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As a marine bacterial pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed in seawater worldwide. It can infect different animals as well as humans, even cause deaths. The highly conserved regions of PDD mcp gene on chromosome and dly gene on plasmid were selected as the target fragments to design the specific primers. Recombinant plasmid standard was prepared based on the primers. With GENECHECKER UF-150 qRT-PCR instrument as the platform, a fluorescence-based quantitative real-time PCR (qRT-PCR) method was established for the detection of PDD. This method can specifically detect PDD and distinguish the highly virulent strains. Additionally, the test results can be qualitatively judged by visualization, while the quantitative detection can be achieved through the standard curve calculation. The minimum limit of detection was 1.0×101 copies µL−1, and the detection time was less than 20 min. In summary, this new method has outstanding advantages, such as strong specificity, high sensitivity, and low site requirements. It is a rapid on-site detection technology for highly virulent PDD strains. [ABSTRACT FROM AUTHOR]

Generally speaking, fish intestinal microbiota is easily affected by food or water environment, and it may be dynamically changed along with body growth. However, it remains unclear whether fish gut microbiota can be affected under any conditions. In the present study, we focused on cultured larval turbot (Scophthalmus maximus) and tracked its artificial breeding process from eggs to larvae in two farms located in different regions of China. Through continuous sampling, we analyzed and compared characteristics of intestinal microbiota in turbot larvae and its correlation with the bacteria in water and food at different developmental stages. The results showed that there was a steady group of microbiota in larval gut, and the highest relative abundance of strain was same between the two farms. This microbiota was established soon after hatching of fertilized eggs. Particularly, the structure of this microbiota was nearly not changeable afterward 3–4 months of development. The bacteria carried by fertilized eggs might play an important role during the formation of this microbiota. In conclusion, our findings suggested that there was a core microbiota represented by Lactococcus sp. in gut of artificially bred turbot larvae. The relative proportion of such strain in gut was higher than 30% at the initial stage of turbot life. [ABSTRACT FROM AUTHOR]

Freeze-drying, continuous passage and ultra-low temperature cryopreservation are often used to preserve pathogens. In this study, Vibrio anguillarum was rejuvenated by intramuscular infection as the initial strain. The difference between cells preserved with different preservation methods and their initial strains were compared with physiological and biochemical methods and through antibiotics resistance analysis. The composition of protectants for freeze-drying V. anguillarum was optimized. We found that the optimal composition of protectants was 8% of trehalose, 12% of skim milk, 8.0% of lactose, 2.0% of sodium citrate, 12.0% of serum and 8.0% of mannitol. The indexes of lysine decarboxylase and urease changed after continuous passage. The urease reaction changed after freeze-drying and freeze-thawing, but the reaction can be restored to the initial after freeze-drying. Based on the antibiotics resistance analyses, the sensitivity of V. anguillarum to different drugs including rifampicin, erythrocin, furazolidone, ceftazidime, lomefloxacin, gentamycin, azithromycin, doxycycline, ampicillin, co-trimoxazole and cefoperazone changed after different treatments, and some of these changes can be restored to the original through activation culture. In sum, compared with cryopreservation and continuous passage, the freeze-drying is more sustainable for the long-term preservation of V. anguillarum, which showed a better effect in maintaining the original characteristics of pathogen. [ABSTRACT FROM AUTHOR]

In this study, the influences of immersion bathing in different concentrations of Bacillus velezensis DY-6 on the body weight gain rate and non-specific immune enzyme activities of the coelom fluid of sea cucumber (Apostichopus japonicus) were determined in order to obtain the optimum bacterial concentration. The gut microbiota change in A. japonicus was then analyzed through high-throughput sequencing during the immersion bathing in B. velezensis DY-6 at the optimum concentration for 49 d. The results illustrate that the body weight growth rate of all bathing groups was higher than that of the control. The highest growth rate (25.3%) was achieved when the bacterial concentration was 1×103 CFU/mL. The activities of non-specific immune enzymes (ACP, AKP, SOD and LZM) of all bathing groups increased, and the activities of the enzymes of groups bathed with the bacterium at 1×103 and 1×104 CFU/mL reached the highest on day 21 and day 28. Taking the growth rate and economic cost into consideration, the optimum concentration of B. velezensis DY-6 was 1×103 CFU/mL. The influences of immersion bathing in B. velezensis DY-6 at 1×103 CFU/mL on the gut microbiota of A. japonicus were then evaluated through 16S rDNA sequencing analysis. Results showed that the gut microbiota changed with the addition of B. velezensis DY-6, and the richness and diversity of the gut microbiota peaked twice on day 14 and day 21, respectively. In association with the non-specific immune enzyme activities and if day 28 was selected as the dividing point, the community structure of the gut microbiota could be obviously divided into two types. The correlation analysis revealed that the non-specific immune enzyme activities were correlated significantly to some gut bacteria (in the phyla Firmicutes, Proteobacteria, and Bacteroidetes) after immersion bathing in B. velezensis DY-6. Our findings will provide the theoretical foundation for probiotic application in sea cucumber farming. [ABSTRACT FROM AUTHOR]

The effects of an exogenous probiotic (Bacillus amyloliquefaciens) on microbial community structure of Branchionus plicatils and Artemia sinica were evaluated in this study during turbot (Scophthalmus maximus) larval breeding. The analysis and comparison of the microfloral composition of live feed with probiotic was conducted using the Illumina HiSeq PE250. The abundance of microbial species and diversity of microflora in live feed with B. amyloliquefaciens were higher than those in the control. The microfloral composition was similar among the three replicate experimental groups of B. plicatils compared with the control after enrichment. Lactococcus, Pseudoalteromonas, and Alteromonas were always dominant. Additionally, some other bacterial species became dominant during the enrichment process. The microbial community during nutrient enrichment of A. sinica was rather similar among the three control replicates. Relative abundance of Cobetia sp., the most dominant species, was 54%–65.2%. Similarity in the microbial community was still high after adding B. amyloliquefaciens. Furthermore, Pseudoalteromonas and Alteromonas replaced Cobetia as the dominant species, and the abundance of Cobetia decreased to 4.3%–25.3%. Mean common ratios at the operational taxonomic unit level were 50%–60% between the two B. plicatils and A. sinica treatments. Therefore, the microbial community structure changed after adding B. amyloliquefaciens during nutrient enrichment of B. plicatils or A. sinica and tended to stabilize. Additionally, the abundance of Vibrio in any kind of live feed was not significantly different from that in the control. These results will help improve the microflora of B. plicatils and A. sinica and can be used to understand the multiple-level transfer role of probiotic species among probiotic products, microflora of live feed, and fish larvae. [ABSTRACT FROM AUTHOR]

Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose (8.0%), skim milk (12.0%), sodium citrate (2.0%), serum (12.0%) and PVP (2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of NaCl was 10.0, 30.0 and between 5.0 and 10.0 g L−1 for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at −80°C or −40°C for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate (79.63%-82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy (SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda. [ABSTRACT FROM AUTHOR]

The effects of different microbes on fermenting feed for sea cucumber ( Apostichopus japonicus) were compared to select the optimal fermentation strain in this study. Saccharomgces cerevisae, Candida utilis, Bacillus subtilis and Geotrichum candidum were independently added into the experimental compound feed, while only saline was mixed with the control feed. The fermentation treatments were inoculated with 10% seed solution under the condition of 25°C and 70% water content, which lasted for 5 days to elucidate the optimal microbe strain for fermenting effect. Physicochemical indexes and sensorial characteristics were measured per day during the fermentation. The indexes included dry matter recovery (DMR), crude protein (CP), the percentage of amino acid nitrogen to total nitrogen (AA-N/tN), the percentage of ammonia nitrogen to total nitrogen (NH-N/tN), and the ratio of fermentation strains and vibrios to the total microbes, color, smell and viscosity. The results showed that DMR, CP and AA-N/tN of the S. cerevisae group reached the highest level on day 3, but the ratio of fermentation strain was second to C. utilis group. In addition, its NH-N/tN and the ratio of vibrios were maintained at low levels, and the sensory evaluation score including smell, color and viscosity was the highest in S. cerevisae group on day 3. Therefore, S. cerevisae could be the optimal strain for the feed fermentation for sea cucumber. This research developed a new production method of fermentation feed for sea cucumber. [ABSTRACT FROM AUTHOR]

The cultivation of the first filial generation of monoploid gametophyte clones of different Laminaria species (hybrid Laminaria) is an effective way of utilizing heterozygous vigor (heterosis). A female gametophyte clone of L. japonica and a male gametophyte clone of L. longissima were hybridized, generating Dongfang No.2 hybrid Laminaria. The parentage of this hybrid Laminaria was determined using AFLP of total DNA, SNP of the ITS region of ribosomal RNA transcription unit and microsatellite DNA variation at two loci. In addition to 167 AFLP bands shared by Dongfang No.2, L. japonica and L. longissima, Dongfang No.2 hybrid Laminaria shared another 70 and 55 bands with L. japonica and L. longissima, respectively, which were obviously more than 11 bands shared by L. japonica and L. longissima. Dongfang No.2 held both ‘T’ and ‘C’ at position 847 of the ITS region, while ‘T’ at this position was specific for L. japonica and ‘C’ for L. longissima, respectively. Dongfang No.2 also held the microsatellite DNA alleles of two parents together at two microsatellite DNA marker loci. These observations clearly proved that Dongfang No.2 is a true hybrid of L. japonica and L. longissiuma. Unfortunately, the origin of the chloroplast of Dongfang No.2 was not determined based on the variation of RuBisCo spacer. More sequence variants of both ITS region and RuBisCo spacer were identified in Dongfang No.2 and most of them did not exist in either L. japonica or L. longissima. The unexpected variants may be due to the mutation of gametophyte clones occurring during their vegetative amplification. [ABSTRACT FROM AUTHOR]