Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Why does the establishment of the starch preferring<Emphasis Type="Italic">Entodinium caudatum</Emphasis> in the rumen decrease the numbers of the fibrolytic ciliate<Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>?

The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.     

Ability of rumen protozoa <Emphasis Type="Italic">Diploplastron affine</Emphasis> to utilize β-glucans

The ability of the rumen ciliates to utilize β-glucans other than cellulose and xylan is currently being recognized. The objective of the present study was to characterize the ability of the ciliate Diploplastron affine to digest some pachyman, laminarin, pustulan, curdlan and lichean. The protozoa were isolated from the rumen of sheep and either grown in vitro or inoculated into the rumen of ciliate-free sheep and maintained in natural conditions. In vitro culture studies showed that the enrichment of culture medium with the examined saccharides results in an increase in the number of ciliates in comparison to the control cultures. The increase was over 36 and 15 % when the growth medium was supplemented with pachyman (1,3-β-glucan) and pustulan (1,6-β-glucan), respectively. A positive correlation was also found between the population density of ciliates and the dose of saccharide supplemented to the growth medium. Enzyme studies were performed using the crude enzyme preparation obtained from ciliates treated with antibiotics. The ability of ciliates to digest the examined β-glucans was tested by the quantification of reducing sugars released from the mentioned substrates during the incubation with crude enzyme preparation. The results showed that D. affine ciliates were able to digest both of them. The mean degradation rate varied between 6.7 and 28.2 μmol/L glucose per mg protein per h for pustulan and lichean, respectively, whereas the digestion velocity was the highest at 5.0–5.5 pH and 45–50°C.     

Chitinolytic enzymes of the rumen ciliate <Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>

The ability of rumen ciliates to digest chitin is clearly recognized. We investigated the chitinolytic system of the rumen ciliate Eudiplodinium maggii. The ciliates were grown in a selectively faunated sheep. They were isolated from the rumen and purified by sedimentation. A crude enzyme preparation was prepared following incubation of ciliates with antibiotics. This was done in order to reduce their contamination with intracellular bacteria. The activity of particular enzymes was examined by quantification of the products released from specific substrates. It was stated that the optimum conditions for the detected activities varied between 4.5 and 5.5 pH, and 45 and 55 °C. β-N-Acetylglucosaminidase was found as an enzyme of the highest activity (4.2 μmol/l released product per mg protein per h). The activities of endochitinase and exochitinase were almost two times lower than that of β-N-acetylglucosaminidase. Zymographic studies revealed the presence of two endochitinases, two exochitinases and two β-N-acetylglucosaminidases in the examined preparation.     

The ability of rumen ciliates, Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum, to use the murein saccharides

Murein polysaccharides may contribute to a considerable part of the dry matter of bacterial cells. Their utilization by protozoa inhabiting the rumen is, however, poorly recognized. The objective of this study was to examine the ability of three species of ciliates, i.e., Eudiplodinium maggii, Diploplastron affine, and Entodinium caudatum of digest, and ferment these saccharides. The cultivation experiments showed that the enrichment of growth medium with bacterial cell wall β-glycans increased the ciliate number (p?<?0.05). A statistically significant increase (p?<?0.01) was followed by a continuous decrease (p?<?0.01) in the percentage of individuals containing β-glycans particles after 4- and 24-h incubation of ciliates with this substrate, respectively. The enzymatic experiments confirmed the ability of the examined protozoa to digest murein. E. caudatum exhibited the highest activity (8.2 unit (U)/mg protein per min), and E. maggii, the lowest (3.0 U/mg protein per min). The production rates of volatile fatty acids by starved and fed ciliate species were 0.7 and 1.6 (E. caudatum)?pmol/ciliate cell per h, 30.5 and 42.5 (E. maggii)?pmol/ciliate cell per h, and 8.3 and 19.2 (D. affine)?pmol/ciliate cell per h (p?<?0.05).     

Enterocin M and its Beneficial Effects in Horses—a Pilot Experiment

Probiotic bacteria or their antimicrobial proteinaceous substances called bacteriocins (enterocins) hold promising prophylactic potential for animal breeding. This study present the results achieved after application of Enterocin M in horses. Enterocin M has never been applied to horses before. Clinically healthy horses (10) were involved in this pilot experiment. They were placed in the stables of the University of Veterinary Medicine and Pharmacy, Ko?ice, Slovakia, with the approval of the University Ethics Committee. The animals were fed twice a day with hay and oats, or alternatively grazed with access to water ad libitum. The experiment lasted 6 weeks. Sampling was performed at the start of the experiment, at day 0–1, at day 21 (3 weeks of Enterocin M application), and at day 42 (3 weeks of cessation). Feces were sampled directly from the rectum and blood from the vena jugularis; the samples were immediately treated and/or stored for analyses. Each horse itself represented a control animal (compared to its status at the start of the experiment, day 0–1). After initial sampling, the horses were administered 100 μl of Ent M (precipitate, 12,800 AU/ml) in a small feed bolus to ensure it was consumed; Ent M was applied for 3 weeks (21 days). Fecal samples were treated using the standard microbial dilution method; phagocytic activity was assessed with standard and flow cytometry; biochemistry and metabolic profiles were tested using commercial kits and standard methods. Administration of Ent M led to mathematical reduction of coliforms, campylobacters (^abP?<?0.05), and significant reduction of Clostridium spp. (^abP?<?0.001, ^bcP?<?0.001); increase of PA values was noted (P?<?0.05, P?<?0.0001); no negative influence on hydrolytic enzyme profile or biochemical blood parameters was noted.     

Seasonal variations of the digestive tract of the Eurasian beaver <Emphasis Type="Italic">Castor fiber</Emphasis>

Forage availability for wild rodents varies with season. In turn, the composition of food can affect morphometric parameters of the digestive tract. This study was performed in Eurasian beavers (Castor fiber) whose population was close to extinction in most Eurasian countries, but has now increased. Due to the previous low number of studies, information about the effect of forage availability on the digestive tract morphology has previously been lacking. This study was performed using beavers captured from the natural environment during three seasons of different forage availability: winter, summer and autumn. It was found that the diet of the beaver varied during the year; in winter it was dominated by woody material consisting of willow shoots, whereas in summer the diet was primarily herbs, grass and leaves. Season also affected the mass of digested contents of the digestive tract. The digestive content increased in the caecum and colon in winter and autumn, when poor-quality food dominated the beaver’s diet. The results indicated that the digestive tract parameters of beavers varied based on the composition of available forage.     

Enterocin M and Sage Supplementation in Post-weaning Rabbits: Effects on Growth Performance,Caecal Microbiota,Fermentation and Enzymatic Activity

The effects of enterocin (Ent) M and sage extract applied separately and in combination were investigated. EntM (E 50 μL/animal/day in water) and sage extract (S 10 μL/animal/day in water) were applied individually and in combination (E+S) to rabbits during 21 days of treatment. The rabbits’ growth was not significantly influenced by the additives. Lower feed conversion (FC) was noted in the experimental groups compared with controls, with the lowest data detected in E. The antimicrobial activity of EntM was noted (in E+S: lactic acid bacteria—P < 0.01; in E, E+S: enterococci, enterobacteria—P > 0.05; in E: clostridia—P > 0.05). The most significant changes in fermentation between weaned and older rabbits were noted in amylolytic activity at day 21 (E P < 0.05; E + S P < 0.05); prolonged reduction effect of sage extract on amylolytic activity was observed. The activity of cellulase, pectinase and xylanase was higher in older than in younger animals. Decrease in lactic acid and volatile fatty acids was noted during EntM administration, with significant effect on propionic acid concentration (E P < 0.05; E+S P < 0.001). The sage extract reduced propionic acid (S P < 0.001) and butyric acid levels (S P < 0.05) and increased the concentrations of butyric, iso-valeric, valeric, caproic acids and lactic acid (P < 0.001). It seems to be that EntM and sage supplementation may improve the economy of rabbit farms (increased FC) and the health status of rabbits (reduction of spoilage microbiota, enhanced enzymatic activities in caecum).

     

Characterisation of Faecal Staphylococci from Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus) and Their Susceptibility to Gallidermin

Our current knowledge of microbiota in wild ruminants is limited. The goal of this study was to evaluate staphylococcal species in red and roe deer for various attributes (haemolysis, DNase, and urease activities; lactic acid and biofilm production; and antibiotic profile) and their susceptibility to gallidermin. Sixteen staphylococcal strains were identified from faeces of 21 free-living animals (9 adult female Cervus elaphus—red deer and 12 young female Capreolus capreolus—roe deer) sampled by the Polish colleagues in the Strzałowo Forest District, Piska Primaeval Forest. The variability in the species of staphylococci was determined. Seven species (Staphylococcus capitis, S. epidermidis, S. haemolyticus, S. hominis, S. pseudintermedius, S. vitulinus and S. warneri) and five clusters/groups of coagulase-negative staphylococci (CoNS) were identified. The strains were generally not haemolytic and Dnase negative; did not form biofilms or only produced low-grade biofilms; exhibited high levels of lactic acid; were urease positive; and were generally susceptible to antibiotics (only two strains were resistant to multiple antibiotics). However, all of the strains were susceptible to the lantibiotic bacteriocin gallidermin, with a minimal inhibitory concentration of 0.0156 μg (up to 6400 AU/ml in arbitrary units). This is the first study to perform a detailed study of the properties of CoNS from roe and red deer.