Daniel L. Merrifield

Microbial manipulations to improve fish health and production--a Mediterranean perspective.

The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts.

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.

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10⁶ CFU g⁻¹ feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.

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).

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).

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 (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.

A high-resolution map of the gut microbiota in Atlantic salmon (Salmo salar): A basis for comparative gut microbial research

Gut health challenges, possibly related to alterations in gut microbiota, caused by plant ingredients in the diets, cause losses in Atlantic salmon production. To investigate the role of the microbiota for gut function and health, detailed characterization of the gut microbiota is needed. We present the first in-depth characterization of salmon gut microbiota based on high-throughput sequencing of the 16S rRNA gene’s V1-V2 region. Samples were taken from five intestinal compartments: digesta from proximal, mid and distal intestine and of mucosa from mid and distal intestine of 67.3 g salmon kept in seawater (12–14 °C) and fed a commercial diet for 4 weeks. Microbial richness and diversity differed significantly and were higher in the digesta than the mucosa. In mucosa, Proteobacteria dominated the microbiota (90%), whereas in digesta both Proteobacteria (47%) and Firmicutes (38%) showed high abundance. Future studies of diet and environmental impacts on gut microbiota should therefore differentiate between effects on mucosa and digesta in the proximal, mid and the distal intestine. A core microbiota, represented by 22 OTUs, was found in 80% of the samples. The gut microbiota of Atlantic salmon showed similarities with that of mammals.

Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case

ABSTRACT In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase.

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.