Renata Miltko

Treponema zioleckii sp. nov., a novel fructan-utilizing species of rumen treponemes

During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.

Why does the establishment of the starch preferring Entodinium caudatum in the rumen decrease the numbers of the fibrolytic ciliate Eudiplodinium maggii

The effect of the establishment ofEntodinium caudatum on the population ofEudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration ofE. maggii were 15.9–38.5 and 11.7–12.4 × 103 cells per g of the rumen contents in the absence and presence ofE. 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 ofE. caudatum. Up to 82–93 % of individuals contained starch grains whenE. 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 byE. 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 observedin vivo in spite of the fact that hay particles up to 42 µm 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 whenE. maggii ciliates were incubatedin 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 ofE. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.

Chitinolytic activity of the sheep rumen ciliate Diploplastron affine

Supplementation of the rumen ciliate Diploplastron affine growth medium with commercial chitin stimulated growth of ciliates and the density of their population was positively correlated with chitin doses (r = 0.95; p < 0.01). The cell-free extracts prepared from bacteria-free ciliates degraded chitin to N-acetyl-d-glucosamine and chitobiose. Three exochitinases, two endochitinases and two β-N-acetylglucosaminidases were identified in the cell-free extract of protozoa. The molar mass of exochitinases was 80, 65 and 30 kDa, and endochitinases 75 and 50 kDa; the molar mass of one of the identified β-N-acetylglucosaminidases was 45 kDa.

The ability of the rumen protozoan Eudiplodinium maggii to utilize chitin

The ability was determined of the rumen ciliate Eudiplodinium maggii to utilize chitin from fungal cell wall. Cultivation experiments shoved that the population concentration (number of ciliates in vitro) was positively correlated with chitin doses. Cell extract prepared from the bacteria-free ciliates degraded colloidal chitin releasing 2.0 μmol reducing sugar per mg protein per h. End products of this reaction were chitotriose and N-acetylglucosamine. Incubation of the bacteria-free ciliates with chitin resulted in an increase in the concentration of acetic, propionic and butyric acids in the incubation medium. The production rate of total volatile fatty acids (VFA) by ciliates incubated with and without chitin was 45.0 and 30.5 pmol VFA per protozoan, respectively, the molar proportion of particular acids remaining unchanged.

Endoparasites of the European beaver (Castor fiber L. 1758) in north-eastern Poland

Abstract Parasitological examination after necropsies of 48 European beavers from Podlaskie and Warmisko-Mazurskie provinces were performed between April 2011 and November 2012. All helminthes were isolated from the contents of the gastro-intestinal tract and their species were determined. In addition, blood samples and faeces were examined. All beavers were infected with six species of parasites. Stichorchis subtriqetrus trematodes were found in 93.7% of animals. They were localized mainly in the caecum, less in the colon, and single juvenile parasites were found in the small intestine. The intensity of infection ranged from two to 893 parasites. Travassosius rufus nematodes (10-4336 specimens) were present in the stomach of 68.7% of the beavers. In the small intestine of four (8.3%) beavers, two-six specimens of Psilotrema castoris were found. This is the first record of this species in Poland and the third of its discovery in the world. Furthermore, in the small intestine of one beaver, two Trichostrongylus capricola nematodes were detected. In the liver of one beaver, pathological changes caused by hydatid cestode Echinococus granulosus occurred. Inflammatory changes of the gastric mucosa caused by Travassosius rufus and of caecum caused by Stichorchis subtriquertus, were observed. Coproscopy was performed with the use of Baermann, flotation, and decantation methods. All results of Baermann method were negative. Examinations with flotation and decantation methods confirmed necropsy findings. Using the flotation method, single oocysts of Eimeria sprehni in one beaver were detected. A blood test conducted by Kingston and Morton method did not reveal the presence of protozoa or microfilariae.

Mureinolytic ability of the rumen ciliate Diploplastron affine

Rumen ciliate protozoa intensively engulf bacteria. However, their ability to utilize murein which is the main polysaccharide of bacterial cell wall has hardly been recognized. The present study concerns the ability of the rumen protozoa Diploplastron affine to digest and ferment murein. The ciliates were isolated from the rumen fluid and grown in vitro or inoculated into the rumen of defaunated sheep. The results of long-term cultivation of protozoa showed a positive correlation between their number and murein content in the culture medium. It was also found that bacteria-free D. affine ciliates incubated with or without murein produced volatile fatty acids at the rate of 12.3 and 8.7 pmol/h per protozoan, respectively, acetic, butyric and propionic acids being the three main acids released to the medium. Enzyme studies performed with the use of protozoan cell extract prepared from bacteria-free ciliates degraded murein at a rate of 25 U/mg protein per h; two mureinolytic enzymes were identified by zymographic technique in the examined preparation.

Effect of Entodinium caudatum on starch intake and glycogen formation by Eudiplodinium maggii in the rumen and reticulum

This study aimed to quantify the engulfed starch and reserve α-glucans (glycogen) in the cells of the ciliates Eudiplodinium maggii, as well the α-glucans in defaunated and selectively faunated sheep. The content of starch inside the cell of ciliates varied from 21 to 183mg/g protozoal DM relative to the rumen fauna composition whereas, the glycogen fluctuated between 17 and 126mg/g dry matter (DM) of this ciliate species. Establishment of the population Entodinium caudatum in the rumen of sheep already faunated with E. maggii caused a drop in both types of quantified carbohydrates. The content of α-glucans in the rumen of defaunated sheep varied from 4.4 to 19.9mg/g DM and increased to 7.4-29.9 or 11.8-33.9mg/g DM of rumen contents in the presence of only E. maggii or E. maggii and E. caudatum, respectively. The lowest content of the carbohydrates was always found just before feeding and the highest at 4h thereafter. The α-glucans in the reticulum varied 7.5-40.1, 14.3-76.8 or 21.9-106.1mg/g DM of reticulum content for defaunated, monofaunated or bifaunated sheep, respectively. The results indicated that both ciliate species engulf starch granules and convert the digestion products to the glycogen, diminishing the pool of starch available for amylolytic bacteria.

Seasonal variations of the digestive tract of the Eurasian beaver Castor fiber

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.

Effect of exogenous butyrate on the gastrointestinal tract of sheep. II. Hydrolytic activity in the rumen and structure and function of the small intestine1

The aim of this study was to determine the effect of exogenous butyrate on the activity of carbohydrate-digesting enzymes in the reticuloruminal digesta and structure and selected functions of the small intestine in sheep. Eighteen rams (30.8 ± 2.1 kg; 12 to 15 mo of age) were fed for 14 d a diet without (CTRL) or with sodium butyrate (BUT; 36 g/kg of offered DM). Butyrate concentration in the reticuloruminal fluid and proximal small intestinal digesta was greater for BUT compared with CTRL (P ≤ 0.05). Amylolytic activity was greater, whereas cellulolytic and xylanolytic activity in the reticuloruminal digesta was less for BUT compared with CTRL (P ≤ 0.04). Relative to BW, small intestinal tissue mass and small intestine length did not differ between treatments (P ≥ 0.15); however, absolute length of the small intestine was greater for BUT compared with CTRL (P = 0.04). In the duodenum, crypt depth tended (P = 0.10) to be greater, whereas in the ileum, crypt depth and muscularis thickness tended (P = 0.10) to be less for BUT compared with CTRL. Mitosis-to-apoptosis ratio in the proximal jejunum was greater for CTRL compared with BUT (P = 0.02). Expression of G-protein-coupled receptor 43 mRNA in the duodenal epithelium was greater for BUT compared with CTRL (P < 0.01). On the other hand, peptide transporter 1 mRNA expression in the distal sections of the small intestine, as well as activity of aminopeptidase A and dipeptidylpeptidase IV, were greater for CTRL (P ≤ 0.05). In summary, exogenous butyrate supplementation in feed affects hydrolytic activity in the rumen, and increased butyrate flow out of the reticulorumen affects both proximal and distal sections of the small intestine in sheep.

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

ABSTRACT 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.