Aeromonas veronii and aerolysin are important for the pathogenesis of motile aeromonad septicemia in cyprinid fish.

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.

Comparison of fecundity and offspring immunity in zebrafish fed Lactobacillus rhamnosus CICC 6141 and Lactobacillus casei BL23.

To increase the knowledge of probiotic effects on zebrafish (Danio rerio), we compare the effects of two probiotic strains, Lactobacillus rhamnosus CICC 6141 (a highly adhesive strain) and Lactobacillus casei BL23 (a weakly adhesive strain), on zebrafish reproduction and their offsprings' innate level of immunity to water-borne pathogens. During probiotics treatments from 7 to 28 days, both the Lactobacillus strains, and especially L. casei BL23, significantly increased fecundity in zebrafish: higher rates of egg ovulation, fertilization, and hatching were observed. Increased densities of both small and large vitellogenic follicles, seen in specimens fed either Lactobacillus strain, demonstrated accelerated oocyte maturation. Feeding either strain of Lactobacillus upregulated gene expression of leptin, kiss2, gnrh3, fsh, lh, lhcgr, and paqr8, which were regarded to enhance fecundity and encourage oocyte maturation. Concomitantly, the gene expression of bmp15 and tgfb1 was inhibited, which code for local factors that prevent oocyte maturation. The beneficial effects of the Lactobacillus strains on fecundity diminished after feeding of the probiotics was discontinued, even for the highly adhesive gut Lactobacillus strain. Administering L. rhamnosus CICC 6141 for 28 days was found to affect the innate immunity of offspring derived from their parents, as evinced by a lower level of alkaline phosphatase activity in early larval stages. This study highlights the effects of probiotics both upon the reproductive process and upon the offsprings' immunity during early developmental stages.

Effects of chito-oligosaccharides supplementation on growth performance, intestinal cytokine expression, autochthonous gut bacteria and disease resistance in hybrid tilapia Oreochromis niloticus ♀ × Oreochromis aureus ♂.

We investigated the effects of incorporating chitinase (ChiB565)-hydrolyzed shrimp shell chitin into the diet of hybrid tilapia (Oreochromis niloticus ♀ × Oreochromis aureus ♂) with regard to production, intestinal immune status and autochthonous gut bacteria, and protection against bacterial pathogen Aeromonas hydrophila. Five experimental diets were formulated by supplementing the basal diet with the hydrolyzed shrimp shell chitin (0.0%, T1 control; 0.8%, T3; 1.6%, T4; or 2.4%, T5) or 0.1% commercial chitosan-oligosaccharides as commercial recommendation dose (T2, positive control). After a 35-day feeding trial, we found no significant difference in weight gain, feed conversion ratio or survival rate in tilapia among all treatment groups. However, the levels of mRNAs encoding the pro-inflammatory protein tumor necrosis factor-α and the stress-response protein heat shock protein 70 were much lower in groups T2, T3, T4 and T5 (p < 0.001). The levels of transforming growth factor-ß were higher in groups T2 and T4 (p < 0.001 and p < 0.0001, respectively). In addition, group T3 and T4 with 0.8% and 1.6% hydrolyzed shrimp shell chitin supplementation respectively changed marginally their autochthonous gut bacteria (0.60 < Cs < 0.80). When challenged with A. hydrophila, the mortality of groups fed chito-oligosaccharides was lower than the control, especially in groups T4 and T5 (p < 0.05). These results indicate that dietary intake of chito-oligosaccharides can improve intestinal health, changed autochthonous gut bacteria, and improve resistance to infection by A. hydrophila, even with higher efficiency than receiving the manufacturer recommended dose of the commercial chitosan-oligosaccharides.

High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture.

Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and ß-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5% (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1ß expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.

Impact of Lactobacillus casei BL23 on the Host Transcriptome, Growth and Disease Resistance in Larval Zebrafish.

In this study, zebrafish were treated with Lactobacillus strains as probiotics from hatching to puberty, and the effect of treatment with L. casei BL23 on the development and immunity response of the host was investigated. Genes that were differentially expressed (DEGs) in the overall body and intestine were detected at 14 days post fertilization (dpf) and 35 dpf, respectively, using whole transcriptome sequencing (mRNAseq). We showed that zebrafish raised by continuous immersion with L. casei BL23 showed a higher final body weight at 14 dpf (P < 0.05), and 35 dpf (P < 0.01). DEGs between L. casei BL23 treatment and control group at 14 dpf were involved in myogenesis, cell adhesion, transcription regulation and DNA-binding and activator. At 35 dpf, 369 genes were DEGs in the intestine after treatment with L. casei BL23, which were involved in such categories as signaling, secretion, motor proteins, oxidoreductase and iron, tight junctions, lipid metabolism, growth regulation, proteases, and humoral and cellular effectors. KEGG analysis showed DEGs to be involved in such pathways as those associated with tight junctions and the PPAR signal pathway. RT-qPCR analysis showed that expression of insulin-like growth factors-I (igf1), peroxisome proliferator activated receptors-α (ppar-α) and -ß (ppar-ß), Vitamin D receptor-α (vdr-α), and retinoic acid receptor-γ (rar-γ) was up-regulated in fish treated with L. casei BL23 at 35 dpf. After 35 days of treatment, the mortality rate in L. casei BL23 treated group was lower than the control after challenge with A. hydrophila (P < 0.05), and the pro-inflammatory cytokine il-1ß, anti-inflammatory cytokine il-10 and complement component 3a (c3a) showed more expression in L. casei BL23 group at 8h after challenge, 24 h after challenge, or both.. Together, these data suggest that specific Lactobacillus probiotic strains can accelerate the development profile and enhance immunity in zebrafish, which supports the rationale of early administration of probiotics in aquaculture.

Effect of Saccharomyces boulardii and Bacillus subtilis B10 on gut microbiota modulation in broilers.

The gut microbiota plays important roles in animal overall health and productiveness. Balancing host gut microbiota by probiotics has been documented. Our previous study showed that Saccharomyces boulardii (Sb) and Bacillus subtilis B10 (Bs) significantly improve growth performance and modulate the intestinal histomorphology in broilers. To increase the knowledge regarding Sb and Bs, this study investigated the effects of these 2 probiotic strains on the gut microbiota in broilers. Three hundred 1-day-old Sanhuang broilers (Chinese cross breed) were randomly divided into 3 groups, each group with 5 replications (n = 20). The control group (CK) was fed a basal diet containing an antibiotic (virginiamycin, 20 mg/kg) and the other 2 groups received Sb and Bs (1 × 108 cfu/kg of feed) in addition to the basal diet. After 72 d of treatment, pyrosequencing revealed that the bacterial communities varied along the section of intestinal tract in the control and Bs groups, but not in the Sb group. No difference in microbial diversity was observed among 3 groups. The major phyla observed along the GI tract of broilers (particularly in the duodenum and cecum) were Firmicutes, Bacteroidetes, Proteobacteria, and Verrucomicrobia, which were considered potentially growth performance-related. Bacteroidetes, Proteobacteria, and Verrucomicrobia were observed at a much higher abundance in the jejunums and ileums of the Sb group (P < 0.05). In addition, the jejunal microbial communities formed 3 different clusters at either the genus level or the category of metabolism among the groups, based on the principal component analyses. These data indicated that Sb and Bs can modulate the microbial ecosystem, and subsequently enhance the health status of broilers.

EPSP of L. casei BL23 Protected against the Infection Caused by Aeromonas veronii via Enhancement of Immune Response in Zebrafish.

Aquaculture is the fastest-growing food production sector in the world, and it supplies nearly 50% of the global food fish supply. However, disease outbreaks have become a major problem in the fish farming industry. The beneficial contribution of probiotic bacteria to aquatic animals' health has been widely described, and they have been widely used in aquaculture for disease control and growth promotion. However, the action of probiotic bacterial components and mechanisms underlying protection against pathogens afforded by probiotic bacteria remain poorly understood. In the present study, we pre-colonized zebrafish larvae (before hatching) with 17 potential probiotic bacterial strains and screened for those possessing anti-infective effects against Aeromonas veronii. We found that Lactobacillus casei BL23 significantly increased the survival of zebrafish larvae upon A. veronii infection. Using a germ-free (GF) zebrafish model and gut microbiota transplant experiment, we showed that L. casei BL23 per se has anti-infective effects in zebrafish larvae, which does not involve microbiota. Furthermore, we identified an exopolysaccharide-protein complex (EPSP) extracted from L. casei BL23 cells, which consisted of a 40-45 KD size protein and an exopolysaccharide composed of α-Rha, α-Glc, ß-GlcNAc, and ß-GalNAc. EPSP significantly increased the survival rate of GF zebrafish at a dose of 10-20 µg/ml after A. veronii infection (P < 0.01). In addition, the EPSP induced a higher expression of TLR1 and TLR2, and modulated the expression profile of pro-inflammatory and anti-inflammatory cytokines in zebrafish liver (ZFL) cells. Our data indicated that the anti-infective effect of EPSP from L. casei BL23 was mediated by enhancement of immune responses in zebrafish, which might involve the TLR1/TLR2 signal pathway.

Anti-Infective Effect of Adhesive Probiotic Lactobacillus in Fish is Correlated With Their Spatial Distribution in the Intestinal Tissue.

ABSTRACR: In this study, we tested the distribution of 49 Lactobacillus strains in the mucus and mucosa of the intestine tissue of zebrafish. We observed a progressive change in the spatial distribution of Lactobacillus strains, and suggested a division of the strains into three classes: mucus type (>70% in mucus), mucosa type (>70% in mucosa) and hybrid type (others). The hybrid type strains were more efficient in protection of zebrafish against Aeromonas hydrophila infection. Three strains representing different distribution types (JCM1149, CGMCC1.2028, and JCM 20300) were selected. The mucosa type strain JCM1149 induced higher intestinal expression of inflammatory cytokines and Hsp70 than the other strains. Furthermore, we used L. rhamnosus GG and its mutant (PB22) lacking SpaCBA pili to investigate the influence of pili on spatial distribution. LGG showed a mucosa type distribution, while PB22 revealed a hybrid distribution and the disease protection was accordingly improved. The different protection ability between LGG and PB22 did not involve the intestinal microbiota, however, LGG induced injury to the mucosa of zebrafish. Collectively, the disease protection activity of Lactobacillus in zebrafish is correlated with their spatial distribution in the intestinal tissue, with strains showing a balanced distribution (hybrid type) more efficient in protection.