Emergency response to emerging disease: AHPND in shrimp

Outbreaks of acute hepatopancreatic necrosis disease (AHPND) have caused great economic losses to many shrimp producing countries in Asia since its first appearance in 2009. The causative agent was first reported in 2013 as specific isolates of Vibrio parahaemolyticus (VPAHPND) that were later found to harbor a plasmid (pVA) encoding the Pir-like binary toxin genes PirvpA and PirvpB. More recent information indicates that pVA plasmid and variants occur in many Vibrio parahaemolyticus serotypes and also in other Vibrio species such as V. campbellii, V. harveyi and V. owensii. Information on such genomic and proteomic studies of different VPAHPND isolates from different countries are reviewed. A cohort study carried out in Thailand in 2014 indicated that AHPND outbreaks account for only a portion of the disease outbreaks reported by shrimp farmers as outbreaks of early mortality syndrome (EMS). It is urgent that the etiology of the other EMS-associated mortalities be investigated and not be overlooked. It is recommended that a regional research network and surveillance program for newly-emerging or re-emerging pathogens be established to speed up the process of diagnosis and the implementation of coordinated control measures and to avoid a repeat of the EMS/AHPND scenario

Mud crab susceptibility to disease from white spot syndrome virus is species-dependent

<p>Abstract</p> <p>Background</p> <p>Based on a report for one species (<it>Scylla serrata</it>), it is widely believed that mud crabs are relatively resistant to disease caused by white spot syndrome virus (WSSV). We tested this hypothesis by determining the degree of susceptibility in two species of mud crabs, <it>Scylla olivacea </it>and <it>Scylla paramamosain</it>, both of which were identified by mitochondrial 16 S ribosomal gene analysis. We compared single-dose and serial-dose WSSV challenges on <it>S. olivacea </it>and <it>S. paramamosain</it>.</p> <p>Findings</p> <p>In a preliminary test using <it>S. olivacea </it>alone, a dose of 1 × 10⁶WSSV copies/g gave 100% mortality within 7 days. In a subsequent test, 17 <it>S. olivacea </it>and 13 <it>S. paramamosain </it>were divided into test and control groups for challenge with WSSV at 5 incremental, biweekly doses starting from 1 × 10⁴and ending at 5 × 10⁶copies/g. For 11 <it>S. olivacea </it>challenged, 3 specimens died at doses between 1 × 10⁵and 5 × 10⁵copies/g and none died for 2 weeks after the subsequent dose (1 × 10⁶copies/g) that was lethal within 7 days in the preliminary test. However, after the final challenge on day 56 (5 × 10⁶copies/g), the remaining 7 of 11 <it>S. olivacea </it>(63.64%) died within 2 weeks. There was no mortality in the buffer-injected control crabs. For 9 <it>S. paramamosain </it>challenged in the same way, 5 (55.56%) died after challenge doses between 1 × 10⁴and 5 × 10⁵copies/g, and none died for 2 weeks after the challenge dose of 1 × 10⁶copies/g. After the final challenge (5 × 10⁶copies/g) on day 56, no <it>S. paramamosain </it>died during 2 weeks after the challenge, and 2 of 9 WSSV-infected <it>S. paramamosain </it>(22.22%) remained alive together with the control crabs until the end of the test on day 106. Viral loads in these survivors were low when compared to those in the moribund crabs.</p> <p>Conclusions</p> <p><it>S. olivacea </it>and <it>S. paramamosain </it>show wide variation in response to challenge with WSSV. <it>S. olivacea </it>and <it>S. paramamosain </it>are susceptible to white spot disease, and <it>S. olivacea </it>is more susceptible than <it>S. paramamosain</it>. Based on our single-challenge and serial challenge results, and on previous published work showing that <it>S. serrata </it>is relatively unaffected by WSSV infection, we propose that susceptibility to white spot disease in the genus <it>Scylla </it>is species-dependent and may also be dose-history dependent. In practical terms for shrimp farmers, it means that <it>S. olivacea </it>and <it>S. paramamosain </it>may pose less threat as WSSV carriers than <it>S. serrata</it>. For crab farmers, our results suggest that rearing of <it>S. serrata </it>would be a better choice than <it>S. paramamosain </it>or <it>S. olivacea </it>in terms of avoiding losses from seasonal outbreaks of white spot disease.</p

Characterisation of Some Immune Genes in the Black Tiger Shrimp, Penaeus monodon

The molecular mechanisms of the immune system in shrimp, Penaeus monodon, are completely unknown, despite its economic importance as an aquaculture species, especially in Asia and Latin America. The genes and their gene products involved in the prophenoloxidase activating system, which is considered to be a non-self recognition and defence system in many invertebrates, have been isolated and characterised in shrimp. These include a zymogen of this cascade, prophenoloxidase (proPO); a cell adhesion protein, peroxinectin and a pattern recognition protein, β-1,3-glucan binding protein (GBP). All proteins are synthesised in shrimp hemocytes, not in the hepatopancreas. The shrimp proPO cDNA clone has 3,002 bp and contains an open reading frame of 2,121 bp encoding a putative polypeptide of 688 amino acids, with a molecular mass of 78.7 kDa. Comparison of amino acids sequences showed that this shrimp proPO was more closely to that of another crustacean, the freshwater crayfish, Pacifastacus leniusculus, than to insect proPOs. Upon activation of the proPO system in shrimp, a cell adhesion activity in the hemolymph is generated. Inhibition of adhesion by an antiserum against the crayfish cell adhesion protein, peroxinectin, revealed that the cell adhesion activity detected in shrimp hemolymph might result from a peroxinectin in shrimp. Indeed, a cDNA clone which encoded shrimp peroxinectin was isolated with an open reading frame of 2,337 bp encoding a putative protein of 778 amino acids including a signal peptide. Two putative integrin-binding motifs (RGD and KGD) are present suggesting that integrin is involved in the adhesion activity. The peroxinectin transcript was slightly reduced in shrimp injected with a β-1,3-glucan or laminarin. Also found in shrimp hemolymph was a 31 kDa-GBP that could bind to β-1,3-glucan polymers such as curdlan and zymosan, but not to LPS. The cDNA sequence of shrimp GBP showed high similarity to that of crayfish LGBP, other insect recognition proteins as well as bacterial and sea urchin glucanases. Shrimp injected with an insoluble β-1,3-glucan, curdlan or heat-killled Vibrio harveyi did not show any significant changes in relevant mRNA levels. An attempt to knock out the LGBP expression by its exogeneous dsRNA was done in a proliferating blood cell culture from the hematopoietic tissue of crayfish. We found that the expression of endogeneous LGBP mRNA could be substantially inhibited by incubation of dsRNA-LGBP in the cell culture. The effect is quick, specific, and also affects the cell behaviours

Research update on emergent shrimp pathogens in Thailand

Abstract only.Recent evidence suggest that the emergent microsporidian, Enterocytozoon hepatopenaei (EHP) is a component cause of white feces syndrome (WFS) in shrimp. The natural WFS shrimp were found to be infected with EHP. At the laboratory level, shrimp induced to be heavily infected with EHP showed no WFS symptom suggesting that the causes of WFS is complex involved with other cause, not only EHP. The other component causes are under investigation. Better understanding of virulence mechanism of EHP infection in shrimp will assist in establishing innovative strategies to reduce its viability and potential infectivity in shrimp farms. Transmission of microsporidia is involved ingestion of spores in the water and the site of initial infection being the gastrointestinal tract. EHP spore is having a thick, protective chitinous wall around the cell membrane that allows them to survive outside their hosts and involve with the microsporidian pathogenesis. Here we describe successful purification of active EHP spores with a novel spore viability assay based on polar-tube extrusion or germination triggered by Phloxin B. The physical conditions such as temperature and PH, and chemical factors such as KMnO4, and chlorine that affect spore germination were examined as a practical guideline for the inactivation of the spores at a farm level. The potential environmental reservoir of EHP were found to be a mussel of the genus Mytilopsis, which is found frequently in the water canal or pipe in the shrimp rearing system. Recent evidence demonstrates that the mussel can be infected by EHP and can transmit EHP to shrimp in the laboratory model

PmRab7 Is a VP28-Binding Protein Involved in White Spot Syndrome Virus Infection in Shrimp

Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membrane-associated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-022-08802-3

Physical maps of plasmids harboring m<i>Pm</i>Rab7 and pVP28 fused with half 3’-agglutinin of <i>S</i>. <i>cerevisiae</i> for surface display by <i>P</i>. <i>pastoris</i>.

<p>Physical maps of plasmids harboring m<i>Pm</i>Rab7 and pVP28 fused with half 3’-agglutinin of <i>S</i>. <i>cerevisiae</i> for surface display by <i>P</i>. <i>pastoris</i>.</p

Yeast agglutination and agglutination inhibition assays.

<p>(A) OD₆₀₀ of His⁺Mut^S yeast cells induced by methanol for 24–96 h, mixed at initial OD₆₀₀ = 0.7 and measured after 1 h. Percent agglutination was calculated from OD₆₀₀ at time zero and after 1 h. (B) Percent inhibition of agglutination by treatment of yeast cells with <i>Pm</i>Rab7 and VP28 antibodies for 1 h prior to agglutination assay. Percent inhibition was calculated as percent of agglutination with and without specific antibody treatment. The values were obtained from three independent experiments. Bar = means ± SD (Some of the SD bars are so small that they cannot be seen in the graph).</p