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

This is the final version of the article. Available from the publisher via the DOI in this record.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.This project was supported by the Agricultural Research Development Agency (ARDA) of Thailand under project CRP5905020530, by the Thailand Research Fund (TRF) under project IRG5980008 and TRG5780032, by the Newton Institutional Links (IL) program to BIOTEC, Thailand and Cefas, UK, and by Mahidol University. PJ would like to thank the Science Achievement Scholarship of Thailand (SAST) for a PhD scholarshi

A nested PCR assay to avoid false positive detection of the microsporidian enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms

PublishedJournal Article© 2016 Jaroenlak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.OI acknowledges support from Agricultural Research Development Agency under project CRP5905020530 and Mahidol University. KS received funding from National Research Council Thailand, Division of Plan Administration and Research Budget/2557-79. PJ is supported by the Science Achievement Scholarship of Thailand (SAST). GDS acknowledges support of DG SANCO of the European Commission, and the UK Department of Environment, Food and Rural Affairs under project FB002. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

New Paradigms to Help Solve the Global Aquaculture Disease Crisis.

Published onlineJournal ArticleThis is the final version of the article. Available from Public Library of Science via the DOI in this record.n/aThe authors (GDS, KS) acknowledge funding administered by the British Council under the Newton Fund Researcher Links Programme, for a UK-Thailand bilateral workshop entitled "Scientific, technological and social solutions for sustainable aquaculture in Thailand: a key player in global aquatic food supply," Bangkok, March 2016. Further funding support is acknowledged from the European Commission (EC) and the UK Department for Environment, Food and Rural Affairs (Defra) under contracts C6928 and FB002 (to GDS and DB); from the Royal Society under a University Research Fellowship (to BAPW); and to the Agricultural Research Development Agency (ARDA) and National Research Council of Thailand (NRCT) (to KS, TWF, and OI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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

The scope of the crustacean immune system for disease control

The culture or wild capture of marine and freshwater shellfish, including crustaceans, is without doubt a key source of protein for a burgeoning world population. Historically the expansion of aquaculture has, however, been accompanied by the increased incidence of economically significant diseases, most notably of viral and bacterial origin. Since the late 1970s great progress has been made in our understanding of the generalized protostome innate immune system. Distinct pathways, pathogen receptor proteins and effector molecules have since been identified that are not ancestral or homologous to those of the deuterostomes, including vertebrates. Within the past decade progress has accelerated with the rapid characterisation of new classes of recognition proteins, immune effectors and regulatory pathways. This paper provides a broad overview of our current understanding of invertebrate immunology, taking the crustacean decapod immune system as its focus. Recent developments in the field are described briefly and their implications and potential considered. These advances offer fundamental new insights in our efforts to understand disease in cultured populations and also to develop knowledge of environmental effects on host/pathogen interactions within a fishery context. Of course, challenges do remain, including the lack of an immortal cell line and the limited publically-available genomic resources. These are considered in this review as priorities for future research effort. With the continued application of more insightful technologies, coupled with associated investment, it is expected that the speed at which some of these issues are resolved will accelerate

Diseases of marine fish and shellfish in an age of rapid climate change

Peer reviewe

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

Compounds from Silicones Alter Enzyme Activity in Curing Barnacle Glue and Model Enzymes

Background: Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. Methodology/Principal Findings: GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Conclusions/Significance: Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties

Selection of shrimp breeders free of white spot syndrome and infectious hypodermal and hematopoietic necrosis

The objective of this work was to select surviving breeders of Litopenaeus vannamei from white spot syndrome virus (WSSV) outbreak, adapted to local climatic conditions and negatively diagnosed for WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV), and to evaluate if this strategy is a viable alternative for production in Santa Catarina, Brazil. A total of 800 males and 800 females were phenotypically selected in a farm pond. Nested-PCR analyses of 487 sexually mature females and 231 sexually mature males showed that 63% of the females and 55% of the males were infected with IHHNV. Animals free of IHHNV were tested for WSSV, and those considered double negative were used for breeding. The post-larvae produced were stocked in nine nursery tanks for analysis. From the 45 samples, with 50 post-larvae each, only two were positive for IHHNV and none for WSSV. Batches of larvae diagnosed free of virus by nested-PCR were sent to six farms. A comparative analysis was carried out in growth ponds, between local post-larvae and post-larvae from Northeast Brazil. Crabs (Chasmagnathus granulata), blue crabs (Callinectes sapidus), and sea hares (Aplysia brasiliana), which are possible vectors of these viruses, were also evaluated. The mean survival was 55% for local post-larvae against 23.4% for post-larvae from the Northeast. Sea hares showed prevalence of 50% and crabs of 67% of WSSV