Gregarine-Like Bodies Accompany EMS

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Gregarine-Like Bodies Accompany EMS

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Abstract: “Accompanying acute hepatopancreatic necrosis disease (AHPND) in cultivated Asian shrimp has been an increasing prevalence of vermiform, gregarine-like bodies within the shrimp hepatopancreas (HP) and midgut. In high quantity they result in white fecal strings and a phenomenon called white feces syndrome (WFS). Light microscopy (LM) of squash mounts and stained smears from fresh HP tissue revealed that the vermiform bodies are almost transparent with widths and diameters proportional to the HP tubule lumens in which they occur. Despite vermiform appearance, they show no cellular structure. At high magnification (LM with 40-100x objectives), they appear to consist of a thin, outer membrane enclosing a complex of thicker, inter-folded membranes.

Transmission electron microscopy (TEM) revealed that the outer non-laminar membrane of the vermiform bodies bore no resemblance to a plasma membrane or to the outer layer of any known gregarine, other protozoan or metazoan. Sub-cellular organelles such as mitochondria, nuclei, endoplasmic reticulum and ribosomes were absent. The internal membranes had a tubular sub-structure and occasionally enclosed whole B-cells, sloughed from the HP tubule epithelium. These internal membranes were shown to arise from transformed microvilli that peeled away from HP tubule epithelial cells and then aggregated in the tubule lumen. Stripped of microvilli, the originating cells underwent lysis. By contrast, B-cells remained intact or were sloughed independently and whole from the tubule epithelium.

When sometimes engulfed by the aggregated, transformed microvilli (ATM) they could be misinterpreted as cyst-like structures by light microscopy, contributing to gregarine-like appearance. The cause of ATM is currently unknown, but formation by loss of microvilli and subsequent cell lysis indicate that their formation is a pathological process. If sufficiently severe, they may retard shrimp growth and may predispose shrimp to opportunistic pathogens. Thus, the cause of ATM and their relationship (if any) to AHPND should be determined.”

Introduction: “The results presented in this manuscript describing transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines was obtained over a period of 6 years in a piecemeal fashion as a series of initially independent, sideline observations made during the course of dedicated research projects on a variety of known shrimp pathogens ranging from viruses to bacteria, fungi and parasites. It was not until very recently that the connections between the independent observations were understood, allowing them to be linked together into a coherent whole. A major activity that helped us to gain understanding of the connections between our piecemeal observations was the intensive research that has been carried out since 2009 and particularly since 2011 on many hundreds of shrimp specimens studied in attempts to determine the cause of acute hepatopancreatic necrosis disease (AHPND), the most recent, serious shrimp pandemic to cause severe losses in Asian shrimp cultivation.”

Discussion: “It may be significant that the increase in prevalence of ATM has been coincidental with the increase in prevalence of AHPND outbreaks. Although this might suggest a possible causal association, there has also been a coincidental increase in prevalence of the hepatopancreatic microsporidian Enterocytozoon hepatopenaei with AHPND, and we now know that this is certainly not a causal relationship, since AHPND is caused by unrelated bacteria [1]. So at least for E. hepatopenaei and AHPND bacteria, it seems likely that some of their increased prevalence has resulted from contamination of broostock and/or post-larvae (PL). This contention is supported by anecdotal evidence from widely separated Thai shrimp farmers who received portions of single batches of PL derived from SPF shrimp stocks but then experienced AHPND outbreaks more-or-less simultaneously. It is also supported by our discovery of endemic E. hepatopenaei in locally held broodstock and PL of SPF P. vannamei stocks that originated from the Americas where this microsporidian has never previously been reported.”

“Altogether the previous information suggests that the biosecurity measures in at least some shrimp hatcheries have not been sufficiently rigorous to exclude contamination by imported and/or local pathogens. Therefore, we must consider two possibilities with respect to ATM. Either they are caused by a new agent that has contaminated SPF stocks in a similar manner to AHPND and E. hepatopenaei, or that they constitute an alternate manifestation of an existing pathogen. For example, it has been reported that AHPND bacteria produce a potent toxin that can cause sloughing of hepatopancreatic tubule epithelial cells [1], and it may be asked whether the same toxin at low doses may cause the formation of ATM in the absence of cell sloughing.

To test this latter possibility, the laboratory infection model recently described [1] may be used with diluted toxin preparations from the causative bacteria. With respect to the existence of a new pathogen, comparative metagenomic analysis of shrimp with and without ATM may be the most useful. Alternatively, the possible involvement of the minute, electron dense bodies described here to be associated with microvillar transformation may be further investigated. For example, it may be possible to separate them physically from tissue homogenates by differential centrifugation and/or filtration for further analysis and for laboratory challenge tests.”

“In conclusion, we have revealed by TEM that vermiform structures superficially resembling gregarines and commonly found now in the HP of Asian cultivated shrimp are not independent organisms but result from the transformation, sloughing and aggregation of microvilli from the HP tubule epithelial cells themselves. The denuded epithelial cells subsequently undergo lysis, indicating that the process has the potential for negative impact on shrimp growth and survival, and in very severe cases can lead to the phenomenon called white feces syndrome (WFS). Further investigation is needed to understand the cause of ATM and evaluate their impact on shrimp production.”

Figure 1. Gross signs of white feces syndrome WFS.

(a) Floating, white fecal strings. (b) White fecal strings on a feeding tray. (c) White intestine of affected shrimp. (d) Golden brown intestine of an affected shrimp. (e) Photomicrograph of fecal string contents. doi:10.1371/journal.pone.0099170.g001

Source: PLOS One.  White Feces Syndrome of Shrimp Arises from Transformation, Sloughing and Aggregation of Hepatopancreatic Microvilli into Vermiform Bodies Superficially Resembling Gregarines.  Siriporn Sriurairatana, Visanu Boonyawiwat, Warachin Gangnonngiw, Chaowanee Laosutthipong, Jindanan Hiranchan and Timothy W. Flegel (email, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pratum Thani, Thailand).  June 9, 2014.

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