False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia

<p>Abstract</p> <p>Background</p> <p>Infectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp <it>Penaeus (Litopenaeus) vannamei </it>are characterized by gross signs of whitened abdominal muscles and by slow mortality reaching up to 70%. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Indonesia. Since then rumours have periodically circulated about IMNV disease outbreaks in other Asian countries. Our findings indicate that these are false rumours.</p> <p>Findings</p> <p>Our continual testing by nested RT-PCR of shrimp samples suspected of IMNV infection from various Asian countries since 2006 has yielded negative results, except for samples from Indonesia. Our results are supported by the lack of official reports of IMNV outbreaks since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that also commonly causes muscle whitening in stressed whiteleg shrimp. Thus, we suspect that most of the false rumours in Asia about IMNV outside of Indonesia have resulted because of muscle cramp syndrome.</p> <p>Conclusions</p> <p>Results from continual testing of suspected IMNV outbreaks in Asian countries other than Indonesia since 2006 and the lack of official country reports of IMNV outbreaks since January 2007, indicate that rumours of IMNV outbreaks in Asian countries outside of Indonesia are false. We suspect that confusion has arisen because muscle cramp syndrome causes similar signs of whitened tail muscles in whiteleg shrimp.</p

Failed shrimp vaccination attempt with yellow head virus (YHV) attenuated in an immortal insect cell line

This short paper on yellow head virus Type-1 (YHV-1) of shrimp describes preliminary research on the potential for using YHV-1 attenuated in insect cells to protect shrimp against yellow head disease (YHD). YHV-1 can cause severe mortality in the cultivated shrimp Penaeus (Penaeus) monodon and Penaeus (Litopenaeus) vannamei.  No practical vaccination has been reported. The C6/36 mosquito cell cultures inoculated with YHV-1 become positive by PCR and by immunocytochemistry (immunopositive) for up to 30 split-cell passages. Shrimp injected with homogenates from low-passage cultures die from typical YHV-1 disease while shrimp injected with homogenates from high passage cultures do not, even though they become PCR positive and immunopositive for YHV-1. This suggested that viral attenuation had occurred during insect-cell passaging, and it opened the possibility of using homogenates from high-passage insect cultures as a vaccine against YHV-1. To test this hypothesis, homogenates from 30th-passage, YHV-positive cultures were injected into shrimp followed by challenge with virulent YHV-1. Controls were injected with homogenate from 30th-passage, naive (normal stock) insect-cell cultures. No shrimp mortality occurred following injection of either homogenate, but shrimp injected with the YHV-1 homogenate became both RT-PCR positive and immunopositive. Upon challenge 10 days later with YHV-1, mortality in shrimp injected with naive insect-cell homogenate was 100% within 7 days post-challenge while 100% mortality in the YHV-1 homogenate group did not occur until day 9 post-challenge. Kaplan-Meier log-rank survival analysis revealed that survival curves for the two groups were significantly different (p < 0.001). The cause of delay in mortality may be worthy of further investigation

White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines.

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

Squash mount of vermiform bodies (ATM) in shrimp hepatopancreatic tissue.

<p>(a) Low magnification photomicrograph showing 3 ATM with the central one inside an HP tubule. (b) Higher magnification photomicrograph showing an ATM containing cyst-like structures later found to be sloughed B-cells. (c) High magnification of an ATM stained with Rose Bengal to more clearly reveal its internal membranous structure.</p

ATM aggregation steps in H&E stained HP tissue sections in comparison to true gregarines.

<p>(a) Small, scattered membrane-lie structures in the HP tubule lumen. (b) More extended membranes beginning to aggregate in the tubule lumen. (c) Tighter aggregation of membranes bound by a continuous outer membrane and taking the shape of ATM. (d) Highly condensed ATM in a tubule lumen. (e) Accumulation of many individual ATM at the center of the HP near the midgut junction. (f) True gregarines clustered near the midgut junction and showing prominent nuclei.</p

HP tissue smear stained with hematoxylin and eosin to reveal ATM.

<p>The photomicrographs clearly show the vermiform morphology and the cyst-like contents inside or free.</p

TEM of an ATM structure containing an enclosed, sloughed and modified B-cell.

<p>Note the single-layer enclosing membrane and the internal complex of sloughed, modified microvilli.</p

TEM of unusual electron-dense particles in HP tubule crypts.

<p>(a) Low magnification of electron-dense particles of highly variable shape in the HP tubule lumen between layers of normal microvilli from facing epithelial cells. (b) High magnification of one of the electron-dense particles between the microvilli on the outside surface of an epithelial cell, possibly prior to cell entry. (c) High magnification of electron dense particles inside an epithelial cell with adjacent microvilli on the cell surface undergoing morphological changes. (d) Low magnification of an epithelial cell containing large numbers of electron dense particles and with microvilli in an advanced stage of transformation.</p

Semi-thin sections of HP tissue stained with toluidine blue.

<p>(a) Cross section of an HP tubule near the distal end showing densely stained particles in crypts formed by folds of the tubule epihelium and showing aggregated, transformed microvilli (ATM) in the tubule lumen. Note that microvillar layers of all the cells are intact. (b) Cross sections of HP tubules showing sloughed, transformed microvilli. (c) Cross section of an HP tubule showing a modified, sloughed B-cell in the tubule lumen with microvilli scattered over its surface. Also seen are tubule epithelial cells with normal microvilli and transformed mivrovilli, and one cell denuded of microvilli, undergoing lysis. (d) High magnification of clustered ATM at the center of the HP clearly showing an outer membrane enclosing multitudes of folded transformed microvilli. Some also contain enclosed, sloughed B-cells. Note many free transformed microvilli fragments surrounding the ATM.</p

TEM of steps in microvillar transformation and sloughing.

<p>(a) Low magnification of HP tubule epithelial cells showing normal and transformed microvilli and two denuded cells undergoing lysis. Also shown is an early stage in the aggregation of transformed and sloughed microvilli surrounded by an enclosing membrane. (b) Low magnification of HP tubule epithelial cells with transformed microvilli peeling from the cell surface, prior to cell lysis. (c) Higher magnification of the field from (b) clearly showing the difference between normal and transformed microvilli. (d) High magnification of HP tubule epithelial cells showing the tubular nature of transformed and peeled microvillar layers.</p