135 37(27) 30(22) 21(15) 19(14) 23(17) 24(17) Total 67(49) 40(29) 47(34)

Table-42 Presence of viruses in shrimp samples showing multiple viral infections

No of samples analyzed

WSSV+MBV+HPV WSSV+MBV WSSV+HPV MBV+HPV

135 21(~16) 6(4) 17(125) 2(1)

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

4 RESULTS

Majority of shrimp (Penaeus monodon) samples (healthy and moribund shrimps)

analyzed in this study were collected during the disease outbreaks Total numbers of

samples analyzed by PCR were 135 of which 67 were post larvae and rest were adult or

juvenile shrimp Of the 135 shrimp samples analyzed 49 (67) were positive for WSSV

(37 in single step PCR (Fig-1) and 30 in nested PCR (Fig-2)) MBV was detected in 29

(40) of the samples of which 21 were found positive in single step PCR (Fig-3) and 19

were positive in nested PCR (Fig-4) HPV was detected in 34 (47) of the samples (23

were found positive in first step PCR (Fig-5) and 24 in nested PCR (Fig-6) Table-41)

Presence of multiple viral infections was observed in many samples (Table-42)

Twenty-one samples (15) showed the presence of triple viral infections (WSSV MBV

and HPV) Dual viral infection with WSSV and HPV were seen in 17 (125) samples

Dual viral infection with WSSV and MBV were found in 6 and with MBV and HPV in 2

samples

41 Loose Shell Syndrome (LSS)

The LSS affected shrimps show hard or leathery shell but not soft and shrunken

tail meat with gap between the shell and the muscle tissue (causing the ldquoloose shellrdquo)

They also go off feed and may have swollen hind gut with whitish fluid The moribund

shrimp exhibited bacterial fungal and algal parasite infections on the exterior (Plate-1)

The infected shrimps move to the pond margins and finally die Studies conducted so far

have not conclusively determined the etiological agent for this syndrome Usually LSS

was noticed during the fourth month of the culture period In many of the farms the

water colour was dark-greenish (Plate-2) and in some farms LSS was also noticed in

normal bluish-grey colour ponds In some farms the pond bottom was degraded and

turned to black colour with rotten smell Affected shrimps were very lethargic and

reduced preening activity Some shrimps were affected so much that Zoothamnium sp

was densely covered the whole of the carapace and shrimp even lost its normal reflexive

activity when disturbed Some shrimps were discoloured to slight yellowish or greenish

due to algal growth

M 1 2 3 4 5

Fig 1 Agarose gel electrophoresis of WSSV one step PCR products Lane M 100bp

DNA ladder plus Lane 1 Positive control Lane 2-3 Samples positive for WSSV Lane

4 Sample negative for WSSV and Lane 5 Negative control

M 1 2 3 4 5

Fig 2 Agarose gel electrophoresis of WSSV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for WSSV Lane 4

Sample negative for WSSV and Lane 5 Negative control

M 1 2 3 4 5

Fig 3 Agarose gel electrophoresis of MBV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 4 Agarose gel electrophoresis of MBV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 5 Agarose gel electrophoresis of HPV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4

Sample negative for HPV and Lane 5 Negative control

1 2 3 4 5 6 M

Fig 6 Agarose gel electrophoresis of HPV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4-5

Sample negative for HPV and Lane 6 Negative control

Table-41 Results of shrimp samples from farms of South West and South East coast analyzed by PCR for WSSV MBV and HPV

WSSV MBV HPV No of Samples analyzed

I step II step I step II step I step II step

135 37(27) 30(22) 21(15) 19(14) 23(17) 24(17) Total 67(49) 40(29) 47(34)

Table-42 Presence of viruses in shrimp samples showing multiple viral infections

No of samples analyzed

WSSV+MBV+HPV WSSV+MBV WSSV+HPV MBV+HPV

135 21(~16) 6(4) 17(125) 2(1)

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

M 1 2 3 4 5

Fig 1 Agarose gel electrophoresis of WSSV one step PCR products Lane M 100bp

DNA ladder plus Lane 1 Positive control Lane 2-3 Samples positive for WSSV Lane

4 Sample negative for WSSV and Lane 5 Negative control

M 1 2 3 4 5

Fig 2 Agarose gel electrophoresis of WSSV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for WSSV Lane 4

Sample negative for WSSV and Lane 5 Negative control

M 1 2 3 4 5

Fig 3 Agarose gel electrophoresis of MBV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 4 Agarose gel electrophoresis of MBV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 5 Agarose gel electrophoresis of HPV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4

Sample negative for HPV and Lane 5 Negative control

1 2 3 4 5 6 M

Fig 6 Agarose gel electrophoresis of HPV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4-5

Sample negative for HPV and Lane 6 Negative control

Table-41 Results of shrimp samples from farms of South West and South East coast analyzed by PCR for WSSV MBV and HPV

WSSV MBV HPV No of Samples analyzed

I step II step I step II step I step II step

135 37(27) 30(22) 21(15) 19(14) 23(17) 24(17) Total 67(49) 40(29) 47(34)

Table-42 Presence of viruses in shrimp samples showing multiple viral infections

No of samples analyzed

WSSV+MBV+HPV WSSV+MBV WSSV+HPV MBV+HPV

135 21(~16) 6(4) 17(125) 2(1)

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Fig 3 Agarose gel electrophoresis of MBV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 4 Agarose gel electrophoresis of MBV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for MBV Lane 4

Sample negative for MBV and Lane 5 Negative control

M 1 2 3 4 5

Fig 5 Agarose gel electrophoresis of HPV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4

Sample negative for HPV and Lane 5 Negative control

1 2 3 4 5 6 M

Fig 6 Agarose gel electrophoresis of HPV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4-5

Sample negative for HPV and Lane 6 Negative control

Table-41 Results of shrimp samples from farms of South West and South East coast analyzed by PCR for WSSV MBV and HPV

WSSV MBV HPV No of Samples analyzed

I step II step I step II step I step II step

135 37(27) 30(22) 21(15) 19(14) 23(17) 24(17) Total 67(49) 40(29) 47(34)

Table-42 Presence of viruses in shrimp samples showing multiple viral infections

No of samples analyzed

WSSV+MBV+HPV WSSV+MBV WSSV+HPV MBV+HPV

135 21(~16) 6(4) 17(125) 2(1)

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Fig 5 Agarose gel electrophoresis of HPV one step PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4

Sample negative for HPV and Lane 5 Negative control

1 2 3 4 5 6 M

Fig 6 Agarose gel electrophoresis of HPV nested PCR products Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples positive for HPV Lane 4-5

Sample negative for HPV and Lane 6 Negative control

Table-41 Results of shrimp samples from farms of South West and South East coast analyzed by PCR for WSSV MBV and HPV

WSSV MBV HPV No of Samples analyzed

I step II step I step II step I step II step

135 37(27) 30(22) 21(15) 19(14) 23(17) 24(17) Total 67(49) 40(29) 47(34)

Table-42 Presence of viruses in shrimp samples showing multiple viral infections

No of samples analyzed

WSSV+MBV+HPV WSSV+MBV WSSV+HPV MBV+HPV

135 21(~16) 6(4) 17(125) 2(1)

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Table-41 Results of shrimp samples from farms of South West and South East coast analyzed by PCR for WSSV MBV and HPV

WSSV MBV HPV No of Samples analyzed

I step II step I step II step I step II step

135 37(27) 30(22) 21(15) 19(14) 23(17) 24(17) Total 67(49) 40(29) 47(34)

Table-42 Presence of viruses in shrimp samples showing multiple viral infections

No of samples analyzed

WSSV+MBV+HPV WSSV+MBV WSSV+HPV MBV+HPV

135 21(~16) 6(4) 17(125) 2(1)

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-1 Loose shell syndrome shrimp

Plate-2 Loose shell syndrome affected pond Note the greenish colour of the pond water

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

The histopathological studies revealed extensive degenerative changes in

hepatopancreas including sloughing of the hepatopancreatic tubules from the basement

membrane (Plate-5) Necrotized areas showing deep basophilia karyorhexis and

pyknosis (Plate-6) were evident in hepatopancreas of most of the animals examined

Deeply basophilic inclusion-like bodies (Plate-7 and Plate-8) were observed in the cells

of tubular epithelium as well as in the inter-tubular space In some hepatopancreatic

tubules areas of focal haemocytic infiltration (Plate-9) with melanized nodular formation

with granulation tissues (Plate-10) were observed Atrophied tubules were characterized

by the absence of storage vacuoles However midgut traversing through hepatopancreas

appeared normal with intact epithelial cells to its basement membrane (Plate-11)

Histological sections also showed moderate to severe necrotic changes in the gills when

compared to normal gills (Plate-12) The changes were characterized by the necrosis of

pillar cells and detachment of cuticular layer from the underlying epithelium (Plate-13)

Many attached epicommensals were also observed in the gill filaments (Plate-13)

Integument appeared normal except the attachment of epicommensals in outer and inner

regions of carapace (Plate-15) in contrast to normal healthy shrimp (Plate-14) Moderate

degeneration of body muscles and heart tissue was also pronounced in all the animals

examined

Out of eleven LSS samples analyzed (Table-43) one sample showed multiple

viral infection with WSSV MBV HPV and LSNV (Fig-7 and Fig-8) Triple viral

infection was noticed in 2 samples one with WSSV MBV and HPV and another with

WSSV MBV and LSNV Five samples showed dual infection with WSSV and MBV and

only three were positive for HPV alone One sample was not infected with any of the

viruses tested except LSNV None of the samples tested for necrotizing hepatopancretitis

(NHP) bacterium by PCR was positive

Ten shrimp samples were collected from ponds showing the symptoms of loose

shell syndrome and subjected for bacteriology From each shrimp hepatopancreas and

hindgut were aseptically removed and streaked onto TCBS and TSAS plates Few

isolated colonies were subjected to biochemical analysis and the biochemical results

revealed the presence of V fluvialis V cincinnatiensis V fischeri V harveyii and V

natriegens

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-3 Histology of the Hepatopancreas of normal subadult shrimp Note the intact tubules (arrows) and different types of cells (B B-cells RR-cells)HampE400x

Plate-4 Histology of the Hepatopancreas of normal subadult shrimp Note the myoepithelial cells (arrowheads) haemocytes (arrow) EE-cells HampE1000x

E

B

R

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-5 Histology of the Hepatopancreas of LSS shrimp Note the detachment of the tubules from basement membrane (arrows) and other necrotic changes HampE400x

Plate-6 Histology of the Hepatopancreas of LSS shrimp Note the necrotized area showing the picnotic stage (arrow) of the nucleus HampE1000x

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-7 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-8 Histology of the Hepatopancreas of LSS shrimp Note deeply basophilic inclusion-like body (arrow) HampE1000x

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-9 Histology of the Hepatopancreas of LSS shrimp Note the degerative changes and accumulation of hemocytes HampE400x

Plate-10 Histology of the Hepatopancreas of LSS shrimp Note the granuloma formation HampE400x

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-11 Histology of the midgut of LSS shrimp Note the intact normal epithelial cells to its basement membrane HampE1000x

Plate-12 Histology of the gills of normal shrimp HampE400x

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-13 Histology of the gills of LSS shrimp Note the severe necrotic changes Arrow indicates Zoothamnium sp in gillsHampE 400x

Plate-14 Histology of integument of normal shrimp Note the different layers of the integument (arrow) with normal subcuticular epithelium (block arrow) and other underlying tissues HampE 400x

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Plate-15 Histology of the integument of LSS shrimp Note the presence of epicommensals (arrows) in inner region of the carapace HampE400x

Plate-16 Histology of the hindgut of whitegut syndrome shrimp HampE100x

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Table-4 3 Presence of viruses in adult shrimp samples showing Loose shell syndrome

Sample WSSV MBV HPV LSNV NHP KLA-1 + + + + - KLA-2 + + + - - ALA-3 - - + - - ALA-4 - - + - - ALA-5 - - + - - KLA-6 - - - + - KLA-7 + + - + - KLA -8 + - - - - ALA-9 + - - - - ALA-10 + - - - - ALA-11 + - - - -

Total 7 3 5 3 0 Table-4 4 Results of the juvenile adult shrimp samples showing Swollen hind gut or white gut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KWA-1 + - + - KWA-2 - + - - KWA-3 + + - - AWA-4 + + + - AWA-5 + + - -

Total 4 4 2 0

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Fig 7 First step RT-PCR for detection of LSNV in Penaeus monodon M 100 bp DNA

ladder Plus (Gene Ruler TM genetix) Lane 1 Positive control Lane 2 Negative

control Lanes 3-5 Penaeus monodon samples with LSS positive for LSNV Lane 6

Normal adult shrimp sample Lane 7 Normal postlarvae of Penaeus monodon

Fig 8 Nested RT-PCR for detection of LSNV in P monodon Lane M 100bp DNA

ladder plus Lane 1 Positive control Lane 2-3 Samples negative for LSNV Lane 4-6

Sample positive for LSNV and Lane 7 Negative control

M 1 2 3 4 5 6 7

200 bp

M 1 2 3 4 5 6 7

150 bp

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

411 Slow growth syndrome

Three shrimps which were collected and preserved in RNA-friendly fixative

during the harvest of the crop were analyzed for LSNV WSSV MBV and HPV All

these shrimps were small in size (6-8g) when compared to other shrimps (25-30g) of the

same crop pond The culture period was 4-45 months After analyses the shrimps were

positive for only LSNV This was confirmed by sequencing the PCR products BLAST

(Altschul et al 1997) analysis of the sequences obtained in this study revealed 98

homology with the sequences of LSNV reported by Sritunyalucksana et al (2006) The

nucleotide sequence from this study was deposited in GenBank Accession Number

EF593037 (Fig-9) This is the first report of LSNV from India To characterize the new

virus (LSNV) closely related virusrsquos (Mushroom bacilliform virus) coat protein was

selected from the GenBank and three sets of primers were developed With these new

sets of primers RT-PCR was done using LSNV RNA as template After analysis there

were nonspecific bands in the gel Even after doing gradient PCR with annealing

temperatures ranging from 40ordm-60 ordmC we did not get any specific bands of 500bp 300bp

or 288bp which were expected from the new set of primers after amplification

42 Swollen Hindgut or White gut syndrome in adult shrimp

The shrimps having white gut syndrome were having bulged hindgut which can

be seen very clearly from outside The color of the hindgut was white or creamish when

compared to normal which was translucent Affected shrimps were of smaller in size and

there was no feed in the gut They were also found lethargic unlike healthy shrimps from

the same pond Pond bottom was turned to black in some of the farms where white gut

shrimps were collected But in some farms pond bottom was clean with normal color

Usually white gut syndrome was noticed during third month of the culture period Out of

five white gut syndrome-affected samples analyzed triple viral infection was seen in only

one sample whereas three samples showed dual viral infection (WSSV and HPV in one

and WSSV and MBV in two samples Table-44) MBV alone was noticed in only one

sample

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

Fig 9 Nucleotide sequence of Laem-Singh virus

My NCBI[Sign In] [Register]

PubMed Nucleotide Protein Genome Structure PMC Taxonomy OMIM Books

Search CoreNucleotide for Go

You need JavaScript to work with this page

1 EF593037 Reports Penaeus monodon R[gi148710407] Links

bull Features bull Sequence

LOCUS EF593037 194 bp mRNA linear VRL 12-JUN-2007 DEFINITION Penaeus monodon RNA virus nonfunctional RNA-dependent RNA polymerase (RdRP) mRNA partial sequence ACCESSION EF593037 VERSION EF5930371 GI148710407 KEYWORDS SOURCE Penaeus monodon RNA virus ORGANISM Penaeus monodon RNA virus Viruses unclassified viruses REFERENCE 1 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Detection of Laem-Singh Virus in cultured Penaeus monodon from India JOURNAL Dis Aquat Org (2007) In press REFERENCE 2 (bases 1 to 194) AUTHORS PrakashaBK KarunasagarI and KarunasagarI TITLE Direct Submission JOURNAL Submitted (07-MAY-2007) Fishery Microbiology College of Fisheries Matsyanagar Mangalore Karnataka 575 002 India FEATURES LocationQualifiers source 1194 organism=Penaeus monodon RNA virus mol_type=mRNA isolation_source=hepatopancreas db_xref=taxon343515 country=India gene lt1gt194 gene=RdRP misc_feature lt1142 gene=RdRP note=nonfunctional RNA-dependent RNA polymerase due to mutation similar to GenBank Accession Number DQ127905 ORIGIN 1 ttgccttctc ccgagtggtc aggtttacgt gcaagagttc tcaggcttca tgaagtcagg

61 cattgtgata ctatctccac caattctcat gcccagatca tgctgcatat gcttgcttgc 121 aagcgctcgg gtgagcccgt gactcctata ttggcctgcg gtgatgacac tattcaagca 181 gctacctcag ccgg

Bacteriological analysis of six shrimp samples collected from ponds showing the

symptoms of swollen hind gut or white gut revealed the presence of V fluvialis V

proteolyticus V carchariae V logei V metschnikovii and Bacillus sp When apparently

normal shrimp from the same culture pond was subjected to bacteriology only V

fluvialis was found to be present

The histopathological studies showed mild vacuolar degeneration of the tegmental

gland (Plate-16 17 18 19) associated with the hind gut when compared to normal shrimp

but the cuticular epithelium appeared normal (Plate-16 17 18) in the moribund shrimp

However hepatopancreas showed extensive degeneration wherein the complete tubular

architecture was lost in most of the tubules (Plate-20) especially the inner part of the

capsule

43 Swollen hindgut (SHG) syndrome in postlarvae

Of the 67 batches of post larval samples analyzed 14 were showing the symptoms

of swollen hind gut Post larvae with SHG showed enlargement and distention of the

hindgut folds and its junction with the midgut (Plate-22) although in some cases swelling

also occurred in the midgut of the sixth abdominal segment in contrast to healthy PL

(Plate-21) The abnormality caused cessation of the rhythmic movements of the hindgutndash

midgut junction resulting to failure of affected post-larvae to excrete fecal pellets

Hind gut appeared normal histologically in all the animals examined (Plate-25)

Some region of the midgut epithelium anterior to the junction of hindgut showed

degenerative changes (Plate-23 24) However severe necrotic changes in any of the cells

associated with the gut were not discernible in any of the animals examined Presence of

obvious bacterial colonies or parasitic infection was also not observed in the hind gut

region (Plate-25) Nevertheless the significant pathology observed was in

hepatopancreas where severely necrotized tubules were evident with occasional deeply

basophilic inclusion-like bodies (Plate-26) The degenerated tubule lumen also showed

aggregation of bacteria-like particles along with the tissue debris in few specimens

Plate-17 Histology of the hindgut of whitegut syndrome shrimp Note normal chitin lining (arrows) and mild degeneration of the tegmental gland (block arrow) HampE400x

Plate-18 Histology of hindgut in Whitegut syndrome shrimp Note normal chitin lining (arrows) and mild degeneration of the tegmental gland (block arrows) HampE 400x

Plate-19 Histology of hindgut in Whitegut syndrome shrimp Note mild degeneration of the tegmental gland (block arrow)HampE 1000x

Plate-20 Histology of the hepatopancreas of whitegut syndrome shrimp Note the severe necrosis of the tubules HampE100x

Plate-21 Sixth abdominal segment of normal postlarvae showing normal hindgut (arrow) 100x

Plate-22 Sixth abdominal segment of the postlarvae showing the swollen hindgut Arrow indicates the swollen hindgut 100X

Plate-23 Histology of Swollen hindgut syndrome in postlarvae HampE 100x

Plate-24 Histology of midgut region of Swollen hindgut syndrome in postlarvae Note the degenerative changes in midgut region anterior to the hindgut-midgut junction HampE 400x

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Bacteriological analysis of six shrimp samples collected from ponds showing the

symptoms of swollen hind gut or white gut revealed the presence of V fluvialis V

proteolyticus V carchariae V logei V metschnikovii and Bacillus sp When apparently

normal shrimp from the same culture pond was subjected to bacteriology only V

fluvialis was found to be present

The histopathological studies showed mild vacuolar degeneration of the tegmental

gland (Plate-16 17 18 19) associated with the hind gut when compared to normal shrimp

but the cuticular epithelium appeared normal (Plate-16 17 18) in the moribund shrimp

However hepatopancreas showed extensive degeneration wherein the complete tubular

architecture was lost in most of the tubules (Plate-20) especially the inner part of the

capsule

43 Swollen hindgut (SHG) syndrome in postlarvae

Of the 67 batches of post larval samples analyzed 14 were showing the symptoms

of swollen hind gut Post larvae with SHG showed enlargement and distention of the

hindgut folds and its junction with the midgut (Plate-22) although in some cases swelling

also occurred in the midgut of the sixth abdominal segment in contrast to healthy PL

(Plate-21) The abnormality caused cessation of the rhythmic movements of the hindgutndash

midgut junction resulting to failure of affected post-larvae to excrete fecal pellets

Hind gut appeared normal histologically in all the animals examined (Plate-25)

Some region of the midgut epithelium anterior to the junction of hindgut showed

degenerative changes (Plate-23 24) However severe necrotic changes in any of the cells

associated with the gut were not discernible in any of the animals examined Presence of

obvious bacterial colonies or parasitic infection was also not observed in the hind gut

region (Plate-25) Nevertheless the significant pathology observed was in

hepatopancreas where severely necrotized tubules were evident with occasional deeply

basophilic inclusion-like bodies (Plate-26) The degenerated tubule lumen also showed

aggregation of bacteria-like particles along with the tissue debris in few specimens

Plate-17 Histology of the hindgut of whitegut syndrome shrimp Note normal chitin lining (arrows) and mild degeneration of the tegmental gland (block arrow) HampE400x

Plate-18 Histology of hindgut in Whitegut syndrome shrimp Note normal chitin lining (arrows) and mild degeneration of the tegmental gland (block arrows) HampE 400x

Plate-19 Histology of hindgut in Whitegut syndrome shrimp Note mild degeneration of the tegmental gland (block arrow)HampE 1000x

Plate-20 Histology of the hepatopancreas of whitegut syndrome shrimp Note the severe necrosis of the tubules HampE100x

Plate-21 Sixth abdominal segment of normal postlarvae showing normal hindgut (arrow) 100x

Plate-22 Sixth abdominal segment of the postlarvae showing the swollen hindgut Arrow indicates the swollen hindgut 100X

Plate-23 Histology of Swollen hindgut syndrome in postlarvae HampE 100x

Plate-24 Histology of midgut region of Swollen hindgut syndrome in postlarvae Note the degenerative changes in midgut region anterior to the hindgut-midgut junction HampE 400x

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Plate-17 Histology of the hindgut of whitegut syndrome shrimp Note normal chitin lining (arrows) and mild degeneration of the tegmental gland (block arrow) HampE400x

Plate-18 Histology of hindgut in Whitegut syndrome shrimp Note normal chitin lining (arrows) and mild degeneration of the tegmental gland (block arrows) HampE 400x

Plate-19 Histology of hindgut in Whitegut syndrome shrimp Note mild degeneration of the tegmental gland (block arrow)HampE 1000x

Plate-20 Histology of the hepatopancreas of whitegut syndrome shrimp Note the severe necrosis of the tubules HampE100x

Plate-21 Sixth abdominal segment of normal postlarvae showing normal hindgut (arrow) 100x

Plate-22 Sixth abdominal segment of the postlarvae showing the swollen hindgut Arrow indicates the swollen hindgut 100X

Plate-23 Histology of Swollen hindgut syndrome in postlarvae HampE 100x

Plate-24 Histology of midgut region of Swollen hindgut syndrome in postlarvae Note the degenerative changes in midgut region anterior to the hindgut-midgut junction HampE 400x

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Plate-19 Histology of hindgut in Whitegut syndrome shrimp Note mild degeneration of the tegmental gland (block arrow)HampE 1000x

Plate-20 Histology of the hepatopancreas of whitegut syndrome shrimp Note the severe necrosis of the tubules HampE100x

Plate-21 Sixth abdominal segment of normal postlarvae showing normal hindgut (arrow) 100x

Plate-22 Sixth abdominal segment of the postlarvae showing the swollen hindgut Arrow indicates the swollen hindgut 100X

Plate-23 Histology of Swollen hindgut syndrome in postlarvae HampE 100x

Plate-24 Histology of midgut region of Swollen hindgut syndrome in postlarvae Note the degenerative changes in midgut region anterior to the hindgut-midgut junction HampE 400x

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Plate-21 Sixth abdominal segment of normal postlarvae showing normal hindgut (arrow) 100x

Plate-22 Sixth abdominal segment of the postlarvae showing the swollen hindgut Arrow indicates the swollen hindgut 100X

Plate-23 Histology of Swollen hindgut syndrome in postlarvae HampE 100x

Plate-24 Histology of midgut region of Swollen hindgut syndrome in postlarvae Note the degenerative changes in midgut region anterior to the hindgut-midgut junction HampE 400x

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Plate-23 Histology of Swollen hindgut syndrome in postlarvae HampE 100x

Plate-24 Histology of midgut region of Swollen hindgut syndrome in postlarvae Note the degenerative changes in midgut region anterior to the hindgut-midgut junction HampE 400x

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Plate-25 Histology of Swollen hindgut syndrome in postlarvae Note normal hind-gut with chitin lining (arrow) and absence of bacterial or parasitic infection HampE 1000x

Plate-26 Histology of Hepatopancreas in Swollen hindgut syndrome in post larvae Note the severely necrotized tubules (arrow) with deeply basophilic inclusion like-body (arrowheads) HampE 100x

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Out of 14 batches of post larvae (showing SHG) analyzed nine were positive for

WSSV and HPV each and only two for MBV (Table-45) Triple viral infections were not

noticed in any of the samples analyzed Dual viral infection with WSSV and HPV was

noticed in six post larval samples whereas dual viral infections with WSSV and MBV

were seen in only two samples Five samples showed infection with HPV alone

Bacteriological analysis of post larval samples showed the presence of V

alginolyticus

44 White Tail Disease (WTD) in fresh water prawn

Freshwater prawns (Macrobrachium rosenbergii) Artemia nauplii Acetes sp

were analyzed for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus

(XSV) by RT-PCR Four berried prawns which were collected from Nethravati estuary

were analyzed by taking few eggs and pleopods separately and subjected to RT-PCR and

found negative for MrNV and XSV Two Artemia nauplii and Artemia flakes were also

found to be negative Six juvenile samples were collected from a farm in Hasson district

were also found negative for both MrNV and XSV Some prawn larvae which were

showing necrosis of tail muscle and abdomen were also found negative for both the

viruses Even some Acetes sp which were collected at the same farm in Nellore also gave

negative result Water sample collected from prawn farm in Hasson was also negative

45 Prevalence of WSSV MBV and HPV in post larvae Prevalence of viruses in post larval samples from West Coast of India was

analyzed by PCR A total of 505 samples were screened for WSSV MBV and HPV

(Table-46) Of these 25 (49) were positive for WSSV 83 (1643) were positive for

MBV and 55 (1089) for HPV In addition to single viral infection dual and triple viral

infections were observed Six samples showed infection with two viruses (WSSV and

MBV) Simultaneous infections with MBV and HPV were found in 11 samples The co-

occurrence of WSSV and HPV was also noticed in 9 samples (178) One sample was

found infected with WSSV MBV and HPV

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Table-4 5 Results of the post larval samples showing Swollen hindgut analyzed for WSSV MBV HPV and LSNV

Sample WSSV MBV HPV LSNV KSP-1 - - + - ASP-2 + - + - ASP-3 - - + - ASP-4 + - + - ASP-5 + - + - ASP-6 + - + - ASP-7 + - + - ASP-8 + - + - ASP-9 + - - - KSP-10 - - - - KSP-11 - - - - KSP-12 + + - - KSP-13 + + - - ASP-14 - - + - ASP-15 - - + - ASP-16 - - - - ASP-17 - + + - ASP-18 - - + -

Total 9 3 12 0

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Table-46 Results of prevalence of viruses in post larvae

Year 2004 2005 2006

Viruses

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

No of samples positive

Prevalence ()

WSSV 4 289 6 363 15 742 MBV 29 2101 27 1636 27 1336 HPV 28 2028 18 109 9 445 WSSV + MBV 1 07 0 0 5 247 MBV + HPV 4 289 7 424 0 0 WSSV + HPV 7 507 1 06 1 049 WSSV+MBV+HPV 0 0 1 06 0 0 No of samples analyzed

138 165 202

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively

Year-wise analysis of the samples showed that in the year 2004 out of 138

samples 4 (289) were positive for WSSV 29 (2101) for MBV and 28 (2028) for

HPV One (07) sample showed dual viral infection with WSSV and MBV

Simultaneous occurrence of MBV and HPV was observed in 4 (289) samples and

WSSV and HPV in 7 (507) samples

In the year 2005 out of 165 samples analyzed six (363) were positive for

WSSV 27 (1636) for MBV and 18 (109) for HPV Seven (424) samples showed

the simultaneous presence of MBV and HPV and one (06) was found to have WSSV

and HPV Triple viral infection (WSSV MBV amp HPV) was noticed in one sample In the

year 2006 of 202 samples analyzed 15 (742) were positive for WSSV 27 (1336)

for MBV and 9 (445) for HPV alone Five (247) samples showed dual viral

infection with WSSV and MBV only one sample for WSSV and HPV but none for MBV

and HPV

The year-wise analysis reveals that there is an increasing trend in the level of

WSSV infection year after year but decreasing trend in the level of individual infections

of MBV and HPV However there is fluctuation in the dual and triple viral infections

year after year Dual viral infection with WSSV and MBV was 07 in 2004 but

decreased to zero in the year 2005 and again increased to 247 in the year 2006 In the

case of dual viral infection with MBV and HPV there was an increase in the rate of

infection from 289 in 2004 to 424 in 2005 but decreased to zero in the year 2006

However there is decreasing trend in the case of dual viral infections with WSSV and

HPV from 507 in 2004 to 06 and 049 in 2005 and 2006 respectively