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Water represents the most important component in the white spot syndrome virus (WSSV) transmission pathway in aquaculture, yet there is very little information. Detection of viruses in water is a challenge, since their counts will often be too low to be detected by available methods such as polymerase chain reaction (PCR). In order to overcome this difficulty, viruses in water have to be concentrated from large volumes of water prior to detection. In this study, a total of 19 water samples from aquaculture ecosystem comprising 3 creeks, 10 shrimp culture ponds, 3 shrimp broodstock tanks and 2 larval rearing tanks of shrimp hatcheries and a sample from a hatchery effluent treatment tank were subjected to concentration of viruses by ultrafiltration (UF) using tangential flow filtration (TFF). Twenty to 100 l of water from these sources was concentrated to a final volume of 100 mL (200–1000 fold). The efficiency of recovery of WSSV by TFF ranged from 7.5 to 89.61%. WSSV could be successfully detected by PCR in the viral concentrates obtained from water samples of three shrimp culture ponds, one each of the shrimp broodstock tank, larval rearing tank, and the shrimp hatchery effluent treatment tank with WSSV copy numbers ranging from 6 to 157 mL −1 by quantitative real time PCR. The ultrafiltration virus concentration technique enables efficient detection of shrimp viral pathogens in water from aquaculture facilities. It could be used as an important tool to understand the efficacy of biosecurity protocols adopted in the aquaculture facility and to carry out epidemiological investigations of aquatic viral pathogens. [ABSTRACT FROM AUTHOR]

White feces syndrome (WFS) has recently been reported as a serious problem for shrimp aquaculture in major shrimp farming nations. WFS affected shrimp show white/golden brown intestine, reduced feed intake, stunted growth and is often associated with loose shell, while WFS affected pond show masses of floating white feces on the surface of the pond. Accumulation of aggregated transformed microvilli (ATM) structures in hepatopancreas (HP) lumen that superficially resemble gregarine are reported to cause WFS. But the causal association of ATM and WFS is unknown. Also, WFS occurrences are significantly associated with incidences of the emerging microsporidian Enterocytozoon hepatopenaei (EHP) in the Penaeus vannamei grow-out system. The present study unveiled the occurrences of EHP spores along with ATM structures in the WFS affected shrimps. The histology section stained with calcofluor white stain (CFW) and semithin section revealed the presence of EHP spores in and around the transformed microvilli. Further, EHP spores were detected in all the individual WFS affected shrimps and white fecal threads by light microscopy and SWP-PCR. All WFS samples tested negative for WSSV, IHHNV, IMNV, and AHPND. None of the Vibrio isolates were found predominant in WFS affected shrimps. In bioassay studies, by feeding with WFS affected HP tissue, white fecal threads were successfully reproduced along with ATM structures and EHP spores. However, purified EHP spores (106 ml−1) failed to elicit WFS in challenged animals. In the clinical experiment, when WFS affected shrimp were reared under optimal laboratory settings, white feces were found to resolve, and the animals excreted normal feces in about 7–10 days, without any interventions. Further, histological studies showed the epithelial cells (E cells or embryonic cells) regeneration ability in WFS resolved shrimp. However, the WFS recovery observed was not a complete recovery since the resolved shrimps continue to shed EHP spores in normal feces. It is observed that EHP is the major pathogen excreted/disseminated via white feces in shrimp. With this study, we postulate that WFS is a clinical condition/shrimp diarrhea associated with severe EHP infection that may be caused by EHP in combination with an unknown agent. • White Feces syndrome (WFS) was successfully reproduced in experimental laboratory conditions. • WFS is shrimp diarrhea through which EHP disseminates the spores into the aquatic environment. • The presence of EHP, along with ATM structures, was revealed in WFS shrimps. • All individual WFS affected shrimps tested EHP positive. • Severe EHP infection in combination with an unknown agent may cause WFS. [ABSTRACT FROM AUTHOR]

Abstract: White spot syndrome virus (WSSV), is a highly virulent rapidly replicating large, enveloped, double-stranded DNA (dsDNA) virus, causing an estimated losses of over US$6billion since its emergence in 1992. As part of the better management practices (BMPs), ploughing, tilling and sun-drying of shrimp culture ponds are advocated for prevention and control of this disease. Despite this, instances of outbreaks of white spot disease (WSD) recur, causing loss to shrimp farmers. Some studies have indicated that WSSV DNA is reported to persist for over 20months in sediments as detected by PCR. Since mere detection of virus using PCR methods does not indicate its viable nature and ability to infect, information on its viability outside the host in water and pond sediment is necessary. Hence, in this study, the viability of WSSV in seawater and shrimp pond sediments under experimentally simulated drainable and non-drainable pond conditions was examined by shrimp infectivity experiments. WSSV with an initial viral load of 1000virionsmL−1 was found to be viable for a period of 12days in seawater of 27ppt salinity, pH of 7.5 at 29–33°C as revealed by its ability to infect juvenile shrimp, whereas, in shrimp pond sediment (with initial viral load of 211,500copiesg−1), the virus was viable and infective up to 19days despite sun-drying. In the case of non-drainable conditions, WSSV (753,600copiesg−1) remained infective for a period of 35days. Although the sediment samples tested nested PCR positive after 19days of sun-drying and 40days under water-logged conditions, shrimps did not develop WSD, suggesting that WSSV was not viable. Over a period of time after 21days under sun-drying and 40days under non-drainable experimental conditions, due to reduction in viral load, sediments were positive only by nested PCR, and by this time, viability of WSSV was almost lost as revealed by shrimp infectivity. Hence, PCR testing of shrimp farm sediment before starting culture as one of the BMPs may help in ensuring biosecurity from WSSV. The information generated here would help in the improvement of better management practices (BMPs) with regard to pond preparation protocols for shrimp aquaculture. [Copyright &y& Elsevier]

[Display omitted] • This LAMP is a closed tube and comparatively simple and specific EHP diagnostic assay. • This LAMP assay is developed based on EHP spore wall protein (SWP) gene sequence. • This LAMP can detect EHP in all clinical samples such as Hepatopancreas, faeces, pond water and soil. • In combination with FTA cards this assay can detect EHP within 1 h (LAMP-40 min, DNA elution – 17 min). • This EHP LAMP can be possibly performed by the farmer himself with a mini dry bath. Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment. [ABSTRACT FROM AUTHOR]