BACKGROUND
the term of monodon slow growth syndrome  has been used by thai shrimp farmers to refer to unusual retarded growth that has occurred in cultivated p. monodon since  <dig>  it has been suggested that an infectious agent may be the cause  <cit> .
this contention was supported by the rapid spread of the problem and preliminary laboratory challenge tests showing that membrane-filtered  tissue extracts from msgs shrimp were able to cause msgs when injected into p. monodon but not when injected into p. vannamei  <cit> .
thai researchers adopted a case definition to distinguish ponds exhibiting msgs from ponds exhibiting slow growth caused by other problems  <cit> .
according to this case definition, an msgs pond must show a coefficient of size variation ≥35% together with freedom from hepatopancreatic parvovirus   or any other severe hepatopancreas infection, plus any  <dig> of the following  <dig> characters: 1) unusually dark color, 2) average daily weight gain of less than  <dig>  g/day at  <dig> months, 3) unusually bright yellow markings, 4) "bamboo-shaped" abdominal segments, and 5) brittle antennae.
these features distinguish msgs from stunted growth caused by monodon baculovirus   or hpv  <cit> .
an earlier survey revealed no correlation between the presence of several known shrimp pathogens  and the problem of msgs ponds  <cit> .
the results suggested that a new pathogen might be the cause of msgs.
separation of the purported agent from shrimp tissue homogenates has been difficult because of the presence of one or more known viruses and because bioassays require long incubation times of up to  <dig> month or more before results can be assessed.
therefore, we adopted a general "shot-gun" strategy to screen msgs cases for the presence of unknown pathogens  <cit> .
briefly, it consisted of obtaining shrimp from msgs case ponds followed by preparation of "viral" extracts for fractionation by gradient centrifugation.
the resulting bands were removed and used for total nucleic acid extraction before shotgun cloning of cdna prepared using random hexamer primers.
since total nucleic acid was used as the template, both rna and dna were amplified.
clones that hybridized with labelled, total dna from normal p. monodon were eliminated and negative hybridization clones were sent for sequencing.
clones identified by blast analysis to contain sequences of shrimp or previously screened pathogens were discarded, and remaining clones were used for in situ hybridization assays with shrimp from msgs ponds and from normal growth ponds.
using this strategy, one new rna virus  was identified  <cit>  but its presence in shrimp was not initially correlated with the occurrence of msgs ponds.
however, subsequent work revealed that stunted shrimp from msgs ponds gave strong in situ hybridization reactions for lsnv in retinal lesions that were not present in large shrimp from msgs ponds or in shrimp from normal growth ponds that gave positive rt-pcr reactions for the presence of lsnv  <cit> .
this led to the conclusion that lsnv was a necessary but not sufficient cause of msgs and suggested that another factor must be combined with lsnv to cause msgs.
one possibility for the additional factor was another pathogen.
to examine this possibility, the shotgun strategy was used for further screening of shrimp from msgs ponds.
as a result, a unique clone was discovered that contained an integrase domain and that appeared to arise from rna of viral-like particles present in tissue homogenates of shrimp from msgs ponds.
RESULTS
density gradient separations from tissue homogenates
based on previous work suggesting that the causative agent of msgs were viral in nature  <cit> , the gills of shrimp from msgs ponds were subjected to homogenization and sucrose density gradient centrifugation that yielded several bands in the range of 10-50%  sucrose .
each band was collected separately and subjected to a second step of purification using 10-40%  cscl gradients.
fractions of the single bands obtained from cscl gradients were placed on coated grids and negatively stained with 2% phosphotungstic acid  at ph  <dig>  for transmission electron microscopy .
this process revealed that a band at 20-25%  cscl gave presumptive viral-like particles.
a third round of gradient separation yielded a distinct band at 21% cscl  <cit>  .
tem of negatively-stained samples from this band revealed the presence of viral-like particles in two diameters of approximately  <dig> nm and  <dig> nm and with rounded to angular shapes indicating possible icosahedral morphology .
these were similar in size and shape to those seen by tem in tissue sections of stunted shrimp from msgs ponds .
shotgun cloning and screening for unknown, non-shrimp clones
from a total of  <dig> clones obtained from shotgun cloning of the nucleic acid extract from the 21% cscl density gradient band,  <dig> did not hybridize with labeled dna from normal shrimp in dot-blot hybridization assays, and these were selected for nucleic acid sequencing.
partial sequencing from both ends revealed that  <dig> clones contained shrimp sequences and  <dig> bacterial sequences.
these clones were not studied further.
of the  <dig> remaining clones, clustal w analysis of insert end-sequences revealed that all contained common sequence regions.
thus,  <dig> of the largest inserts of approximately  <dig>  kb called b <dig>  and c <dig>  were subjected to single-pass sequencing.
this revealed that the sequence of c <dig> fell completely within the sequence of b <dig>  primers were designed based on the consensus sequence for c010/b <dig> to produce amplicons of  <dig> bp from the 5' end ,  <dig> bp from the middle  and  <dig> bp from the 3' end   of the sequence using pcr and rt-pcr amplification with dnase-treated and rnase-treated total nucleic acid extracts from the 21% cscl band.
the expected amplicons were obtained only by rt-pcr from the dnase-treated rna extract .
an additional rt-pcr reaction was carried out using the forward primer of probe  <dig> and the reverse primer of probe  <dig> to obtain a predicted amplicon of  <dig> bp  that was cloned and sequenced from both strands.
all the data was combined to obtain the final consensus sequence of  <dig> bp shown in figure  <dig> .
this was subjected to a blastn search that revealed no significant identity to known nucleic acid sequences except for 100% identity in  <dig> bases near the 3' end of the sequence with thrombospondin of penaeus  japonicas .
a frame + <dig> translation of the  <dig> bp sequence yielded a single uninterrupted sequence of  <dig> deduced amino acids that had no known homology to any protein or translated protein sequence at genbank except for an integrase protein of bacteriophage sh <dig> of acinetobacter johnsonii  near the 3' end, but at low identity .
the deduced amino acids at positions 1894- <dig>  in the c-terminal portion of the sequence had homology to a conserved domain for phage integrases and dna breaking enzymes in the dna_bre_c family, as outlined in grey in figure  <dig> and shown in alignment with similar sequences in figure  <dig>  thus, for convenience, the sequence in figure  <dig> was called an integrase containing element .
the acronym ice as used herein should not be confused with that used for "integrating and conjugative elements " in bacteria  <cit> .
our several attempts to extend the ice sequence by either 3' or 5' race failed.
lsnv also in the 21% cscl gradient band
total nucleic acid extracts from the 21% cscl gradient band also gave positive rt-pcr reactions for lsnv.
attempts to remove lsnv reactivity by further density gradient centrifugation failed.
therefore, it is important to understand that the inocula used for all challenge tests consisted of a mixture of at least lsnv and ice.
in addition to lsnv, the extract was tested and found negative for ihhnv by pcr.
northern blot of rna from the 21% cscl gradient band
when rna extracted from the 21% cscl gradient band was subjected to northern blot analysis using ice probe  <dig>  a positive hybridization signal at approximately  <dig>  kb was obtained for ice, while no signal was detected in the rna extracted from spf shrimp .
pcr screening for ice and lsnv in shrimp from msgs ponds
when one-step rt-pcr protocols for ice probe  <dig> and for lsnv were used with template rna extracted from gills of shrimp from ponds that fit the msgs case definition, it was revealed that most of the shrimp tested gave positive reactions for both lsnv and ice .
by contrast, domesticated p. monodon specimens in families with no history of association with msgs and shrimp from normal growth ponds were always negative for ice even though some were positive for lsnv .
sample
chanthaburi/
laem-singh district chanthaburi/
nakornsrithammarat
nakornsrithammarat/
chanthaburi
in situ hybridization tests for ice and lsnv in shrimp from msgs ponds
using a rhodamine-labeled probe  prepared from ice and a fluorescence probe  prepared from a fragment of lsnv for in situ hybridization tests with lymphoid organ  tissue of shrimp from msgs ponds resulted in positive results for both lsnv and ice .
viral-like particles in lo tissue positive for ice and lsnv by in situ hybridization
when parallel tissue samples from shrimp showing positive hybridization with ice probes were subjected to examination by transmission electron microscopy , non-enveloped, icosahedral viral-like particles approximately  <dig> nm and  <dig> nm diameter were seen, similar to those previously shown for samples from msgs ponds .
shrimp challenge tests using inoculum from the 21% cscl gradient band
in an attempt to fulfil koch's postulates as modified by rivers , two challenge tests were carried out.
in the first challenge, primary inoculum was prepared from the original 21% cscl gradient band and injected into juvenile p. monodon that were maintained for  <dig> days before analysis and also for preparation of inoculum  for a second challenge test.
in the second challenge test, the secondary inoculum was injected into a second set of test shrimp, also maintained for  <dig> days before analysis.
the size of the domesticated shrimp used for the first challenge was  <dig>  ±  <dig>  g while that for the second was  <dig>  ±  <dig>  g. some of the stock shrimp were positive for lsnv by rt-pcr .
at the end of  <dig> days in the first challenge experiment, there was no significant difference in survival between the test  and control  groups.
one-step or nested rt-pcr  revealed that all test and many control shrimp  were positive for lsnv by nested rt-pcr, although the overall lsnv reactions tended to be more prevalent  and stronger  than those from the control shrimp when using the same amount of total rna template.
by contrast, only the test shrimp were positive for ice by either one-step or nested rt-pcr.
with respect to other parameters, there were no significant differences  in mean length, weight or survival between the test and control shrimp although the means for weight and length in the test group were lower than those in the control groups  and the statistics software package advised caution in interpreting the negative t-test results because the power for the tests was low .
in addition, the test group showed a greater tendency for abnormally dark coloration and brittle antennae than did the control group.
at the end of  <dig> days in the second challenge experiment, there was again no significant difference in survival between the test  and control  groups.
as with the first test, there was a tendency for abnormally dark coloration and brittle antennae in the test but not the control group .
also, one-step or nested rt-pcr  revealed that all of the test shrimp and many of the control shrimp were positive for lsnv by nested rt-pcr, while only the test shrimp were positive for ice by either one-step or nested rt-pcr .
however, in the second challenge experiment, the test group had significantly lower mean weight , weight gain , length  and length gain  than the control group .
for totals, the numbers with asterisks in each row were significantly different .
in situ hybridization tests with experimentally challenged shrimp
an arbitrary selection of  <dig> shrimp each from the control and test groups from the second challenge experiment was subjected to in situ hybridization assays for ice and lsnv.
because of the previously described link between lsnv retinopathy and msgs, the assays focused on tissues of the eyes and eyestalks in addition to the lo and gills.
results for the lo and gills showed that control shrimp positive for lsnv by rt-pcr gave positive hybridization results in the cell cytoplasm for lsnv only , while those negative by rt-pcr gave negative hybridization results for both ice and lsnv .
by contrast, the test shrimp gave positive hybridization results in the cell cytoplasm of lo cells for both ice and lsnv .
the fluorescence for both probes was evenly distributed in the cytoplasm, did not completely overlap and was most intense in normal lo tubules, as opposed to lo spheroids.
the eyes of both test and control shrimp showed no retinopathy, but those of the control shrimp that tested either negative or positive for lsnv were negative for both lsnv and ice  while test shrimp showed positive in situ hybridization reactions for both ice and lsnv .
these results supported the previously described link between retinopathy and lsnv  <cit> .
in addition to the lo and eyes, gills of the test shrimp only were also positive for both ice and lsnv .
discussion
the nature of ice
here we have shown the presence of an integrase containing element  that is located together with laem-singh virus  in the cytoplasm of cells of the lymphoid organ , gills and the sub-retinal, fasciculated zone of the eyes of black tiger shrimp exhibiting gross signs of disease referred to as monodon slow growth syndrome .
since ice was isolated from a density gradient band derived from gill tissue homogenates of shrimp from an msgs pond and since the gradient band contained two sizes of viral-like particles  it is possible that ice originated from these particles.
since the extracts containing ice also contained genomic material from lsnv, and since the diameter of unenveloped lsnv particles has been estimated at approximately  <dig> nm from thin tissue sections  <cit> , it is reasonable to suggest that ice may have originated from the particles  <dig> nm in diameter.
if so, the positive rt-pcr reactions indicated that the particles contained the ice fragment in the form of rna.
although this is reminiscent of extra small virus  that has been described from the giant freshwater prawn macrobrachium rosenbergii  <cit>  as a possible satellite virus  <cit> , the ice sequence shared no significant identity to the xsv genome sequence or any other known satellite virus sequence.
nor does the xsv genome or other known satellite genomes contain integrase genes  <cit> .
similarly, the ice sequence showed no significant homology to the genomes of unenveloped rna viruses with integrase genes that can be found in the families pseudoviridae  <cit>  and metaviridae  <cit>  that are related to enveloped viruses in the family retroviridae  <cit> .
in the pseudoviridae and metaviridae viral-like particles are sometimes produced in insect hosts, but the ones reported are much larger than  <dig> nm.
in addition, phylogenetic comparisons among the reverse-transcribing viruses are usually based on their reverse transcriptase  sequences and the lack of an rt sequence from ice made such a comparison impossible.
ice also contained no group-specific antigen-like  sequence for structural proteins, another common feature of viral-like members of the families pseudoviridae and metaviridae.
if the problem of unsuccessful 3' and 5' race reactions could be resolved with ice, rt and gag sequences might be revealed and allow standard phylogenetic comparisons.
on the other hand, the northern blot result indicated that the full size of the rna fragment containing the ice target sequence was not a great deal larger than the ice clone raising the question as to whether it would be sufficiently large to contain rt and gag sequences.
although ice clearly contained an integrase domain with a conserved catalytic triad h-r-h and a conserved tyrosine  in a binding region that may be involved in host genome insertion  <cit> , the  <dig> amino-acid integrase domain showed homology only to integrases or recombinases of bacteria and bacteriophages and not viral-like members of the families pseudoviridae and metaviridae.
this is also problematic.
one might ask whether the ice sequence arose from bacteria in the shrimp homogenates.
we believe this to be unlikely since inspection by light microscopy and tem revealed no bacteria in the parallel tissue sections that were positive for ice by in situ hybridization.
thus, the nature of ice and its phylogenetic relationship to other viral-like entities known to occur in invertebrates is an enigma.
association of ice with lsnv and msgs
despite the uncertainty regarding the nature and phylogenetic relationships of ice, we have shown that there is a strong tendency for it to be associated with lsnv in shrimp samples from msgs ponds, and in shrimp challenged with inoculum from a density gradient band derived from tissue homogenates of shrimp from msgs ponds.
furthermore, in situ hybridization analysis revealed that ice and lsnv occur together in lo and eyestalk tissues previously shown to give positive reactions for lsnv in stunted shrimp from msgs ponds .
in no case were shrimp found that were positive for ice in the absence of lsnv.
since lsnv was proposed to be a necessary but insufficient cause of msgs based on its association with retinal lesions only in small shrimp from msgs ponds, the co-association of ice with lsnv in such lesions suggests that it may also be causally linked to msgs.
although the inability to clearly separate ice from lsnv for laboratory challenge tests makes it difficult to prove this causal relationship, our results using inoculum positive for ice and lsnv to challenge shrimp already positive for lsnv did support the argument that they may be co-causes of msgs.
this is because the control shrimp already infected with lsnv did not show retarded growth, darkening or in situ positive retinal tissues that were seen in the challenged shrimp, even though the lo of the control shrimp were positive for lsnv.
it is true that the ice and lsnv positive reactions in the test shrimp were not associated with retinal lesions in our challenge tests, but this may have been due to the age of the shrimp at the time of challenge or other experimental parameters that differed from those in shrimp ponds.
in addition, it cannot be ruled out that the inoculum contained yet another viral-like agent that was not identified but moved together with lsnv and ice.
thus, final proof that lsnv and ice are causally associated with msgs will probably have to await the advent of infectious clones of ice and lsnv so that perfectly controlled single and dual challenge tests can be carried out.
unfortunately, complete sequences of neither ice nor lsnv are yet available.
CONCLUSIONS
an ice clone was derived from a process of differential centrifugation that would have eliminated bacteria from the final cscl gradient, and the gradient band contained no bacterial cells.
the nucleic acid clone derived from that band had no sequence identity to any known bacterium, and pcr and rt-pcr tests with that nucleic acid revealed that the clone arose from rna, not dna.
further, an in situ hybridization probe derived from that sequence gave positive hybridization results in the cytoplasm of cells of the lymphoid organ and eyes of shrimp where no bacterial cells were seen by either light microscopy or by electron microscopy, but where viral-like particles were seen by electron microscopy.
finally, the inoculum for the challenge trials was prepared with ultrafiltration steps that would have removed bacterial cells.
altogether, we may conclude that the ice element is not from a bacterium, but that its nature remains elusive.
despite this ambiguity, we found that ice occurred together with lsnv only in shrimp from msgs ponds and only in shrimp showing gross signs of msgs after injection with a preparation containing ice and lsnv.
ice was never found in the absence of lsnv although lsnv was sometimes found in normal shrimp in the absence of ice.
this association between ice and lsnv suggests that they may act together as component causes of msgs.
however, conclusive proof that they cause msgs is not possible without single and combined bioassays using purified preparations of both ice and lsnv.
since the discovery of lsnv in  <dig>  it has been added to the list of viruses to be excluded from domesticated, specific pathogen free  stocks of penaeus monodon in thailand and it has been recommended that shrimp farmers avoid stocking post larvae rt-pcr positive for lsnv to avoid msgs.
in the same way, we recommend that ice be added to the list of excludable agents in development of spf stocks, since it is not highly prevalent in wild stocks of p. monodon that might be sourced as founders for such stocks.
in addition, the availability of rt-pcr methods makes it possible to carry out continual monitoring in research programs to determine whether there is a positive correlation between msgs and simultaneous presence of lsnv and ice in cultivated p. monodon.

 the term of monodon slow growth syndrome has been used by thai shrimp farmers to refer to unusual retarded growth that has occurred in cultivated p. monodon since it has been suggested that an infectious agent may be the cause. this contention was supported by the rapid spread of the problem and preliminary laboratory challenge tests showing that membrane-filtered tissue extracts from msgs shrimp were able to cause msgs when injected into p. monodon but not when injected into p. vannamei. thai researchers adopted a case definition to distinguish ponds exhibiting msgs from ponds exhibiting slow growth caused by other problems. according to this case definition, an msgs pond must show a coefficient of size variation ≥35% together with freedom from hepatopancreatic parvovirus or any other severe hepatopancreas infection, plus any of the following characters: 1) unusually dark color, 2) average daily weight gain of less than g/day at months, 3) unusually bright yellow markings, 4) "bamboo-shaped" abdominal segments, and 5) brittle antennae. these features distinguish msgs from stunted growth caused by monodon baculovirus or hpv. an earlier survey revealed no correlation between the presence of several known shrimp pathogens and the problem of msgs ponds. the results suggested that a new pathogen might be the cause of msgs. separation of the purported agent from shrimp tissue homogenates has been difficult because of the presence of one or more known viruses and because bioassays require long incubation times of up to month or more before results can be assessed. therefore, we adopted a general "shot-gun" strategy to screen msgs cases for the presence of unknown pathogens. briefly, it consisted of obtaining shrimp from msgs case ponds followed by preparation of "viral" extracts for fractionation by gradient centrifugation. the resulting bands were removed and used for total nucleic acid extraction before shotgun cloning of cdna prepared using random hexamer primers. since total nucleic acid was used as the template, both rna and dna were amplified. clones that hybridized with labelled, total dna from normal p. monodon were eliminated and negative hybridization clones were sent for sequencing. clones identified by blast analysis to contain sequences of shrimp or previously screened pathogens were discarded, and remaining clones were used for in situ hybridization assays with shrimp from msgs ponds and from normal growth ponds. using this strategy, one new rna virus was identified but its presence in shrimp was not initially correlated with the occurrence of msgs ponds. however, subsequent work revealed that stunted shrimp from msgs ponds gave strong in situ hybridization reactions for lsnv in retinal lesions that were not present in large shrimp from msgs ponds or in shrimp from normal growth ponds that gave positive rt-pcr reactions for the presence of lsnv. this led to the conclusion that lsnv was a necessary but not sufficient cause of msgs and suggested that another factor must be combined with lsnv to cause msgs. one possibility for the additional factor was another pathogen. to examine this possibility, the shotgun strategy was used for further screening of shrimp from msgs ponds. as a result, a unique clone was discovered that contained an integrase domain and that appeared to arise from rna of viral-like particles present in tissue homogenates of shrimp from msgs ponds.  density gradient separations from tissue homogenates based on previous work suggesting that the causative agent of msgs were viral in nature, the gills of shrimp from msgs ponds were subjected to homogenization and sucrose density gradient centrifugation that yielded several bands in the range of 10-50% sucrose. each band was collected separately and subjected to a second step of purification using 10-40% cscl gradients. fractions of the single bands obtained from cscl gradients were placed on coated grids and negatively stained with 2% phosphotungstic acid at ph for transmission electron microscopy. this process revealed that a band at 20-25% cscl gave presumptive viral-like particles. a third round of gradient separation yielded a distinct band at 21% cscl. tem of negatively-stained samples from this band revealed the presence of viral-like particles in two diameters of approximately nm and nm and with rounded to angular shapes indicating possible icosahedral morphology. these were similar in size and shape to those seen by tem in tissue sections of stunted shrimp from msgs ponds. shotgun cloning and screening for unknown, non-shrimp clones from a total of clones obtained from shotgun cloning of the nucleic acid extract from the 21% cscl density gradient band, did not hybridize with labeled dna from normal shrimp in dot-blot hybridization assays, and these were selected for nucleic acid sequencing. partial sequencing from both ends revealed that clones contained shrimp sequences and bacterial sequences. these clones were not studied further. of the remaining clones, clustal w analysis of insert end-sequences revealed that all contained common sequence regions. thus, of the largest inserts of approximately kb called b and c were subjected to single-pass sequencing. this revealed that the sequence of c fell completely within the sequence of b primers were designed based on the consensus sequence for c010/b to produce amplicons of bp from the 5' end, bp from the middle and bp from the 3' end of the sequence using pcr and rt-pcr amplification with dnase-treated and rnase-treated total nucleic acid extracts from the 21% cscl band. the expected amplicons were obtained only by rt-pcr from the dnase-treated rna extract. an additional rt-pcr reaction was carried out using the forward primer of probe and the reverse primer of probe to obtain a predicted amplicon of bp that was cloned and sequenced from both strands. all the data was combined to obtain the final consensus sequence of bp shown in figure. this was subjected to a blastn search that revealed no significant identity to known nucleic acid sequences except for 100% identity in bases near the 3' end of the sequence with thrombospondin of penaeus japonicas. a frame + translation of the bp sequence yielded a single uninterrupted sequence of deduced amino acids that had no known homology to any protein or translated protein sequence at genbank except for an integrase protein of bacteriophage sh of acinetobacter johnsonii near the 3' end, but at low identity. the deduced amino acids at positions 1894- in the c-terminal portion of the sequence had homology to a conserved domain for phage integrases and dna breaking enzymes in the dna_bre_c family, as outlined in grey in figure and shown in alignment with similar sequences in figure thus, for convenience, the sequence in figure was called an integrase containing element. the acronym ice as used herein should not be confused with that used for "integrating and conjugative elements " in bacteria. our several attempts to extend the ice sequence by either 3' or 5' race failed. lsnv also in the 21% cscl gradient band total nucleic acid extracts from the 21% cscl gradient band also gave positive rt-pcr reactions for lsnv. attempts to remove lsnv reactivity by further density gradient centrifugation failed. therefore, it is important to understand that the inocula used for all challenge tests consisted of a mixture of at least lsnv and ice. in addition to lsnv, the extract was tested and found negative for ihhnv by pcr. northern blot of rna from the 21% cscl gradient band when rna extracted from the 21% cscl gradient band was subjected to northern blot analysis using ice probe a positive hybridization signal at approximately kb was obtained for ice, while no signal was detected in the rna extracted from spf shrimp. pcr screening for ice and lsnv in shrimp from msgs ponds when one-step rt-pcr protocols for ice probe and for lsnv were used with template rna extracted from gills of shrimp from ponds that fit the msgs case definition, it was revealed that most of the shrimp tested gave positive reactions for both lsnv and ice. by contrast, domesticated p. monodon specimens in families with no history of association with msgs and shrimp from normal growth ponds were always negative for ice even though some were positive for lsnv. sample chanthaburi/ laem-singh district chanthaburi/ nakornsrithammarat nakornsrithammarat/ chanthaburi in situ hybridization tests for ice and lsnv in shrimp from msgs ponds using a rhodamine-labeled probe prepared from ice and a fluorescence probe prepared from a fragment of lsnv for in situ hybridization tests with lymphoid organ tissue of shrimp from msgs ponds resulted in positive results for both lsnv and ice. viral-like particles in lo tissue positive for ice and lsnv by in situ hybridization when parallel tissue samples from shrimp showing positive hybridization with ice probes were subjected to examination by transmission electron microscopy, non-enveloped, icosahedral viral-like particles approximately nm and nm diameter were seen, similar to those previously shown for samples from msgs ponds. shrimp challenge tests using inoculum from the 21% cscl gradient band in an attempt to fulfil koch's postulates as modified by rivers, two challenge tests were carried out. in the first challenge, primary inoculum was prepared from the original 21% cscl gradient band and injected into juvenile p. monodon that were maintained for days before analysis and also for preparation of inoculum for a second challenge test. in the second challenge test, the secondary inoculum was injected into a second set of test shrimp, also maintained for days before analysis. the size of the domesticated shrimp used for the first challenge was ± g while that for the second was ± g. some of the stock shrimp were positive for lsnv by rt-pcr. at the end of days in the first challenge experiment, there was no significant difference in survival between the test and control groups. one-step or nested rt-pcr revealed that all test and many control shrimp were positive for lsnv by nested rt-pcr, although the overall lsnv reactions tended to be more prevalent and stronger than those from the control shrimp when using the same amount of total rna template. by contrast, only the test shrimp were positive for ice by either one-step or nested rt-pcr. with respect to other parameters, there were no significant differences in mean length, weight or survival between the test and control shrimp although the means for weight and length in the test group were lower than those in the control groups and the statistics software package advised caution in interpreting the negative t-test results because the power for the tests was low. in addition, the test group showed a greater tendency for abnormally dark coloration and brittle antennae than did the control group. at the end of days in the second challenge experiment, there was again no significant difference in survival between the test and control groups. as with the first test, there was a tendency for abnormally dark coloration and brittle antennae in the test but not the control group. also, one-step or nested rt-pcr revealed that all of the test shrimp and many of the control shrimp were positive for lsnv by nested rt-pcr, while only the test shrimp were positive for ice by either one-step or nested rt-pcr. however, in the second challenge experiment, the test group had significantly lower mean weight, weight gain, length and length gain than the control group. for totals, the numbers with asterisks in each row were significantly different. in situ hybridization tests with experimentally challenged shrimp an arbitrary selection of shrimp each from the control and test groups from the second challenge experiment was subjected to in situ hybridization assays for ice and lsnv. because of the previously described link between lsnv retinopathy and msgs, the assays focused on tissues of the eyes and eyestalks in addition to the lo and gills. results for the lo and gills showed that control shrimp positive for lsnv by rt-pcr gave positive hybridization results in the cell cytoplasm for lsnv only, while those negative by rt-pcr gave negative hybridization results for both ice and lsnv. by contrast, the test shrimp gave positive hybridization results in the cell cytoplasm of lo cells for both ice and lsnv. the fluorescence for both probes was evenly distributed in the cytoplasm, did not completely overlap and was most intense in normal lo tubules, as opposed to lo spheroids. the eyes of both test and control shrimp showed no retinopathy, but those of the control shrimp that tested either negative or positive for lsnv were negative for both lsnv and ice while test shrimp showed positive in situ hybridization reactions for both ice and lsnv. these results supported the previously described link between retinopathy and lsnv. in addition to the lo and eyes, gills of the test shrimp only were also positive for both ice and lsnv. discussion the nature of ice here we have shown the presence of an integrase containing element that is located together with laem-singh virus in the cytoplasm of cells of the lymphoid organ, gills and the sub-retinal, fasciculated zone of the eyes of black tiger shrimp exhibiting gross signs of disease referred to as monodon slow growth syndrome. since ice was isolated from a density gradient band derived from gill tissue homogenates of shrimp from an msgs pond and since the gradient band contained two sizes of viral-like particles it is possible that ice originated from these particles. since the extracts containing ice also contained genomic material from lsnv, and since the diameter of unenveloped lsnv particles has been estimated at approximately nm from thin tissue sections, it is reasonable to suggest that ice may have originated from the particles nm in diameter. if so, the positive rt-pcr reactions indicated that the particles contained the ice fragment in the form of rna. although this is reminiscent of extra small virus that has been described from the giant freshwater prawn macrobrachium rosenbergii as a possible satellite virus, the ice sequence shared no significant identity to the xsv genome sequence or any other known satellite virus sequence. nor does the xsv genome or other known satellite genomes contain integrase genes. similarly, the ice sequence showed no significant homology to the genomes of unenveloped rna viruses with integrase genes that can be found in the families pseudoviridae and metaviridae that are related to enveloped viruses in the family retroviridae. in the pseudoviridae and metaviridae viral-like particles are sometimes produced in insect hosts, but the ones reported are much larger than nm. in addition, phylogenetic comparisons among the reverse-transcribing viruses are usually based on their reverse transcriptase sequences and the lack of an rt sequence from ice made such a comparison impossible. ice also contained no group-specific antigen-like sequence for structural proteins, another common feature of viral-like members of the families pseudoviridae and metaviridae. if the problem of unsuccessful 3' and 5' race reactions could be resolved with ice, rt and gag sequences might be revealed and allow standard phylogenetic comparisons. on the other hand, the northern blot result indicated that the full size of the rna fragment containing the ice target sequence was not a great deal larger than the ice clone raising the question as to whether it would be sufficiently large to contain rt and gag sequences. although ice clearly contained an integrase domain with a conserved catalytic triad h-r-h and a conserved tyrosine in a binding region that may be involved in host genome insertion, the amino-acid integrase domain showed homology only to integrases or recombinases of bacteria and bacteriophages and not viral-like members of the families pseudoviridae and metaviridae. this is also problematic. one might ask whether the ice sequence arose from bacteria in the shrimp homogenates. we believe this to be unlikely since inspection by light microscopy and tem revealed no bacteria in the parallel tissue sections that were positive for ice by in situ hybridization. thus, the nature of ice and its phylogenetic relationship to other viral-like entities known to occur in invertebrates is an enigma. association of ice with lsnv and msgs despite the uncertainty regarding the nature and phylogenetic relationships of ice, we have shown that there is a strong tendency for it to be associated with lsnv in shrimp samples from msgs ponds, and in shrimp challenged with inoculum from a density gradient band derived from tissue homogenates of shrimp from msgs ponds. furthermore, in situ hybridization analysis revealed that ice and lsnv occur together in lo and eyestalk tissues previously shown to give positive reactions for lsnv in stunted shrimp from msgs ponds. in no case were shrimp found that were positive for ice in the absence of lsnv. since lsnv was proposed to be a necessary but insufficient cause of msgs based on its association with retinal lesions only in small shrimp from msgs ponds, the co-association of ice with lsnv in such lesions suggests that it may also be causally linked to msgs. although the inability to clearly separate ice from lsnv for laboratory challenge tests makes it difficult to prove this causal relationship, our results using inoculum positive for ice and lsnv to challenge shrimp already positive for lsnv did support the argument that they may be co-causes of msgs. this is because the control shrimp already infected with lsnv did not show retarded growth, darkening or in situ positive retinal tissues that were seen in the challenged shrimp, even though the lo of the control shrimp were positive for lsnv. it is true that the ice and lsnv positive reactions in the test shrimp were not associated with retinal lesions in our challenge tests, but this may have been due to the age of the shrimp at the time of challenge or other experimental parameters that differed from those in shrimp ponds. in addition, it cannot be ruled out that the inoculum contained yet another viral-like agent that was not identified but moved together with lsnv and ice. thus, final proof that lsnv and ice are causally associated with msgs will probably have to await the advent of infectious clones of ice and lsnv so that perfectly controlled single and dual challenge tests can be carried out. unfortunately, complete sequences of neither ice nor lsnv are yet available.  an ice clone was derived from a process of differential centrifugation that would have eliminated bacteria from the final cscl gradient, and the gradient band contained no bacterial cells. the nucleic acid clone derived from that band had no sequence identity to any known bacterium, and pcr and rt-pcr tests with that nucleic acid revealed that the clone arose from rna, not dna. further, an in situ hybridization probe derived from that sequence gave positive hybridization results in the cytoplasm of cells of the lymphoid organ and eyes of shrimp where no bacterial cells were seen by either light microscopy or by electron microscopy, but where viral-like particles were seen by electron microscopy. finally, the inoculum for the challenge trials was prepared with ultrafiltration steps that would have removed bacterial cells. altogether, we may conclude that the ice element is not from a bacterium, but that its nature remains elusive. despite this ambiguity, we found that ice occurred together with lsnv only in shrimp from msgs ponds and only in shrimp showing gross signs of msgs after injection with a preparation containing ice and lsnv. ice was never found in the absence of lsnv although lsnv was sometimes found in normal shrimp in the absence of ice. this association between ice and lsnv suggests that they may act together as component causes of msgs. however, conclusive proof that they cause msgs is not possible without single and combined bioassays using purified preparations of both ice and lsnv. since the discovery of lsnv in it has been added to the list of viruses to be excluded from domesticated, specific pathogen free stocks of penaeus monodon in thailand and it has been recommended that shrimp farmers avoid stocking post larvae rt-pcr positive for lsnv to avoid msgs. in the same way, we recommend that ice be added to the list of excludable agents in development of spf stocks, since it is not highly prevalent in wild stocks of p. monodon that might be sourced as founders for such stocks. in addition, the availability of rt-pcr methods makes it possible to carry out continual monitoring in research programs to determine whether there is a positive correlation between msgs and simultaneous presence of lsnv and ice in cultivated p. monodon. 
bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of msgs, and in situ hybridization assays revealed ice and lsnv together in lo, eyes and gills. ice and lsnv were found together only in shrimp from msgs ponds and only in shrimp showing gross signs of msgs after injection with a preparation containing ice and lsnv. using a universal shotgun cloning protocol, a novel rna, integrase-containing element was found in giant tiger shrimp from msgs ponds. in situ hybridization probes and rt-pcr tests revealed that ice and laem-singh virus occurred together in lymphoid organs of shrimp from msgs ponds but not in shrimp from normal ponds. tissue homogenates of shrimp from msgs ponds yielded a fraction that gave positive rt-pcr reactions for both ice and lsnv and showed viral-like particles by transmission electron microscopy. from 2001- monodon slow growth syndrome caused severe economic losses for thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. ice was never found in the absence of lsnv although lsnv was sometimes found in normal shrimp in the absence of ice. in newly discovered laem-singh virus was proposed as a necessary but insufficient cause of msgs, and this stimulated the search for the additional component cause of msgs in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species. the results suggest that ice and lsnv may act together as component causes of msgs, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ice and lsnv. viral-like particles similar to those seen in tissue extracts from natural infections were also seen by tem. despite this ambiguity, it is recommended in the interim that ice be added to the agents such as lsnv already listed for exclusion from domesticated stocks of the black tiger shrimp.
 from 2001- monodon slow growth syndrome caused severe economic losses for thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. in newly discovered laem-singh virus was proposed as a necessary but insufficient cause of msgs, and this stimulated the search for the additional component cause of msgs in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.  using a universal shotgun cloning protocol, a novel rna, integrase-containing element was found in giant tiger shrimp from msgs ponds. in situ hybridization probes and rt-pcr tests revealed that ice and laem-singh virus occurred together in lymphoid organs of shrimp from msgs ponds but not in shrimp from normal ponds. tissue homogenates of shrimp from msgs ponds yielded a fraction that gave positive rt-pcr reactions for both ice and lsnv and showed viral-like particles by transmission electron microscopy. bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of msgs, and in situ hybridization assays revealed ice and lsnv together in lo, eyes and gills. viral-like particles similar to those seen in tissue extracts from natural infections were also seen by tem.  ice and lsnv were found together only in shrimp from msgs ponds and only in shrimp showing gross signs of msgs after injection with a preparation containing ice and lsnv. ice was never found in the absence of lsnv although lsnv was sometimes found in normal shrimp in the absence of ice. the results suggest that ice and lsnv may act together as component causes of msgs, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ice and lsnv. despite this ambiguity, it is recommended in the interim that ice be added to the agents such as lsnv already listed for exclusion from domesticated stocks of the black tiger shrimp. 