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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.

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High magnification in situ hybridization of LO from bioassay. High magnification example of an in situ hybridization reaction using a rhodamine-labeled ICE probe (red) together with an FITC-labeled LSNV probe (green) with lymphoid organ (LO) tissue of a P. monodon specimen from challenge test 1 that was positive by RT-PCR for both ICE and LSNV. This is a photomicrograph of a single confocal microscope image layer showing that the fluorescence distribution for ICE and LSNV is in the cell cytoplasm and not the nuclei. A lower magnification photomicrograph showing brighter fluorescence from multiple confocal microscope image layers is shown in Additional file 4. a) Phase contrast image; b) Image of LSNV fluorescence; c) Image of ICE fluorescence; d) Combined images with a magnified insert showing that the fluorescence distribution for the two signals is single (green or red) and combined (yellow). Note that the fluorescence is located in the cell cytoplasm and is most intense in the normal tubules of the LO and much less intense in the spheroids (i.e., the circular groups of cells surrounded by a ring of sheath cells that are most clearly visible in the combined image d). Negative control images from a normal shrimp specimen are shown in Additional file 3.
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Figure 8: High magnification in situ hybridization of LO from bioassay. High magnification example of an in situ hybridization reaction using a rhodamine-labeled ICE probe (red) together with an FITC-labeled LSNV probe (green) with lymphoid organ (LO) tissue of a P. monodon specimen from challenge test 1 that was positive by RT-PCR for both ICE and LSNV. This is a photomicrograph of a single confocal microscope image layer showing that the fluorescence distribution for ICE and LSNV is in the cell cytoplasm and not the nuclei. A lower magnification photomicrograph showing brighter fluorescence from multiple confocal microscope image layers is shown in Additional file 4. a) Phase contrast image; b) Image of LSNV fluorescence; c) Image of ICE fluorescence; d) Combined images with a magnified insert showing that the fluorescence distribution for the two signals is single (green or red) and combined (yellow). Note that the fluorescence is located in the cell cytoplasm and is most intense in the normal tubules of the LO and much less intense in the spheroids (i.e., the circular groups of cells surrounded by a ring of sheath cells that are most clearly visible in the combined image d). Negative control images from a normal shrimp specimen are shown in Additional file 3.

Mentions: An arbitrary selection of 5 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 (not shown), while those negative by RT-PCR gave negative hybridization results for both ICE and LSNV (Additional file 3). By contrast, the test shrimp gave positive hybridization results in the cell cytoplasm of LO cells for both ICE and LSNV (Figure 8 and Additional file 4). 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 (Additional file 5) while test shrimp showed positive in situ hybridization reactions for both ICE and LSNV (Figure 9). These results supported the previously described link between retinopathy and LSNV [8]. In addition to the LO and eyes, gills of the test shrimp only were also positive for both ICE and LSNV (Figure 10).


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)

High magnification in situ hybridization of LO from bioassay. High magnification example of an in situ hybridization reaction using a rhodamine-labeled ICE probe (red) together with an FITC-labeled LSNV probe (green) with lymphoid organ (LO) tissue of a P. monodon specimen from challenge test 1 that was positive by RT-PCR for both ICE and LSNV. This is a photomicrograph of a single confocal microscope image layer showing that the fluorescence distribution for ICE and LSNV is in the cell cytoplasm and not the nuclei. A lower magnification photomicrograph showing brighter fluorescence from multiple confocal microscope image layers is shown in Additional file 4. a) Phase contrast image; b) Image of LSNV fluorescence; c) Image of ICE fluorescence; d) Combined images with a magnified insert showing that the fluorescence distribution for the two signals is single (green or red) and combined (yellow). Note that the fluorescence is located in the cell cytoplasm and is most intense in the normal tubules of the LO and much less intense in the spheroids (i.e., the circular groups of cells surrounded by a ring of sheath cells that are most clearly visible in the combined image d). Negative control images from a normal shrimp specimen are shown in Additional file 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: High magnification in situ hybridization of LO from bioassay. High magnification example of an in situ hybridization reaction using a rhodamine-labeled ICE probe (red) together with an FITC-labeled LSNV probe (green) with lymphoid organ (LO) tissue of a P. monodon specimen from challenge test 1 that was positive by RT-PCR for both ICE and LSNV. This is a photomicrograph of a single confocal microscope image layer showing that the fluorescence distribution for ICE and LSNV is in the cell cytoplasm and not the nuclei. A lower magnification photomicrograph showing brighter fluorescence from multiple confocal microscope image layers is shown in Additional file 4. a) Phase contrast image; b) Image of LSNV fluorescence; c) Image of ICE fluorescence; d) Combined images with a magnified insert showing that the fluorescence distribution for the two signals is single (green or red) and combined (yellow). Note that the fluorescence is located in the cell cytoplasm and is most intense in the normal tubules of the LO and much less intense in the spheroids (i.e., the circular groups of cells surrounded by a ring of sheath cells that are most clearly visible in the combined image d). Negative control images from a normal shrimp specimen are shown in Additional file 3.
Mentions: An arbitrary selection of 5 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 (not shown), while those negative by RT-PCR gave negative hybridization results for both ICE and LSNV (Additional file 3). By contrast, the test shrimp gave positive hybridization results in the cell cytoplasm of LO cells for both ICE and LSNV (Figure 8 and Additional file 4). 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 (Additional file 5) while test shrimp showed positive in situ hybridization reactions for both ICE and LSNV (Figure 9). These results supported the previously described link between retinopathy and LSNV [8]. In addition to the LO and eyes, gills of the test shrimp only were also positive for both ICE and LSNV (Figure 10).

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