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

Evidence for a novel coding sequence overlapping the 5'-terminal ~90 codons of the Gill-associated and Yellow head okavirus envelope glycoprotein gene

The genus Okavirus (order Nidovirales) includes a number of viruses that infect crustaceans, causing major losses in the shrimp industry. These viruses have a linear positive-sense ssRNA genome of ~26-27 kb, encoding a large replicase polyprotein that is expressed from the genomic RNA, and several additional proteins that are expressed from a nested set of 3'-coterminal subgenomic RNAs. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF that overlaps the 5' end of the envelope glycoprotein encoding sequence, ORF3, in the +2 reading frame. The new ORF has a strong coding signature and, in fact, is more conserved at the amino acid level than the overlapping region of ORF3. We propose that translation of the new ORF initiates at a conserved AUG codon separated by just 2 nt from the ORF3 AUG initiation codon, resulting in a novel 86 amino acid protein

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 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

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

Gross signs of white feces syndrome WFS.

<p>(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.</p