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Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has caused severe damage to the shrimp farming industry of Vietnam. Probiotics are chosen as a prophylactic method to mitigate the outbreak of diseases. We report in this article the isolation and evaluation of the effect of a potential probiotic, Bacillus subtilis BLD01, which enhances the survival rates and changes gut microbiota of whiteleg shrimp (Penaeus vannamei) after challenge with AHPND V. parahaemolyticus. After seven days of the challenge, the treatment where shrimps were fed with B. subtilis BLD01 strain (106 CFU/g) and challenge with AHPND V. parahaemolyticus (106 CFU/mL) showed high survival rates of 71% as compared to 33% in the treatment where shrimp were given standard feed without probiotics supplementation and challenged with AHPND V. parahaemolyticus. 16S rRNA amplicon data of the gut microbiomes of shrimps in four treatments were carried out using Illumina sequencing. A total of 231 436 reads were obtained, and a total of 14 phyla, 28 classes, 142 genera were revealed. The most abundant phyla in all subjects were Proteobacteria and Bacteroidetes. The sequence number of the Vibrio genus was the highest with 28% in treatment without B. subtillis BLD01 in-feed addition and shrimps challenged with AHPND V. parahaemolyticus. The sequence number of Bacillus genus was the highest with 3% in the treatment with B. subtillis BLD01 addition and without AHPND V. parahaemolyticus challenge. These results contribute to confirming the mechanism of action of B. subtilis against V. parahaemolyticus in the experimental model, creating a scientific basis for the development and use of probiotics products applied in shrimp farming in Vietnam.

Diseases in shrimp farms in the Mekong Delta of Vietnam cause significant crop losses and are therefore of great concern to producers. Once a pond becomes infected, it is difficult to prevent spread of the disease to nearby shrimp farming areas. Thus, predicting the occurrence of disease is an essential part of reducing the risk for shrimp farmers. In this study, we applied an integrated geographic information system and machine learning system to predict three serious diseases of shrimp, namely, acute hepatopancreatic necrosis, white spot syndrome disease, and Enterocytozoon hepatopenaei infection, based on data collected from shrimp farms in the Tra Vinh, Bac Lieu, Soc Trang, and Ca Mau provinces of Vietnam. We first constructed a map showing the distribution of these diseases using the locations of affected farms, and then we conducted spatial analysis to acquire the geographical features of the affected locations. This latter information was combined with environmental factors and clinical signs to form the set of independent variables affecting the outbreak of diseases. The neural network model outperformed the logistic regression, random forest, and gradient boosting methods in terms of predicting infection to estimate the probability of disease occurrence in farmed areas. Acute hepatopancreatic necrosis disease infected farms downstream of the Co Chien and Hau Rivers of Tra Vinh and west of Ca Mau. Enterocytozoon hepatopenaei infection is distributed in Soc Trang Province, while white spot syndrome virus has spread to the coastal districts of Soc Trang and Bac Lieu Provinces, where it is highly associated to water from a complex canal system.

Predicting the outbreak of disease is essential when managing shrimp farms. Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus is a serious disease in shrimp. It is essential that shrimp farmers on the east coast of the Mekong Delta detect the disease as early as possible, because the mortality rate can reach 100%. Here, we used machine learning to predict AHPND development based on data collected since 2010 from shrimp farms in Tra Vinh, Ben Tre, Bac Lieu, and Ca Mau provinces. We initially hypothesized that the dependent variable, AHPND, was affected by 31 independent variables, but ultimately used 15 key variables to train the models. Logistic regression, artificial neural network, decision tree, and K-nearest neighbor analyses were performed, and the accuracy of the predictions was evaluated using hold-out and cross-validation tests. Logistic regression, as the most stable algorithm, was thus used to predict AHPND outbreaks in shrimp farms.

Gut bacteria comprise a complex bacterial community related to many functions in a host. The stability of gut bacteria plays important models in the health and immunology of a host. Many studies on intestine bacteria constructed via cultivation and Denaturation Gradient Gel Electrophoresis (DDGE) methods have proved a limited efficiency. In order to tackle these drawbacks, the next generation sequencing method was developed on 16S-rRNA-based sequences (Metabarcoding). The composition of bacterial communities was revealed based on the analysis of 16S rRNA sequences of intestine bacteria in Litopenaeus vannamei ponds in comparison with microbial communities in a Penaeus monodon pond and a muscle of shrimp. These results showed that the dominant phyla of intestine bacteria in Litopenaeus vannamei were Proteobacteria (49.3–57.4%), Firmicutes (15.6–34.4%) and Bacteroidetes (0.1–16.9%). Rhizobium(0.4%-26.1%), Vibrio(0–23.9%) and Spongiimonas(0–16,7%) were dominant genera in Litopenaeus vannamei gut. A higher proportion of Fusobacterium (10%), a shrimp pathogen group, was found in a disease shrimp pond (ST4) in comparison with a low growth shrimp pond (ST3) (0%) and a healthy shrimp pond (ST1) (0.6%). Vibrio was marked as shrimp pathogen genus accounted for 22.3% of total genera in ST4 in comparison with 2.4% in ST3 and 3.5% in ST1. Interestingly, a higher percentage of Vibrio rotiferianus (7.98%) was found in ST4 compared to ST3 (1%) and ST1 (0%). Fusobacterium and Vibrio will be the objects for the next experiments to discover shrimp pathogens specifically.

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.

1 Faculty of Veterinary Medicine, Department of Virology, Parasitology and Immunology, Gent University, Salisburylaan 133, 9820 Merelbeke, Belgium 2 Department of Aquatic Biology and Pathology, College of Aquaculture and Fisheries, Cantho University, Campus 2, 3-2 Street, Ninh Kieu District, Cantho City, Vietnam 3 Laboratory of Aquaculture & Artemia Reference Center, Faculty of Bioscience Engineering Department of Animal Production, Ghent University, Rozier 44, B-9000 Gent, Belgium

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.

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 (mean10(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 increased10(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.

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.

In the course of an international research project on hazard analysis of antimicrobial resis- tance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquacul- ture environments was sent by the central laboratory to the other participating laboratories. In each lab- oratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were per- formed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of sub- cultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distrib- uted in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global aver- aging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indi- cated that the use of either ISA or MH medium in DD testing has a limited impact on the method's preci- sion among different laboratories. In conclusion, the current study has provided a validated SOP to pro- mote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive break- points should be specifically designed and validated for aquaculture drugs and organisms.

We compared Edwardsiella ictaluri from striped catfish in Vietnam with US channel catfish isolates. Biochemical analyses and sequencing of the 16S rRNA gene confirmed that the Vietnamese isolates were E. ictaluri. Comparison using rep-PCR fingerprinting demonstrated no significant differences between the isolates, but plasmid analysis indicated that the Vietnamese isolates grouped into 4 plasmid profiles, each different from the typical pEI1 and pEI2 plasmid profile found in the US isolates. Sequencing plasmids representative of the 4 profiles indicated that all contained derivatives of the E. ictaluri plasmid pEI1, whereas only 1 contained a plasmid derivative of the E. ictaluri plasmid pEI2. The pEI2 encoded type III secretion effector, EseI, and its chaperone, EscD, were found to be present on the chromosome in isolates lacking a pEI2 derivative. In addition, 1 isolate carried a 5023 bp plasmid that does not have homology to either pEI1 or pEI2. Furthermore, Vietnamese isolates were PCR positive for the type III and type VI secretion system genes esrC and evpC, respectively, and the urease enzyme, but were PCR-negative for the putative type IV secretion system gene virD4. A monoclonal antibody against the lipopolysaccharide of E. ictaluri ATCC 33202 did not react with the Asian isolates or with the more recent US isolates. Antibiotic resistance patterns were variable and did not correlate to the presence of any particular plasmid profile. Finally, the Vietnamese isolates were avirulent and had a significantly reduced capacity for intracellular replication within head-kidney-derived channel catfish macrophages.

For studying shrimp immunity, in vitro haemocyte cultures are essential. Despite various reported attempts, well-described and reproducible culture techniques are lacking. The current work aimed to establish two in vitro haemocyte culture systems for Penaeus (Litopenaeus) vannamei. Haemocyte suspensions were either seeded in conventional Nunc® Nunclon™? Surface 24-well cell culture plates with glass cover slips (haemocytes in attachment) or in Nunc® Hydrocell Surface 24-well cell culture plates (haemocytes in suspension). The culture medium was based on L-15 (Leibovitz), and was further supplemented with L-glutathione and protease inhibitors in an attempt to improve haemocyte survival. Parameters such as number of living adherent and non-adherent single cells, number and average diameter of clusters and survival of cells inside clusters were evaluated. Additionally, live-cell imaging videos were recorded. It was found that haemocytes cultured for 1 h on glass coverslips in Nunc® Nunclon™? Surface plates could be separated in two cell fractions: adherent or non-adherent. Shrimp haemocytes cultured in Nunc® Hydrocell Surface plates remained in suspension and over time formed cell clusters which melanised. L-glutathione supplementation clearly improved haemocyte survival up to 48 h and delayed clustering and melanisation; addition of protease inhibitors did not. To validate the system, the phagocytic and antibacterial activities of adherent haemocytes towards Vibrio campbellii were evaluated. After 1 h of co-culture, 11.5 ± 0.14% of haemocytes showed phagocytosis with an average of 2.4 ± 0.1 bacteria internalised per haemocyte. Furthermore, haemocytes clearly demonstrated an antibacterial activity. It was concluded that these systems were reproducible and could keep haemocytes functionally active during the time required for the study of innate immune processes. Consequently, these techniques represent powerful tools for studying a variety of cell-mediated and humoral immune responses of shrimp in vitro.

The aquaculture sector in Vietnam began in the early 1960s with small scale extensive culture systems. The rapid growth of this sector has been achieved during the last two decades as a direct result of its diversification on farming practices and adaptation to the production of exportable species at increased levels of intensification. Foremost amongst these is the catfish culture in the Mekong Delta in Southern part of Vietnam.

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 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa 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. (c) 2007 Elsevier Ltd. All rights reserved.

In the present study, samples of pond water, sediment and farmed species were collected at 12 fish and shrimp farms in Malaysia, Thailand and Vietnam to determine the biodiversity and environmental distribution of chloramphenicol-resistant (CmR) mesophilic heterotrophs in Southeast Asian aquaculture sites. Following isolation on Iso-Sensitest agar supplemented with 35mug ml(-1) Cm and dereplication using (GTG)(5)-PCR fingerprinting, 557 genotypically unique CmR strains were subjected to polyphasic identification. The 557 mesophilic heterotrophic CmR isolates represented 18 different genera largely dominated by the genera Escherichia (40.2%), Pseudomonas (11.7%), Acinetobacter (11.1%), Klebsiella (7.5%) and Bacillus (5.9%). A total of 439 CmR isolates were further assigned to 31 described species or species groups, mainly including organisms that have been associated with various human opportunistic infections such as Escherichia coli (n=219), Pseudomonas putida (n=47), Klebsiella pneumoniae (n=38) and Acinetobacter baumannii (n=23). Strains of Escherichia, and most notably, of E. coli, were the only common group of CmR heterotrophs irrespective of country, sample type or farm type. Together with other predominant but less widespread groups such as acinetobacters and pseudomonads, the results of this biodiversity study suggest that E. coli can be regarded as a potential indicator of Cm resistance in Southeast Asian aquaculture environments.

The sediment and water inter-phase layer in aquaculture ponds is a dynamic zone that plays a crucial role in maintaining the ecological balance of the pond system. An accurate sediment profile of the soft-watery layer that includes several important parameters can only be determined by using an adequate sampling technique. Because most of the commercially available grabs and soil corers currently used do not fulfill these requirements, a new, simple device for sediment sampling was designed in the framework of the European Union (EU)-funded ASIARESIST project. The sampler was used to collect samples from the upper 5 to 10 cm soil layer for bacteriological analysis from aquaculture ponds in Malaysia, Thailand and Vietnam. The sampler consisted of a three-piece PVC pipe that could be sterilized and had a total length of 230 cm. Each section was threaded at the end except for the top of the first and last section that tapered into a sharp point at an angle of 45°. When dismantled into sections, the sampler was convenient to transport and to reassemble at the site of sampling. The sediment sampler proved to be effective in obtaining intact profiles of bottom sediment, including soft sediments, was user-friendly and could be easily constructed at low cost.