Impacts of Lactobacillus plantarum in Depuration for Reducing Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

This study investigated potential application of lactic acid bacteria (LAB) in depuration for reducing Vibrio parahaemolyticus in oysters. Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against V. parahaemolyticus in vitro, was added to artificial seawater for depuration of Pacific oysters (Crassostrea gigas) inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) to levels of about 10⁴ MPN/g at 15 ± 1 and 10 ± 1°C. Application of L. plantarum ATCC 8014 treatment (10⁷ CFU/mL) in oyster depuration did not enhance reductions of V. parahaemolyticus in oysters depurated at 15 ± 1°C but significantly decreased (p < 0.05) levels of V. parahaemolyticus in oysters depurated at 10 ± 1°C after 5 days (3.40 log reductions) when compared with controls (2.75 log reductions). It is not clear if a competitive exclusion by LABs to compete with V. parahaemolyticus binding sites in oyster tissues plays a role in the reduction of V. parahaemolyticus in the oysters. Further studies utilizing different types of LABs in oyster depuration might provide additional knowledge for application of LAB in depuration for decontaminating V. parahaemolyticus in oysters.     

Impact of Feeding Status on the Efficacy of Depuration for Decontaminating Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

Vibrio parahaemolyticus causes acute human gastroenteritis and is often linked to consumption of raw oysters. Previous investigations indicated that refrigerated seawater depuration at 12.5°C could significantly reduce V. parahaemolyticus contamination in Pacific oysters; however, further optimization is necessary to achieve the regulatory target of >3.52 log most probable number (MPN)/g reduction. The current study investigated influences of algal feeding on efficacy of depuration to reduce V. parahaemolyticus in raw oysters. A V. parahaemolyticus cocktail (10290, 10292, 10293, BE 98–2029, 027-1c1) was mixed in artificial seawater (70 L) to inoculate oysters (n = 35) at 4–5 log MPN/g. Inoculated oysters were subjected to depuration with feed (algae = 0.036 ml/gram of oyster) and without feed at 12.5°C. Oysters (n = 5) were analyzed for V. parahaemolyticus using a three-tube MPN method after 0, 1, 3, 5, and 6 days of depuration. Depuration (6 days) achieved average V. parahaemolyticus reductions of 2.75 log MPN/g and 3.03 log MPN/g in the fed and unfed systems, respectively; however, feeding status did not significantly impact the efficacy of depuration to reduce V. parahaemolyticus in Pacific oysters. Further optimization of depuration is necessary to achieve the regulatory target for V. parahaemolyticus decontamination in raw oysters.     

Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone)

Five different Vibrio parahaemolyticus strains (SH8, SH108, SH58, AH5 and GD10) isolated from the hepatopancreas of moribund shrimp in farms of mainland China were identified and capable of inducing massive mortality of Penaeus (Litopenaeus) vannamei. The immersion challenge results with five isolates indicated variance of virulence, while only GD10 caused massive sloughing of tubule epithelial cells which was recognized as the most significant symptom of AHPND. Differences in immune responses were detected of P. vannamei during 48 h post‐infection (p.i.) by injection or immersion challenge with V. parahaemolyticus (SH8, SH108 and GD10) isolates. When injected SH8 and SH108 isolates, the expression of lysozyme (LSZ) showing statistically significant upregulation at 16 and 48 h p.i. and that of Toll‐like receptors (TLR) showed statistically significant upregulation at 48 h p.i. When immersion challenge with the GD10 isolate, TLR were upregulated after 8 h p.i. challenge with 10⁴ cfu mL^?1; however, LSZ was downregulated when challenged with 10³ cfu mL^?1. The results suggested that LSZ and TLR serve as crucial molecular markers of innate immunity in shrimp against V. parahaemolyticus infection. LSZ is a vital marker for acute bacterial infection, while TLR serves as a crucial marker for chronic infection.     

Antibacterial effect of short‐chain fatty acids on gnotobiotic Artemia franciscana nauplii against Vibrio parahaemolyticus

The present study was undertaken to determine the antibacterial effect of short‐chain fatty acids (SCFA) such as acetic, propionic and butyric acids on gnotobiotic Artemia franciscana nauplii against Vibrio parahaemolyticus. An initial experiment was undertaken to determine the antibacterial effect of different concentrations (1, 10 and 100 mM) of individual SCFA against V. parahaemolyticus through bacterial growth study at pH 6. In this study, the pathogen growth was completely inhibited at 100 mM concentration of all the tested SCFA. In another investigation, the different pH (5–7) on growth inhibitory effect of 20 mM SCFA was tested against V. parahaemolyticus through bacterial growth study. In this study, the pathogen growth was highly inhibited at pH 5 in all the tested SCFA. The Artemia nauplii were gnotobiotically reared in 20 mM SCFA supplemented medium and the mortality was recorded at an every 6 h interval up to 60 h after challenged with V. parahaemolyticus. The reduction in mortality (%) of A. franciscana nauplii over control was ranged from 25.36% to 38.56% in all the tested SCFA.     

Impact of a reduced water salinity on the composition of Vibrio spp. in recirculating aquaculture systems for Pacific white shrimp (Litopenaeus vannamei) and its possible risks for shrimp health and food safety

Tropical shrimp, like Litopenaeus vannamei, in land‐based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable.     

Green fluorescent protein visualization of Vibrio parahaemolyticus infections in Indian white shrimp Fenneropenaeus indicus (H Milne Edwards)

The pathways by which pathogens invade Fenneropenaeus indicus, the potential colonization in various tissues and the disease transmission mechanisms are unclear. The aims of the present study were to visualize the colonization and pathogenesis of GFP‐tagged Vibrio parahaemolyticus, in various tissues of F. indicus to evaluate the pathogen interaction. Among the three strains isolated, a virulent strain VpDAHV2 was tagged with green fluorescent protein (GFP‐VpDAHV2) and validated for both its growth characteristics and its virulence as a genuine model for F. indicus infection. VpDAHV2 was positive for toxR and tlh genes and negative for tdh genes. CLSM images revealed that maximum colonization was observed in the haemolymph of the F. indicus challenged with GFP‐VpDAHV2. The haemolymph was the primary site for the colonization of GFP‐VpDAHV2 in F. indicus. The enteric localization occurred independently of the flagellum or motility of GFP‐VpDAHV2 through the intestinal route. The F. indicus infection model suggests that the haemolymph and the intestine represent the sites of infection by GFP‐VpDAHV2, and hence are the active sites of pathogen interactions. GFP tagging of V. parahaemolyticus is a new and systemic approach to determine the presence of bacteria in vivo for the confirmation of host pathogen interactions in aquaculture studies.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

Selection of lactic acid bacteria as candidate probiotics and in vivo test on Artemia nauplii

Lactic acid bacteria (LAB) were isolated from rotifer cultures in a marine hatchery to search for potential probiotics for marine animals. Fifteen strains were first selected among a total of 55, according to antibacterial activity against Vibrio sp. Among eight strains identified as Lactobacillus casei, four were highly adhesive, suggesting some ability for surface colonization. The other strains were identified as Lactobacillus plantarum, Lactobacillus dextrinicus, and Leuconostoc sp. To validate probiotic potential, Artemia were challenged against pathogenic Vibrio alginolyticus, with or without one of six selected LAB strains. The six strains protected Artemia against the pathogen, to some extent on condition that nutrient enrichment was provided. La. casei BR51 and X2 were preferred, as they were efficient even in the absence of nutrient supply. La. casei X2 was finally selected as candidate probiotic, due to the best growth performances of Artemia, with or without the pathogen.     

Evidence of fish and human pathogens associated with doctor fish (Garra rufa,Heckel, 1843) used for cosmetic treatment

Doctor fish (Garra rufa, Heckel, 1843) are increasingly used for cosmetic treatment raising particular concerns regarding the potential transmission of infections to clients. Investigations of microbial causes undertaken in two outbreaks of mortality among G. rufa used for cosmetic treatment revealed the presence of multiple bacteria, including both fish and human pathogens such as Aeromonas veronii, A. hydrophila, Vibrio cholerae, Shewanella putrefaciens, Mycobacterium marinum and M. goodii. This range of bacteria indicates an intense microbial proliferation involving multiple pathogens, most likely induced by the poor health condition of the fish. Most of the detected pathogens are well‐known agents of zoonosis. Indeed, M. goodii is an emerging nosocomial human pathogen that has never been detected in fish to date, nor in other animals. This first detection of M. goodii associated with fish infection points out a new zoonotic potential for this pathogen. These findings point out that handling, poor environmental conditions and the presence of fish pathogens, that can compromise the immune system of fish, can result in a mixed microbial proliferation and increase the spread of waterborne bacteria, including zoonosis agents. Accordingly, the microbiological surveillance of fish used for cosmetic treatment is extremely important, particularly in association with mortality outbreaks.     

Molecular identification and distribution of mangrove oysters ( Crassostrea) in an estuarine ecosystem in Southeast Brazil: implications for aquaculture and fisheries management

This study investigated the existing species of Crassostrea in the natural environment, farming systems and artificial spat collectors at a protected estuarine area in Brazil, through PCR‐RFLP of mitochondrial 16S rRNA. Among 450 samples collected in the natural environment, 303 were C. brasiliana and 147 C. rhizophorae. Oysters present in the rocky subtidal zone were C. brasiliana. However, both species occurred on mangrove roots in the intertidal zone. Farm‐raised samples included only C. brasiliana. It was observed that attached specimens in commercial collectors had a banding pattern distinct from C. brasiliana and C. rhizophorae, indicating the presence of a third species in the estuary. The 16S rRNA sequence analysis showed that these specimens are clustered with the oysters from Pacific and Indian Oceans, and genetically close to the oysters of Beihai, China (0.3% genetic distance). Oysters obtained from the seed capture showed 17.8% distance of in relation to C. brasiliana, 17.6% for C. rhizophorae and 10.3% for C. gigas, demonstrating high genetic divergence from these species. The occurrence of an exotic species in the Cananéia estuary may have strong ecological and economic implications which require new guidelines for farming, conservation and sustainable fisheries management for the native oyster species.     

Assignment of parentage in triploid species using microsatellite markers with null alleles,an example from Pacific oysters (Crassostrea gigas)

Triploid production in aquaculture is increasing because of their more profitable growth and reproduction traits. Triploids are mostly produced through mass spawning techniques, meaning that exact pedigree is unknown. The ability to trace the pedigree of high performing triploids would allow selection of broodstock to perpetuate triploids of greater economic value. This study aimed to develop a method of determining parental assignment in triploids and test its accuracy on triploid oysters. Using a likelihood approach and accounting for null allele frequencies, a method was developed which proved to be efficient at determining the pedigree of triploid oysters. This method was able to provide accurate pedigree on simulated data and two commercial cohorts of triploid oysters. The analysis of the triploid cohorts showed that mass spawning to produce triploid oysters, like that for diploid and tetraploids, results in a strong bias in parental contributions, with the effective population size being 34‐49% lower than the census population. This highlights the need for pedigree control in breeding programs and indicates that the ability to determine parentage of triploids will be a valuable tool for breeding programs.     

Ostreid herpesvirus OsHV‐1 μVar in Pacific oysters Crassostrea gigas (Thunberg 1793) of the Wadden Sea,a UNESCO world heritage site

The Wadden Sea is an extensive wetland area, recognized as UNESCO world heritage site of international importance. Since the mid‐1990s, the invasive Pacific oyster Crassostrea gigas (Thunberg 1793) population in the area has grown exponentially, having a distinct impact on the ecosystem. The recent spread of the emerging oyster pathogen Ostreid herpesvirus OsHV‐1 μVar worldwide and specifically in the oyster culture areas in the south of the Netherlands raised the question whether the virus may also be present in the Wadden Sea. In the summer of 2012 juvenile Pacific oysters were collected from five locations in the Dutch Wadden Sea. The virus was shown to be present in three of the five locations by real‐time PCR and sequencing. It was concluded that OsHV‐1 μVar has settled itself in Pacific oyster reefs in the Wadden Sea. These results and the recent discoveries of OsHV‐1 microvariants in Australia and Korea indicate that OsHV‐1 μVar and related variants might be more widespread than can be deduced from current literature. In particular in regions with no commercial oyster culture, similar to the Wadden Sea, the virus may go undetected as wild beds with mixed age classes hamper the detection of mortality among juvenile oysters.     

Siltation increases the susceptibility of surface‐cultured eastern oysters (Crassostrea virginica) to parasitism by the mudworm Polydora websteri

Mudworm (Polydora websteri) parasitism is known to result in unsightly mud blisters in eastern oysters (Crassostrea virginica), resulting in reduced product quality and increased vulnerability to illness and environmental stress. While typically a concern only for bottom‐grown oysters, an abnormal severe outbreak of P. websteri in surface‐cultured oysters in New Brunswick, Canada, was reported in 2013, along with an anecdotally reported concurrent increase in siltation. Although heavier loads of silt are reported to increase P. websteri infestations in oysters, studies report mixed effects of siltation to this regard. Here, we report the results of a field experiment testing the effect of siltation on P. websteri recruitment in surface‐grown oysters at an aquaculture site in Richibucto, New Brunswick. Live oysters of similar size were deployed at the aquaculture site and were left to collect P. websteri recruits under one of two siltation treatments (high vs. low) for 50 days. Results suggested that P. websteri recruitment correlated with metrics of oyster size (shell weight, length, width, depth and surface area). When P. websteri counts were standardized for oyster shell morphometry, P. websteri recruitment was significantly higher in oysters exposed to the high siltation treatment, accumulating approximately 1.5× as many P. websteri as oysters exposed to the low siltation treatment. Our results suggest that higher amounts of siltation on surface‐cultured oysters can result in increased rates of P. websteri parasitism. Enhanced cleaning regimes may help to alleviate the impacts of P. websteri in surface‐grown oysters, although other mitigation strategies exist.     

Volatile Organic Compounds Profile Obtained from Processing Steps of Pacific Oysters (Crassostrea gigas) as Perspective for Food Industry

ABSTRACT

In the heat treatment of oysters, many aroma compounds are lost in the cooking steam and water. In this study, aroma compound profiles from oyster processing were identified by gas chromatography- mass spectrometry (GC-MS). The compounds were extracted by headspace-solid phase microextraction (HS-SPME), while the best process conditions such as temperature, extraction time, and desorption time were studied. The aroma compounds were obtained from raw oysters, from oysters after thermal treatment, from water released from raw oysters, from cooking steam, and from oyster boiling juice. All compounds were identified and semi-quantified by GC-MS. The major oyster aroma compounds were 1-octen-3-ol, hexanal, and benzaldehyde. Most of the components from oysters were lost both to the cooking steam and to the oyster boiling juice. The concentration range of volatile organic compounds transferred for oyster boiling juice between 14 and 90 mg L^?1 shows that the residues from oyster processing have great technological potential, and they can be recovered for application as aromas by the food industry.     

Identification of potential general markers of disease resistance in Exopalaemon carinicauda via three‐round challenge selection with an acute hepatopancreatic necrosis disease‐causing strain of Vibrio parahaemolyticus

Sixteen candidate disease‐resistant parameters were selected through which to evaluate the acute hepatopancreatic necrosis disease (AHPND)‐resistant capability of Exopalaemon carinicauda after three generations of selection for AHPND‐causing strain of Vibrio parahaemolyticus (VP_AHPND) resistance in our previous study. However, these parameters required further verification. In this study, another AHPND‐resistant E. carinicauda series was obtained through a short‐term selection procedure, consisting of three virulent challenge rounds of selection (about three‐week interval for each challenge) with VP_AHPND infection. After this selection, the survival rate at 144 hr post infection (hpi) increased from 23.33% to 37.78% and the observed 48‐hr LD₅₀ of VP_AHPND to shrimp increased from 10^5.5 cfu/ml to 10^6.5 cfu/ml. Then, the immune response of this AHPND‐resistant E. carinicauda was studied using the 16 candidate AHPND‐resistant parameters selected for in our previous study. The improved VP_AHPND clearance rate in hpi, increased total haemocyte counts, haemocyanin concentration, alkaline phosphatase activity and expressions of six immune‐related genes (Tollip and ALF in haemocytes and hepatopancreas; lysozyme, crustin and cathepsin B in hepatopancreas; and LGBP in haemocytes) at 24 hpi after the three‐round challenge selection suggest that these immune parameters may be reliable markers for the evaluation of the physiological status and potential AHPND‐resistant phenotypes in E. carinicauda.     

Rearing equipment on the quality aspects and growth performance of Crassostrea gigas (Thunberg, 1793): Implications for marketability

Shell characteristics and consequent marketability of the oyster Crassostrea gigas are influenced by several factors, and most important of which are the rearing methods and gears. In this study, we investigated the effects of two different gears on the quality aspect (shell shape, weight meat percentage, backward shell shape, epibionts and blisters) and growth rate (weight and length) of C. gigas by adopting the innovative Ortac Oyster Farming System, during the field grow‐out phase of production. Aspect and growth of oysters farmed in Ortac and floating bags were compared. After 14 weeks, the aspect of the animals was influenced by the gear, resulting in a lower quality in the Ortac (i.e. worse shell shape, lower weight meat percentage, higher percentage of backwards and blisters) than in the floating bag, while the incidence of epibionts was less in the Ortac. Regarding the growth performance, total wet weight gained in the Ortac was lower (~28.3 g) than in the floating bag (~32.4 g; p < .05). These results highlight the efficiency of the floating bag as oyster rearing system in the Mediterranean basin and suggest the need to alter the Ortac gear to these environmental conditions.     

Administration of probiotic Bacillus licheniformis induces growth,immune and antioxidant enzyme activities,gut microbiota assembly and resistance to Vibrio parahaemolyticus in Litopenaeus vannamei

This study aimed to elucidate the effects of dietary Bacillus licheniformis ATCC 11946 (BL) on the growth, immune and antioxidant activities, intestinal morphology and microbiota, and susceptibility to Vibrio parahaemolyticus (VP_AHPND) in juvenile pacific white shrimp, Litopenaeus vannamei (LV). Juvenile LV (initial weight = 0.63 ± 0.001) were fed diets containing varying BL concentrations (0 (BL0), 10⁶ (BL1), 10⁷ (BL2) and 10⁸ (BL3) CFU/g feed) for 8 weeks. Growth performance, immune and antioxidant enzyme activities, and intestinal morphology significantly improved in the probiotic‐treated groups than the untreated. Regardless of the treatment group, the two dominant phyla were Proteobacteria and Bacteroidetes, whereas the two dominant genera were Ruegeria and Vibrio. Increasing inclusion of probiotics in diets led to significant increase in beneficial bacterial genera (Ruegeria, and Pseudoalteromonas) and a significant decrease in some known opportunistic pathogens (Vibrio, Tenacibaculum, Photobacterium, Kangiella and Spongiimonas) with the BL3 group witnessing the best. A 7‐day challenge study with VP_AHPND showed significantly high protection in the probiotic‐treated groups, with the BL3, BL2 and BL1 obtaining 51%, 34% and 22% relative percentage survival, respectively. In conclusion, BL at 10⁸ CFU/g feed should be used to help in shrimp production since it attained the most significant improvement.