Inonotus obliquus containing diet enhances the innate immune mechanism and disease resistance in olive flounder Paralichythys olivaceus against Uronema marinum

The present study describes the effect of diet supplementation with Chaga mushroom, Inonotus obliquus extract at 0%, 0.01%, 0.1%, and 1.0% levels on the innate humoral (lysozyme, antiprotease, and complement), cellular responses (production of reactive oxygen and nitrogen species and myeloperoxidase), and disease resistance in olive flounder, Paralichythys olivaceus against Uronema marinum. The lysozyme activity and complement activity significantly increased in each diet on weeks 2 and 4 against pathogen. The serum antiprotease activity and reactive nitrogen intermediates production significantly increased in fish fed with 0.1% and 1.0% diets from weeks 1-4. However, reactive oxygen species production and myeloperoxidase activity significantly increased in 1.0% and 2.0% diets on weeks 2 and 4. In fish fed with 0.1% and 1.0% diets and challenged with U. marinum the cumulative mortality was 50% and 40% while in 0% and 0.01% diets the mortality was 85% and 55%. The results clearly indicate that supplementation diet with I. obliquus at 0.1% and 1.0% level positively enhance the immune system and confer disease resistance which may be potentially used as an immunoprophylactic in finfish culture.     

Evaluation of non-specific immune components from the skin mucus of olive flounder (Paralichthys olivaceus)

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish under intensive aquaculture and at prolonged low temperatures from relevant infections due to inadequate adaptive immune responses. In the present study we aimed to understand the mucosal immunity of an economically important mariculture fish, olive flounder (Paralichthys olivaceus) by evaluating the immune components from its skin mucus. The activities of lysozyme (233.33+/-171.82 units mg(-1)), trypsin-like protease (42.84+/-1.249 units mg(-1)), alkaline phosphatase (0.376+/-0.005 units mg(-1)) and esterase (0.170+/-0.006 units mg(-1)) were detected in the skin mucus. Transferrin was identified by MALDI-TOF/MS analysis. ELISA and immunoblot assays using anti-flounder IgM monoclonal antibody showed the presence of a significant level (1.80+/-0.001, n=3) of monomer immunoglobulin M (IgM) with approximate molecular weight of 160 and 25 kDa under non-denaturing and denaturing states, respectively. Skin mucus showed strong antibacterial activity against tested fish pathogenic bacteria. In addition, skin mucus successfully agglutinated (HA titre 2(8)), but completely failed to haemolyse, rabbit erythrocytes. In conclusion, the major immune components of the skin mucus, identified in the present study, are possibly involved in the broad spectrum non-specific immunity of olive flounder.     

Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss)

We analysed the effect of probiotic strains on the cellular and humoral immune responses of rainbow trout (Oncorhynchus mykiss), and their capacity to prevent furunculosis during a challenge trial. Probiotic strains (Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202) were administered orally to fish for 2 weeks at 10(6) CFU g(-1) of feed. In comparison to untreated control fish, the phagocytic activity of head kidney leukocytes and the alternative complement activity in serum were significantly greater in all probiotic groups at the end of the second week. With the exception of the group fed with Lactobacillus sakei, superoxide anion production was also significantly increased in the probiotic groups. Analysis of lysozyme activity did not exhibit any significant difference in the probiotic and control groups. Fifteen days after the start of the probiotic feeding, fish were challenged with Aeromonas salmonicida ssp. salmonicida. The fish supplemented with probiotics exhibited survival rates ranging from 97.8% to 100%, whereas survival was 65.6% in fish not treated with the probiotics. These results demonstrate that probiotic supplementation to fish can reduce the severity of furunculosis, and suggest that this reduction may be associated with enhanced humoral and cellular immune response.     

Probiotics and herbal mixtures enhance the growth, blood constituents, and nonspecific immune response in Paralichthys olivaceus against Streptococcus parauberis

The present study was reported the effect of probiotics and herbals mixture supplementation diet on growth, blood constituents, and nonspecific immune response in olive flounder Paralichthys olivaceus against Streptococcus parauberis on weeks 1, 2, 4, 6, 8, 10, and 12 after injected intraperitoneally (i.p.) with 50 μl of PBS (phosphate buffer saline) containing S. parauberis (2.1 × 10? CFU ml?1). The initial weight did not significantly increased in supplementation diet group from 1 to 4 weeks, whereas it was significantly increased from weeks 6 to 12 as compared to fish fed without supplementation diet. The serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) activities significantly increased from weeks 4 to 12 in infected fish fed with supplementation diet compared to fish fed without supplementation diet. However, the total protein (TP) and glucose (GLU) levels were significantly increased in infected fish fed with supplementation diet after 6 weeks. The phagocytic, respiratory burst, complement, and lysozyme activities significantly enhanced in infected fish fed with supplementation diet from weeks 4 to 12 as compared to fish fed without supplementation diet. These results suggested that different probiotics and herbals mixture supplementation diet enhanced the growth, blood biochemical constituents, and nonspecific immunity in olive flounder against S. parauberis.     

The use of innate immune responses as biomarkers in a programme of integrated biological effects monitoring on flounder (Platichthys flesus) from the southern North Sea

Immunological biomarkers that reflect the effects of exposure to environmental contaminants in coastal marine habitats were sought in European flounder (Platichthys flesus) from five locations in the German Bight with different anthropogenic impacts. During a 2-year period of sampling, innate immune responses were monitored from a total of 331 individual flounder of a body length of 18 to 25 cm. From the fish, plasma lysozyme, phagocytosis and respiratory burst activity of head kidney leucocytes were analysed and implemented as part of an integrated biological effects monitoring programme. As the measurements of the parameters applied here varied within wide ranges at some locations, spatial differences could not always be established, but some general trends could be drawn: plasma lysozyme activity was decreased in flounder contaminated with DDT adducts and some PCBs, while cellular functions such as phagocytosis and respiratory burst were stimulated by some chlorinated hydrocarbons. Correlation analysis also revealed connections not only between the parameters applied here and some contaminants but also with some biochemical parameters used as biomarkers in pollution monitoring: in flounder with decreased integrity of hepatocyte lysosomal membranes, immune functions also were impaired, and plasma lysozyme as well as phagocytosis activity of head kidney cells were impaired when the activity of cytochrome P450 1A was induced. The data presented here indicate that innate immune responses may be useful parameters to monitor cellular functions in a battery of biomarkers of different levels of biological organisation. Communicated by H. v. Westernhagen, A. Diamant     

Short- and long-term effects of a dietary yeast beta-glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax)

The present study investigated the immunomodulatory activity of Ergosan, an algal extract containing alginic acid, and Macrogard, a yeast extract containing beta-glucans, on innate and specific immunity in sea bass (Dicentrarchus labrax). Four cycles of experimental feeding using normal fish feed formulation (control group) supplemented with Ergosan (0.5%) or Macrogard (0.1%) were performed at 60-day intervals (15 days of treatment+45 days of suspension). Serum complement, lysozyme, total proteins and heat shock protein (HSP) concentrations were measured at 15, 30 and 45 days from the end of the first 15-day feeding cycle (short term) and 45 days after the end of each feeding cycle over a 35-week period (long term). The percentage of B- and T-lymphocytes in peripheral blood leucocytes and gut were measured over long-term trial. Significant elevation (P < 0.05) in serum complement activity occurred in sea bass fed with alginic acid and glucans, at 15 days from the end of first cycle of treatment. Significant elevation (P < 0.05) in serum lysozyme, gill and liver HSP concentration were observed in the same experimental groups at 30 days from the end of treatment, whereas a significant increase (P < 0.05) of complement activity was only observed in fish that received an Ergosan diet. At 45 days from the end of treatment, complement, lysozyme and HSP concentration did not differ among groups. Over the long-term period, no significant differences were observed in innate and specific immune parameters, survival, growth performances and conversion index in treated and control fish. A dramatic decrease of both innate and acquired immune parameters was observed during the winter season in all groups, followed by a partial recovery when water temperature increased. Reduction in complement and lysozyme activities was significatively correlated (p < 0.01) to water temperature variation. The results suggested the potential of alginic acid and beta-glucans to activate some innate immune responses in sea bass, and particularly under conditions of immunodepression related to environmental stress.     

Effects of phenanthrene on growth and basic physiological functions of the olive flounder, Paralichthys olivaceus

Flounder were exposed to waterborne phenanthrene (0.5, 1 and 2 μM) for 4 weeks to test effects of waterborne phenanthrene on growth and hematological properties of the olive flounder (Paralichthys olivaceus). The average weight gain (WG) of flounder was significantly decreased in fish exposed to phenanthrene at 2.0 μM for 2 weeks, whereas WGs of fish treated by 1.0 and 2.0 μM phenanthrene for 4 weeks were significantly decreased. However, hepatosomatic index (HSI) and condition factor (CF) of flounder were not significantly affected by phenanthrene exposure. Red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Ht), the mean corpuscular hemoglobin (MCH) and the mean corpuscular hemoglobin concentration (MCHC) mean levels were decreased with an increase in exposure time of phenanthrene to the fish, but the level of the mean corpuscular volume (MCV) was increased. Plasma bilirubin concentrations were significantly increased following exposure to waterborne phenanthrene (2.0 μM) for 2 and 4 weeks; however, there were no significant changes in plasma total cholesterol in fish of all treated groups compared to control. The phenanthrene-exposed groups (≥1.0 μM) showed significantly higher mean plasma lysozyme activity. Kidney lysozyme activity of fish exposed to phenanthrene (≥1.0 μM) was also significantly higher than that of control fish. The central finding from these data is that olive flounder exposed to waterborne phenanthrene at more than 1.0 μM are likely to experience negative impacts on fish health and basic physiological functions.     

Immunomodulatory effect of probiotics enriched diets on Uronema marinum infected olive flounder

The effect of five probiotics, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus sakei, Bacillus subtilis, and Saccharomyces cerevisiae as individual and mixed enriched diet on the seasonal prevalence, activity and intensity of Uronema marinum infection in olive flounder Paralichthys olivaceus is reported. The growth performance, feed efficiency, blood biochemistry, survival rate, and non-specific immune response of U. marinum infected olive flounder on week 0, 1, 2, 4, 6, and 8 were quantified. The prevalence and infection intensity reached a peak from June to December and then it declined from December to March. The scuticocidal activity in the serum was significantly higher when fed with L. plantarum, L. acidophilus, and S. cerevisiae diets on weeks 2-8. All enriched diets significantly enhanced the weight gain significantly between week 6 and 8; the feed efficiency registered a significantly increase from week 4 to 8 when compared to infected fish fed with control diet. Infected fish fed with L. plantarum-supplemented diet had higher survival rate than with other enriched diets. The serum aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) levels significantly increased when fed with L. plantarum, L. acidophilus or S. cerevisiae-supplemented diet. Total protein (TP) and glucose (GLU) level significantly increased with any enriched diet from week 4 to 8. The superoxide anion production and serum lysozyme activity registered a significant increase when fed with L. plantarum, L. acidophilus, and S. cerevisiae-supplemented diet from week 4-8. The present study concludes that L. plantarum, L. acidophilus, and S. cerevisiae-supplemented diets act as immunostimulants enhancing the growth, feed efficiency, blood biochemistry, survival rate, and non-specific immune response in U. marinum infected olive flounder.     

Oral immunization of olive flounder (Paralichthys olivaceus) with recombinant live viral hemorrhagic septicemia virus (VHSV) induces protection against VHSV infection

A recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP) that has enhanced green fluorescent protein (EGFP) gene instead of NV gene was previously generated using reverse genetics technology. In this study, potential of the rVHSV-ΔNV-EGFP to be used as a live oral vaccine candidate was assessed. The presence of the recombinant virus in internal organs of orally administered olive flounder (Paralichthys olivaceus) was analyzed by semi-quantitative RT-PCR. Although the recombinant VHSV-specific band was detected only when the number of PCR cycle was increased to 35, the band was detected from internal organs, such as kidney, spleen, and liver of fish that were reared at either 15 °C or 20 °C till even 20 days, suggesting that a few orally administered rVHSV-ΔNV-EGFP might be transported to internal organs, and might keep weak replication ability in the organs. VHSV-neutralizing activity was induced by oral immunization of olive flounder with the NV gene knock-out recombinant VHSV not only in skin and intestinal mucus but also in serum, suggesting that mucosal and systemic adaptive immune responses were elicited by oral immunization. In challenge experiment, groups of fish immunized with 10?, 10?, and 2 × 10? PFU of rVHSV-ΔNV-EGFP/fish showed 25%, 50%, and 70% of relative percent survival (RPS), respectively. The RPSs were elevated to 60%, 75%, and 90% by a boost immunization in fish boost immunized with 10?, 10?, and 2 × 10? PFU of rVHSV-ΔNV-EGFP, respectively. The cumulative mortality of fish in the control groups was 100%. Conclusionly, the present results demonstrate that the NV gene knock-out recombinant VHSV administered orally to olive flounder can induce dose- and boosting-dependent VHSV-neutralizing antibody in mucus and serum, and can provide a high protection in olive flounder against a virulent VHSV challenge.     

No beneficial effects evident for Enterococcus faecium NCIMB 10415 in weaned pigs infected with Salmonella enterica serovar Typhimurium DT104

Salmonella enterica serovar Typhimurium DT 104 is the major pathogen for salmonellosis outbreaks in Europe. We tested if the probiotic bacterium Enterococcus faecium NCIMB 10415 can prevent or alleviate salmonellosis. Therefore, piglets of the German Landrace breed that were treated with E. faecium (n = 16) as a feed additive and untreated controls (n = 16) were challenged with S. Typhimurium 10 days after weaning. The presence of salmonellae in feces and selected organs, as well as the immune response, were investigated. Piglets treated with E. faecium gained less weight than control piglets (P = 0.05). The feeding of E. faecium had no effect on the fecal shedding of salmonellae and resulted in a higher abundance of the pathogen in tonsils of all challenged animals. The specific (anti-Salmonella IgG) and nonspecific (haptoglobin) humoral immune responses as well as the cellular immune response (T helper cells, cytotoxic T cells, regulatory T cells, γδ T cells, and B cells) in the lymph nodes, Peyer's patches of different segments of the intestine (jejunal and ileocecal), the ileal papilla, and in the blood were affected in the course of time after infection (P < 0.05) but not by the E. faecium treatment. These results led to the conclusion that E. faecium may not have beneficial effects on the performance of weaned piglets in the case of S. Typhimurium infection. Therefore, we suggest a critical discussion and reconsideration of E. faecium NCIMB 10415 administration as a probiotic for pigs.     

Effects of dietary β-1,3-glucan, chitosan or raffinose on the growth, innate immunity and resistance of koi (Cyprinus carpio koi)

This study was performed to determine the efficacy of three immunomodulators viz., β-1,3 glucan, chitosan and raffinose on the innate immune response of koi, Cyprinus carpio koi. Kois were divided into 4 groups and each group was fed with diets supplemented with or without immunostimulant for 56 days. Total leukocyte counts (WBC), the non-specific humoral (lysozyme, alternative complement pathway and superoxide dismutase) and cellular (phagocytic capacity and respiratory burst activity) responses were determined and compared with controls (no supplement) after 7, 14, 21 and 56 days of feeding. The results of 8 weeks feeding trial showed that β-1,3 glucan supplementation significantly enhanced koi growth, whereas other immunostimulants did not. Variation in the levels of responses was?evident among different supplements. Compared with chitosan or raffinose, β-1,3 glucan could maintain the immunity of kois at a higher level during the experimental period. However, continuously applying β-1,3 glucan, chitosan or raffinose into the diet caused immunity fatigue in koi. No significant change in alternative complement pathway (ACP) activity was observed for any of the three supplements over the four different periods. After feeding for 14 days, the total leukocyte count (WBC), respiratory burst activity and superoxide dismutase (SOD) activity of the kois fed with chitosan or raffinose continuously remained relatively unchanged, subsequently decreased on the 56th day, but SOD did not. Meanwhile, lysozyme activity was no longer significantly higher on the 7th day, and for phagocytic capacity on the?14th day. After 56 days, these three immunostimulants groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with Aeromonas veronii. These results indicated that dietary intake containing immunostimulants could enhance the immune responses of koi and improve its resistance to infection by A.veronii. Especially supplementation with β-1,3 glucan to the kois for 56 days showed considerable improvement in the growth, survival and immune response of the kois.     

CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

Unmethylated cytosine-phosphate-guanine (CpG) dinucleotides flanked by specific bases in bacterial DNA induce a favorable immune response by acting as danger signals to the host. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) also act like the unmethylated CpG oligonucleotides in bacterial DNA. In the present study, we investigated the effects of synthetic CpG-ODN on the protection of olive flounder (Paralichthys olivaceus) against infection by Philasterides dicentrarchi, a pathogen of scuticociliatosis, through two consecutive experiments (trial I and II). Fish were intraperitoneally (i.p.) injected with CpG-ODN 1668 or GpC-ODN 1720 at different doses (3 microg in trial I and 10 microg in trial II), and after one week the fish were i.p. challenged with P. dicentrarchi. In both trial I and II, fish injected with CpG-ODN 1668 showed significantly higher serum scuticocidal activity than fish injected with PBS alone, while the scuticocidal activity disappeared by heat-inactivation. This result suggests that CpG-ODN might activate an alternative pathway of complement of olive flounder, and complement-mediated killing might be an important innate immune factor in the resistance against P. dicentrarchi infection. Although the cumulative mortality was largely different between trials I and II, the relative survival rate of fish injected with a high dose of CpG-ODN 1668 was considerably higher than that of fish injected with a low dose of this ODN, while the relative survival rate was not different between fish injected with the high dose and low dose of GpC-ODN 1720. The results of the present study suggest that CpG-ODNs may be used as potential immunostimulants to lessen cultured fish loss caused by scuticociliates.     

Effect of dietary supplementation with Suaeda maritima on blood physiology, innate immune response, and disease resistance in olive flounder against Miamiensis avidus

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder.     

Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus)

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure. The tissue expression analysis of PoPLD showed that the mRNA of PoPLD was predominantly expressed in the brain, gullet, muscle, stomach, head kidney, pyloric caeca, intestine and gill. The expression of the PoPLD gene was examined in various tissues of flounder by RT-PCR following stimulation with LPS and compared also with that of the inflammatory cytokines IL-1beta and IL-8 in various tissues of the stimulated flounder. This provides indirect evidence that PLD1 might have a relevant role in immune responses against pathogens and in inflammation. In addition, the recombinant protein of PoPLD (GFP-PoPLD), which demonstrated a phosphatidylcholine (PC)-hydrolyzing activity, was partially localized as a distinct ring-shaped form surrounding the rim of the nucleus in EPC cells. Together, our results suggest that PoPLD is similar to the mammalian PLD1 isoform, is generally widespread within olive flounder tissue, might have a relevant role in the fish immune system against pathogens and specifically may be localized in the subcellular membranes of the nuclear rim in EPC cells.     

Effect of the probiotic Enterococcus faecium NCIMB10415 on cell numbers of total Enterococcus spp., E. faecium and E. faecalis in the intestine of piglets

Sows and their piglets were fed a diet supplemented with or without the probiotic E. faecium NCIMB10415 (also known as SF68). Piglets were sacrificed 14, 28, 35 and 56 days after birth and DNA from intestinal segments was extracted and purified. A real time PCR assay was used to distinguish Enterococcus spp. (16s rDNA based), E. faecium (Efaafm gene), E. faecalis (Efaafs gene) as well as the probiotic strain (unique plasmid sequence). Extracts of autoclaved sow feces inoculated with E. faecium and E. faecalis cultures were used to calibrate real time PCR results. The probiotic strain was detected in 14 day old suckling piglets before the piglets had access to the starter diet. In piglets of the probiotic group, probiotic E. faecium cell counts were always a significant proportion of total E. faecium cells in stomach digesta (4-20%), however only a small fraction of the total Enterococcus spp. cell number on day 14 and 28 in all intestinal segments (0.1-0.7%). Compared to control samples, the probiotic E. faecium strain significantly (p < or = 0.05) decreased the amount of total Enterococcus spp. and E. faecalis cells in the colon of 14 day old suckling piglets as well as in jejunum and colon samples one week after weaning. E. faecium cell counts were not modified on any sampling day or intestinal segment. This study showed that the presence of probiotic E. faecium NCIMB10415 coincided with reduced total E. faecalis, but not total E. faecium cell numbers in the intestine of piglets. In view of unchanged cell numbers and ratios in sow feces, modifications must have taken place within the intestine of suckling piglets.     

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.     

Effects of Enterococcus faecium and dried whey on broiler performance, gut histomorphology and intestinal microbiota

The experiment was conducted to study the effects of supplementing a broiler starter diet with the probiotic Enterococcus faecium NCIMB 10415 and dried whey (80% lactose) on chick performance, gut histomorphology and intestinal microbiota. One-day-old male Ross 308 strain broiler chickens were fed diets containing: (i) control feed, (ii) control + 3.5% dried whey, (iii) control + 0.2% E. faecium, and (iv) control + 3.5% dried whey + 0.2% E. faecium. Birds were maintained in battery brooders confined in an environmentally controlled experimental room. The experiment lasted for 21 days. Birds fed E. faecium or E. faecium + dried whey exhibited significantly improved weight gain and feed conversion rate (FCR). Weight gain and FCR of treatment groups 1-4 were 628.7, 657.8, 690.9, 689.3 and 1.218, 1.193, 1.107, 1.116, respectively. Lactic acid bacteria counts in both the ileal content and excreta were significantly affected by dietary treatment. Supplementation of the E. faecium and dried whey separately and in combination increased lactic acid bacteria colonization in the ileal content from 4.2 to 5.0, 7.8 and to 5.1 log cfu/g, respectively (treatments 1-4). Similarly, supplementation of dried whey and E. faecium separately and in combination increased lactic acid bacteria in the excreta from 5.3 to 5.5, 8.0 and to 7.2 log cfu/g, respectively. Addition of the probiotic E. faecium increased villus height in the ileum (p < 0.05). Thus, supplementation of E. faecium enhanced broiler chick performance with respect to weight gain and FCR. No additive effect of E. faecium and dried whey was detected. Further studies are needed to investigate the relationship between E. faecium and dried whey with respect to gut histomorphology.