It is well known that healthy gut microbiota is essential to promote host health and well‐being. The intestinal microbiota of endothermic animals as well as fish are classified as autochthonous or ...indigenous, when they are able to colonize the host's epithelial surface or are associated with the microvilli, or as allochthonous or transient (associated with digesta or are present in the lumen). Furthermore, the gut microbiota of aquatic animals is more fluidic than that of terrestrial vertebrates and is highly sensitive to dietary changes. In fish, it is demonstrated that a dietary form (live feeds or pelleted diets), b dietary lipid (lipid levels, lipid sources and polyunsaturated fatty acids), c protein sources (soybean meal, krill meal and other meal products), d functional glycomic ingredients (chitin and cellulose), e nutraceuticals (probiotics, prebiotics, synbiotics and immunostimulants), f antibiotics, g dietary iron and h chromic oxide affect the gut microbiota. Furthermore, some information is available on bacterial colonization of the gut enterocyte surface as a result of dietary manipulation which indicates that changes in indigenous microbial populations may have repercussion on secondary host–microbe interactions. The effect of dietary components on the gut microbiota is important to investigate, as the gastrointestinal tract has been suggested as one of the major routes of infection in fish. Possible interactions between dietary components and the protective microbiota colonizing the digestive tract are discussed.
The intestinal microbiota and morphology of tilapia (Oreochromis niloticus) were investigated after the application of a multi-species probiotic containing Lactobacillus reuteri, Bacillus subtilis, ...Enterococcus faecium and Pediococcus acidilactici (AquaStar® Growout). Tilapia (55.03 ± 0.44 g) were fed either a control diet or a probiotic diet (control diet supplemented with AquaStar® Growout at 5 g kg⁻¹). After four and eight weeks, culture-dependent analysis showed higher levels of lactic acid bacteria (LAB), enterococci and Bacillus spp. in the mucosa and digesta of fish fed AquaStar® Growout. At week four, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) revealed a higher similarity within the probiotic fed replicates than replicates of the control group; after eight weeks, the compositional dissimilarity of the microbiome profiles between the groups was greater than the dissimilarities within each group (P < 0.05). High-throughput sequencing revealed that the probiotic treatment significantly reduced the number of operational taxonomic units and species richness in the digesta. Significantly higher proportions of reads belonging to Proteobacteria and Cyanobacteria were detected in the control group whereas the probiotic-fed fish displayed a significantly higher abundance of reads assigned to the Firmicutes (which accounted for >99 % of reads). Bacillus, Cetobacterium and Mycobacterium were the dominant genera in the digesta of control fish whereas Bacillus, Enterococcus and Pediococcus were the largest constituents in probiotic-fed fish. The addition of AquaStar® Growout to tilapia diets led to increased populations of intraepithelial leucocytes, a higher absorptive surface area index and higher microvilli density in the intestine. These data suggest that AquaStar® Growout can modulate both the intestinal microbiota and morphology of tilapia.
The growth performance, immunological status, intestinal morphology and microbiology of tilapia, Oreochromis niloticus, were investigated after dietary administration of the commercial probiotic ...AquaStar® Growout. Tilapia (29.02 ± 0.33 g) were split into five treatments; control (CON), 1.5 g kg−1 probiotic (PRO-1.5), 3 g kg−1 probiotic (PRO-3), pulsed probiotic feeding (PRO-PULSE) or an initial probiotic feed followed by control feeding (PRO-INI). After six weeks of experimental feeding, fish fed PRO-3 displayed significantly higher final weight, weight gain and SGR compared to the CON or PRO-INI treatments. Supplementation of the probiotic at this dose induced an up-regulation of intestinal caspase-3, PCNA and HSP70 mRNA levels compared to the CON fed fish. Immuno-modulatory pathways were also affected; significantly higher expression of TLR2, pro-inflammatory genes TNFα and IL-1β, and anti-inflammatory genes IL-10 and TGFβ suggest that the probiotic may potentiate a higher state of mucosal tolerance and immuno-readiness. Histological appraisal revealed significantly higher numbers of intraepithelial leucocytes in the intestine of PRO-3 fed fish compared with treatments CON, PRO-PULSE and PRO-INI but not PRO-1.5. Additionally, fish receiving PRO-3 had a significantly higher abundance of goblet cells in their mid-intestine when compared with fish from all other treatments. Together, these data suggest that continuous provision of AquaStar® Growout at 3 g kg−1 can improve tilapia growth and elevate the intestinal immunological status of the host.
•AquaStar® Growout improves the growth performance of juvenile tilapia.•AquaStar® Growout can augment mucosal tolerance.•AquaStar® Growout improves immune readiness.
A trial was conducted to determine the effect of 6% hydrolysed wheat gluten (HWG) inclusion in a low-fishmeal diet on growth performance and intestinal microbiota and morphology of Asian seabass ...(Lates calcarifer). Fish (initial average weight of 36.3g) were allocated into floating cages (eight replicates per diet, 30 fish per cage). They were fed either a fishmeal-based diet (positive, diet code: POS; 36% fishmeal), or a diet based on a mix of animal and plant proteins (negative, diet code: NEG). The fishmeal inclusion rate in this diet was reduced to 6%, with the major protein sources being soybean meal and animal by-products. The third diet (diet code: HWG) was similar to the NEG diet with the exception that 6% HWG was included in replacement of the other proteins. At the end of the 48day trial, fish were sampled for intestinal microbiology and histology. Growth parameters were also assessed. Final body weight, SGR and daily feed intake were not significantly different across diets. However, the feeding rate was significantly affected, with the lowest rate observed in the POS treatment and the highest with the NEG treatment. The FCR was significantly lower for both POS and HWG fed fish than for the NEG fed fish. High throughput sequencing revealed that the majority of reads derived from the mucosa samples belonged to members of Proteobacteria (70.3% of the reads), Cyanobacteria (10.0%) and Firmicutes (7.6%). In the digesta reads were mainly assigned to Proteobacteria (34.5%), Fusobacteria (34.5%), and Firmicutes (22.6%). The alpha diversity did not differ among dietary treatments. Some differences in OTU relative abundances were obtained between diets, however, the overall community was not modified to a large extent by HWG. Histological appraisal revealed that the HWG fed fish exhibited significantly higher posterior intestinal perimeter ratio than that of the POS treatment. Overall, including HWG in a low fishmeal diet positively affects feed efficiency. Concomitantly the absorptive surface area of the posterior intestine was improved while the intestinal microbiota, described comprehensively here for the first time in Asian seabass, was similar to the microbiota of other healthy carnivorous marine fish species. The mechanisms involved in these changes may be related to the high glutamine content and to the high protein digestibility of HWG.
Fishmeal used to be the major source of proteins, especially for marine fish and salmonids. Nevertheless, its inclusion in diets has been reduced. Some commercial fish feeds may contain less than 10% fishmeal, largely replaced by a mix of animal and plant proteins. Our manuscript demonstrates that hydrolysed wheat gluten may be used to formulate high quality low fishmeal diets.
•We compare intestine microbiota and morphology of Asian seabass fed with two diets.•We examine benefits of including hydrolysed wheat gluten in a low fishmeal diet.•We describe the taxonomic composition of Asian seabass intestinal microbiota.
To assess Pediococcus acidilactici as a dietary supplement for on-growing red tilapia (Oreochromis niloticus). Tilapia were fed either a control diet or control diet supplemented with Ped. ...acidilactici at 10⁷ CFU g⁻¹ for 32 days. Ped. acidilactici colonized the intestinal tract and significantly affected the intestinal microbial communities. PCR-DGGE revealed direct antagonism of gastric Ped. acidilactici with an endogenous uncultured bacterium during a period of reverting to nonsupplemented feeding. Light microscopy revealed that gut integrity and leucocyte levels were unaffected by Ped. acidilactici; however, blood leucocyte levels and serum lysozyme activity were elevated after 14-days' feeding. No significant improvements in growth performance were observed at the end of the trial (day 32), but survival was significantly higher in the probiotic group. The study demonstrates that oral supplementation of Ped. acidilactici modulates intestinal bacterial communities in on-growing red tilapia and also stimulates some aspects of the nonspecific immune response. To our knowledge this is the first study assessing the effects of probiotics on the gut microbiota of tilapia using culture-independent methods. Such methods are crucial to understand the mechanisms which underpin and mediate host benefits.
A study was conducted to investigate the effect of mannan oligosaccharide (MOS) on the gut microbiota and intestinal morphology of rainbow trout under commercial farming conditions. Juvenile (mean ...initial BW 38.2 +/- 1.7 g) and subadult (111.7 +/- 11.6 g) trout were fed 2 dietary treatments for 111 and 58 d, respectively. The control treatment consisted of a standard commercial diet, and the MOS treatment consisted of the control diet supplemented with 0.2% MOS. Morphology of the anterior and the posterior intestine was examined with light and electron microscopy. Light microscopy demonstrated increased gut absorptive surface area in the subadult MOS group. Additionally, electron microscopy revealed an increase in microvilli length and density in the subadult MOS group compared with the control (P < 0.05). However, no significant improvements were detected in the juvenile group. Culture-based evaluation of the intestinal microbiota showed that MOS significantly reduced (P < 0.05) the viable intestinal bacterial populations (by approximately 2 log scales in all cases). Levels of Aeromonas/Vibrio spp. were significantly decreased (P < 0.05) in the juvenile MOS group (9% of the total microbiota) compared with the juvenile control group (37%). Additionally, analysis of microbial communities was conducted using denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA. The denaturing gradient gel electrophoresis fingerprinting revealed an alteration of bacterial populations; analysis of similarity, similarity percentages, and nonmetric multidimensional scaling analysis showed that MOS reduced species richness and increased similarity of bacterial populations found within the subadult and juvenile groups. The current study shows that MOS modulates intestinal microbial communities, which subsequently improve gut morphology and epithelial brush border.
The aim of this study was to determine the best synbiotic combination (based on growth and short chain fatty acids production) between Pediococcus acidilactici and five prebiotics in vitro, inulin, ...fructooligosaccharide (FOS), xylooligosaccharide (XOS), galactooligosaccharide (GOS), isomaltooligosaccharide (IMO). The best in vitro growth under aerobic and anaerobic culture condition was observed in the treatment, P. acidilactici + GOS. Furthermore, the study of short chain fatty acids (SCFA) production at the end of exponential growth phase revealed that the major SCFA produced was propionic acid in all synbiotic treatments except for the P. acidilactici + GOS treatment where butyric acid production was significantly higher compared with the other treatments. Based on the results of in vitro studies, P. acidilactici + GOS that were selected as synbiotic and rainbow trout fingerlings (15.04 ± 0.52 g) were fed on diet supplemented with the selected synbiotic. After 8 weeks of feeding, growth performance, intestinal microbiota and physiological responses were evaluated. Growth performance was significantly improved by synbiotic feeding. No significant differences of haematological parameters were revealed between the different treatments. Level of presumptive autochthonous lactic acid bacteria was elevated, while total autochthonous bacterial level remained unaffected. These results showed that P. acidilactici + GOS can be considered as an efficient synbiotic for rainbow trout.
Aims
The aim of this study was to determine the intestinal microbiota of pirarucu (Arapaima gigas) in different growth stages (adult and fingerlings) and to isolate and identify potential probiotic ...bacteria.
Methods and Results
High‐throughput sequencing analysis of the intestinal contents revealed that the majority of sequences belonged to the Proteobacteria, Fusobacteria and Firmicutes phyla. At the genus level, the greatest number of sequences belonged to Bradyrhizobium in adult fish, while Cetobacterium was the most abundant in juvenile fish. Twenty‐three lactic‐acid bacteria (LABs) were isolated on MRS agar from healthy juvenile fish. The isolates were tested in vitro for probiotic properties. Two isolates (identified as strains of Lactococcus lactis subsp. lactis and Enterococcus faecium) displayed antagonism against all 10 pathogens tested, were nonhaemolytic and maintained good viability for at least 3 weeks when supplemented to fish diets. The presence of a number of antibiotic resistance genes (ARGs), conferring resistance to erythromycin, tetracycline and chloramphenicol, was investigated by PCR.
Conclusions
The absence of ARGs investigated the potential to antagonize pathogens, and favourable growth and survival characteristics indicate that these autochthonous isolates have the potential to be considered probiotics, which will be studied in future in vivo experiments.
Significance and Impact of the Study
This study has demonstrated, for the first time, the normal microbiota in the A. gigas intestine during different life stages and the presence of LAB strains. It also demonstrated LAB antibiotic resistance and antagonistic behaviour against pathogens isolated from the same fish.
A feeding trial was conducted to determine the effect of dietary administration of Pediococcus acidilactici MA18/5M and short chain fructooligosaccharides (scFOS) on Atlantic salmon (Salmo salar L.) ...intestinal health. Salmon (initial average weight 250 g) were allocated into triplicate sea pens and were fed either a control diet (commercial diet: 45% protein, 20% lipid) or a synbiotic treatment diet (control diet + P. acidilactici at 3.5 g kg−1 and 7 g kg−1 scFOS) for 63 days. At the end of this period, fish were sampled for intestinal microbiology, intestinal histology and the expression of selected immune-related genes (IL1β, TNFα, IL8, TLR3 and MX-1) in the intestine.
Compared to the control fish, the total bacterial levels were significantly lower in the anterior mucosa, posterior mucosa and posterior digesta of the synbiotic fed fish. qPCR revealed good recovery (log 6 bacteria g−1) of the probiotic in the intestinal digesta of the synbiotic fed fish and PCR-DGGE revealed that the number of OTUs, as well as the microbial community diversity and richness were significantly higher in the anterior digesta of the synbiotic fed fish than the control. Compared to the control fed fish, the mucosal fold (villi) length and the infiltration of epithelial leucocytes were significantly higher in the anterior and posterior intestine, respectively, in the synbiotic group. Real-time PCR demonstrated that all of the genes investigated were significantly up-regulated in the anterior and posterior intestine of the synbiotic fed salmon, compared to the control group. At the systemic level, serum lysozyme activity was significantly higher in the synbiotic fed fish and growth performance, feed utilisation and biometric measurements (condition factor, gutted weight and gut loss) were not affected.
Together these results suggest that the synbiotic modulation of the gut microbiota has a protective action on the intestinal mucosal cells, improving morphology and stimulating the innate immune response without negatively affecting growth performance or feed utilization of farmed Atlantic salmon.
•Dietary synbiotic application modulates the gut microbiota of Atlantic salmon.•Dietary synbiotic application modulates the Atlantic salmon intestinal immune response.•Modulation of the intestinal microbiota and immune status did not have adverse affects on Atlantic salmon growth performance.
The application of probiotics in aquaculture has received concerted research efforts but the localised intestinal immunological response of fish to probiotic bacteria is poorly understood. Therefore, ...a study was conducted to evaluate the probiotic effect of Pediococcus acidilactici on Nile tilapia (Oreochromis niloticus) with specific emphasis on intestinal health and probiotic levels as well as system level responses such as growth performance, feed utilization and haemato-immunological parameters under non-challenged conditions. Fish (9.19 ± 0.04 g) were fed either a control diet or a P. acidilactici supplemented diet (at 2.81 × 10(6) CFU g(-)(1)) for six weeks. At the end of the study the probiotic was observed to populate the intestine, accounting for ca. 3% (1.59 × 10(5) CFU g(-)(1)) of the cultivable intestinal bacterial load. Real-time PCR indicated that the probiotic treatment may potentiate the immune-responsiveness of the intestine as up-regulation of the gene expression of the pro-inflammatory cytokine TNFα was observed in the probiotic fed fish (P < 0.05). Light microscopy observations revealed elevated intraepithelial leucocyte (IEL) levels in the intestine of P. acidilactici fed tilapia after six weeks (P < 0.05) of feeding and a trend towards elevated goblet cells was also observed after six weeks feeding (P = 0.08). Concomitantly at week six, along with elevated IELs and elevated TNFα mRNA levels in the intestine, an increased abundance of circulating neutrophils and monocytes were observed in fish fed the probiotic supplemented diet (P < 0.05). This haemopoietic expansion of innate immune cells could be reflective of an elevated state of immuno-readiness. Together these results suggest that the probiotic has a protective action on the intestinal mucosal cells, stimulating the innate immune response after feeding for a period of six weeks. These immunological modulations did not impair growth performance or the remaining haematological and zootechnical parameters compared to the control group (P > 0.05).
PDF
