Integration of histopathology and molecular tools in shrimp health surveillance: A case study from Colombia.

Pacific white shrimp Penaeus vannamei that were pre-exposed to Taura syndrome virus (TSV) and then challenged with yellow head virus (YHV) acquired partial protection from yellow head disease (YHD). Experimental infections were carried out using specific-pathogen-free (SPF) shrimp which were first exposed per os to TSV; at 27, 37 and 47 d post infection they were then challenged by injection with 1 × 104 copies of YHV per shrimp (designated the TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a positive control. Survival analyses comparing the TSV-YHV and YHV (positive control) groups were conducted, and significant survival rates were found for all the time groups (p < 0.001). A higher final survival was found in the TSV-YHV group (mean 55%) than in the positive control (0%) (p < 0.05). Duplex reverse transcription quantitative PCR was used to quantify both TSV and YHV. Lower YHV copy numbers were found in the TSV-YHV group than in the positive control in pleopods (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.001) and lymphoid organ (LO) samples (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.01). In situ hybridization assays were conducted, and differences in the distribution of the 2 viruses in the target tissues were found. The foci of LO were infected with TSV but were not infected with YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could, in part, explain the absence of YHD in the Americas, where P. vannamei are often raised in farms where TSV is present.

Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry

Accurate and rapid detection of six viral pathogens in penaeid shrimp by using a novel multiplex PCR.

Crustacean diseases in European legislation: Implications for importing and exporting nations

Low viral loads and lymphoid organ spheroids are associated with yellow head virus (YHV) tolerance in whiteleg shrimp Penaeus vannamei

A one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as penaeid rod shaped DNA virus (PRDV), hypodermal and haematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV) and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR, RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid, where specific amplification is obtained for PRDV, IHHNV, TSV and YHV in 60 min under isothermal conditions at 63 o C employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed that all of these viruses can be detected up to 100 copies of template DNA, rendering it ten-fold more sensitive than conventional LAMP assay.

Vector or reservoir species of three diseases of crustaceans listed in the Animal Health Law were identified based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Crustacean species on or in which Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV) were identified, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected crustaceans was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90–100%) that WSSV, TSV and YHV will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or aquaculture establishments or by water supply can possibly transmit WSSV, TSV and YHV.

Penaeus vannamei farming is one of the profitable businesses of the aquaculture sector in India. As a result of excessive farming, shrimps were susceptible to various exotic, transboundary, and emerging pathogens. The purpose of this study is to monitor the prevalence of shrimp pathogens in Karnataka. A total of 91 samples of P. vannamei post larvae and juveniles were collected from April 2022 to November 2022 in Karnataka. Samples were screened for pathogens as listed in Office of Internationale des Epizootics (OIE) or World Organisation for Animal Health (WOAH) which include infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND), hepatopancreatic parvovirus (HPV), infectious myonecrosis virus (IMNV), yellow head virus (YHV), taura syndrome virus (TSV), white spot syndrome virus (WSSV), and other diseases such as decapod iridescent virus-1(DIV-1), and monodon baculovirus (MBV). Out of 91 samples, 5 (5.5%) samples were positive for WSSV, 26 (28.6%) samples were positive for EHP, and 2 (2.2%) samples had co-infection caused by EHP and WSSV. In this study, we have reported a high prevalence of EHP than WSSV in all three coastal districts of Karnataka. Farmers were using specific pathogen-free (SPF) seeds for culture but still, their cultures are getting infected with the same pathogens which indicates poor pond preparation and bio-security. So we strongly recommend that farmers have to follow good management practices and bio-security to increase the productivity and sustainability of P. vannamei farming in India.

An improved Taura syndrome virus (TSV) RT-PCR using newly designed primers

Viral diseases are one of the constraints affecting the sustainability of the shrimp farming industry worldwide. The common causes of viral infections include white spot syndrome virus, infectious hypodermal and haematopoietic necrosis virus, hepatopancreatic parvovirus, Penaeus monodon nudivirus, decapod iridescent virus 1, Taura syndrome virus, yellow head virus, infectious myonecrosis virus and Macrobrachium rosenbergii nodavirus. Accurate diagnostic methods are necessary for preventing the spread of these transboundary diseases. Many molecular diagnostic methods for shrimp viral diseases have been developed and are currently in use, including conventional polymerase chain reaction (PCR), nested PCR, loop‐mediated isothermal amplification PCR, recombinase polymerase assay and real‐time PCR. Although the World Organization for Animal Health (founded as OIE) recommends several PCR methods for shrimp disease diagnosis, there has been a lack of efforts to comparatively evaluate the performance of the available methods, resulting in a significant knowledge gap. Therefore, in this review, we compare various PCR assays and commercial PCR‐based diagnostic kits that have been developed and/or published previously, and discuss pros and cons of each of these methods. Our objective of this review is to offer enhanced clarity and a comprehensive overview of molecular diagnostic methods for shrimp viral diseases. This endeavour would serve as an important resource for researchers engaged in relevant studies and professionals within the shrimp‐farming industry, providing valuable guidance and insights.

Costs and benefits of freedom from shrimp diseases in the European Union

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.

In recent years, several pathogens have emerged and caused heavy mortality in Asia. In this study, we investigated the prevalence of pathogens in specimens of whiteleg shrimp (Penaeus vannamei) collected from greenhouse ponds. In total, six pathogens were tested in these shrimp, three of which, including Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus and infectious hypodermal and haematopoietic necrosis virus (IHHNV), were detected in shrimp aquaculture, with EHP being the most prevalent; Taura syndrome virus (TSV), White spot syndrome virus (WSSV) and yellow head virus (YHV) were not detected in these samples. Ultrastructure examination revealed the presence of a large number of EHP spores infecting hepatopancreatic epithelial cells of shrimp. Body weight (BW), body length (BL) and BW/BL ratio, revealed growth retardation among EHP-infected shrimp, as indicated by the significantly lower values of BW, BL and BW/BL ratio in these shrimps than those in EHP-uninfected shrimp (P<0.05). Together, these data indicated that EHP causes severe growth retardation of shrimp and is a major pathogen of aquacultured shrimp culturing in greenhouse ponds in Jiangsu Province; effective measures should be undertaken to control its spread.

Historic emergence, impact and current status of shrimp pathogens in Asia

Necrotizing hepatopancreatitis (NHP) is a severe bacterial disease affecting penaeid shrimp aquaculture. NHP is caused by the gram-negative, pleomorphic, obligately intracellular NHP-bacterium (NHPB) that targets the hepatopancreas tissue of shrimp. NHPB is classified as an α -proteobacterium and is related to members of the Rickettsia. First reported in 1985 in a Texas shrimp farm, NHP has since affected shrimp farming in several North and South American countries, causing mortalities up to 95% and devastating economic losses to aquaculture crops. Because NHPB remains unculturable through traditional in vitro methods, the development of an in vivo cultivation system of NHPB in susceptible, specific pathogen-free Litopenaeus vannamei has allowed advancement of experimental research, including NHPB population biology and transmission dynamics. Current research on NHPB is discussed relative to management strategies of NHP disease in a shrimp pond. Compared to other important shrimp pathogens, NHPB is geographically limited, like that of yellowhead virus (YHV), but in contrast to the global distribution of Taura syndrome virus (TSV) and white spot syndrome virus (WSSV). Additionally, NHPB is less virulent than TSV and WSSV, and the transmission dynamics of TSV and YHV are much more complicated than that of NHPB and WSSV in penaeid shrimp.