The effect of enterocin A/P dipeptide on growth performance, glutathione-peroxidase activity, IgA secretion and jejunal morphology in rabbits after experimental methicillin-resistant Staphylococcus epidermidis P3Tr2a Infection.

The increasing frequency of methicillin-resistant (MR) staphylococci in humans and animals need special attention for their difficult treatment and zoonotic character, therefore novel antimicrobial compounds on a natural base against antibiotic-resistant bacteria are requested. Currently, bacteriocins/enterocins present a new promising way to overcome this problem, both in prevention and treatment. Therefore, the preventive and medicinal effect of dipeptide enterocin EntA/P was evaluated against MR Staphylococcus epidermidis SEP3/Tr2a strain in a rabbit model, testing their influence on growth performance, glutathione-peroxidase (GPx) enzyme activity, phagocytic activity (PA), secretory (s)IgA, and jejunal morphometry (JM). Eighty-eight rabbits (aged 35 days, meat line M91, both sexes) were divided into experimental groups S (SEP3/Tr2a strain; 1.0 × 105 CFU/mL; dose 500µL/animal/day for 7 days, between days 14 and 21 to simulate the pathogen attack), E (EntA/P; 50 µL/animal/day, 25,600 AU/mL in two intervals, for preventive effect between days 0 and 14; for medicinal effect between days 28 and 42), E + S (EntA/P + SEP3/Tr2a; preventive effect; SEP3/Tr2a + EntA/P; medicinal effect) and control group (C; without additives). Higher body weight was recorded in all experimental groups (p < 0.001) compared to control data. The negative influence/attack of the SEP3Tra2 strain on the intestinal immunity and environment was reflected as decreased GPx activity, worse JM parameters and higher sIgA concentration in infected rabbits. These results suggest the promising preventive use of EntA/P to improve the immunity and growth of rabbits, as well as its therapeutic potential and protective role against staphylococcal infections in rabbit breeding.

Development and evaluation of a fish feed mixture containing the probiotic Lactiplantibacillus plantarum prepared using an innovative pellet coating method.

Introduction: Due to the intensification of fish farming and the associated spread of antimicrobial resistance among animals and humans, it is necessary to discover new alternatives in the therapy and prophylaxis of diseases. Probiotics appear to be promising candidates because of their ability to stimulate immune responses and suppress the growth of pathogens. Methods: The aim of this study was to prepare fish feed mixtures with various compositions and, based on their physical characteristics (sphericity, flow rate, density, hardness, friability, and loss on drying), choose the most suitable one for coating with the selected probiotic strain Lactobacillus plantarum R2 Biocenol™ CCM 8674 (new nom. Lactiplantibacillus plantarum). The probiotic strain was examined through sequence analysis for the presence of plantaricin- related genes. An invented coating technology based on a dry coating with colloidal silica followed by starch hydrogel containing L. plantarum was applied to pellets and tested for the viability of probiotics during an 11-month period at different temperatures (4°C and 22°C). The release kinetics of probiotics in artificial gastric juice and in water (pH = 2 and pH = 7) were also determined. Chemical and nutritional analyses were conducted for comparison of the quality of the control and coated pellets. Results and discussion: The results showed a gradual and sufficient release of probiotics for a 24-hour period, from 104 CFU at 10 mi up to 106 at the end of measurement in both environments. The number of living probiotic bacteria was stable during the whole storage period at 4°C (108), and no significant decrease in living probiotic bacteria was observed. Sanger sequencing revealed the presence of plantaricin A and plantaricin EF. Chemical analysis revealed an increase in multiple nutrients compared to the uncoated cores. These findings disclose that the invented coating method with a selected probiotic strain improved nutrient composition and did not worsen any of the physical characteristics of pellets. Applied probiotics are also gradually released into the environment and have a high survival rate when stored at 4°C for a long period of time. The outputs of this study confirm the potential of prepared and tested probiotic fish mixtures for future use in in vivo experiments and in fish farms for the prevention of infectious diseases.

Feeding-Regime-Dependent Intestinal Response of Rainbow Trout after Administration of a Novel Probiotic Feed.

Intensive fish farming is associated with a high level of stress, causing immunosuppression. Immunomodulators of natural origin, such as probiotics or phytoadditives, represent a promising alternative for increasing the immune function of fish. In this study, we tested the autochthonous trout probiotic strain L. plantarum R2 in a newly developed, low-cost application form ensuring the rapid revitalization of bacteria. We tested continuous and cyclic feeding regimes with regard to their effect on the intestinal immune response and microbiota of rainbow trout. We found that during the continuous application of probiotic feed, the immune system adapts to the immunomodulator and there is no substantial stimulation of the intestinal immune response. During the cyclic treatment, after a 3-week break in probiotic feeding and the reintroduction of probiotics, there was a significant stimulation of the gene expression of molecules associated with both cellular and humoral immunity (CD8, TGF-ß, IL8, TLR9), without affecting the gene expression for IL1 and TNF-α. We can conclude that, in aquaculture, this probiotic feed can be used with a continuous application, which does not cause excessive immunostimulation, or with a cyclic application, which provides the opportunity to stimulate the immunity of trout, for example, in periods of stress.

Effects of the Probiotic Enterococcus faecium on Muscle Characteristics of Chickens.

The use of antibiotics in farm animals is one of the main reasons for the development of resistant bacterial strains (e.g., zoonotic pathogens). Therefore, save alternatives are needed. Here, we examined how post-hatch application (day one to seven of life) of the probiotic Enterococcus faecium AL41 (EF) affects the development and tissue properties of the broiler pectoralis major muscle (PM). Expression of regulators, namely IGF-1, PAX7, and MYF5, was also investigated. At day 1 (n = 6), and days 5, 8, and 12 (n = 10), muscle samples were taken from control and EF supplemented chicks. From day 5 on, myonuclei number per fiber was elevated in EF chicks. Improved capillarization (from day 8), larger myofibers, increased body and PM weights (day 12) were found in the EF group. Part of our findings is explainable by higher intramuscular expression of IGF-1 and lower MYF5 expression in EF birds. In both groups IGF-1 expression decreases with age, thereby increasing the cellular myogenic potential. However, a strong increase in PAX7 expression and more PAX7-positive nuclei were found in EF chicks at day 12. We conclude that EF supplementation improves PM growth and health due to positive effects on bioavailability and fusion capacity of SATC progeny and better tissue perfusion.

The Effect of Enterococcus faecium AL41 on the Acute Phase Proteins and Selected Mucosal Immune Molecules in Broiler Chickens.

Probiotic bacteria, including the Enterococcus faecium strain, can improve intestinal mucosal health by several mechanisms, including modulation of the immune response, as well as by improving the protective function of the epithelial barrier. In this study, we tested the effect of Enterococcus faecium AL41 on the acute phase proteins response (blood), gene expression of selected molecules of mucosal immunity (immunoglobulin A, mucin-2, insulin-like growth factor 2) and mucus production (all parts of the small intestine) in broilers. Eighty broiler chicks were divided into two groups: a control and E. faecium AL41 (birds were inoculated with AL41 for 7 days) group. The whole experiment lasted 11 days. Our results revealed that the administration of E. faecium AL41 had no substantial effect on the concentrations of acute phase proteins, but we recorded a significant increase in ß- and γ-globulin fractions at the end of the experiment, which may indicate an improvement in the immune status. A significant prolonged stimulatory effect of E. faecium AL41 on the relative expression of molecules (immunoglobulin A, mucin-2) as well as on the dynamic of mucus production in the chicken intestine was observed. In addition, AL41 significantly reduced the total number of enterococci in the cecum and faeces.

Thymol in fattening rabbit diet, its bioavailability and effects on intestinal morphology, microbiota from caecal content and immunity.

To our knowledge, no study has been carried out to evaluate the effects of thymol sustained administration on gut health and immune response in terms of its bioavailability in the rabbit organism. A total of 48 rabbits were randomly divided at 35 days of age into two dietary treatment groups (C/control or T/thymol at 0.025% addition). Thymol was added for 21 days and then withdrawn for 7 days. Thymol content in faeces (p < 0.001) and caecal content (p < 0.05) was significantly higher than in plasma during the thymol addition and withdrawal (2442 ± 451.4, 881.9 ± 231.3 ng/g DM and 46.2 ± 28.4 ng/ml, respectively; 149.5 ± 40.54, 45.76 ± 12.44 ng/g DM and 2.73 ± 0.45 ng/ml, respectively). Increasing of villi height to crypt depth ratio in small intestinal wall (p < 0.01), phagocytic activity in blood (p < 0.0001) and lactic acid bacteria in caecal appendix (p < 0.01) and faeces (p < 0.05) was still presented after withdrawn of thymol. The thymol at this concentration demonstrated its biological properties and was able to positively affect gut health and immune response of rabbits.

Effect of enterocin M and durancin ED26E/7 supplementation on blood parameters, immune response and jejunal morphometry in rabbits.

Natural feed additives application in rabbit nutrition can help to control and prevent digestive disturbances and improve gut health and immunity around the critical weaning period. While probiotics are frequently used in rabbits, in vivo administration of bacteriocins is often limited. Therefore, the present study evaluates the effect of enterocin EntM, durancin EntED26E/7 and their combination on serum biochemistry, phagocytic activity and jejunal morphometry of rabbits. Eighty rabbits (aged 35 days, meat line M91, both sexes) were divided into experimental groups E (EntM; dose 50 µl/animal/day, activity 25,600 AU/ml), D (EntED26E/7; 50 µl/animal/day, 12,800 AU/ml), E + D (50 µl EntM + 50 µl EntED26E/7 /animal/day) and control group (C). Additives were administrated in drinking water for 21 days. Both enterocins positively influenced tested serum parameters, with emphasis on durancin EntED26E/7 administration, alone and/or in combination with EntM. Increased total proteins (E, D: p < 0.001), urea (D: p < 0.001), albumin (D: p < 0.05) and triglycerids (E, D, E+D: p < 0.001) were found. Hypocholesterolaemic effect of both additives was recorded (p < 0.001), with the lowest HDL concentration in E + D. The most of tested hepatic enzymes were positively influenced by enterocins combination (E + D; p < 0.001). The lowest AST was noted in group D (p < 0.001). Mineral profile was also improved (p < 0.001), with the highest values in D. Oxidative stress, was not evoked during enterocins application. Both additives showed a tendency to improve phagocytic activity (prolonged effect of EntED26D/7; D, E+D: p < 0.05) and jejunal morphometry parameters (increased villus cut surface; E, D, E+D; p < 0.001). Diet supplementation with EntM and mostly with EntED26E/7 can improve serum biochemistry, phagocytic activity and jejunal morphometry.

Production of Intestinal Mucins, sIgA, and Metallothionein after Administration of Zinc and Infection of Ascaridia galli in Chickens: Preliminary Data.

The effect of inorganic zinc and Ascaridia galli infection was studied on MUC1, MUC2 (mucin), sIgA (secretory immunoglobulin A), and metallothionein in the intestines of broilers. Thirty-five-day-old chickens (n = 24), COBB 500 breed, were included in a 14-day experiment. Chickens were divided into 4 groups of 6 chickens each: control ©, Ascaridia galli (AG), Zinc group (Zn), and combined group (AG + Zn). Samples from the intestine for determination of MUC1, MUC2, sIgA, and metallothionein were taken at 7 and 14 days during necropsy. Samples from the jejunum for determination of MUC1, MUC2, sIgA, and metallothionein were taken at 7 and 14 days during necropsy. The results demonstrated that 12 days' administration of inorganic zinc increased production of MUC1 (p < 0.0001) and MUC2 (p < 0.001) in the Ascaridia galli-infected group (Ag + Zn) in comparison to control (C). The beneficial effect of zinc was also revealed in the production of sIgA (p < 0.0001) in the combined group (AG + Zn) at 7 days. The concentration of metallothionein increased mainly in the zinc group (p < 0.01) of first sampling and was upregulated in Zn and AG + Zn groups. The obtained data indicate the use of inorganic zinc as a suitable immunomodulator of intestinal immunity in Ascaridia galli-infected chickens.

Can Enterocin M in Combination with Sage Extract Have Beneficial Effect on Microbiota, Blood Biochemistry, Phagocytic Activity and Jejunal Morphometry in Broiler Rabbits?

The present study investigated the effects of enterocin (Ent) M and sage extract alone and also in combination on the gut microflora, phagocytic activity, blood biochemistry, and morphometry of rabbits. Sixty-four rabbits (aged five weeks, M91 meatline, both sexes) were divided into three experimental groups: E (EntM; 50 µL/animal/day), S (sage; 10 µL/animal/day), and E + S (EntM + sage) groups and control group (C). The additives were administered in drinking water for a period of 21 days. Dietary supplementation of EntM and sage significantly reduced the coliforms (E: p < 0.001; S: p < 0.001; E + S: p < 0.001) in feces, while simultaneous addition of EntM and sage decreased enterococci (E + S: p < 0.0001), lactic acid bacteria (E + S: p < 0.01), and coagulase-positive staphylococci (E + S: p < 0.0001) in the appendix. Sage addition reduced HDL (S: p < 0.001) and LDL cholesterol (S: p < 0.001; E + S: p < 0.001), LDL/HDL ratio (S: p < 0.001; E + S: p < 0.01), and increased urea (S: p < 0.01; E + S: p < 0.001) and creatinine (S: p < 0.001; E + S: p < 0.001) in serum. EntM and sage application, alone or in combination, improve the jejunal morphometry (p < 0.0001) in rabbits.

Enterococci Isolated from Trout in the Bukovec Water Reservoir and Cierny Váh River in Slovakia and Their Safety Aspect.

The aim of this study was to investigate enterococci as lactic acid bacteria and as part of Firmicutes phylum. We focused on the virulence factor, biofilm formation, and antibiotic resistance and also on lactic acid production and enterocin gene detection. Intestinal samples were taken from 50 healthy trout (3 Salmo trutta and 47 Salmo gairdneri) collected in April 2007, 2010, and 2015 from different locations at the Bukovec water reservoir and the Cierny Váh River in Slovakia. Twenty pure colonies were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification system based on protein fingerprints, and then seven identified strains were also phenotyped. Based on the identification methods used, the identified enterococci (7) belong taxonomically to four different enterococcal species: Enterococcus durans, E. faecium, E. mundtii, and E. thailandicus. They were hemolysis, DNase, and gelatinase negative with acceptable enzymatic activity. They did not form biofilm and were mostly susceptible to antibiotics. All strains produced lactic acid amounting to 1.78 ± 0.33 mmol/l on average and possessed the gene for enterocin A production. This is the first study reporting more detailed properties of enterococci from trout in Slovakian wild water sources, and it produces new possibilities for studying microbiota in trout.

Resistance and tolerance to mixed nematode infections in chicken genotypes with extremely different growth rates.

Fast growing broilers are less able to cope with fitness related challenges. As the allocation of metabolic resources may be traded off between performance and defence functions in parasitized hosts, we hypothesized that fast growing broilers are more sensitive to mixed nematode infections compared with slower growing genotypes under the same environmental conditions. Therefore, we compared male birds of genotypes selected for either meat production (Ross-308, R) or egg production (Lohmann Brown Plus, LB) or for both purposes (Lohmann Dual, LD), to assess their resistance and tolerance to mixed nematode infections with Ascaridia galli and Heterakis gallinarum. While infections reduced feed intake in all three genotypes, feed conversion efficiency was not affected. Infections impaired growth performance only in R birds, indicating lower tolerance in the fast growing genotype compared with slower growing LB and LD genotypes. Impaired tolerance in R birds was associated with a relative nutrient scarcity due to an infection-induced lower feed intake. Resistance to experimentally induced infections depended on host genotype as well as on the worm species involved. Overall, the A. galli burden was higher in R than LB, whereas the burden of LD was not different from that of R and LB. In contrast, the H. gallinarum burden of first generation worms was similar in the three genotypes. Susceptibility to re-infection with H. gallinarum was higher in LB than in LD, whereas very low levels of re-infection were observed in R birds. Our data collectively suggest that resistance and tolerance to mixed nematode infections are sensitive to growth rate in chickens. These differences amongst genotypes may partly be associated with a mismatch between the actual nutrient supply and genotype-specific nutrient requirements.

The Influence of Feed-Supplementation with Probiotic Strain Lactobacillus reuteri CCM 8617 and Alginite on Intestinal Microenvironment of SPF Mice Infected with Salmonella Typhimurium CCM 7205.

Alginite is a non-ore raw material arising by fossilization of accumulated organic (algae) and inorganic material, particularly clay, carbonates, quartz, and amorphous modification of silicic acid in the aqueous environment. Humic acids as a component of organic portion of alginite are known for very good buffering ability which allows them to stabilise pH throughout the digestion system of animals, stimulate receptors of the immune system in intestinal villi against pathogenic bacteria, and support proliferation and activity of beneficial bacteria (lactobacilli, bifidobacteria, and similar). Our investigations focused on the influence of a probiotic strain in combination with alginite on intestinal microenvironment of SPF mice infected with Salmonella Typhimurium. The 66 female mice (BALB/c) used in our study were divided to four experimental groups, control NC1, control NC2 (alginite), IC (alginite + Salmonella Typhimurium CCM 7205NAL), LAB (Lact. reuteri CCM 8617 + alginite + Salm. Typhimurium CCM 7205NAL). The group supplemented with Lact.reuteri CCM 8617 and alginite showed significant reduction in growth of Salm. Typhimurium in mice faeces at 24 and 72 h (P < 0.001) post infection. The supplementation of additives affected positively also nitrogen, enzymatic, hepatic and energy metabolism of mice. The demonstrable positive influence of additives alleviated the negative impact of Salm. Typhimurium infection on the morphology investigated in the jejunum and ileum of LAB group of mice. The livers of mice treated with both alginite and Lact.reuteri CCM 8617 showed marked reduction of overall inflammation, hepatocyte necrosis and size of typhoid nodules.

Relation to enterocins of variable Aeromonas species isolated from trouts of Slovakian aquatic sources and detected by MALDI-TOF mass spectrometry.

Aeromonads represent bacteria thought to be primarily mostly autochthonous to aquatic environments. This study was focused on the relation with antibiotics and enterocins of identified Aeromonas species isolated from the intestine of trouts living in Slovakian aquatic sources. Intestinal samples from 50 trouts (3 Salmo trutta and 47 Salmo gairdnerii) were collected in April of years 2007, 2010, and 2015 from trouts of different water sources in Slovakia (pond Bukovec near Kosice, river Cierny Váh). Due to the MALDI-TOF mass spectrometry evaluation, 25 strains were proposed to the genus Aeromonas involving nine different species (Aeromonas bestiarum-nine strains, Aer. salmonicida-four strains, Aer. encheleia, Aer. eucrenophila, Aer. molluscorum, Aer. media, Aer. sobria, Aer. popoffii, Aer. veronii). Phenotypic evaluation of individual strains confirmed their species identification. Twenty-five strains of different Aeromonas species were sensitive to azithromycin, amikacin, mecillinam, mezlocillin, piperacillin, gentamicin, chloramphenicol, and tetracycline. On the other side, they were resistant to carbenicillin and ticarcillin. The growth of Aer. bestiarum R41/1 was inhibited by treatment with Ent M and Ent 2019 (inhibition activity 100 AU/mL). Aer. bestiarum R47/3 was inhibited by eight enterocins (100 AU/mL). It is the first study testing enterocins to inhibit the growth of Aeromonas species from trouts.

Ontogenic Changes of Villus Growth, Lactase Activity, and Intestinal Glucose Transporters in Preterm and Term Born Calves with or without Prolonged Colostrum Feeding.

Oral glucose supply is important for neonatal calves to stabilize postnatal plasma glucose concentration. The objective of this study was to investigate ontogenic development of small intestinal growth, lactase activity, and glucose transporter in calves (n = 7 per group) that were born either preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery) or spontaneously born and fed colostrum for 4 days (TC). Tissue samples from duodenum and proximal, mid, and distal jejunum were taken to measure villus size and crypt depth, protein concentration of mucosa and brush border membrane vesicles (BBMV), total DNA and RNA concentration of mucosa, mRNA expression and activity of lactase, and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter 2 (GLUT2) in mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and immunochemical localization of GLUT2 in the enterocytes were determined. Villus height in distal jejunum was lower in TC than in T. Crypt depth in all segments was largest and the villus height/crypt depth ratio in jejunum was smallest in TC calves. Concentration of RNA was highest in duodenal mucosa of TC calves, but neither lactase mRNA and activity nor SGLT1 and GLUT2 mRNA and protein expression differed among groups. Localization of GLUT2 in the apical membrane was greater, whereas in the basolateral membrane was lower in TC than in T and PT calves. Our study indicates maturation processes after birth for mucosal growth and trafficking of GLUT2 from the basolateral to the apical membrane. Minor differences of mucosal growth, lactase activity, and intestinal glucose transporters were seen between PT and T calves, pointing at the importance of postnatal maturation and feeding for mucosal growth and GLUT2 trafficking.

Diet effects on glucose absorption in the small intestine of neonatal calves: importance of intestinal mucosal growth, lactase activity, and glucose transporters.

Colostrum (C) feeding in neonatal calves improves glucose status and stimulates intestinal absorptive capacity, leading to greater glucose absorption when compared with milk-based formula feeding. In this study, diet effects on gut growth, lactase activity, and glucose transporters were investigated in several gut segments of the small intestine. Fourteen male German Holstein calves received either C of milkings 1, 3, and 5 (d 1, 2, and 3 in milk) or respective formulas (F) twice daily from d 1 to d 3 after birth. Nutrient content, and especially lactose content, of C and respective F were the same. On d 4, calves were fed C of milking 5 or respective F and calves were slaughtered 2h after feeding. Tissue samples from duodenum and proximal, mid-, and distal jejunum were taken to measure villus size and crypt depth, mucosa and brush border membrane vesicles (BBMV) were taken to determine protein content, and mRNA expression and activity of lactase and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter (GLUT2) were determined from mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and BBMV were determined, as well as immunochemically localized GLUT2 in the intestinal mucosa. Villus circumference, area, and height were greater, whereas crypt depth was smaller in C than in F. Lactase activity tended to be greater in C than in F. Protein expression of SGLT1 was greater in F than in C. Parameters of villus size, lactase activity, SGLT1 protein expression, as well as apical and basolateral GLUT2 localization in the enterocytes differed among gut segments. In conclusion, C feeding, when compared with F feeding, enhances glucose absorption in neonatal calves primarily by stimulating mucosal growth and increasing absorptive capacity in the small intestine, but not by stimulating abundance of intestinal glucose transporters.

Beneficial effect of lantibiotic nisin in rabbit husbandry.

Nisin is a bacteriocin marketed as Nisaplin. The aim of our work was to test its in vivo effect in a rabbit model; its effect on phagocytic activity (PA) and morphometry has not so far been studied. Post-weaning rabbits (48), 5 weeks old (both sexes, Hycole breed), were divided into the experimental (E) and the control groups (C), 24 animals in each. They were fed a commercial diet with access to water ad libitum. Rabbits in E had nisin additionally administered to their drinking water (500 IU-20 µg per animal/day) for 28 days. The experiment lasted 42 days. On day 28, significant decrease in coagulase-positive (CoPS) staphylococci and coliforms was noted (p < 0.01) in faeces of group E compared with C. Pseudomonads and clostridiae were also significantly reduced (p < 0.001; p < 0.05) and slight decrease was also in CoNS and enterococci. On day 42, coliforms were still significantly reduced (p < 0.001) in faeces; slight decrease in CoPS and pseudomonads was noted. In the caecum, significant decrease in pseudomonads (p < 0.05) was noted on day 28; slight decrease in coliforms. In the appendix slight decrease in coliforms, pseudomonads was obtained on both days. PA was increased significantly in E on days 28, 42 (p < 0.001). Biochemical parameters were not influenced; nor were volatile fatty acids or lactic acid in the chymus. Nisin application did not evoke oxidative stress. In group E, an increase in average body weight gain (about 9.4 %) was noted. The villus height/crypt depth ratio was not influenced; that is, resorption surface and functionality of mucosa were not influenced.

Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen epithelium to high-energy diet.

Ruminal vacuolar H(+)-ATPase (vH(+)-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH(+)-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH(+)-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH(+)-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger to intracellular pH (pH(i)) regulation was investigated. REC isolated from h/c-fed sheep keep their pH(i) at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pH(i) by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP](i) were reduced after foliomycin-induced vH(+)-ATPase inhibition. Our results provide the first evidence for a role of vH(+)-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pH(i) changes to signaling pathways regulating this process.

The vacuolar-type H-ATPase in ovine rumen epithelium is regulated by metabolic signals.

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H(+)-ATPase (vH(+)-ATPase) activity was investigated. Using fluorescent spectroscopy, basal pH(i) of REC was measured to be 7.3 +/- 0.1 in HCO(3) (-)-free, glucose-containing NaCl medium. MI induced a strong pH(i) reduction (-0.44 +/- 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A (-0.30 +/- 0.03 versus -0.21 +/- 0.03 pH units). Treatment with foliomycin, a specific vH(+)-ATPase inhibitor, decreased REC pH(i) by 0.21 +/- 0.05 pH units. After MI induction, this effect was nearly abolished (-0.03 +/- 0.02 pH units). In addition, membrane-associated localization of vH(+)-ATPase B subunit disappeared. Metabolic control of vH(+)-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability.

Molecular identification, immunolocalization, and functional activity of a vacuolar-type H(+)-ATPase in bovine rumen epithelium.

In this study, we have studied the expression, localization, and functionality of vacuolar-type H(+)-ATPase (vH(+)-ATPase) and Na(+)/K(+)-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pH(i)) of control rumen epithelial cells (REC; 7.06 +/- 0.07), application of inhibitors selective for vH(+)-ATPase (foliomycin) and Na(+)/K(+)-ATPase (ouabain) reduced pH(i) by 0.10 +/- 0.03 and 0.18 +/- 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results from qRT-PCR and immunoblotting clearly confirm the expression of vH(+)-ATPase B subunit in REC. However, the amount of Na(+)/K(+)-ATPase mRNA and protein is tenfold and 11-fold of those of vH(+)-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na(+)/K(+)-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum, with the highest abundance in basal cells. In contrast, the vH(+)-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum of the epithelium. Furthermore, vH(+)-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H(+) pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes.