Ecological factors determining establishment of cellulolytic bacteria and protozoa in the rumens of meroxenic lambs

Ecological factors that control the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic animals. Axenic lambs received dilutions of rumen liquor from either conventional lambs and sheep (pool A) or meroxenic lambs (pool B). The total number of bacteria established in the rumen was between 10(9) and 5 x 10(10) g-1. In lambs inoculated with dilutions (10(-6), 10(-7), 10(-8)) of pool A, cellulolytic bacteria did not become established. However, subsequent inoculation with Bacteroides succinogenes, resulted in colonization in lambs that had received 10(-6) and 10(-7) dilutions of pool A. However, B. succinogenes became established in only one of three lambs that received the 10(-8) dilution. Similar results were obtained for the protozoan Entodinium sp. With pool B, lambs were inoculated earlier and cellulolytic bacteria were established directly from the 10(-6) and 10(-7) inocula. Polyplastron multivesiculatum establishment occurred readily when inoculated into the lambs which had received the 10(-6) dilution of pool B. Results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early animal inoculation.     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.     

Phylogeny of the Rumen Ciliates Entodinium, Epidinium and Polyplastron (Litostomatea: Entodiniomorphida) Inferred from Small Subunit Ribosomal RNA Sequences

ABSTRACT. Three complete 18S ribosomal RNA gene sequences from the rumen ciliates, Entodinium coudatum (1,639 bp), Epidinium caudarum (1,638 bp), and Polyplastron multivesiculatum (1,640 bp) were determined and confrimed in the opposite direction. Trees produced using maximum parsimony and distance-matrix methods (lest squares and neighbour-joining). with strong bootstrap support, depict the rumen ciliates as a monophyletic group, Entodinium caudatum is the earliest branching rumen ciliate. However, Entodiniwn simplex does not pair with En. caudatum , but rather with Polyplastron multivesiculatum. Signature sequences for these rumen ciliates reveal that the published SSrRNA gene sequence from En. simplex is in fact a Polyoplastron species. The free-living haptorian ciliates, Loxophyllum, Homalozoon and Spathidium (Subclass Hoptoria), are monophyletic and are the sister group to the rumen cilates. The litostomes (class Litostomatea), consisting of the haptorians and the rumen ciliates, are also a monophyletic group.     

Effect of rumen ciliates on the digestive utilization of various carbohydrate-rich diets and on the end-products formed in the rumen. II. Utilization of inulin, saccharose and lactose

Three diets rich in inulin, saccharose and lactose, respectively, were given to 10 rumen-fistulated sheep. Two animals were defaunated, two were inoculated with either Polyplastron multivesibulatum or Entodinium sp., and two others were inoculated with both. The latter two were bred in conventional conditions. All animals ingested the same amounts of carbohydrates in the three diets (21-22 g/kg P0.75/day). Dietary nitrogen content was similar (table 1). The ciliate population was improved with the inulin diet (fig. 1; table 2). With a mixed population, the Entodinium improved with the inulin diet (fig 1; table 2). With a mixed population, the Entodinium sp. genus was always predominant. Holotrich protozoa (mainly Isotricha) in the rumen of the conventional sheep represented 15 to 30% of the total ciliate biomass, indicating that they were able to metabolize these soluble sugars. We also observed that P. multivesiculatum can ferment cellulose and all the soluble carbohydrates proposed in these diets. However, Entodinium sp. development occurred mainly in the presence of the sugard produced during carbohydrate hydrolysis by other ciliates or bacteria. The highest organic matter digestibility, noted in faunated animals (table 3) was confirmed by the VFA concentration in the rumen (table 4). This could be explained either by an activation of bacterial metabolism due to predation or by the direct effect of ciliates on fermentations, or both. Modifications in the VFA composition varied with ciliate inoculation, showing that ciliate metabolism may vary with the nature of the energy in the diet or that the observed results depended on various opposite effects in which the intensity of each component was influenced by the diet. In general, the acetic acid molar proportion increased and propionic acid decreased when there was a considerable Entodinium sp. population. The effect on butyric acid was low with these diets. Higher ammonia and lactic acid concentrations were observed in the rumen of faunated than defaunated sheep, irrespective of the ciliate inoculum.     

Two-step freezing procedure for cryopreservation of rumen ciliates,an effective tool for creation of a frozen rumen protozoa bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

Xylanases and carboxymethylcellulases of the rumen protozoa Polyplastron multivesiculatum, Eudiplodinium maggii and Entodinium sp

Endoglucanase and xylanase activities of three rumen protozoa, Polyplastron multivesiculatum, Eudiplodinium maggii, and Entodinium sp. were compared qualitatively by zymograms and quantitatively by measuring specific activities against different polysaccharides. A set of carboxymethylcellulases and xylanases was produced by the large ciliates whereas no band of activity was observed for Entodinium sp. in zymograms. Specific activity of endoglucanases from P. multivesiculatum (1.3 micromol mg prot(-1) min(-1)) was twice that of E. maggii, whereas xylanase specific activity (4.5 micromol mg prot(-1) min(-1)) was only half. Very weak activities were observed for Entodinium sp. A new xylanase gene, xyn11D, from P. multivesiculatum was reported and its gene product compared to 33 other family 11 xylanases. Phylogenetic analysis showed that xylanase sequences from rumen protozoa are closely related to those of bacteria.     

Effect of rumen ciliates on the digestion of different carbohydrates in sheep. I.--Utilization of cell wall carbohydrates (cellulose and hemicellulose) and of starch

Two diets rich in cell-wall carbohydrates or starch were given to 10 rumen-fistulated sheep; two sheep were defaunated and the others were inoculated with Polyplastron multivesiculatum (P) or Entodinium sp. (E), or both (P + E), or with conventional fauna. Ciliate biomass was greater when the animals were fed a high starch diet than when the diet was rich in cell-wall carbohydrates (table 2). With both diets, the Entodinium genus in the mixed fauna sampling predominated. We showed that Polyplastron was directly involved in cell-wall carbohydrate breakdown, while Entodinium capacity to digest cellulose remained low. We noted that with a diet rich in cellulose and hemicellulose, bacterial cellulolytic activity was improved by the presence of ciliates in the rumen but was decreased with the "starch" diet (table 3). The greater VFA concentration observed in the faunated animals expressed ciliate effect on the fermentations as well as activation of bacterial metabolism. With a high starch diet, the Entodinium sp. ciliates may have a buffering effect on the pH values in the rumen by limiting bacterial fermentation after food intake and by prolonging starch digestion during the day (table 4). The composition of the VFA mixture was modified by ciliate inoculation. The molar proportion of butyric acid always increased, while that of acetic and propionic acids evolved differently according to the diets and the ciliates (table 4). The higher ammonia concentration in the rumen liquor observed in faunated animals (table 4) could be explained either by the breakdown of both feed and bacterial proteins ingested by ciliates or by a lower ammonia nitrogen incorporation by fewer bacteria. Statistical analyses were used to explain the specific effect of P and E and also the interactions between them and between each of them and the diets.     

Highly efficient galvanotaxis apparatus for cleaning and concentrating rumen ciliates

Galvanotaxis was shown to be an efficient method for cleaning and concentrating rumen ciliate protozoa whose harvesting (centrifugation of large volumes of in vitro cultures followed by repeated washing of the sediment to remove plant debris) is time consuming. We suggested the use of a new galvanotaxis apparatus (a small-capacity two-way glass stopcock) to improve cell yield in concentrating the rumen ciliate protozoa and cleaning them from impurities. Migration of the ciliates (Entodinium caudatum, Entodinium furca monolobum and Diploplastron affine) into the cathode compartment under different electric currents (0, 5, 10, and 15 mA) and intervals (5, 10, 20, and 30 min) was evaluated. The lethal current level was 20 mA. Cell yield was 9-81%, depending on ciliate species, migration time and current. The migration time significantly affected both E. caudatum and D. affine. The electric current-migration time interplay was shown to be significant in both E. caudatum and D. affine. The advantages and disadvantages of the tested apparatus were determined; the two-way glass stopcock was very convenient for both cleaning and concentrating rumen ciliate in vitro cultures by galvanotaxis.     

The metabolism of starch, glucose, amino acids, purines, pyrimidines and bacteria by the rumen ciliate Polyplastron multivesiculatum.

The large rumen ciliate protozoon Polyplastron multivesiculatum grown in vitro engulfed a wide range of bacteria (from a population density of 10(9) bacteria ml(-1)) at a rate of 1500 to 137000 bacteria h(-1) protozoon(-1). No evidence was found for the preferential engulfment of bacteria of rumen origin. Except for Proteus mirabilis none of the bacteria were digested with the liberation of soluble materials into the medium. Glucose and amino acids were taken up rapidly by P. multivesiculatum compared with the rate of uptake by Entodinium caudatam. Glucose was incorporated into protozoal polysaccharide and into bacteria associated with the protozoa and was used for the synthesis of a wide range of amino acids. Evidence showed that bacteria and free amino acids at the concentrations found in the rumen could supply the protein requirements of the protozoa for division at least once each day.     

The effects of organic selenium supplementation on the rumen ciliate population in sheep

The effect of selenium supplementation on the rumen protozoan population of sheep was demonstrated. Both the total and generic counts of rumen ciliates in sheep fed a diet with basal Se content (70 microg/kg dry matter) were compared to those of animals given feed supplemented with inorganic (disodium selenite) or organic Se (selenized yeast) (310 microg/kg dry matter). The genera of Entodinium, Isotricha, Dasytricha, Ophryoscolex, Diploplastron and Polyplastron occurred in all sheep except for the control, in which Ophryoscolex was not observed. The population of Ophryoscolex caudatus f. tricoronatus was significantly higher in sheep supplemented with organic Se than in animals given inorganic Se (by 160 %). Supplementation of feed with selenized yeast induced significant growth in the Diploplastron population (by 63 %) while no change occurred in sheep given selenite. The populations of Dasytricha ruminantium and Polyplastron multivesiculatum were higher than control in both Se-supplemented groups. The ciliate population of Entodinium spp. was not influenced by Se supplements. Our results suggest a protective effect of Se feed supplementation on the development of some rumen ciliate species in young ruminants.     

Hydrolysis of Fraction 1 leaf protein and casein by rumen entodiniomorphid protozoa

Cell-free extracts of fourteen individual species of rumen ciliate protozoa and of mixed rumen ciliates degraded Fraction 1 leaf protein. For Entodinium caudatum and Eudiplodinium maggii the optimum pH was 3·2. The maximum rates of proteolysis (in µmol acid soluble-tyrosine formed/mg protein/h) were 0·16 to 5·7 with protozoa grown in vivo and 0·38 to 6·4 with protozoa grown in vitro. The highest rates were obtained with Entodinium caudatum and E. simplex and the lowest with the cellulolytic species grown in vivo. K _m values (mg/ml) ranged from 0·42 to 19 with protozoa grown in vivo and 0·35 to 13·3 with protozoa grown in vitro. All single species (with one exception) whether grown in vivo or in vitro degraded Fraction 1 leaf protein faster (1·4 to 21 times) than casein. Partial inhibition of the activity of Entodinium caudatum was obtained with pepstatin and N-ethylmaleimide and almost complete inhibition with leupeptin suggesting the presence of 'carboxyl' and 'thiol' enzymes.     

Rumen Ciliate Fauna of Alaskan Moose (Alces americana), Musk-Ox (Ovibos moschatus) and Dall Mountain Sheep (Ovis dalli)*

Total numbers, generic distribution and percentage species distribution were determined for the ciliate protozoa in rumen contents obtained from Alaskan moose (Alces americana), musk-ox (Ovibos moschatus) and Dall mountain sheep (Ovis dalli). The musk-ox has a fauna somewhat similar to that previously observed in reindeer and caribou. In contrast, only protozoa in the genus Entodinium were observed in moose, while Dall mountain sheep have a fauna unique among Alaskan ruminants studied to date. Other than Entodinium exiguum which was common to all animals, only 2 additional species of Entodinium, observed in the moose and musk-ox, occurred in more than one animal species. Four new species of protozoa are described, Entodinium dalli sp.n., Entodinium constrictum sp.n. and Polyplastron alaskum sp.n. from the Dall mountain sheep and Entodinium alces sp.n. from moose.     

Effect of Absence of Ciliate Protozoa From the Rumen on Microbial Activity and Growth of Lambs

A survey of the components of the rumen ciliate population in a series of adult sheep, raised in the Faculty of Agriculture, University of Alexandria, has shown that a mixture of Entodinium, Isotricha, Ophryoscolex, Diplodinium, and Polyplastron species was found in the rumen contents of Egyptian sheep; no Epidinium and a negligible number of Dasytricha ruminantium were also observed. The microbial population, reducing sugars, ammonia, volatile fatty acids (VFA) production, and growth rate of 14 lambs inoculated with whole rumen contents from a mature sheep were compared over a 6-month period with those of 13 lambs maintained under the same conditions, except that they were strictly isolated from other ruminants. Certain large oval organisms and large numbers of flagellates and Oscillospira were frequently observed in the rumen contents of the isolated lambs. The reducing sugars, ammonia, and VFA levels, measured before and at intervals after feeding, in the inoculated lambs showed a pronounced rise above the values found in the ciliate-free animals. The propionic acid-acetic acid ratio in the rumen contents of the faunated lambs was considerably higher than in the nonfaunated controls. The inoculated lambs grew faster than the isolated lambs. Differences in weight gain which ranged from 15 to 17% were statistically significant. The inoculated animals impressed the observers by their good appearance which was superior to that of the ciliate-free lambs. It was, therefore, concluded that the rumen ciliate protozoa are essential for the metabolism and growth of young lambs.     

Potential of galvanotaxis to separation and cleaning of rumen ciliates

The ability of rumen ciliate protozoa to move in a unidirectional electrical field from the anode to the cathode was tested in large-volume electromigration equipment; the aim was to concentrate the microorganisms and clean them of impurities. During galvanotaxis in the freshly harvested rumen fluid and at a voltage of 10 V (I=0.8 mA), cells of Isotricha (Isotricha prostoma, Isotricha intestinalis) were the first to swim towards the cathode; 1 min later, they were followed by Dasytricha ruminantium. Entodiniomorphous ciliates (small Entodiniae as well as large species) displayed minimum movement towards the cathode. The yield of electromigration of Entodinium caudatum from the in vitro culture ranged within 2-6% when using 75-100 V (10 mA) and 60 V (5 mA) voltage, respectively. At 10 V (0.8 mA), E. caudatum did not move towards the cathode. It is shown that the behavior of Trichostomatids (Dasytricha and Isotricha) facilitates separation by means of the tested large-volume equipment. Concentration and cleaning of Entodiniomorphous ciliates using galvanotaxis in the tested equipment proved to be ineffective.     

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble ¹⁴C from ¹⁴C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoal population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter , while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and thzis effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

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.     

Effect of insulin on in vitro fermentation activity of microrganism community of rumen ciliate Entodinium caudatum culture

The influence of insulin (17.4 nmol l-1) on total gas and methane production, the concentration of total and individual fatty acids and dry matter degradability was investigated in the rumen ciliate culture of Entodinium caudatum. The experimental groups consisted of control group (without insulin) and two groups with insulin application--single shot and long-term application (over 30 days). Fermentation activity of each experimental group was observed on two subgroups: whole protozoan culture (protozoa plus bacteria) and bacterial fraction (bacteria without protozoa). Long-term application of insulin significantly increased methane production and DM degradability in the whole protozoan culture. Total VFA concentration was significantly increased by long-term as well as single-dose application of insulin (by 255% and 158%, respectively). The growth of the protozoa was not influenced by insulin treatments. It can be concluded that the fermentation activity of the community of the rumen ciliate Entodinium caudatum culture was marked stimulated by application of insulin.     

Some rumen ciliates have endosymbiotic methanogens

Abstract Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F₄₂₀ autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H₂ evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.