Limits...
The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei.

Tangprasittipap A, Srisala J, Chouwdee S, Somboon M, Chuchird N, Limsuwan C, Srisuvan T, Flegel TW, Sritunyalucksana K - BMC Vet. Res. (2013)

Bottom Line: More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS).Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS.Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI rd, Bangkok 10400, Thailand.

ABSTRACT

Background: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS.

Results: Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS.

Conclusions: The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

Show MeSH

Related in: MedlinePlus

Photomicrographs of microsporidian spores in hepatopancreatic cells. Microsporidian spores (arrows) inside B cells of the hepatopancreatic tubule epithelium (left) and free in the tubule lumen (right). Such cells were present in the section in low numbers, giving a misleading impression of the extent of the severe microsporidian infection evident by in situ hybridization as seen in Figure 2. The magnification bar applies to both images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3717009&req=5

Figure 3: Photomicrographs of microsporidian spores in hepatopancreatic cells. Microsporidian spores (arrows) inside B cells of the hepatopancreatic tubule epithelium (left) and free in the tubule lumen (right). Such cells were present in the section in low numbers, giving a misleading impression of the extent of the severe microsporidian infection evident by in situ hybridization as seen in Figure 2. The magnification bar applies to both images.

Mentions: As previously reported for infections of E. hepatopenaei in P. monodon, the number of hepatopancreatic cells showing spore formation in P. vannamei (Figure 2) was small, giving a superficial impression that the extent of the infections was very limited. The size of the spores (approximately 1 μm in length and less than 1 μm in width) and cytoplasmic location were also similar to those previously described for E. hepatopenaei in P. monodon. However, differences in P. vannamei included spore formation exclusively in B cells (Figure 2) and extensive infection of the medial and proximal tubule epithelial cells of the hepatopancreas in the absence of spores, as revealed by in situ hybridization (Figure 3). This was not the situation for previous reports on E. hepatopenaei in P. monodon, where relatively few cells produced spores or showed recognizable plasmodia, and only those cells were positive by in situ hybridization [12,13]. The cells giving positive in situ hybridization reactions in P. vannamei were restricted to the central region of the HP and did not extend to the distal region composed of E-cells. In the transitional zone between the medial and distal cells, pinpoint positive in situ hybridization reactions suggested that early infection stages occurred as the HP cells differentiated from E cells into B, F and R cells. Negative control slides using the GFP-DIG-labled probe gave no positive in situ hybridization reactions (not shown). At low and medium magnification by H&E staining (Figure 3a,c,e), there were no distinctive cytoplasmic features that revealed the microsporidian elements giving rise to positive in situ hybridization reactions in adjacent tissue sections. At the highest magnification by H&E staining (oil emersion lens), basophilic, cytoplasmic inclusions of highly variable, shape, size and number (Figure 3g) were present in the H&E stained cells and some of these may have been of microsporidian origin but they could not be unequivocally distinguished from other normal, basophilic cytoplasmic structures of the host. Because of this phenomenon, histopathological evaluation of the severity of these infections by H&E staining might be misleading, if the criterion used was the number of cells showing spores or other easily recognizable microsporidian structures.


The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei.

Tangprasittipap A, Srisala J, Chouwdee S, Somboon M, Chuchird N, Limsuwan C, Srisuvan T, Flegel TW, Sritunyalucksana K - BMC Vet. Res. (2013)

Photomicrographs of microsporidian spores in hepatopancreatic cells. Microsporidian spores (arrows) inside B cells of the hepatopancreatic tubule epithelium (left) and free in the tubule lumen (right). Such cells were present in the section in low numbers, giving a misleading impression of the extent of the severe microsporidian infection evident by in situ hybridization as seen in Figure 2. The magnification bar applies to both images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3717009&req=5

Figure 3: Photomicrographs of microsporidian spores in hepatopancreatic cells. Microsporidian spores (arrows) inside B cells of the hepatopancreatic tubule epithelium (left) and free in the tubule lumen (right). Such cells were present in the section in low numbers, giving a misleading impression of the extent of the severe microsporidian infection evident by in situ hybridization as seen in Figure 2. The magnification bar applies to both images.
Mentions: As previously reported for infections of E. hepatopenaei in P. monodon, the number of hepatopancreatic cells showing spore formation in P. vannamei (Figure 2) was small, giving a superficial impression that the extent of the infections was very limited. The size of the spores (approximately 1 μm in length and less than 1 μm in width) and cytoplasmic location were also similar to those previously described for E. hepatopenaei in P. monodon. However, differences in P. vannamei included spore formation exclusively in B cells (Figure 2) and extensive infection of the medial and proximal tubule epithelial cells of the hepatopancreas in the absence of spores, as revealed by in situ hybridization (Figure 3). This was not the situation for previous reports on E. hepatopenaei in P. monodon, where relatively few cells produced spores or showed recognizable plasmodia, and only those cells were positive by in situ hybridization [12,13]. The cells giving positive in situ hybridization reactions in P. vannamei were restricted to the central region of the HP and did not extend to the distal region composed of E-cells. In the transitional zone between the medial and distal cells, pinpoint positive in situ hybridization reactions suggested that early infection stages occurred as the HP cells differentiated from E cells into B, F and R cells. Negative control slides using the GFP-DIG-labled probe gave no positive in situ hybridization reactions (not shown). At low and medium magnification by H&E staining (Figure 3a,c,e), there were no distinctive cytoplasmic features that revealed the microsporidian elements giving rise to positive in situ hybridization reactions in adjacent tissue sections. At the highest magnification by H&E staining (oil emersion lens), basophilic, cytoplasmic inclusions of highly variable, shape, size and number (Figure 3g) were present in the H&E stained cells and some of these may have been of microsporidian origin but they could not be unequivocally distinguished from other normal, basophilic cytoplasmic structures of the host. Because of this phenomenon, histopathological evaluation of the severity of these infections by H&E staining might be misleading, if the criterion used was the number of cells showing spores or other easily recognizable microsporidian structures.

Bottom Line: More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS).Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS.Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI rd, Bangkok 10400, Thailand.

ABSTRACT

Background: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS.

Results: Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS.

Conclusions: The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

Show MeSH
Related in: MedlinePlus