Infectious Hypodermal And Hematopoietic Necrosis Research Articles

Studies on infectious myonecrosis virus (IMNV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) in cultured penaeid shrimp in Egypt.

The present study aimed to diagnose infectious myonecrosis virus (IMNV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) among cultured penaeid shrimp (Penaeus semisulcatus, n = 120) collected from private farms in 2 Egyptian provinces (Damietta and North Sinai) along the Mediterranean coast. The collected shrimp were subjected to clinical examination, histopathology, molecular characterization, and phylogenetic analysis. Most of the shrimp infected with IMNV showed a distinctive appearance resembling cooked shrimp and white necrosis on distal abdominal segments and tail fans. Simultaneously, IHHNV-infected cases displayed opaque abdominal muscles, white milky to buff mottling on the shell, and a pathognomonic runt-deformity syndrome. Histopathological examination of infected specimens revealed muscular edema, hemocyte infiltration, deformities, Zenker's necrosis, and eosinophilic intra-nuclear inclusion bodies (Cowdry type A). PCR results gave predictable amplicon sizes of 139 and 81 bp and confirmed the presence of IMNV and IHHNV with a total prevalence of 37.5 and 25%, respectively. A homology search by BLAST analysis showed that the retrieved isolates putatively belonged to IMNV and IHHNV based on 96.3 to 97% nucleotide identity to the corresponding open reading frame gene of each virus. The phylogenetic analysis clearly showed genetic similarity and cross-lineage between our isolates and other isolates from Egypt, the USA, Brazil, Indonesia, China, Korea, Taiwan, and Ecuador. In conclusion, gross inspection and histopathology may aid in the diagnosis of viral diseases; however, molecular tools are indispensable for confirming a possible infection. The current study recommends strict regulations during live shrimp transportation and implementing health control certificates over all imports and exports, especially in developing countries, including Egypt.

Reduced transmission of IHHNV to Penaeus monodon from shrimp pond wastewater filtered through a polychaete-assisted sand filter (PASF) system

A polychaete-assisted sand filter (PASF) can aid water recirculation systems for shrimp culture ponds by removing large amounts of nutrients and suspended solids and chemically treating wastewater. By ingesting and degrading organic matter, Perinereis helleri polychaetes reared in a PASF can accumulate and potentially remove infectious hypodermal and hematopoietic necrosis virus (IHHNV) present in wastewater from ponds rearing virus-infected Penaeus monodon shrimp. Reported here are data showing that filtering pond wastewater through a PASF reduces its potential to transmit IHHNV. The trial employed 3 groups of 4 tanks each containing 20 P. monodon randomly selected from a pond in which only low-level IHHNV infection was evident at moderate prevalence (IHHNV-low Pond 2). Over a 2 week period, each tank group was supplied with wastewater from either the same pond (Pond 2) or IHHNV-high Pond 1, in which IHHNV was 100% prevalent at ~104-fold higher infection loads, either directly or after being filtered through a PASF. IHHNV real-time qPCR data on total nucleic acid (TNA) extracted from pleopods of 35 P. monodon selected at random from each group identified an elevated IHHNV infection prevalence (91%) in shrimp tested from tanks supplied directly with wastewater from Pond 1. In comparison, IHHNV was detected at a much reduced prevalence and lower loads among shrimp tested from tanks supplied with wastewater from Pond 2 (33%) or from Pond 1 after it had been filtered through a PASF (31%). The IHHNV prevalence and load data indicate that a PASF can play a useful role in reducing the potential for shrimp pond wastewater to transmit IHHNV infection to naïve P. monodon.

Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of enterocytozoon hepatopenaei (EHP)

The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP) causes retardation of shrimp growth, leading to significant financial losses in shrimp aquaculture. Therefore, the development of an efficient and sensitive detection method will be conducive to the prevention and control of the shrimp parasite. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) method that can detect EHP within 15 min at a constant temperature of 38.5 °C. The detection limit of this EHP RPA was 10 copies/μL of DNA molecules per reaction. The specificity of EHP RPA was tested, and the assay did not cross-react with white spot syndrome virus (WSSV), shrimp hemocyte iridescent virus (SHIV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), or Vibrio parahaemolyticus. Field and clinical applicability of this assay was evaluated using 61 field samples. The coincidence rate of the detection results for the clinical samples between RPA and qPCR was 95.1 %. In summary, the real-time RPA analysis provides an efficient and sensitive detection method for EHP.

Major viral diseases in culturable penaeid shrimps: a review

The practice and development of the culture of commercially important penaeid shrimp results in the occurrence of infectious and non-infectious diseases worldwide. Most of the diseases are caused by the opportunistic microorganisms which are part of the microflora and fauna of the penaeid shrimp. Once the pathogens get a favourable condition, it will cause diseases to the host organisms. Most of the organisms are ubiquitous and are found and reported all over the cultured area in the world. The pathogens are the bacteria, viruses, parasites and protozoans. Viruses are the most important disease-causing agents in penaeid shrimps. The penaeid viruses are widely spread in geographical areas of shrimp culture. The shipment of broodstock and post larvae from one geographical region to another region often results in spreading of viral diseases. The penaeid viruses are the Whispovirus, white spot syndrome virus (WSSV), Penaeus monodon baculovirus (monodon baculovirus), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV), Taura syndrome virus (TSV), yellow head virus (YHV), white tail disease (WTD), covert mortality nodavirus (CMNV), Laem–Singh virus (LSNV) and shrimp haemocyte iridescent virus (SHIV). The review examines and highlights the aquaculture penaeid shrimp viral diseases in detail and concludes the need and importance of severe disease surveillance of the global countries through networking and coordination to safeguard the aquaculture practice for our future generation.

Polychaetes (Perinereis helleri) reared in sand beds filtering nutrients from shrimp (Penaeus monodon) culture ponds can transiently carry IHHNV

A polychaete-assisted sand filter (PASF) system has been developed to help remove nutrients from aquaculture pond wastewater whilst also producing polychaetes that are highly prized as bait by recreational anglers and as a dietary supplement to improve the fecundity of shrimp broodstock. Whilst rearing polychaetes in PASF beds offers potential to reduce impacts of sourcing them from the wild, the use of wastewater from ponds rearing shrimp such as Penaeus monodon will present a biosecurity risk of viruses being transferred to, and potentially amplified in, the worms. To assess such risks for transmitting infectious hypodermal and haematopoietic necrosis virus (IHHNV), groups of 3 or 4 PASF beds seeded with sand worm (Perinereis helleri) juveniles were supplied with wastewater from ponds of P. monodon with either high-load or low-load IHHNV infections. TaqMan real-time qPCR identified low loads of IHHNV (≤878 IHHNV DNA copies 200 ng−1 TNA) in most worms from PASF beds supplied wastewater from the high-load pond. IHHNV was either not detected or detected at the qPCR test sensitivity limits in worms from beds supplied wastewater from the low-load pond. Purging harvested worms of their gut contents in clean filtered seawater for 2 days significantly reduced IHHNV loads. Reverting PASF beds to clean seawater for 8 weeks before harvest also significantly reduced worm loads of IHHNV. Daily additions of a commercial probiotic to the sand bed surface for 4 weeks prior to clean seawater application provided no discernible benefit to IHHNV clearance. While clearly demonstrated to be capable of carrying IHHNV, the remediation measures examined suggest potential to ameliorate the infection transmission risks of P. helleri reared in PASF beds supplied with shrimp pond wastewater as a nutrient source.

Transcriptome analysis of Procambarus clarkii infected with infectious hypodermal and haematopoietic necrosis virus

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii.

Heritability and genetic correlations for infectious hypodermal and hematopoietic necrosis virus load, body weight at harvest, and survival rate in Pacific white shrimp (Litopenaeus vannamei)

AbstractHeritability and genetic correlations for body weight at harvest size (BW), grow‐out survival (SU), and log‐transformed infectious hypodermal and hematopoietic necrosis virus (IHHNV) load (VLln) in Litopenaeus vannamei were estimated. Data were obtained in 2012 from a shrimp breeding population in Mexico using 12,440 shrimp for BW and 16,814 shrimp for SU, and quantitative polymerase chain reaction determinations from 160 full‐sib families were obtained for VLln. DNA was extracted from pooled muscle samples of six shrimp per family for a total of 960 individuals. Heritability estimates for BW, SU, and VLln at the family mean and at individual levels were 0.24 ± 0.08, 0.02 ± 0.01, 0.42 ± 0.45, and 0.08 ± 0.11, respectively. Genetic correlation estimates of BW with SU, BW with VLln, and VLln with SU were 0.40, −0.04, and −0.57, respectively. Our results suggest that VLln has genetic variation and a favorable genetic association with BW and SU. No serious drawbacks were found from selection responses estimated using single‐trait and multitrait indices to increase BW and SU and decrease VLln. Favorable correlated responses for IHHNV tolerance estimated as BW/VLln and SU/VLln were also found. This study offers new insights into the possibility of using IHHNV viral load as a possible selection criterion in L. vannamei breeding programs.

Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay

Shrimp hemocyte iridescent virus (SHIV), which was first identified in white leg shrimp (Litopenaeus vannamei) in China in 2014, can cause extensive shrimp mortality and major economic losses in the shrimp farming industry in China. In this study, a novel real-time isothermal recombinase polymerase amplification (RPA) assay was developed using a TwistAmp exo kit for SHIV detection. First, five primers and a probe were designed for the major capsid protein gene (GenBank: KY681039.1) according to the TwistDx manual; next, the optimal primers were selected by a comparison experiment. The primers and probe were specific for SHIV and did not react with shrimp white spot syndrome virus (WSSV), shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp enterocytozoon hepatopenaei (EHP), and macrobrachium rosenbergii nodavirus (MrNV) samples, as well as pathogens of acute hepatopancreatic necrosis disease (AHPND). The RPA assay reached a detection limit of 11 copies per reaction according to probit regression analysis. In addition, RPA assay detected the positive plasmid samples at concentration of 1000 copies/μL within 16.04 ± 0.72 min at a single low operation temperature (39 °C). The results proved that the proposed RPA method was an accurate, sensitive, affordable, and rapid detection tool that can be suitably applied for the diagnosis of SHIV in field conditions and in resource-poor settings.

Delivery of plasmid DNA to shrimp hemocytes by Infectious hypodermal and hematopoietic necrosis virus (IHHNV) nanoparticles expressed from a baculovirus insect cell system

Virus-like particles (VLPs) are potential containers for delivery of therapeutic agents at the nanoscale. In this study, the capsid protein of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was expressed in a baculovirus insect cell system. The 37-kDa recombinant protein containing the hexahistidine residues (His Tag) at N-terminal was purified using immobilized metal affinity chromatography (IMAC) and assembled into VLPs with a diameter of 23 ± 3 nm analyzed by transmission electron microscopy. We also verified that disassembly/reassembly of IHHNV-VLPs was controlled in the presence and absence of DTT. The efficiency of IHHNV-VLPs to encapsulate plasmid DNA was about 48.2%, and the VLPs encapsulating the pcDNA3.1(+)-EGFP plasmid DNA could recognize the primary shrimp hemocytes and deliver the loaded plasmid into cells by detection of expressed enhanced green fluorescent protein (EGFP). These results implied that the IHHNV-VLPs might be a good candidate for packaging and delivery of expressible plasmid DNA, and may produce an antiviral product in shrimp cells for gene therapy.

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.

Diversity of single-stranded DNA containing viruses in shrimp.

Over the past four decades, shrimp aquaculture has turned into a major industry providing jobs for millions of people worldwide especially in countries with large coastal boundaries. While the shrimp industry continues to expand, the sustainability of shrimp aquaculture has been threatened by the emergence of diseases. Diseases caused by single-stranded DNA containing viruses, such as infectious hypodermal and hematopoietic necrosis virus (IHHNV) and hepatopancreatic parvovirus (HPV), have caused immense losses in shrimp aquaculture since the early 1980s. In fact, the disease outbreak in the blue shrimp (Penaeus stylirostris) caused by IHHNV in early 1980s ultimately led to the captive breeding program in shrimp being shifted from P. stylirostris to the white shrimp (Penaeus vannamei), and today P. vannamei is the preferred cultured shrimp species globally. To date, four single-stranded DNA viruses are known to affect shrimp; these include IHHNV, HPV, spawner-isolated mortality virus (SMV) and lymphoidal parvo-like virus (LPV). Due to the economic losses caused by IHHNV and HPV, most studies have focused on these two viruses, and only IHHNV is included in the OIE list of Crustacean Diseases. Hence this review will focus on IHHNV and HPV. IHHNV and HPV virions are icosahedral in morphology measuring 20-22nm in size and contain a single-stranded DNA (ssDNA) of 4-6kb in size. Both IHHNV and HPV are classified into the sub-order Brevidensoviruses, family Densovirinae. The genome architecture of both viruses are quite similar as they contain two completely (as in IHHNV) or partially overlapping (as in HPV) non-structural and one structural gene. Histopathology and polymerase chain reaction (PCR)-based methods are available for both viruses. Currently, there is no anti-viral therapy for any viral diseases in shrimp. Therefore, biosecurity and the use of genetically resistant lines remains as the corner stone in the management of viral diseases. In recent years, gene silencing using the RNA interference (RNAi) approach has been reported for both IHHNV and HPV via injection. However, the delivery of RNAi molecules via oral route remains a challenge, and the utility of RNAi-based therapy has yet to be materialized in shrimp aquaculture.

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond-reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection.

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow-out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV-positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m-PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m-PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510bp for WSSV and EHP, respectively.

Diseases of shrimps and natural methods to protect their health

Shrimp farming is an economically important part of aquaculture. Shrimps constitute the largest portion of shellfish intended for human consumption. According to the World Organization for Animal Health (OIE), viral diseases pose the most serious threat to shrimp farming. There are currently six viral and two bacterial diseases on the OIE list. The most serious viral diseases are white spot disease (WSD), Taura syndrome (TS), infection with yellow head virus genotype 1 (YHV), infectious hypodermal and haematopoietic necrosis (IHHN), infectious myonecrosis (IMN) and white tail disease (WTD), whereas the most dangerous bacterial diseases are acute hepatopancreatic necrosis disease (AHPND) and necrotizing hepatopancreatitis (NHP). All these diseases result in high mortality, thus limiting shrimp production and causing large economic losses. Therefore, effective preparations are constantly sought to control infectious diseases in shrimps. Probiotics and immunostimulants, thanks to their demonstrated antibacterial and antiviral effects, as well their positive impact on the immune system, may play a crucial role in protecting the health of shrimps.

Nanocontainer designed from an infectious hypodermal and hematopoietic necrosis virus (IHHNV) has excellent physical stability and ability to deliver shrimp tissues.

BackgroundA virus-like particle (VLP) is an excellent tool for a compound delivery system due to its simple composition, symmetrical structure and self-assembly. Its surface modification both chemically and genetically is established, leading to the target-specific delivery and improved encapsulation efficiency. However, its physical stabilities against many harsh conditions that guarantee long term storage and oral administration have been much less studied.MethodsIHHNV-VLPs were reconstructed from recombinant IHHNV capsid protein in E. coli. Their physical properties against three strong physical conditions including long term storage (0–30 days) in 4 °C, physical stabilities against broad ranged pH (4–9) and against three types of digestive enzymes were tested. Disassembly and reassembly of VLPs for encapsidating an enhanced green fluorescent protein tagged plasmid DNA (EGFP-VLPs) were controlled by the use of reducing agent (DTT) and calcium specific chelating agent (EGTA). Lastly, delivering ability of EGFP-VLPs was performed in vivo by intramuscular injection and traced the expression of GFP in the shrimp tissues 24 hr post-injection.ResultsUpon its purification, IHHNV-VLPs were able to be kept at 4 °C up to 30 days with only slight degradation. They were very stable in basic condition (pH 8–9) and to a lesser extent in acidic condition (pH 4–6) while they could stand digestions of trypsin and chymotrypsin better than pepsin. As similar with many other non-enveloped viruses, the assembly of IHHNV-VLPs was dependent on both disulfide bridging and calcium ions which allowed us to control disassembly and reassembly of these VLPs to pack EGFP plasmid DNA. IHHNV-VLPs could deliver EGFP plasmids into shrimp muscles and gills as evident by RT-PCR and confocal microscopy demonstrating the expression of GFP in the targeted tissues.DiscussionThere are extensive data in which capsid proteins of the non-enveloped viruses in the form of VLPs are constructed and used as nano-containers for therapeutic compound delivery. However, the bottleneck of its application as an excellent delivery container for oral administration would rely solely on physical stability and interacting ability of VLPs to the host cells. These properties are retained for IHHNV-VLPs reported herein. Thus, IHHNV-VLPs would stand as a good applicable nanocontainer to carry therapeutic agents towards the targeting tissues against ionic and digestive conditions via oral administration in aquaculture field.

Investigation on the infectious nature of Running Mortality Syndrome (RMS) of farmed Pacific white leg shrimp, Penaeus vannamei in shrimp farms of India

Shrimp aquaculture in India has made significant strides especially after the introduction of Pacific white shrimp in the year 2009. However, intensification of culture practice has exacerbated several disease issues. Apart from infectious diseases, the Indian shrimp aquaculture has been witnessing certain syndromes, affecting productivity. One such syndrome associated with significant morbidity and mortality is popularly termed as running mortality syndrome (RMS) by shrimp farmers. Since 2011, RMS has been widely prevalent in the shrimp farms in Andhra Pradesh (AP) and Tamil Nadu (TN). The affected shrimp show patches of whitish musculature in the abdominal segments as a clinical sign with continuous low-level mortalities, especially after about 35–40 days of shrimp culture. We tried to investigate if this kind of mortality is infectious in nature. Investigations conducted in 34 farms comprising 25 RMS affected and 9 healthy farms were tested negative for all the major OIE listed and other pathogens such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), hepatopancreatic parvo virus (HPV), infectious myonecrosis (IMNV), Taura syndrome virus (TSV), yellow head virus (YHV), and Penaeus vannamei noda virus (PvNV). Bacteriological examination of haemolymph and hepatopancreas of RMS affected shrimp showed the predominance of Vibrio spp., such as Vibrio parahaemolyticus and Vibrio azureus. Histopathological examination of the hepatopancreas was found to be largely normal, except for haemocytic infiltration in the abdominal segments. Hemolymph of affected shrimp was observed to have less concentration of major and trace minerals than healthy ones. Bioassay through feeding RMS affected shrimp tissue to healthy shrimp and co-habitation experiment of healthy shrimp with the affected animals failed to induce RMS. When maintained in water with optimal physico-chemical parameters, affected shrimps showed recovery within 6–7 days. On the other hand, environmental parameters of pond waters such as total ammonia nitrogen, nitrite nitrogen and turbidity were relatively higher than the optimal values in RMS affected farms. Multiple correspondence statistical analysis of critical factors indicated running mortality to be associated with high stocking densities, high nitrite-N, and high turbidity. Though the study could not identify any specific known aetiological agent associated with RMS affected shrimp, failure to reproduce the syndrome by bioassay, the recovery of affected shrimp under the optimal environmental conditions and the positive correlation with critical environmental parameters and the stocking densities to mortality rates clearly suggest RMS to be a pond ecosystem or pond management associated syndrome rather than infectious in nature and thus can be overcome through best management practices.

Reduced growth performance of Black Tiger shrimp (Penaeus monodon) infected with infectious hypodermal and hematopoietic necrosis virus

In Australia, Infectious hypodermal and hematopoietic necrosis virus (IHHNV) occurs endemically in Black tiger shrimp (Penaeus monodon) and has been detected at high prevalence in farmed shrimp. Here we examined the role of high IHHNV infection prevalence and loads in reduced growth and survival in P. monodon reared under commercial conditions in 0.16 ha research ponds. TaqMan real-time quantitative (q)PCR testing of wild broodstock from the East Coast of Queensland identified lower and higher IHHNV infection loads among females used to generate 2 cohorts of progeny. Tracking of IHHNV loads in eggs and juveniles sampled progressively over grow-out identified these to be substantially higher and to increase more rapidly in 2 ponds stocked with a batch of postlarvae (PL) pooled from 3 females with high-level IHHNV infection at the time they spawned compared to 2 neighbouring ponds stocked with PL pooled from 4 females, of which only 1 possessed IHHNV at a relatively high load. From 120 days of culture (DOC) onwards, the growth performance in the IHHNV-high ponds began to progressively reduce compared to that in the IHHNV-low ponds. When harvested over 2 weeks starting at 155 DOC, the averaged harvest yield of the 2 IHHNV-high ponds was the equivalent of 3.72 t/ha lower than the 2 IHHNV-low ponds, and estimated survival was also markedly lower (79.5–84.5% compared to 95.9–99.8%). As real-time qPCR testing identified no involvement of either Gill-associated virus (GAV) or Yellow head virus genotype 7 (YHV7), other viruses known to cause such production losses in Australia, the data support a sustained high IHHNV infection burden as the primary contributor to the reduced growth and survival. Using a basic extrapolation of yield, stocking low-level IHHNV PL has resulted in an increase of farm gate value of $67,000 per hectare compared to stocking high-level IHHNV PL. Among other problems such as severe shell deformities that acute IHHNV infection has recently been identified to cause in locally-farmed P. monodon, the potential impacts on growth and survival identified here reinforce the value in screening-based selection of IHHNV-free/low broodstock for use in hatcheries and in breeding programs employing specific pathogen-free stocks, and strategies to select for IHHNV resistance/tolerance.

A novel real-time PCR approach for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) in the freshwater crayfish Procambarus clarkii.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infects many crustacean hosts, including cultured penaeid shrimp. In the present study, we aimed to develop a novel sensitive SYBR Green-based real-time PCR method to specifically amplify DNA fragments of IHHNV. Our newly developed real-time PCR method with a 195-bp amplicon specifically detected IHHNV and showed no cross reaction with white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), infectious myonecrosis virus (IMNV) and yellow-head virus (YHV). This method could detect as low as one single copy of IHHNV plasmid DNA, more sensitive than other SYBR Green-based real-time PCR methods and less expensive and more convenient than the TaqMan probe-based real-time PCR. Moreover, our data using the newly designed method showed that 80% of IHHNV-fed Procambarus clarkii samples were IHHNV positive. Our findings further confirmed that P. clarkii can be infected by IHHNV.

A fast and visual method for duplex shrimp pathogens detection with high specificity using rapid PCR and molecular beacon

Molecular diagnosis of genome is one of the major methods for pathogens detection. The commonly used PCR method can realize an exponential amplification of the target gene but is time-consuming. In this work, we proposed a duplex and visual method using rapid PCR combined with molecular beacons to specifically detect two kinds of shrimp pathogens in one reaction tube. We only need to observe the fluorescence change of the reaction tube with naked eye to determine the result. A home-made automatic transfer equipment allows reaction tubes shuttling quickly between two water baths to achieve rapid PCR amplification. A simple device was also designed to present the detection results easily determined with naked eye. This duplex and visual detection method is fast, low-cost and of high specificity. From DNA extraction to results judgment, only 15 min was enough. Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Vibrio parahaemolyticus (VP) are two common shrimp pathogens which were chosen as our detection objects. This method may give a possibility to conduct end-point visual duplex detection, which may make a positive influence on the pathogen prevention.

Real-time PCR tests to specifically detect IHHNV lineages and an IHHNV EVE integrated in the genome of Penaeus monodon.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) can cause mass mortalities in western blue shrimp Penaeus stylirostris, runt deformity syndrome in Pacific white shrimp P. vannamei and scalloped abdominal shell deformities in black tiger shrimp P. monodon. In P. monodon, however, PCR-based diagnosis of IHHNV can be complicated by the presence of a chromosome-integrated, non-replicating endogenous viral element (EVE). To facilitate high-throughput screening of P. monodon for IHHNV infection and/or EVE sequences, here we report real-time PCR tests designed to specifically detect IHHNV Lineage I, II and III but not EVE Type A sequences and vice versa. Using 108 dsDNA copies of plasmid (p)DNA controls containing either IHHNV or EVE-Type A sequences, both tests displayed absolute specificity. The IHHNV-q309 PCR reliably detected down to ≤10 copies of pDNA, at which levels a 309F/R PCR amplicon was just detectable, and the presence of an IHHNV-EVE sequence did not significantly impact its sensitivity. The IHHNV-qEVE PCR was similarly sensitive. Testing of batches of P. monodon clinical samples from Vietnam/Malaysia and Australia identified good diagnostic concordance between the IHHNV-q309 and 309F/R PCR tests. As expected for a sequence integrated into host chromosomal DNA, IHHNV-qEVE PCR Ct values were highly uniform among samples from shrimp in which an EVE was present. The highly specific and sensitive IHHNV-q309 and IHHNV-qEVE real-time PCR tests described here should prove useful for selecting broodstock free of IHHNV infection and in maintaining breeding populations of P. monodon specific pathogen free for IHHNV, and if desired, also free of IHHNV-EVE sequences.

Detection and quantification of shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR.

Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R2 = 0.998) in the range of 4 × 109 to 4 × 100 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei, Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA)-1. Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load.