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

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.

Figure 1

Clustal-W alignment of microsporidian small subunit rRNA sequences. The ssu rRNA sequence of Enterocytozoon hepatopenaei from P. monodon (Pm-Entero)(Genbank accession no. KF362129) and the microsporidian from P. vannamei (Pv-Entero) (Genbank accession no. KF362130) are compared with the matching region of the ssu rRNA gene of E. bieneusi (GenBank AY257180). Regions of 100% identity between Pv-Entero and Pm-Entero are outlined in grey background while 100% identity among all three species is indicated by asterisks.

Figure 2

Agarose gels showing nested PCR microsporidian-specific amplicons using 100 ng of total DNA template from hepatopancreatic tissue obtained from P. vannamei not challenged (A) or orally challenged (B) with the microsporidian. Lane 1-3:Three specimens 2 days post-challenge; Lane 4-6: Three specimens 4 days post-challenge, Lane 7-9: Three specimens 7 days post-challenge; Lane 10: plasmid positive control. The amplicon sizes for the 1^st and 2^nd-step PCR are 779 and 176 bp, respectively.

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.

Figure 4

Photomicrographs of infected hepatopancreatic tissue of P. vannamei. The adjacent sections of shrimp tissue stained with H&E (column 1) and with the in situ hybridization probe (column 2) showing that hepatopancreatic cells of P. vannamei infected with the microsporidian cannot be easily detected by H&E staining even though extensive infection is revealed by in situ hybridization (dark brown to black staining). (a/b) Low magnification showing that positive reactions are restricted to the medial and proximal tubule epithelial cells of the shrimp hepatopancreas (HP) while the distal E cells are negative. Note that B cells dominate in the infected region. (c/d) Medium high magnification showing pinpoint positive, in situ hybridization reactions in the region of the HP adjacent to the distal E cells region. (e/f) High magnification clearly showing the difficulty in identifying infected cells by H&E staining but their clear revelation by in situ hybridization. (g/h) Very high magnification, emphasizing the features described in (e/f).