Confirmatory test of active IHHNV infection in shrimp by immunohistochemistry and IHHNV-LongAmp PCR.

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection.

Molecular and cellular characterization of four putative nucleotide transporters from the shrimp microsporidian Enterocytozoon hepatopenaei (EHP).

Microsporidia are obligate intracellular parasites that lost several enzymes required in energy production. The expansion of transporter families in these organisms enables them to hijack ATP from hosts. In this study, nucleotide transporters of the microsporidian Enterocytozoon hepatopenaei (EHP), which causes slow growth in economically valuable Penaeus shrimp, were characterized. Analysis of the EHP genome suggested the presence of four putative nucleotide transporter genes, namely EhNTT1, EhNTT2, EhNTT3, and EhNTT4. Sequence alignment revealed four charged amino acids that are conserved in previously characterized nucleotide transporters. Phylogenetic analysis suggested that EhNTT1, 3, and 4 were derived from one horizontal gene transfer event, which was independent from that of EhNTT2. Localization of EhNTT1 and EhNTT2 using immunofluorescence analysis revealed positive signals within the envelope of developing plasmodia and on mature spores. Knockdown of EhNTT2 by double administration of sequence specific double-stranded RNA resulted in a significant reduction in EHP copy numbers, suggesting that EhNTT2 is crucial for EHP replication in shrimp. Taken together, the insight into the roles of NTTs in microsporidian proliferation can provide the biological basis for the development of alternative control strategies for microsporidian infection in shrimp.

Shrimp genome sequence contains independent clusters of ancient and current Endogenous Viral Elements (EVE) of the parvovirus IHHNV.

BACKGROUND: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). RESULTS: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called "non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV. CONCLUSIONS: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

Propionigenium and Vibrio species identified as possible component causes of shrimp white feces syndrome (WFS) associated with the microsporidian Enterocytozoon hepatopenaei.

White feces syndrome (WFS) in cultivated shrimp is characterized by white shrimp midguts (intestines) and white fecal strings that float as mats on pond surfaces. The etiology of WFS is complex, but one type called EHP-WFS is associated with the microsporidian Enterocytozoon hepatopenaei (EHP). The hepatopancreas (HP), midgut and fecal strings of EHP-WFS shrimp exhibit massive quantities of EHP spores together with mixed, unidentified bacteria. In EHP-WFS ponds, some EHP-infected shrimp show white midguts (WG) and produce white feces while other EHP-infected shrimp in the same pond show grossly normal midguts (NG) and produce no white feces. We hypothesized that comparison of the microbial flora between WG and NG shrimp would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this, we selected a Penaeus vannamei cultivation pond exhibiting severe WFS and used microscopic and microbial profiling analyses to compare WG and NG samples. Histologically, EHP was confirmed in the HP and midgut of both WG and NG shrimp, but EHP burdens were higher and EHP tissue damage was more severe in WG shrimp. Further, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium burden in the HP of WG shrimp (9364 copies/100 ng DNA) was significantly higher (P = 1.1 × 10-5) than in NG shrimp (12 copies/100 ng DNA). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as potential component causes of EHP-WFS in P. vannamei.

False mussels (Mytilopsis leucophaeata) can be mechanical carriers of the shrimp microsporidian Enterocytozoon hepatopenaei (EHP).

Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite causing hepatopancreatic microsporidiosis (HPM) in cultivated shrimp in Asian countries. One strategy to control EHP is to identify and eliminate biological reservoir(s) in shrimp ponds. Several marine and brackish-water organisms, including false mussels (Mytilopsis) have been reported to test positive for EHP using the PCR method. Thus, we tested Thai false mussel Mytilopsis leucophaeata collected from the 6 ponds with EHP-infected shrimp for the presence of EHP using SWP-PCR. Results revealed the sampled mussels from all 6 ponds were PCR positive. Subsequent bioassays were carried out to study EHP transmission between mussels and shrimp. Firstly, the naïve mussels were cohabitated with EHP-infected shrimp and all mussels were SWP-PCR positive at day 20 post cohabitation. One batch of such PCR-positive mussels was transferred for cohabitation with naïve shrimp and 37.5% EHP-positive shrimp were observed within 10 days. Tissue analysis of the SWP-PCR-positive mussels using light microscopy, in situ hybridization technique and electron microscopy did not confirm EHP infection. In summary, there was no evidence demonstrating that Mytilopsis leucophaeata was itself infected with EHP. However, the false mussels were apparently capable of carrying infectious spores for some period after ingestion and serving as a mechanical or passive carrier. The results support previous reports warning of the danger of feeding living or fresh bivalves to broodstock shrimp in hatcheries or shrimp in rearing ponds without prior heating or freezing.

Shrimp Parvovirus Circular DNA Fragments Arise From Both Endogenous Viral Elements and the Infecting Virus.

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

Shrimp protected from a virus by feed containing yeast with a surface-displayed viral binding protein.

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp.

Caspase-3, a shrimp phosphorylated hemocytic protein is necessary to control YHV infection.

By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process.

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii.

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).

Co-expression of double-stranded RNA and viral capsid protein in the novel engineered Escherichia coli DualX-B15(DE3) strain.

BACKGROUND: Viruses cause significant economic losses to shrimp aquaculture worldwide. In severe cases, they can lead to 100% mortality within a matter of days, hence the aquaculture industry requires antiviral strategies to minimize economic impacts. Currently, a double-stranded RNA (dsRNA)-based platform has been proven effective at a laboratory scale. The bottleneck for its industrialization is the lack of low-cost, efficient and practical delivery approaches. In an effort to bridge the gap between laboratory and farm applications, virus-like particles (VLP) have been used as nanocarriers of dsRNA. However, the implementation of this approach still suffers from high costs and a lengthy procedure, co-expression of subunits of VLP or capsid proteins (CPs) and dsRNA can be the solution for the problem. CP and dsRNA are traditionally expressed in two different E. coli hosts: protease-deficient and RNase III-deficient strains. To condense the manufacturing of dsRNA-containing VLP, this study constructed a novel E. coli strain that is able to co-express viral capsid proteins and dsRNA in the same E. coli cell. RESULTS: A novel bacterial strain DualX-B15(DE3) was engineered to be both protease- and RNase III-deficiency via P1 phage transduction. The results revealed that it could simultaneously express recombinant proteins and dsRNA. CONCLUSION: Co-expression of viral capsid proteins and dsRNA in the same cell has been shown to be feasible. Not only could this platform serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics, it may be applicable for other applications that requires co-expression of recombinant proteins and dsRNA.

Immunopathogenesis of hematopoietic tissues in response to Vibrio parahaemolyticus (VPAHPND) infection in Macrobrachium rosenbergii.

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VPAHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC50 dose of VPAHPND; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VPAHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VPAHPND-infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6-12 h and declined by 24 h. This evident related to the almost VPAHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.

The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control.

Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei, by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures.

Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA).

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.

Shewanella khirikhana sp. nov. - a shrimp pathogen isolated from a cultivation pond exhibiting early mortality syndrome.

Early mortality syndrome (EMS) in cultivated shrimp is of complex aetiology. One of the causes is acute hepatopancreatic necrosis disease (AHPND) caused by unique Vibrio isolates that carry two Pirvp toxin genes, but other causes of EMS remain mostly unexplained. Here, we describe the discovery of a Shewanella isolate TH2012T from an EMS/AHPND outbreak pond and demonstrate its virulence for shrimp (the mean lethal concentration of 105 colony-forming units per millilitre by immersion challenge) accompanied by distinctive histopathology, particularly of the ventral nerve cord and lymphoid organ but also including the digestive tract. On the basis of its complete genome sequence, multilocus phylogenetic trees, digital DNA-DNA hybridization analysis and differential phenotypic characteristics, we propose that Shewanella isolate TH2012T represents a novel species, separated sufficiently from the type strains S. litorisediminis and S. amazonensis to justify naming it Shewanella khirikhana sp. nov. Analysis of the TH2012T genome revealed no homologues of the Pirvp toxin genes but revealed a number of other potential virulence factors. It constitutes the first Shewanella isolate reported to be pathogenic to shrimp.

Cell surface transglutaminase required for nodavirus entry into freshwater prawn hemocytes.

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F1-ATP synthase ß-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.

The hematopoietic organ of Macrobrachium rosenbergii: Structure, organization and immune status.

The hematopoietic organ (HO) of the giant freshwater prawn Macrobrachium rosenbergii is a discrete, whitish mass located in the epigastric region of the cephalothorax, posterior to the brain. It is composed of hematopoietic cells arranged in a thick layer of numerous lobules that surround a central hemal sinus from which they are separated by a thin sheath. At the center of the sinus is the muscular cor frontale. The lobules extend radially outward from the sinus in three developmental zones. Basal Zone 1 nearest the sinus contains large hematopoietic stem cells with euchromatic nuclei that stain positive for proliferation cell nuclear antigen (PCNA). Zone 2 contains smaller, actively dividing cells as indicated by positive 5-bromo-20-deoxyuridine (BrdU) staining. Distal Zone 3 contains small, loosely packed cells with heterochromatic nuclei, many cytoplasmic granules and vesicles indicating that they will eventually differentiate into hemocytes and enter circulation. Three main arteries, namely the ophthalmic and the 2 branches of the antennary, connect the heart to the HO. Use of India ink and 0.1 µm fluorescent micro-beads injected into the heart revealed that the cor frontale could immediately remove foreign particles from hemolymph by filtration. Fluorescent beads were also detected in the hematopoietic tissue at 30 min after injection, indicating that it could be penetrated by foreign particles. However, the fluorescent signal completely disappeared from the whole HO after 4 h, indicating its role in removal of foreign particles. In conclusion, the present study demonstrated for the first time the detailed histological structures of the HO of M. rosenbergii and its relationship to hematopoiesis and removal of foreign particles from hemolymph.

Mendelian inheritance of endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp.

Previous work has shown that non-retroviral endogenous viral elements (EVE) are common in crustaceans, including penaeid shrimp. So far, they have been reported for infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). For the latter, it was shown that shrimp sperm were positive for an EVE of WSSV called EVE366, suggesting that it was heritable, since shrimp sperm (non-motile) do not contain mitochondria. However, to prove this hypothesis that EVE366 was heritable and located in chromosomal DNA, it was necessary to carry out mating tests to show that EVE366 could be detected in parental shrimp and distributed in their offspring in a Mendelian fashion. To do this, we analyzed two shrimp crosses using polyacrylamide gels with a multiple-allele, microsatellite marker Pmo11 as a quality control for single allele detection. In both crosses, all of the shrimp (parents and siblings) were positive for 2 Pmo11 alleles as expected. In Cross 1, the female was PCR-positive for EVE366 while the male was negative, and in Cross 2, both the female and male were PCR-positive for EVE366. Individual analysis of the offspring of Cross 1 revealed a distribution of 1:1 for EVE366, indicating that the EVE366-positive female parent was heterozygous for EVE366. In the second cross, the distribution of EVE366 in the offspring was 3:1, indicating that both PCR-positive parents were heterozygous for EVE366. These results supported the hypothesis that EVE366 was present in shrimp chromosomal DNA and was heritable in a Mendelian fashion. This work provides a model to screen for heritable EVE in shrimp and shows that selection of one parent heterozygous for an EVE and the other negative for it can result in approximately half of the siblings positive and half negative for that EVE as expected. Dividing the siblings of such a cross into an EVE positive group and an EVE negative group followed by challenge with the originating lethal virus should reveal whether or not possession of that specific EVE results in any significant protection against disease caused by the homologous virus.

Complete Genome Sequence of Shewanella sp. Strain TH2012, Isolated from Shrimp in a Cultivation Pond Exhibiting Early Mortality Syndrome.

Here, we present the complete genome sequence of a Shewanella isolate, TH2012, from a shrimp pond in which shrimp exhibited early mortality syndrome (EMS)/acute hepatopancreatic necrosis disease (AHPND). The complete genome of TH2012 has a prophage-like element and a number of potential virulence factors, making TH2012 a possible contributing factor to EMS outbreaks.

Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP).

BACKGROUND: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. RESULTS: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. CONCLUSIONS: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

A Natural Vibrio parahaemolyticus ΔpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.