A new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as acute ...hepatopancreatic necrosis syndrome (AHPNS). Affecting both Pacific white shrimp Penaeus vannamei and black tiger shrimp P. monodon, the disease has caused significant losses in Southeast Asian shrimp farms. AHPNS was first classified as idiopathic because no specific causative agent had been identified. However, in early 2013, the Aquaculture Pathology Laboratory at the University of Arizona was able to isolate the causative agent of AHPNS in pure culture. Immersion challenge tests were employed for infectivity studies, which induced 100% mortality with typical AHPNS pathology to experimental shrimp exposed to the pathogenic agent. Subsequent histological analyses showed that AHPNS lesions were experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas. Bacterial isolation from the experimentally infected shrimp enabled recovery of the same bacterial colony type found in field samples. In 3 separate immersion tests, using the recovered isolate from the AHPNS-positive shrimp, the same AHPNS pathology was reproduced in experimental shrimp with consistent results. Hence, AHPNS has a bacterial etiology and Koch's Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus.
Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp ...industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.
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The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13‑028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities ...in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10(5) CFU ml(-1) and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.
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•The EHP histology includes basophilic inclusions within hepatopancreas cells.•We developed a specific EHP in situ hybridization assay using digoxigenin-labeled probe.•An EHP specific ...PCR was developed targeting its 18S rRNA gene region.•By PCR, EHP was detected in the infected shrimp, their feces and water samples.•By PCR, EHP was detected in some Artemia biomass.
A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Biosecurity, as it is being applied to shrimp aquaculture, may be defined as the practice of exclusion of specific pathogens from cultured aquatic stocks in brood stock facilities, hatcheries, and ...farms, or from entire regions or countries for the purpose of disease prevention. To make a biosecurity program a functional concept in shrimp aquaculture, the relevant risks should be identified and the appropriate biosecurity measures put into practice to mitigate those risks. Examples of biosecurity measures put into place for this purpose may include such basics as site selection when the intent is to locate a new shrimp culture facility in an area where certain diseases are not enzootic. Standard facilitylfarm operating procedures can be adapted to minimize the risks of disease introduction and spread within a facility through such concepts as pretreatment of all source water, and reduced or “zero” water exchange. Stocking shrimp culture facilities with domesticated shrimp stocks that are free of specific diseases (“Specific Pathogen Free” or SPF) and or with stocks resistant to specific disease agents (SPR) is perhaps the most important single component of a biosecurity program. The example set by the development of domesticated SPF stocks of Litopenaeus vannamei has helped to make biosecure shrimp culture feasible. The development of these and other SPF stocks, and the diagnostic methods to develop and monitor them for specific diseases and disease causing agents, have been milestones in the development of the international shrimp farming industry in recent years, and it has contributed to the species rivaliig Penaeus monodon as the dominant farmed shrimp species. The regular monitoring (surveillance) of shrimp stocks in biosecure culture facilities is a necessary component of a biosecurity plan, as is having in place a contingency plan for disease containment and eradication should a breach occur in the physical and managerial components of a biosecure facility and a targeted disease occur.
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Acute hepatopancreatic necrosis disease (AHPND) has caused severe mortalities in farmed penaeid shrimp throughout SE Asia and Mexico. The causative agent of AHPND is the marine bacterium Vibrio ...parahaemolyticus, which secretes PirA- and PirB-like binary toxin that caused deterioration in the hepatopancreas of infected shrimp. The genes responsible for the production of this toxin are located in a large plasmid residing within the bacterial cells. We analyzed the plasmid sequence from the whole genome sequences of AHPND-V. parahaemolyticus isolates and identified 2 regions that exhibit a clear geographical variation: a 4243-bp Tn3-like transposon and a 9-bp small sequence repeat (SSR). The Tn3-like transposon was only found in the isolates from Mexico and 2 unspecified Central American countries, but not in SE Asian isolates from China, Vietnam, and Thailand. We developed PCR methods to characterize AHPND-V. parahaemolyticus isolates as either Mexican-type or SE Asian-type based on the presence of the Tn3-like transposon. The SSR is found within the coding region of a hypothetical protein and has either 4, 5, or 6 repeat units. SSRs with 4 repeat units were found in isolates from Vietnam, China, and Thailand. SSRs with 5 repeat units were found in some Vietnamese isolates, and SSRs with 6 repeat units were only found in the Mexican isolates.
A quantitative polymerase chain reaction (qPCR) assay was developed, based on a TaqMan probe, to detect and quantify a virulence plasmid harbored by the bacterium Vibrio parahaemolyticus which can ...cause acute hepatopancreatic necrosis disease (AHPND). The assay uses a pair of PCR primers, which amplify a 135-bp DNA fragment, and a TaqMan probe selected from the plasmid pirA-like gene. This qPCR assay reacted with AHPND-pathogenic isolates of V. parahaemolyticus collected from Vietnam and Mexico, but not with non-pathogenic strains of Vibrio spp. For quantification, a plasmid (pVpPirA-1) containing the target pirA-like gene was constructed, purified and serially diluted to be used as a standard. With this standard, the qPCR assay was then used to quantify the virulence plasmid in shrimp samples collected from different farms. Up to 5.8×105 copy per mg tissue were detected in AHPND-affected shrimp collected from Vietnam. Lower quantities, up to 1.5×104 copies per mg of tissues were detected in affected shrimp collected from a Chinese farm. In the laboratory bioassays, similar plasmid quantities, 1.8×103 to 4.7×106 copies of plasmid per mg of tissues were found in the moribund/dead shrimp, 3.5×102 to 2.2×106 copies of plasmid per mL were detected in the water samples. This assay is specific with high sensitivity (10 copies of virulence plasmid) and can be used to detect AHPND-pathogenic V. parahaemolyticus in shrimp and water samples.
•We developed a qPCR method to detect and quantify AHPND (EMS).•This assay is specific with high sensitivity (10 copies of virulence plasmid).•This method can be used for quantification in water and shrimp samples from fields.
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The causative agent of myonecrosis affecting cultured Penaeus vannamei in Brazil was demonstrated to be a virus after purification of the agent from infected shrimp tissues. Purified viral particles ...were injected into specific pathogen-free P. vannamei, resulting in a disease that displayed the same characteristics as those found in the original shrimp used for purification. The virus was named infectious myonecrosis virus (IMNV). The viral particles were icosahedral in shape and 40 nm in diameter, with a buoyant density of 1·366 g ml-1 in caesium chloride. The genome consisted of a single, double-stranded (dsRNA) molecule of 7560 bp. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 5' ORF (ORF 1, nt 136-4953) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsRNA-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kDa. The 3' ORF (ORF 2, nt 5241-7451) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsRNA virus family that infects invertebrate hosts.
A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by
Lo et al. (1996) and outlined as the recommended PCR diagnostic assay ...in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (
OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.
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