Limits...
A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp.

Panphut W, Senapin S, Sriurairatana S, Withyachumnarnkul B, Flegel TW - BMC Vet. Res. (2011)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centex Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

ABSTRACT

Background: From 2001-2003 monodon slow growth syndrome (MSGS) 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 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.

Results: Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) 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 (TEM). 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.

Conclusions: 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.

Show MeSH

Related in: MedlinePlus

PCR and RT-PCR results from density gradient band. (A) Electrophoresis gel showing PCR and RT-PCR results using primers for Probe 1 (397 bp), Probe 2 (642 bp) and Probe 3 (399 bp) to carry out PCR and RT-PCR reactions with total nucleic acid template extracted from the 21% CsCl band. Lanes 1-6: PCR reactions carried out using template treated with DNase (Lanes 1-3) and RNase (Lanes 4-6) and showing none of the expected amplicons. Lanes 7-12: RT-PCR reactions (Probes 1-3 sequentially) carried out using template treated with DNase (Lanes 7-9) and RNase (Lanes 10-12) and showing amplicons with DNase treatment but not RNase treatment, indicating that the ICE amplicons originated from RNA. Lanes +C, +M and +B = Positive controls for Probes 1 to 3, respectively. Lanes M = DNA ladder marker. (B) Electrophoresis gel showing the predicted 1958 bp amplicon obtained using the forward primer of Probe 1 and the reverse primer of Probe 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3117699&req=5

Figure 3: PCR and RT-PCR results from density gradient band. (A) Electrophoresis gel showing PCR and RT-PCR results using primers for Probe 1 (397 bp), Probe 2 (642 bp) and Probe 3 (399 bp) to carry out PCR and RT-PCR reactions with total nucleic acid template extracted from the 21% CsCl band. Lanes 1-6: PCR reactions carried out using template treated with DNase (Lanes 1-3) and RNase (Lanes 4-6) and showing none of the expected amplicons. Lanes 7-12: RT-PCR reactions (Probes 1-3 sequentially) carried out using template treated with DNase (Lanes 7-9) and RNase (Lanes 10-12) and showing amplicons with DNase treatment but not RNase treatment, indicating that the ICE amplicons originated from RNA. Lanes +C, +M and +B = Positive controls for Probes 1 to 3, respectively. Lanes M = DNA ladder marker. (B) Electrophoresis gel showing the predicted 1958 bp amplicon obtained using the forward primer of Probe 1 and the reverse primer of Probe 3.

Mentions: From a total of 180 clones obtained from shotgun cloning of the nucleic acid extract from the 21% CsCl density gradient band, 31 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 10 clones contained shrimp sequences and 4 bacterial sequences. These clones were not studied further. Of the 17 remaining clones, CLUSTAL W analysis of insert end-sequences revealed that all contained common sequence regions. Thus, 2 of the largest inserts of approximately 2.2 kb called B004 (2250 bp) and C010 (2223 bp) were subjected to single-pass sequencing. This revealed that the sequence of C010 fell completely within the sequence of B004. Primers were designed based on the consensus sequence for C010/B004 to produce amplicons of 397 bp from the 5' end (Probe 1), 642 bp from the middle (Probe 2) and 399 bp from the 3' end (Probe 3) (Table 1) 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 (Figure 3A). An additional RT-PCR reaction was carried out using the forward primer of Probe 1 and the reverse primer of Probe 3 to obtain a predicted amplicon of 1958 bp (Figure 3B) that was cloned and sequenced from both strands. All the data was combined to obtain the final consensus sequence of 2233 bp shown in Figure 4 (GenBank accession number FJ498866). This was subjected to a Blastn search that revealed no significant identity to known nucleic acid sequences except for 100% identity in 46 bases near the 3' end of the sequence with thrombospondin of Penaeus (Marsupenaeus) japonicas (GenBank AB121211). A frame +1 translation of the 2233 bp sequence yielded a single uninterrupted sequence of 744 deduced amino acids that had no known homology to any protein or translated protein sequence at GenBank except for an integrase protein of bacteriophage SH046 of Acinetobacter johnsonii (E value 1 × e-4) near the 3' end, but at low identity (29%). The deduced amino acids at positions 1894-2025 (44 amino acids) 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 4 and shown in alignment with similar sequences in Figure 5. Thus, for convenience, the sequence in Figure 4 was called an integrase containing element (ICE). The acronym ICE as used herein should not be confused with that used for "integrating and conjugative elements (ICEs)" in bacteria [10]. Our several attempts to extend the ICE sequence by either 3' or 5' RACE failed.


A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp.

Panphut W, Senapin S, Sriurairatana S, Withyachumnarnkul B, Flegel TW - BMC Vet. Res. (2011)

PCR and RT-PCR results from density gradient band. (A) Electrophoresis gel showing PCR and RT-PCR results using primers for Probe 1 (397 bp), Probe 2 (642 bp) and Probe 3 (399 bp) to carry out PCR and RT-PCR reactions with total nucleic acid template extracted from the 21% CsCl band. Lanes 1-6: PCR reactions carried out using template treated with DNase (Lanes 1-3) and RNase (Lanes 4-6) and showing none of the expected amplicons. Lanes 7-12: RT-PCR reactions (Probes 1-3 sequentially) carried out using template treated with DNase (Lanes 7-9) and RNase (Lanes 10-12) and showing amplicons with DNase treatment but not RNase treatment, indicating that the ICE amplicons originated from RNA. Lanes +C, +M and +B = Positive controls for Probes 1 to 3, respectively. Lanes M = DNA ladder marker. (B) Electrophoresis gel showing the predicted 1958 bp amplicon obtained using the forward primer of Probe 1 and the reverse primer of Probe 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117699&req=5

Figure 3: PCR and RT-PCR results from density gradient band. (A) Electrophoresis gel showing PCR and RT-PCR results using primers for Probe 1 (397 bp), Probe 2 (642 bp) and Probe 3 (399 bp) to carry out PCR and RT-PCR reactions with total nucleic acid template extracted from the 21% CsCl band. Lanes 1-6: PCR reactions carried out using template treated with DNase (Lanes 1-3) and RNase (Lanes 4-6) and showing none of the expected amplicons. Lanes 7-12: RT-PCR reactions (Probes 1-3 sequentially) carried out using template treated with DNase (Lanes 7-9) and RNase (Lanes 10-12) and showing amplicons with DNase treatment but not RNase treatment, indicating that the ICE amplicons originated from RNA. Lanes +C, +M and +B = Positive controls for Probes 1 to 3, respectively. Lanes M = DNA ladder marker. (B) Electrophoresis gel showing the predicted 1958 bp amplicon obtained using the forward primer of Probe 1 and the reverse primer of Probe 3.
Mentions: From a total of 180 clones obtained from shotgun cloning of the nucleic acid extract from the 21% CsCl density gradient band, 31 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 10 clones contained shrimp sequences and 4 bacterial sequences. These clones were not studied further. Of the 17 remaining clones, CLUSTAL W analysis of insert end-sequences revealed that all contained common sequence regions. Thus, 2 of the largest inserts of approximately 2.2 kb called B004 (2250 bp) and C010 (2223 bp) were subjected to single-pass sequencing. This revealed that the sequence of C010 fell completely within the sequence of B004. Primers were designed based on the consensus sequence for C010/B004 to produce amplicons of 397 bp from the 5' end (Probe 1), 642 bp from the middle (Probe 2) and 399 bp from the 3' end (Probe 3) (Table 1) 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 (Figure 3A). An additional RT-PCR reaction was carried out using the forward primer of Probe 1 and the reverse primer of Probe 3 to obtain a predicted amplicon of 1958 bp (Figure 3B) that was cloned and sequenced from both strands. All the data was combined to obtain the final consensus sequence of 2233 bp shown in Figure 4 (GenBank accession number FJ498866). This was subjected to a Blastn search that revealed no significant identity to known nucleic acid sequences except for 100% identity in 46 bases near the 3' end of the sequence with thrombospondin of Penaeus (Marsupenaeus) japonicas (GenBank AB121211). A frame +1 translation of the 2233 bp sequence yielded a single uninterrupted sequence of 744 deduced amino acids that had no known homology to any protein or translated protein sequence at GenBank except for an integrase protein of bacteriophage SH046 of Acinetobacter johnsonii (E value 1 × e-4) near the 3' end, but at low identity (29%). The deduced amino acids at positions 1894-2025 (44 amino acids) 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 4 and shown in alignment with similar sequences in Figure 5. Thus, for convenience, the sequence in Figure 4 was called an integrase containing element (ICE). The acronym ICE as used herein should not be confused with that used for "integrating and conjugative elements (ICEs)" in bacteria [10]. Our several attempts to extend the ICE sequence by either 3' or 5' RACE failed.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centex Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

ABSTRACT

Background: From 2001-2003 monodon slow growth syndrome (MSGS) 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 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.

Results: Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) 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 (TEM). 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus