A study in the light and electron microscope of the extruded peristome and related structures of the rumen ciliate entodinium caudatum

A study in the light and electron microscope of sections of the rumen ciliate Entodinium caudatum was undertaken in an attempt to elucidate the structure of the peristome when in the extruded state The conformation of the ciliary band on the peristome and the structure of the lining of the buccal cavity are described. The structure and position of a tuft of specialized sheet like extensions of the cell surface is also described.     

The cultivation of the rumen ciliate Entodinium bursa in the presence of Entodinium caudatum.

The rumen ciliate protozoon Entodinium bursa has been grown in vitro in the presence of bacteria and Entodinium caudatum for over a year at population densities of 100 to 200 ml-1. The medium contained potassium phosphate, prepared fresh rumen fluid, cysteine, wholemeal flour (or rice starch), dried grass and a culture of the spineless form of Entodinium caudatum. Entodinium bursa has an obligate requirement for this protozoon and died within 48 h in its absence. During growth from a 2% inoculum, the mean generation time of E. bursa was 6 h. Entodinium bursa engulfed 1-5 to 2-5 E. caudatum organisms h-1, and when E. caudatum was in excess it developed caudal spines for the first time in 17 years; these spined forms were engulfed much less readily than the spineless organisms.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

C oleman , G.S. & H all , F.J. 1984. The uptake and utilization of Entodinium caudatum , bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa. Journal of Applied Bacteriology 56 , 283–294.
Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum , ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus mega-terium, Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bouis , although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and i>Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro , although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa . Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

Electron microscopy of the rumen ciliate Entodinium caudatum, with special reference to the engulfment of bacteria and other particulate matter

A study in the electron microscope of thin sections of the rumen ciliate Entodinium caudatum was undertaken in an attempt to elucidate the mode of engulfment of particulate matter. This protozoon engulfed bacteria, polystyrene latex particles and olive oil into membrane-lined vesicles in the protozoal endoplasm. Particles of palladium black were also taken up into the endoplasm, but due to the toxic nature of this material it was not possible to demonstrate vesicle formation with certainty. The initial uptake of bacteria may be into large sacs containing many organisms which were subsequently taken into the endoplasm in vesicles that contained only one bacterium each. The evidence obtained in this investigation has been used to distinguish between two different mechanisms for the digestion of bacteria and utilization of the amino acids from the bacterial protein for the synthesis of protozoal protein.     

Bacterial Ingestion by the Rumen Ciliates Entodinium and Diplodinium

SYNOPSIS. In cattle fed a high-starch diet, species of Entodinium and Diplodinium ingested associated ruminal bacteria. Stained preparations of diluted rumen contents showed Entodinium caudatum, E. minimum, E. dubardi , (syn. E. simplex ), E. longinucleatum, E. bursa, E. nanellum, E. exiguum , and E. vorax contained gram-positive diplococci. Starch grains with adherent gram-positive diplococci were observed within Entodinium spp. Diplodinium ecaudatum forma ecaudatum, D. ecaudatum forma caudatum, D. neglectum and an unidentified species of Diplodinium also ingested ruminal diplococci. Bacteria were isolated from mixed species of Entodinium by washing and culturing the protozoa in a starch feed-extract agar medium. The strains isolated from the ciliates were gram-positive diplococci, 0.8 times 1–1.5 μm, which attached themselves to starch granules and were able to digest the starch. Conclusive evidence of bacterial ingestion by the oligotrichs was obtained by providing the bacterial cultures to Entodinium species ( E. dubardi and E. minimum ) which had been starved 24 hr. Gram-stained preparations showed the ciliates readily ingested the bacteria. The amylolytic cocci utilized by Entodinium spp. were identified as Streptococcus bovis.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum, ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus megaterium. Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bovis, although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro, although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa. Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

The 3' untranslated region of messages in the rumen protozoan Entodinium caudatum

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.     

Effect of pH on viability of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei in vitro

Cultures of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei were grown and transferred in poorly buffered media prepared using different concentrations of sodium bicarbonate and a nitrogen gas phase. By transferring every 12 or 24 h, culture pH was gradually decreased until the protozoa disappeared. The cultures were transferred by placing half of the culture into an equal volume of fresh medium, resulting in pH fluctuations similar to those in the rumen, resulting from fermentation, eating, and saliva production. All four species appeared to maintain their concentrations around pH 5.8, but numbers decreased as pH values fell below 5.6. The four species were similar in that they all survived above pH 5.3. These results differ from previous reports in which Entodinium species appeared to be more tolerant to low pH than all other species of rumen ciliates. No adaptation to low pH was observed in Epidinium caudatum cultures after recovery from pH 5.4 medium containing only one or two viable cells.     

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.     

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.     

In vitro determination of generation times for Entodinium exiguum, Ophryoscolex purkynjei and Eudiplodinium maggii

ABSTRACT. Most previously reported generation times for rumen ciliate protozoa are longer than would be required to prevent their being flushed out of the rumen. In an earlier study from this lab, using a sequential transfer procedure, generation times between 12 and 13 h were determined for both Epidinium caudatum and Entodinium caudatum . This would permit these species to be maintained in a rumen with a fluid volume turnover rate as rapid as twice a day. In this study, generation times were estimated for Entodinium exiguum (13.2 h), Eudiplodinium maggii (26.8 h), and Ophryoscolex purkynjei (29 h), by sequential transfer at both 12 and 24 h time periods. The generation time for E. exiguum is lower than reported for this and other Entodinium species as determined by logarithmic growth from a small inoculum, but similar to that obtained for Ent. caudatum using sequential transfer. Eudiplodinium maggii and O. purkynjei generation times are similar to previous estimates of 24- and 24–48 h, respectively. However, it was observed that after an adaptation period of 36 to 48 h (generally 3–4 transfers) cell concentrations decreased and generation times were markedly decreased, i.e. 12.2 h for Ent. exiguum , 15.0 h for E. maggii and 12.8 h for O. purkynjei . In a separate study, varying both the concentration of Epidinium and the quantity of substrate fed per cell had no effect on generation time.     

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.     

Distinctive archaebacterial species associated with anaerobic rumen protozoan <Emphasis Type="Italic">Entodinium caudatum</Emphasis>

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.     

Preliminary study on the requirements of Entodinium exiguum and E. caudatum for live or dead bacteria when cultured in vitro

Whether live bacteria are required to culture the rumen protozoa Entodinium exiguum and E. caudatum in vitro was studied. Treatments were protozoa plus antibiotics (PA), PA plus autoclaved bacteria (PAB) or protozoa plus live bacteria (PLB). Generation times at 24 h were 22.8 and 31.0 h for E. exiguum and E. caudatum. Protozoal concentrations were unaffected by the absence of bacteria up to 48 h. After 72 h, E. exiguum, concentrations were higher in PLB than PA or PAB. With E. caudatum differences between PLB and PA were only observed at 96 h. Thus, a requirement for live bacteria appears to be manifested in culture periods longer than 48 (E. exiguum) and 72 (E. caudatum) h. Although differences between PLB and PAB indicate a metabolic dependence for bacteria or a long-term antibiotic effect, non-significant differences between PAB and PA suggest that the effect is also related to a nutritive bacterial contribution.     

An NAD(+)-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan, Entodinium caudatum

An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.     

The uptake and metabolism of bacteria, amino acids, glucose and starch by the spined and spineless forms of the rumen ciliate Entodinium caudatum

Spined and spineless forms of Entodinium caudatum were obtained by growth in vivo in the presence and absence, respectively, of Entodinium bursa. Washed suspensions of both forms engulfed all the bacteria tested although the spined form took them up 1.3 to 1.9 times more rapidly per unit volume of protozoon than did the spineless form. Buytrivibrio fibrisolvens and Selenomonas ruminantium were rapidly digested by the spined form after engulfment. Free amino acids were taken up on average 3.1 times and glucose approximately 60 times faster per unit volume of protozoon by the spined form. Limited amounts of protein were synthesized by the spined form from glucose and starch but engulfed bacteria and, to a lesser extent, free amino acids were probably the prinicpal sources of protein for growth of both forms.     

In vitro excystation and structure of Sarcocystis suicanis Erber, 1977, sporocysts

Methods previously described for artificially excysting coccidia were applied to oocysts and sporocysts of Sarcocystis suicanis recovered from the intestinal mucosa of experimentally infected dogs. Observations with a scanning electron microscope showed that sporocysts consisted of 4 plates, each with a series of accessory veinations; plate margins joined at obtuse angles; plates separated at their margins and curled inward, allowing the sporozoites to escape. Transmission electron microscopy suggested that the sporocyst wall is ultrastructurally similar to that of other coccidia.     

The catabolism of phosphatidylethanolamine by the rumen protozoon Entodinium caudatum and its conversion into the N-(1-carboxyethyl) derivative

1. The N-(2-hydroxyethyl)alanine esterified to phosphatidic acid in anaerobic ciliate rumen protozoa has the l configuration. 2. Labelling experiments with Entodinium caudatum cultures using [(32)P]P(i) [2-(14)C]ethanolamine and (32)P- and (14)C-labelled phosphatidylethanolamine show that phosphatidylethanolamine is the direct lipid precursor of the N-(2-hydroxyethyl)alanine-containing phospholipid. 3. Labelling experiments with [(14)C]starch, [(14)C]lactate and [(14)C]pyruvate with E. caudatum cultures indicate that a three-carbon glycolytic intermediate is probably the precursor of the N-(1-carboxyethyl) grouping which substitutes on the amino group of phosphatidylethanolamine. 4. [(32)P]phosphatidylethanolamine is catabolized by E. caudatum forming initially glycerylphosphorylethanolamine and subsequently glycerophosphate and P(i). A little phosphorylethanolamine formed may possibly arise from bacterial enzymes ingested by the protozoa.     

The effect of sterols and haemin on the growth of the rumen ciliate Ophryoscolex caudatus and some other Entodiniomorphid protozoa

Seven isolates of Ophryoscolex caudatus have been cultured anaerobically in vitro (at a population density of 56/ml) for an average of 18 months each in the presence of bacteria on a reduced buffered salts medium containing prepared fresh rumen fluid with the daily addition of ground wheat and dried grass and with twice weekly dilution of the culture with an equal volume of fresh medium. The ground wheat and dried grass could be replaced by ground wheat coated with β-sitosterol, stigmasterol, ergosterol or α-spinasterol and with β-sitosterol the population density increased to 110/ml. Haemin further increased the population density obtained in the presence of sterol by 9–160%. The population density of cultures of Epidinium ecaudatum caudatum was also increased by sterols and haemin, that of Polyplastron multivesiculatum by sterols only, and some sterols and haemin, under certain conditions, increased that of Entodinium caudatum.     

The aminoethylphosphonate-containing lipids of rumen protozoa

1. A method is presented for identifying and estimating the aminoethylphosphonate (ciliatine)-containing phospholipids in a complex mixture. 2. Evidence was obtained that the phospholipids of a pure culture of Entodinium caudatum and a mixed rumen protozoa sample contain diglyceride ciliatine, and a plasmalogen ciliatine was detected in the latter. 3. A ninhydrin-positive sphingolipid was isolated from rumen protozoa. Although chromatographically homogeneous on silica gel it contains two components, which were provisionally identified as ceramide ciliatine and ceramide phosphorylethanolamine. 4. A detailed phospholipid analysis of E. caudatum and rumen protozoa is presented. They contain no phosphatidylserine or cardiolipin, but an unidentified phosphoglyceride containing a zwitterionic amino acid is present.