A laboratory challenge model for evaluating enyterocytozoon hepatopenaei susceptibility in selected lines of pacific whiteleg shrimp Penaeus vannamei.

Here we report for the first time a laboratory challenge model for Enterocytozoon hepatopenaei (EHP) to determine the difference of two Specific Pathogen Free (SPF) lines of Penaeus vannamei shrimp. These lines were experimentally challenged using EHP-infected fecal strings as inoculum. Real-time PCR and histopathology assays were performed to confirm EHP infection and evaluate differences in EHP susceptibility in the two genetic lines screened. Although the histopathology of the hepatopancreas tissue showed EHP lesions in both challenged groups, the histological lesions were more pronounced in one of the SPF lines. Quantitative PCR results revealed that animals displaying less hepatocellular damage have lower EHP load compared to animals displaying more pronounced pathological changes. There was no significant difference in final survival at 36 days post-infection in these lines with survival ranging between 80 and 100%. The data showed that mortality as an endpoint metric is not a suitable parameter to determine genetic susceptibility to EHP. Instead, histopathological changes in hepatopancreas, EHP load of the same tissue, and growth retardation would be better metrics to screen EHP susceptibility in P. vannamei. The results show the feasibility of screening genetic lines of P. vannamei for EHP resistance/tolerance using fecal string as an inoculum and, assessing histopathological changes, EHP load, and weight as indicators of resistance.

Differentially Expressed Genes in Hepatopancreas of Acute Hepatopancreatic Necrosis Disease Tolerant and Susceptible Shrimp (Penaeus vannamei).

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pirA and pirB in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei. This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei.

Novel infectious myonecrosis virus (IMNV) genotypes associated with disease outbreaks on Penaeus vannamei shrimp farms in Indonesia.

Infectious myonecrosis virus (IMNV) is one of the most pathogenic viruses that affect Penaeus vannamei shrimp. In 2018, IMNV was reported in grow-out ponds of P. vannamei in Situbondo, Indonesia. Diseased animals displayed clinical signs of infectious myonecrosis (IMN) characterized by white discoloration of skeletal muscle. Histopathology of affected shrimp revealed lesions that are pathognomonic of IMNV infection. The major capsid protein (MCP) gene was amplified and sequenced from representative samples showing IMN pathology. Multiple alignment of predicted amino acid sequences of the MCP gene with known IMNV genotypes in the GenBank database revealed three unique genotypes, SB-A, SB-B and SB-C,in Situbondo samples. The number of amino acid changes in SB-A, SB-B and SB-C compared to known IMNV genotypes ranged from 7-710, including the isolate SB-B, which contains deletion of 622 aa. A phylogenetic analysis using homologous sequences from Brazil and Indonesia showed that these three isolates represent new IMNV genotypes.

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 20-22 nm in size and contain a single-stranded DNA (ssDNA) of 4-6 kb 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.