Siriporn Sriurairatana

The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in southern Thailand

Abstract The occurrence, development and histopathology of monodon baculovirus (MBV) in larvae, postlarvae (PL) and broodstock of Penaeus monodon was studied over a period of 1 year in southern Thailand. In histological samples of captured male and female broodstock obtained from the Andaman Sea, the incidence of individuals with MBV occlusion bodies (OBs) was approximately 5.7% during the period. Absence of MBV virions in electron micrographs of oocytes of MBV-infected females, mature eggs and nauplii indicated that the virus may not be transmitted transovarially. Together, these facts suggest that elimination of MBV from hatcheries by screening of quarantined wild broodstock would be feasible. In the hatchery, baculovirus OBs began to be clearly identifiable in the hepatopancreas of larvae at the third zoeal stage. During all subsequent larval and PL developmental stages, there was a high incidence of infected individuals, some with extremely large numbers of OBs. The average incidence of PL positive for OBs in histological specimens approached 100% during the period of the study in a survey that included eight commercial PL producers on both the Gulf of Thailand and the Andaman Sea. However, the incidence of OB-positive animals gradually fell as the shrimp grew older and OBs were often undetectable in healthy juveniles derived from infected PL within approximately 2 weeks of stocking in grow-out ponds. In some instances, it was possible to follow groups of PL from the nursery stage through to harvest of market-size shrimp. Average survival data for such shrimp followed in a large number of ponds over a period of 1 year indicated that MBV was well tolerated by Penaeus monodon if other rearing conditions were optimal. However, in the event of environmental or other stress, the incidence of animals showing large numbers of MBV particles increased dramatically.

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.

Unstable Lysogeny and Pseudolysogeny in Vibrio harveyi Siphovirus-Like Phage 1†

ABSTRACT Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.

Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification

The integration of loop-mediated isothermal amplification (LAMP) and DNA-functionalized AuNPs as visual detection probes (LAMP-AuNPs) was developed and applied for the detection of white spot syndrome virus (WSSV) from Penaeid shrimp in this study. The principle of this combination assay relies on the basis of stability characteristics of the DNA-functionalized AuNPs upon hybridization with the complementary target DNA toward salt-induced aggregation. If the detected target DNA is not complementary to the ssDNA probes, the DNA-functionalized AuNPs will be aggregated due to the screening effect of salt, resulting in the change of solution color from red to blue/gray and shift of the surface plasmon peak to longer wavelength. While the DNA-functionalized AuNPs are perfectly matched to the detected target DNA, the color of solution still remains red in color and no surface plasmon spectral shift. This assay provides simply technique, time-saving and its detection results could be achieved qualitatively and quantitatively by visualization using the naked eye due to the colorimetric change and by measurement using the UV-vis spectroscopy due to the surface plasmon spectral shift, respectively. In this study, LAMP-AuNPs assay was successfully developed with the detection of WSSV-LAMP generated product at 0.03 μg/reaction, and showed the sensitivity of 2 × 10(2) copies WSSV plasmid DNA, that is comparable to the most sensitive method reported to date. The LAMP-AuNPs assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of WSSV. This technique has a potential as a routine method for assessing the infectious diseases in Penaeid shrimp not only for WSSV, but also for other shrimp pathogens, and can be useful tool in field conditions for the diagnosis or surveillance programs.

Impact of yellow head virus outbreaks in the whiteleg shrimp, Penaeus vannamei (Boone), in Thailand.

Abstract Yellow head virus (YHV) is known as a major pathogen in the black tiger shrimp, Penaeus (Penaeus) monodon. It can also cause serious mortality in farmed whiteleg shrimp, Penaeus (Litopenaeus) vannamei. However, there is no published information on the economic and/or production impact of the disease in P. vannamei. Shrimp with gross signs of YHV disease (faded body colour and 60–70% mortality) were observed in 20 study farms rearing P. vannamei in the central part of Thailand from the end of 2007 through early 2008. The estimated economic loss for these farms according to the Thai Animal Aquaculture Association was approximately US

Mosquito cells accommodate balanced, persistent co-infections with a densovirus and Dengue virus

3 million. Detailed sequence analysis of RT‐PCR amplicons from shrimp in all the study ponds revealed the presence of YHV Type 1b (YHV‐1b) alone (characterized by a 162‐bp deletion in the ORF3 region encoding the structural gene for gp116) and the absence of YHV Type 1a (YHV‐1a), the original YHV type reported from Thailand. Despite the large 162‐bp deletion (= 54 deduced amino acids) in the gp116 structural gene, histopathology of YHV‐1b infections was identical to that of YHV‐1a infections, and electron microscopy revealed that YHV‐1b virions were morphologically indistinguishable from those previously reported for YHV‐1a. In addition, an existing commercial RT‐PCR detection kit and an immunochromatographic test strip for the detection of YHV were proven to have been valid tests for both YHV‐1b and YHV‐1a. The source of the virus for these outbreaks was unlikely to have been the post‐larvae used to stock the ponds, as they were derived from domesticated specific pathogen‐free stocks free of YHV. Thus, it is possible that they originated from an unknown, natural reservoir.

White Feces Syndrome of Shrimp Arises from Transformation, Sloughing and Aggregation of Hepatopancreatic Microvilli into Vermiform Bodies Superficially Resembling Gregarines

To study persistent viral co-infections in arthropods, we first produced stable, persistently infected C6/36 mosquito cell cultures by serial passage of exponentially growing whole cells infected with either a densovirus (AalDNV) or Dengue virus (DEN-2). We then obtained stable, persistent co-infections by reciprocal super-challenge and similar passaging. Persistently infected cultures did not differ from naïve-cell cultures in growth rate and cell morphology. Nor did they differ in high production of both viruses with high infection rates for naïve C6/36 cells. Immunocytochemistry revealed that 99-100% of the cells were coinfected but that super-infection order had some effect on antigen distribution for the two viruses. Our results combined with existing field information and previously published experimental work suggest that the capacity to support stable, viral co-infections may be a general phenomenon for arthropod cells, and that they may be achieved easily and rapidly by serial passaging of whole cultured cells. Such persistent infections would facilitate studies on interactions between co-infecting viruses.

A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp

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.

Complete Genome Sequence of Virulence-Enhancing Siphophage VHS1 from Vibrio harveyi

BackgroundFrom 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.ResultsUsing a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and in situ hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM.ConclusionsICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ICE and LSNV. Despite this ambiguity, it is recommended in the interim that ICE be added to the agents such as LSNV already listed for exclusion from domesticated stocks of the black tiger shrimp.

Non-virulence of a recombinant shrimp nidovirus is associated with its non structural gene sequence and not a large structural gene deletion

ABSTRACT Vibrio harveyi siphophage 1 (VHS1) is a tailed phage with an icosahedral head of approximately 66 nm in diameter and an unornamented, flexible tail of approximately 153 nm in length. When Vibrio harveyi 1114GL is lysogenized with VHS1, its virulence for the black tiger shrimp (Penaeus monodon) increases by more than 100 times, and this coincides with production of a toxin(s) associated with shrimp hemocyte agglutination. Curiously, the lysogen does not show increased virulence for the whiteleg shrimp (Penaeus [Litopenaeus] vannamei). Here we present and annotate the complete, circular genome of VHS1 (81,509 kbp; GenBank accession number JF713456). By software analysis, the genome contains 125 putative open reading frames (ORFs), all of which appear to be located on the same DNA strand, similar to the case for many other bacteriophages. Most of the putative ORFs show no significant homology to known sequences in GenBank. Notable exceptions are ORFs for a putative DNA polymerase and putative phage structural proteins, including a portal protein, a phage tail tape measure protein, and a phage head protein. The last protein was identified as a component of the species-specific toxin mixture described above as being associated with agglutination of hemocytes from P. monodon.