Wenshu Liu

Effects of dietary microencapsulated sodium butyrate on growth, intestinal mucosal morphology, immune response and adhesive bacteria in juvenile common carp ( Cyprinus carpio ) pre-fed with or without oxidised oil

The aim of the present study was to investigate the effects of different dietary sustained-release microencapsulated sodium butyrate (MSB) products (0 (non-supplement), 1·5 and 3·0 h) for a control or oxidised soyabean oil (SBO) diet on fish production, intestinal mucosal condition, immunity and intestinal bacteria in juvenile common carp (Cyprinus carpio). Dietary MSB increased weight gain and reduced the feed conversion ratio within the control and oxidised SBO groups. Gut mucosa was damaged in the oxidised SBO group fed without MSB, in contrast to a normal appearance found in fish fed the MSB1·5 and MSB3·0 diets in the oxidised SBO group. Microvillus density increased in fish fed the MSB1·5 and MSB3·0 diets in the oxidised SBO group (P< 0·001); however, microvillus density was affected by the different pre-fed diets in the midgut (P< 0·001) and by the different sustained-release times of MSB in the distal gut (DG) (P= 0·003). The interaction between the pre-fed diets and the sustained-release times of dietary MSB was significant for the relative gene expression levels of gut heat shock protein-70 (HSP70), pro-inflammatory cytokines (IL-1β and TNF-α) and anti-inflammatory cytokines (transforming growth factor-β) within each gut segment, except for HSP70 in the DG and IL-1β in the foregut. Modulation of adherent bacterial communities within each gut segment investigated was not obvious when the common carp were fed the diets with MSB, as similarity coefficients of >0·79 were observed. These results indicated that MSB can be used as a dietary supplement to repair or prevent intestinal damage in carp fed oxidised SBO.

Comparison of adhesive gut bacteria composition, immunity, and disease resistance in juvenile hybrid tilapia fed two different Lactobacillus strains

This study compares the effects of two Lactobacillus strains, highly adhesive Lactobacillus brevis JCM 1170 (HALB) and less-adhesive Lactobacillus acidophilus JCM 1132 (LALB), on the survival and growth, adhesive gut bacterial communities, immunity, and protection against pathogenic bacterial infection in juvenile hybrid tilapia. During a 5-week feeding trial the fish were fed a diet containing 0 to 10(9) cells/g feed of the two Lactobacillus strains. Samples of intestine, kidney, and spleen were taken at the start and at 10, 20, and 35 days for analysis of stress tolerance and cytokine gene mRNA levels and to assess the diversity of adhesive gut bacterial communities. A 14-day immersion challenge with Aeromonas hydrophila NJ-1 was also performed following the feeding trial. The results showed no significant differences in survival rate, weight gain, or feed conversion in the different dietary treatments. The adhesive gut bacterial communities were strikingly altered in the fish fed either the HALB or the LALB, but the response was more rapid and substantial with the adhesive strain. The two strains induced similar changes in the patterns (upregulation or downregulation) of intestinal, splenic or kidney cytokine expression, but they differed in the degree of response for these genes. Changes in intestinal HSP70 expression levels coincided with changes in the similarity coefficient of the adhesive gut bacterial communities between the probiotic treatments. The highest dose of the HALB appeared to protect against the toxic effects of immersion in A. hydrophila (P < 0.05). In conclusion, the degree to which Lactobacillus strains adhere to the gut may be a favorable criterion in selecting probiotic strain for aquaculture.

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-β 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.

Supplemental diagnosis of a myxozoan parasite from common carp Cyprinus carpio: Synonymy of Thelohanellus xinyangensis with Thelohanellus kitauei

Thelohanellus kitauei Egusa et Nakajima, 1981, was described from common carp Cyprinus carpio L. in Japan. In China, a myxosporean infecting the intestinal tissue of the same host species was described as Thelohanellus xinyangensis Xie, Gong, Xiao, Guo, Li et Guo, 2000, despite many similarities to T. kitauei. To examine the potential conspecificity of these species, a morphological and molecular investigation of T. xinyangensis was conducted. Comparing myxospore morphology, the mean spore length and width of each species is not identical between species, but ranges of dimensions overlap. These data are more suggestive of intraspecific variation than distinct species. Comparison of relative ratios of spore length to polar capsule length and spore width to polar capsule width of T. xinyangensis and T. kitauei reveal no differences and scanning electron microscopy reveals a smooth spore surface of T. xinyangensis, which is consistent with that of T. kitauei. Most convincingly, DNA sequences of the small subunit ribosomal RNA (ssrRNA) gene of the two species were identical. From the morphological and molecular biological data, we propose T. xinyangensis from the intestine of common carp is not a distinct species and is synonymous with T. kitauei.

Abrupt suspension of probiotics administration may increase host pathogen susceptibility by inducing gut dysbiosis

In this study, we investigated the risk associated with suspension of probiotics administration in tilapia, an animal model that may mimic immune-compromised conditions in humans. Tilapias were fed for 14 days using a probiotics-supplemented diet, followed by a three-day suspension of probiotics treatment and a subsequent challenge by Aeromonas hydrophila. Unexpectedly, the suspension of a probiotic strain Lactobacillus plantarum JCM1149 significantly triggered susceptibility of the host to A. hydrophila. We further observed that suspension of JCM1149 resulted in host gut microbiota dysbiosis and the subsequent disorder in the intestinal metabolites (bile acids, amino acids, and glucose) and damage in the intestinal epithelium, giving rise to a condition similar to antibiotics-induced gut dysbiosis, which collectively impaired tilapia’s gut health and resistance to pathogenic challenges. Additionally, we determined that JCM1149 adhered relatively poorly to tilapia intestinal mucosa and was rapidly released from the gastrointestinal tract (GIT) after suspension, with the rapid loss of probiotic strain probably being the direct cause of gut dysbiosis. Finally, three other probiotic Lactobacillus strains with low intestinal mucosa binding activity showed similar rapid loss phenotype following administration suspension, and induced higher host susceptibility to infection, indicating that the risk is a generic phenomenon in Lactobacillus.

Lactobacillus planarum subsp. plantarum JCM 1149 vs. Aeromonas hydrophila NJ-1 in the anterior intestine and posterior intestine of hybrid tilapia Oreochromis niloticus ♀ × Oreochromis aureus ♂: An ex vivo study

To investigate the ex vivo interactions of probiotic-pathogen-host in warm-water fish, hybrid tilapia (Oreochromis niloticus♀ × Oreochromis aureus♂) were sacrificed to isolate anterior and posterior intestine for incubation with phosphate-buffered saline (PBS; pH 7.2) as the control, Lactobacillus plantarum JCM 1149 at 1.0 × 10(9) CFU/ml, Aeromonas hydrophila NJ-1 at 1.0 × 10(8) CFU/ml, or the both combination. Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint and consequent sequence analysis confirmed anterior intestine sac was more prone to the colonization of L. plantarum JCM 1149 and A. hydrophila NJ-1 than the posterior part. L. plantarum JCM 1149 and A. hydrophila NJ-1 inhibited the population each other in anterior or posterior sac, indicating their competition for the colonization. The induced expression of HSP70, IL-1β and TNF-α in the anterior sac by the addition of L. plantarum JCM 1149 or A. hydrophila NJ-1 demonstrated the activity and a local immune response of ex vivo anterior sac. Compared with posterior intestine, higher population colonization and more sensitive immune response of anterior sac indicated differential patterns for the probiotic-pathogen-host interactions. Scanning electronic microscopy (SEM) observation showed that pathogen A. hydrophila NJ-1 damaged the anterior intestine, which was alleviated by the pretreatment of L. plantarum JCM 1149, showing its probiotic effect.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model

BACKGROUND Essential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host. OBJECTIVE The purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model. METHODS Juvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d. RESULTS NE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1β (Il1β; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1β (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05). CONCLUSION NE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

Effects of partially replacing dietary soybean meal or cottonseed meal with completely hydrolyzed feather meal (defatted rice bran as the carrier) on production, cytokines, adhesive gut bacteria, and disease resistance in hybrid tilapia (Oreochromis niloticus ♀ × Oreochromis aureus ♂).

We formulated experimental diets for hybrid tilapia to investigate the effects of replacing dietary soybean meal (SBM) or cottonseed meal (CSM) by completely hydrolyzed feather meal (defatted rice bran as the carrier; abbreviated as CHFM), with emphasis on fish growth, the composition of adhesive gut bacteria, intestinal and hepatic immune responses, and disease resistance. A series of four isonitrogenous (33% crude protein) and isolipidic (6% crude lipid) diets were formulated to replace the isonitrogenous percentages of CSM or SBM by 6% or 12% CHFM. Quadruplicate groups of healthy and uniformly sized hybrid tilapia were assigned to each experimental diet. Fish were hand fed three times a day for 8 weeks at a rearing temperature of 25-28 °C. The growth performance of hybrid tilapia fed diets with partial replacement of dietary SBM or CSM with CHFM was comparable to the group of fish fed the control diet. The CHFM-containing diets affected the intestinal autochthonous bacterial community in similar ways. All CHFM-containing diets stimulated the expression of heat shock protein 70 in the intestine but suppressed its expression in the liver. Only the CHFM6/SBM diet stimulated the expression of interleukin-1β in intestine, and no effects were observed in all diets to the expression of interleukin-1β in liver. Thus, regarding the immune response in the intestine and liver, CHFM is a good alternative protein source that induces less stress in the host. CHFM did not affect disease resistance to Aeromonas hydrophila infection in hybrid tilapia. These data suggest that CHFM is a good alternative to partially replace SBM and CSM in tilapia feed.

Effects of dietary Lactobacillus plantarum and AHL lactonase on the control of Aeromonas hydrophila infection in tilapia.

This study addressed the effects of dietary Lactobacillus plantarum or/and N‐acylated homoserine lactonase (AHL lactonase) on controlling Aeromonas hydrophila infection in juvenile hybrid tilapia (Oreochromis niloticus♀ × O. aureus ♂). Fish were fed Lb. plantarum subsp. plantarum strain JCM1149 (108 CFU/g feed) or/and AHL lactonase AIO6 (4 U/g) and were exposed to a chronic challenge of A. hydrophila NJ‐1 (105 cells/mL) for 14 days. Intestinal (foregut) alkaline phosphatase (IAP) activities were evaluated 1 day post challenge to reflect the resistance of fish against A. hydrophila infection. Parallel groups of fish with the same dietary assignments while unchallenged were also included to investigate the effect of dietary Lb. plantarum or/and AIO6 supplementation on gut health of tilapia. The results showed that IAP activity was significantly lower in fish fed with diets supplemented with Lb. plantarum JCM1149 or the combination of Lb. plantarum JCM1149 and AIO6, indicating enhanced resistance against A. hydrophila. Light microscopy and transmission electron microscopy images of foregut revealed damage caused by A. hydrophila NJ‐1, but dietary Lb. plantarumJCM1149 or/and AIO6 significantly alleviated the damages. Compared to the fish immersed in A. hydrophila NJ‐1, dietary Lb. plantarum JCM1149 or AIO6 could maintain the microvilli length in the foregut of tilapia. However, among the unchallenged groups of fish, the microvilli length in the foregut of tilapia fed AIO6 (singly or combination) and the microvilli density of tilapia fed AIO6 (singly) were significantly lower than those of the control, though the microvilli density in the combination treatment was significantly improved. Additionally, the dietary Lb. plantarum JCM1149 could down‐regulate the expression of stress‐related gene in the gut after the acute phase. In conclusion, the dietary Lb. plantarum JCM1149 is recommended to control the A. hydrophila infection in tilapia.