Current status of infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) in the Peruvian and Ecuadorian shrimp industry.

Infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a crustacean disease that caused large-scale mortality in Penaeus stylirostris, deformity and growth retardation in Penaeus vannamei and Penaeus monodon. We surveyed the presence of IHHNV in three major shrimp-producing regions in Ecuador, namely Guayas, El Oro, and Esmeralda. The data show that IHHNV is endemic (3.3-100% prevalence) to shrimp farms in these regions. The whole genome sequences of representative circulating IHHNV genotypes in Ecuador and Peru showed that these genotypes formed a separate cluster within the Type II genotypes and were divergent from other geographical isolates of IHHNV originating in Asia, Africa, Australia, and Brazil. In experimental bioassays using specific pathogen-free (SPF) P. vannamei, P. monodon, and P. stylirostris and representative IHHNV isolates from Ecuador and Peru, the virus did not cause any mortality or induce clinical signs in any of the three penaeid species. Although IHHNV-specific Cowdry type A inclusion bodies were histologically detected in experimentally challenged P. vannamei and P. monodon and confirmed by in situ hybridization, no such inclusions were observed in P. stylirostris. Moreover, P. vannamei had the highest viral load, followed by P. monodon and P. stylirostris. Based on IHHNV surveillance data, we conclude that the currently farmed P. vannamei lines in Ecuador are tolerant to circulating IHHNV genotypes. The genome sequence and experimental bioassay data showed that, although the currently circulating genotypes are infectious, they do not induce clinical lesions in the three commercially important penaeid species. These findings suggest a potentially evolving virus-host relationship where circulating genotypes of IHHNV co-exist in equilibrium with P. vannamei raised in Peru and Ecuador.

Development of a Recombinase Polymerase Amplification (RPA) assay for acute hepatopancreatic necrosis disease (AHPND) detection in Pacific white shrimp (Penaeus vannamei).

Acute hepatopancreatic necrosis disease (AHPND) is currently the most important bacterial disease of shrimp that has caused enormous losses to the shrimp industry worldwide. The causative agent of AHPND are Vibrio spp. Carrying plasmids containing the pirA and pirB genes which encode binary toxins, PirAB. Currently, AHPND is mostly diagnosed by PCR-based platforms which require the use of sophisticated laboratory instrumentation and are not suitable for a point-of-care diagnostics. Therefore, the availability of an alternative method based on isothermal amplification would be suitable for AHPND detection outside a laboratory setting and extremely useful at a pond side location. Isothermal amplification is based on the nucleic acid amplification at a single temperature and does not require the use of a thermal cycler. In this study, we developed an isothermal Recombinase Polymerase Amplification (RPA) assay for AHPND detection targeting both pirA and pirB genes, simultaneously and evaluated the specificity and sensitivity of the assay. The assay could detect AHPND without any cross-reaction with other microbial pathogens and Specific Pathogen Free (SPF) shrimp. The limit of detection of the assay was 5 copies of pirAB genes. To evaluate the reliability of the assay in detecting AHPND, DNA from Penaeus vannamei shrimp displaying acute and chronic infection were analyzed by the RPA assay and the results were compared with SYBR Green real-time PCR assay. While there was a 100% conformity between the two assay while detecting acute phase infection, RPA appeared to be more sensitive in detecting chronic phase infection. The data suggest that RPA assay described here would be a reliable method in detecting AHPND outside a standard laboratory setting.

Development of an indirect Enzyme Linked Immunoassay (iELISA) using monoclonal antibodies against Photorhabdus insect related toxins, PirAVp and PirBVp released from Vibrio spp.

An acute hepatopancreatic necrosis disease (AHPND) causes serious losses to the global shrimp industry. The etiologic agent of AHPND is Vibrio spp. carrying a large plasmid which encodes a binary toxin, PirAB. Currently, AHPND is diagnosed by PCR based methods that detect the presences of both pirA and pirB genes. However, the bacterial strains containing the pirA and pirB genes do not always express the binary toxin, resulting in mis-estimation of the virulence of bacterial strains containing pirA and pirB genes. Thus, the immuno based assay (i.e. ELISA) is a promising approach to detect PirAVp and PirBVp. In the present study, a total of forty monoclonal antibodies clones (mAb) against PirAVp (20 mAbs) and PirBVp (20 mAbs) were screened by western blot analysis to select four mAb clones that show the strongest immunoreactivity in indirect ELISA (iELISA). The four selected mAbs (i.e. 1B9 and 5E9 against PirAVp; 7B7 and 7B9 against PirBVp) detected specifically Vibrio spp. causing AHPND. In addition, four selected mAbs were able to detect either PirAVp or PirBVp down to 0.008 ng/µl. A double blind assay using thirty AHPND-infected and six SPF shrimp Penaeus vannamei were analyzed by iELISA to determine the detection sensitivity of the assay. The results showed that iELISA was able to accurately detect 29 out of 30 AHPND infected shrimp. These finding indicated that iELISA is a reliable method to detect PirAVp and PirBVp toxins in infected shrimp and will be a useful tool in AHPND diagnosis and in studying the role of binary toxins in AHPND pathogenesis.

A comparative study of Enterocytozoon hepatopenaei (EHP) challenge methods in Penaeus vannamei.

The microsporidium Enterocytozoon hepatopenaei (EHP) is considered as an emerging pathogen threating the shrimp industry worldwide. It is an intracellular parasite that has been associated with retarded growth syndrome and white feces syndrome in shrimp. Although the impact of EHP to the shrimp industry is well known, many aspects of host-pathogen interactions are not well understood. A major limitation in the study of EHP is the lack of a reliable method to produce large quantities of inoculum rapidly and reproducibly. The present study was designed to compare different challenge methods including intramuscular injection, oral administration, co-habitation, hepatopancreas (HP) injection and reverse gavage. The results showed that the HP injection and the reverse gavage are two promising methods to infect shrimp rapidly and generate inoculum in a reproducible manner starting with a limited amount of inoculum. Therefore, the HP injection and reverse gavage were chosen for a scale-up study. Histopathology results showed that EHP proliferated in the epithelial cells of the HP in shrimp challenged via direct injection of inoculum into HP and reverse gavage treatments. In accordance with the histopathology results, the qPCR data showed that EHP loads in the challenged shrimp increased significantly with the HP injection and reverse gavage methods. Furthermore, the histopathological and quantification results indicate that HP injection and reverse gavage are two novel methods that can be used in EHP-challenge studies and for rapidly generating viable EHP inoculum.

Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB.

Acute hepatopancreatic necrosis disease (AHPND), also known as Early mortality syndrome (EMS), is a recently emerged lethal disease that has caused major economic losses in shrimp aquaculture. The etiologic agents are Vibrio spp. that carry Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB. A multiplex SYBR Green real-time PCR was developed that detects pirA, pirB, and two internal control genes, the shrimp 18S rRNA and the bacterial 16S rRNA genes in a single reaction. The pirB primers amplify the 3'-end of the pirB gene allowing the detection of Vibrio spp. mutants that contain a complete deletion of pirA and the partial deletion of pirB. The assay also detects mutants that contain the entire pirA gene and the deletion of the pirB gene. Since both toxin genes are needed for disease development, this assays can distinguish between pathogenic strains of Vibrio spp. that cause AHPND in shrimp and mutants that do not cause disease. The amplicons for pirA, pirB, 18S rRNA and 16S rRNA showed easily distinguishable melting temperatures of 78.21 ±â€¯0.18, 75.20 ±â€¯0.20, 82.28 ±â€¯0.34 and 85.41 ±â€¯0.21 °C respectively. Additionally, a duplex real-time PCR assay was carried out by designing TaqMan probes for the pirA and pirB primers. The diagnostic sensitivity and specificity was compared between the SYBR Green and TaqMan assays. Both assays showed similar sensitivity with a limit of detection being 10 copies for pirA and pirB, and neither assays showed any cross reaction with other known bacterial and viral pathogens in shrimp. The high sensitivity of both assays make them suitable for the detection of low copies of the pirA and pirB genes in AHPND causing Vibrio spp. as well as for detecting non-pathogenic mutants.

Characterization of a Kunitz-type protease inhibitor (MjKuPI) reveals the involvement of MjKuPI positive hemocytes in the immune responses of kuruma shrimp Marsupenaeus japonicus.

Serine proteases and their inhibitors play vital roles in biological processes. Serine protease inhibitors, including Kunitz-type protease inhibitors play important roles not only in physiological process (i.e. blood clotting and fibrinolysis) but also in immune responses. In this study, we characterized a Kunitz-type protease inhibitor, designated MjKuPI, from kuruma shrimp Marsupenaeus japonicus. An expression profile showed that MjKuPI was mainly expressed in hemocytes. Immunostaining revealed that some hemocytes expressed MjKuPI (MjKuPI(+) hemocytes) and others did not (MjKuPI(-) hemocytes). Injection of shrimp with Vibrio penaeicida and white spot syndrome virus (WSSV) upregulated the mRNA level of MjKuPI, and a flow cytometry analysis revealed that the proportion of MjKuPI(+) hemocytes increased significantly 24 h after injection. Together, these results suggest that MjKuPI and MjKuPI(+) hemocytes have a role in the innate immune system of kuruma shrimp.