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Infectious hypodermal and hematopoietic necrosis virus (IHHNV) can cause mass mortalities in western blue shrimp Penaeus stylirostris, runt deformity syndrome in Pacific white shrimp P. vannamei and scalloped abdominal shell deformities in black tiger shrimp P. monodon. In P. monodon, however, PCR-based diagnosis of IHHNV can be complicated by the presence of a chromosome-integrated, non-replicating endogenous viral element (EVE). To facilitate high-throughput screening of P. monodon for IHHNV infection and/or EVE sequences, here we report real-time PCR tests designed to specifically detect IHHNV Lineage I, II and III but not EVE Type A sequences and vice versa. Using 108 dsDNA copies of plasmid (p)DNA controls containing either IHHNV or EVE-Type A sequences, both tests displayed absolute specificity. The IHHNV-q309 PCR reliably detected down to ≤10 copies of pDNA, at which levels a 309F/R PCR amplicon was just detectable, and the presence of an IHHNV-EVE sequence did not significantly impact its sensitivity. The IHHNV-qEVE PCR was similarly sensitive. Testing of batches of P. monodon clinical samples from Vietnam/Malaysia and Australia identified good diagnostic concordance between the IHHNV-q309 and 309F/R PCR tests. As expected for a sequence integrated into host chromosomal DNA, IHHNV-qEVE PCR Ct values were highly uniform among samples from shrimp in which an EVE was present. The highly specific and sensitive IHHNV-q309 and IHHNV-qEVE real-time PCR tests described here should prove useful for selecting broodstock free of IHHNV infection and in maintaining breeding populations of P. monodon specific pathogen free for IHHNV, and if desired, also free of IHHNV-EVE sequences.

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The efficacy of the novel aquaculture feed ingredient Novacq TM to improve resilience against viral infection and mortality in Black Tiger shrimp, Penaeus monodon, was examined. Juvenile 4-6 g shrimp were fed either a control diet or treatment diet which included 10% Novacq TM for a 26 d conditioning period. Control and treatment shrimp were subsequently each divided into a no injection, saline injection or GAV challenge injection sub-treatment with each having four replicate tanks of ten shrimp. After injection, shrimp survival in all six treatments was monitored daily over a 14 d experimental bioassay period. Two of the four replicate tanks for each treatment were pleopod sampled for later GAV load quantification at day 0 and 14 of the experimental period (i.e. 'less handling'; survival comparison), whilst the other two replicate tanks were pleopod sampled for later GAV load quantification at days 0, 3, 7, 10 and 14 (i.e. 'more handling'; GAV load comparison). In the survival comparison, shrimp fed Novacq TM had significantly higher survival rates (P >0.05) when GAV challenged compared to shrimp not fed Novacq TM . Similarly, shrimp fed Novacq TM had marginally higher survival rates when injected with saline and compared to their respective control. Shrimp that received no injection showed the same survival rates irrespective of diet. In the GAV load comparison GAV loads were generally lower in shrimp fed Novacq TM when compared to their respective GAV challenge or saline injection controls. Survival and GAV load data indicate that Novacq TM improves resilience against viral infection and mortality in Black Tiger shrimp, Penaeus monodon.

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A polychaete-assisted sand filter (PASF) system has been developed to help remove nutrients from aquaculture pond wastewater whilst also producing polychaetes that are highly prized as bait by recreational anglers and as a dietary supplement to improve the fecundity of shrimp broodstock. Whilst rearing polychaetes in PASF beds offers potential to reduce impacts of sourcing them from the wild, the use of wastewater from ponds rearing shrimp such as Penaeus monodon will present a biosecurity risk of viruses being transferred to, and potentially amplified in, the worms. To assess such risks for transmitting infectious hypodermal and haematopoietic necrosis virus (IHHNV), groups of 3 or 4 PASF beds seeded with sand worm (Perinereis helleri) juveniles were supplied with wastewater from ponds of P. monodon with either high-load or low-load IHHNV infections. TaqMan realtime qPCR identified low loads of IHHNV (≤878 IHHNV DNA copies 200 ng −1 TNA) in most worms from PASF beds supplied wastewater from the high-load pond. IHHNV was either not detected or detected at the qPCR test sensitivity limits in worms from beds supplied wastewater from the low-load pond. Purging harvested worms of their gut contents in clean filtered seawater for 2 days significantly reduced IHHNV loads. Reverting PASF beds to clean seawater for 8 weeks before harvest also significantly reduced worm loads of IHHNV. Daily additions of a commercial probiotic to the sand bed surface for 4 weeks prior to clean seawater application provided no discernible benefit to IHHNV clearance. While clearly demonstrated to be capable of carrying IHHNV, the remediation measures examined suggest potential to ameliorate the infection transmission risks of P. helleri reared in PASF beds supplied with shrimp pond wastewater as a nutrient source.

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