Virulent bacteriophage of Edwardsiella ictaluri isolated from kidney and liver of striped catfish Pangasianodon hypophthalmus in Vietnam.

Striped catfish Pangasianodon hypophthalmus farmed in the Mekong Delta, Vietnam, represents an important contribution to Vietnamese aquaculture exports. However, these fish are affected by frequent disease outbreaks across the entire region. One of the most common infections involves white spots in the internal organs, caused by the bacterium Edwardsiella ictaluri. In this study, a virulent phage specific to E. ictaluri, designated MK7, was isolated from striped catfish kidney and liver samples and characterized. Morphological analysis indicates probable placement in the family Myoviridae with a 65 nm icosahedral head and a 147 %%CONV_ERR%% 19 nm tail. A double-stranded DNA genome of approximately 34 kb was predicted by restriction fragment analysis following digestion with SmaI. The adsorption affinity (ka) of the MK7 phage was estimated as 1.6 %%CONV_ERR%% 10-8 ml CFU-1 min-1, and according to a 1-step growth curve, its latent period and burst size were ~45 min and ~55 phage particles per infected host cell, respectively. Of the 17 bacterial strains tested, MK7 only infected E. ictaluri, although other species of Edwardsiella were not tested. E. ictaluri was also challenged in vitro, in both broth and water from a striped catfish pond and was inactivated by MK7 for 15 h in broth and 51 h in pond water. Thus, initial characterization of phage MK7 indicates its potential utility as a biotherapeutic agent against E. ictaluri infection in striped catfish. This is the first report of a lytic phage specific to an important striped catfish pathogen.

Indel-II region deletion sizes in the white spot syndrome virus genome correlate with shrimp disease outbreaks in southern Vietnam.

Sequence comparisons of the genomes of white spot syndrome virus (WSSV) strains have identified regions containing variable-length insertions/deletions (i.e. indels). Indel-I and Indel-II, positioned between open reading frames (ORFs) 14/15 and 23/24, respectively, are the largest and the most variable. Here we examined the nature of these 2 indel regions in 313 WSSV-infected Penaeus monodon shrimp collected between 2006 and 2009 from 76 aquaculture ponds in the Mekong Delta region of Vietnam. In the Indel-I region, 2 WSSV genotypes with deletions of either 5950 or 6031 bp in length compared with that of a reference strain from Thailand (WSSV-TH-96-II) were detected. In the Indel-II region, 4 WSSV genotypes with deletions of 8539, 10970, 11049 or 11866 bp in length compared with that of a reference strain from Taiwan (WSSV-TW) were detected, and the 8539 and 10970 bp genotypes predominated. Indel-II variants with longer deletions were found to correlate statistically with WSSV-diseased shrimp originating from more intensive farming systems. Like Indel-I lengths, Indel-II lengths also varied based on the Mekong Delta province from which farmed shrimp were collected.

Pathogenicity of gill-associated virus and Mourilyan virus during mixed infections of black tiger shrimp (Penaeus monodon).

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean >10(9) viral RNA copies µg(-1) total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ∼10(5) RNA copies µg(-1) total RNA) and increased >10(4)-fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection.

Mixed-genotype white spot syndrome virus infections of shrimp are inversely correlated with disease outbreaks in ponds.

Outbreaks of white spot syndrome virus (WSSV) in shrimp culture and the relationship between the virus and virulence are not well understood. Here, we provide evidence showing that WSSV mixed-genotype infections correlate with lower outbreak incidence and that disease outbreaks correlate with single-genotype infections. We tested 573 shrimp samples from 81 shrimp ponds in the Mekong delta with outbreak or non-outbreak status. The variable number tandem repeat (VNTR) loci of WSSV were used as molecular markers for the characterization of single- and mixed-genotype infections. The overall prevalence of mixed-genotype WSSV infections was 25.7 %. Non-outbreak ponds had a significantly higher frequency of mixed-genotype infections than outbreak ponds for all VNTR loci, both at the individual shrimp as well as at the pond level. The genetic composition of WSSV populations appears to correlate with the health status of shrimp culture in ponds. The causal relationship between genotypic diversity and disease outbreaks can now be experimentally approached.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: identification and functional characterisation of Dicer-1.

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629bp in length, including a 5' untranslated region (UTR) of 130bp, a 3' UTR of 77bp, and an open reading frame of 7422bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P<0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P>0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp.