Characterization of microbiota in Arapaima gigas intestine and isolation of potential probiotic bacteria.

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.

Dietary administration of a commercial mixed-species probiotic improves growth performance and modulates the intestinal immunity of tilapia, Oreochromis niloticus.

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-1ß, and anti-inflammatory genes IL-10 and TGFß 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.

Modulation of the intestinal microbiota and morphology of tilapia, Oreochromis niloticus, following the application of a multi-species probiotic.

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.

Effects of dietary ß-(1,3)(1,6)-D-glucan supplementation on growth performance, intestinal morphology and haemato-immunological profile of mirror carp (Cyprinus carpio L.).

In recent years, aquaculture research has focused on probiotics, prebiotics, and ß-glucans, in order to improve health status and growth performance. Information regarding the effects of ß-glucan on growth performance and intestinal immunity of mirror carp (Cyprinus carpio L.) is scarce. An experiment was therefore conducted to investigate the effects of a yeast ß-glucan preparation (MacroGard(®) ) on growth performance, intestinal morphology and haemato-immunological indices of mirror carp. Carp (initial weight 11.1 ± 0.0 g) were fed highly purified diets supplemented with 0% (control), 0.1%, 1% or 2% MacroGard(®) for 8 weeks. Fish fed diets containing 1% and 2% MacroGard(®) showed significant improvements in weight gain, specific growth rate and feed conversion ratio compared to fish fed both the control and the 0.1% MacroGard(®) containing diet. Histological appraisal of the intestine showed a significantly higher infiltration of leucocytes into the epithelial layer of fish fed diets supplemented with 1% and 2% MacroGard(®) in the anterior intestine compared to fish fed the control and 0.1% MacroGard(®) diet. This effect was not observed in the posterior intestine. There were no significant differences in the intestinal absorptive surface area and number of goblet cells in either intestinal region. At the end of the experiment, the haematological status of the fish was examined. Compared to control fed fish, the haematocrit value was significantly elevated in fish fed the 2% MacroGard(®) diet. Furthermore, the blood monocyte fraction was significantly higher in fish fed the 1% and 2% MacroGard(®) diets. No significant changes were observed in the other blood parameters assessed. The present study shows that high dietary ß-glucan inclusion increases growth performance without detrimental effects on the health indicators assessed. Increased intraepithelial leucocytes in the anterior intestine may indicate a localized immune response; no detrimental effects on intestinal morphology were observed.

Dietary synbiotic application modulates Atlantic salmon (Salmo salar) intestinal microbial communities and intestinal immunity.

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 (IL1ß, 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.

Effects of a dietary ß-(1,3)(1,6)-D-glucan supplementation on intestinal microbial communities and intestinal ultrastructure of mirror carp (Cyprinus carpio L.).

AIM: To assess the effects of dietary Saccharomyces cerevisiae ß-(1,3)(1,6)-D-glucan supplementation (MacroGard(®)) on mirror carp (Cyprinus carpio L.) intestinal microbiota and ultrastructure of the enterocyte apical brush border. METHODS AND RESULTS: Carp were fed either a control diet or diets supplemented with 0.1, 1 or 2% w/w MacroGard(®). Culture-dependent microbiology revealed that aerobic heterotrophic bacterial levels were unaffected by dietary MacroGard(®) after 2 and 4 weeks. No effects were observed on the allochthonous lactic acid bacteria (LAB) populations at either time point; however, reduced autochthonous LAB populations were observed at week 4. PCR-DGGE confirmed these findings through a reduction in the abundance of autochthonous Lactococcus sp. and Vagococcus sp. in MacroGard(®)--fed fish compared with the control-fed fish. Overall, sequence analysis detected microbiota belonging to the phyla Proteobacteria, Firmicutes, Fusobacteria and unidentified uncultured bacteria. DGGE analyses also revealed that dietary MacroGard(®) reduced the number of observed taxonomical units (OTUs) and the species richness of the allochthonous microbiota after 2 weeks, but not after 4 weeks. In contrast, dietary MacroGard(®) reduced the number of OTUs, the species richness and diversity of the autochthonous microbiota after 2 weeks, and those parameters remained reduced after 4 weeks. Transmission electron microscopy revealed that intestinal microvilli length and density were significantly increased after 4 weeks in fish fed diets supplemented with 1% MacroGard(®). CONCLUSIONS: This study indicates that dietary MacroGard(®) supplementation modulates intestinal microbial communities of mirror carp and influences the morphology of the apical brush border. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge, this is the first study to investigate the effects of ß-(1,3)(1,6)-D-glucans on fish gut microbial communities, using culture-independent methods, and the ultrastructure of the apical brush border of the enterocytes in fish. This prebiotic-type effect may help to explain the mechanisms in which ß-glucans provide benefits when fed to fish.

Probiotic Pediococcus acidilactici modulates both localised intestinal- and peripheral-immunity in tilapia (Oreochromis niloticus).

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).

Haemato-immunological and growth response of mirror carp (Cyprinus carpio) fed a tropical earthworm meal in experimental diets.

An investigation was conducted to evaluate the effect of feeding a tropical earthworm meal (Perionyx escavatus) on the haemato-immunological response and growth performance of mirror carp (Cyprinus carpio). Fish were fed diets for a total of 88 days, fishmeal served as the main protein source in the control diet. Two remaining diets consisted of fishmeal fixed at 33.65% provision of protein and the remaining 66.35% protein was provided by soybean meal (SBM diet) or P. excavatus meal (EW diet). Compared to control and SBM fed fish (7.69 ± 0.28 and 5.92 ± 0.31 g/dl, respectively), a significant increase in haemoglobin was measured in EW fed fish (9.57 ± 0.24 g/dl). Consequently significant elevations were also observed in mean corpuscular haemoglobin (MCH; 79.13 ± 4.59 pg) and mean corpuscular haemoglobin concentration (MCHC; 22.69 ± 0.54 pg) in EW fed fish. On the contrary, compared to control and SBM fed carp total leukocyte levels (2.72 ± 0.17 and 3.10 ± 0.17 × 10(4)/mm(3), respectively) were significantly decreased in the EW group (2.15 ± 0.14 × 10(4)/mm(3)). Moreover at day 14 and 21 post immunisation with bacterin isolated from Aeromonas hydrophila fish fed the EW diet displayed a significant reduction in respiratory burst activity (RBA) compared to control and SBM fed fish. After 60 days of feeding, fish fed EW diet showed a significant elevation in final body weight compared to fish fed a fishmeal based diet (control treatment) and fish fed a soybean meal based diet. Similar improvements were observed in feed utilisation efficiency. The present study shows that feeding P. excavatus meal to mirror carp decreases some aspects of the innate immune response, but at the same time gives rise to significant enhancement of growth and feed utilisation efficiency.

Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae.

A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout-associated microbiota may provide a defensive barrier against L. garvieae.

Effect of dietary Ulva and Spirulina on weight loss and body composition of rainbow trout, Oncorhynchus mykiss (Walbaum), during a starvation period.

A study was conducted to determine the effect of dietary algal supplementation on weight loss and proximate composition in rainbow trout, Oncorhynchus mykiss, during starvation. An algae-free control diet (C) and four experimental diets, including varying levels of raw Ulva meal (5% = U5; 10% = U10) and Spirulina meal (5% = S5; 10% = S10) were formulated. Fish were fed to satiation for 12 weeks and then subjected to a 3-week starvation period. Body weight, viscerosomatic index (VSI, %), hepatosomatic index (HSI, %), dress-out (DO, %) and chemical composition of carcass were analysed at the end of each starvation week. The accumulated weight loss of the fish fed dietary algae was almost 50% less than the control group (p < 0.05) after 2 and 3 weeks starvation. No significant differences in weight loss were observed among the fish fed different algal diets during the starvation period. Total body protein and ash contents remained constant in all groups throughout the starvation period. Fish in all treatments lost a significant level of total lipids and changes of liver fatty acid profiles were also observed. Results indicate that low level inclusion of algae in aquafeeds may have economical advantages in terms of reducing weight loss in fish when subjected to a short-term fasting period, that sometimes is necessary after a heavy rainfall or when fish stocks are held back to meet market demand.

The effect of Pediococcus acidilactici on the gut microbiota and immune status of on-growing red tilapia (Oreochromis niloticus).

AIM: To assess Pediococcus acidilactici as a dietary supplement for on-growing red tilapia (Oreochromis niloticus). METHODS AND RESULTS: Tilapia were fed either a control diet or control diet supplemented with Ped. acidilactici at 10(7) 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Dietary mannan oligosaccharide supplementation modulates intestinal microbial ecology and improves gut morphology of rainbow trout, Oncorhynchus mykiss (Walbaum).

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.

Soybean meal alters autochthonous microbial populations, microvilli morphology and compromises intestinal enterocyte integrity of rainbow trout, Oncorhynchus mykiss (Walbaum).

Abstract Rainbow trout were fed either a diet containing fishmeal (FM) as the crude protein source or a diet containing 50% replacement with soybean meal (SBM) for 16 weeks. An enteritis-like effect was observed in the SBM group; villi, enterocytes and microvilli were noticeably damaged compared with the FM group. The posterior intestine microvilli of SBM-fed fish were significantly shorter and the anterior intestine microvilli significantly less dense than the FM-fed fish. Electron microscopy confirmed the presence of autochthonous bacterial populations associated with microvilli of both fish groups. Reduced density of microvilli consequently led to increased exposure of enterocyte tight junctions, which combined with necrotic enterocytes is likely to diminish the protective barrier of the intestinal epithelium. No significant differences in total viable counts of culturable microbial populations were found between the groups in any of the intestinal regions. A total of 1500 isolates were tentatively placed into groups or genera, according to standard methods. Subsequent partial 16S rRNA sequencing revealed species that have not been identified from the rainbow trout intestine previously. Compared with the FM group levels of Psychrobacter spp. and yeast were considerably higher in the SBM group; a reduction of Aeromonas spp. was also observed.