The Cytology of Sheep Rumen Ciliates. II. Ultrastructure of Eudiplodinium maggii1

The anterior adoral zone of syncilia (AZS) of Eudiplodinium maggii is mounted on an extrusible peristome within a vestibulum. The peristome contains cytopharyngeal components derived from the infraciliature. These components include a crescent-shaped palisade of nematodesmata, two types of sub-membrane cytopharyngeal ribbons, and an ensheathing fibrous layer enclosing a phagoplasmic zone containing the other components. A convoluted esophagus is continuous with and extends from the posterior of the cytopharynx adjacent to the macronucleus. A posterior cytoproct has specialized cytoplasm around it and associated myoneme-like elements. The skeletal plate is composed of finely granular platelets and lies under the cortex ventral to the macronucleus. The endoplasm is separated from the ectoplasm by a fibrous boundary layer. The cortex has an external glycocalyx, a membranous layer, epiplasm, and microtubular and microfilament layers. The AZS infraciliature is of the usual cntodiniomorph type, kinetosomes linked by a sub-kinetosomal rod and with associated bifurcated kinetodesma, postciliary and transverse microtubules-the latter extending into the cytopharynx—nematodesmata, and a fibrous reticulum. A possible vestigial, somatic infraciliature consisting of short, barren kinetosomes with associated basal and cortex-directed microtubules and a periodic incomplete fiber, is found subcortically throughout the cell.     

The Cytology of Sheep Rumen Ciliates. III. Ultrastructure of the Genus Entodinium (Stein)1

This study examines previously undescribed general and cytopharyngeal features of the genus Entodinium. The cytopharynx contains three types of microtubular ribbons underlying the cytostomal membrane as well as a loose palisade of nematodesmata. A protoesophagus composed of microtubular bundles associated with a fibrous wall lies internally to one side of an extrusible peristome on which the adoral zone of syncilia (AZS) is mounted. Macronuclear structures are very similar to those of other ophryoscolecids. The micronucleus has chromatin bodies forming a compact mass but lacks the thick wall found in other species. A tubular spongiome surrounds the contractile vacuole and the cytoproct is relatively undifferentiated. Cortical structure follows the usual five-layered ophryoscolecid pattern with subcortical barren kinetosomes arranged into indistinct kineties. The infraciliature of the AZS has kinetosomes set upon a subkinetal rod and with associated bifurcated kinetodesmata and transverse microtubules, some of which extend into the cytopharynx. Components newly described for Entodinium are the one to three postciliary microtubules and the interkinetosomal centro-lateral strand, all of which are present in other species of ophryoscolecid ciliates. The infraciliature of the paralabial ciliary tuft shows similar components to that of the main AZS, but lacks the subkinetal rod. The microtubular components of the cytopharynx are discussed in relation to the “alimentary” structures in other ophryoscolecids, and a relationship of these structures to dietary differences is suggested.     

The Rumen Ciliate Epidinium in Primary Degradation of Plant Tissues

Pieces of lucerne stem suspended in a sheep rumen in nylon bags were removed after different time intervals and examined by scanning electron microscopy. By 15 min large numbers of the ciliate protozoan Epidinium Crawley were attached to damaged areas of the stem, although a complex protozoal fauna was present in the rumen contents. Highest concentrations were on cortex and phloem tissues, with densely packed protozoa forming a complete ring around the transversely cut end of the stem between the epidermis and the vascular cylinder. Within 2 h there was extensive degradation of thin-walled tissues, as indicated by the amount of exposed vascular cylinder. Epidermis was not degraded but slid down the side of the stem as the underlying tissues were removed. This rapid degradation of plant tissue is explained by direct ingestion of tissues by the protozoa on the plant fragments. Many of the epidinia attached to the stem pieces were ingesting phloem elements and chlorophyllous tissue. The rumen protozoa have not previously been shown to participate on this scale in the physical degradation of plant material.     

Phylogenetic Study of Methanogens Associated with Rumen Ciliates

The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated. Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted. Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture. The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109. Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer. Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences. RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation. The association of Methanobrevibacter spp. with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M. smithii. Received: 16 February 1999 / Accepted: 20 April 1999     

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.     

Molecular cloning,expression, and characterization of an endo-beta-1,4-glucanase cDNA from Epidinium caudatum1

An endo-beta-1,4-glucanase gene (epi3) from the rumen ciliated protozoan Epidinium caudatum was cloned from a cDNA library constructed by using the lambda ZAP II vector. The enzymatic activity of the gene product was detected by the Congo red assay, using carboxymethyl cellulose (CMC) as substrate. The nucleotide sequence of epi3 revealed 1,253 nucleotides with an open reading frame for a protein (Epi3) of 356 amino acids (Mr -41,014). Epi3 shows high homology with family 5 endoglucanase genes and with genes from protozoa isolated from sources other than the rumen. The specific activity of Epi3 produced in Escherichia coli was 5.544, 2.754, and 0.295 mmol of glucose min(-1) mg(-1) protein when the substrates used were CMC, beta-glucan, and xylan, respectively. A beta-1,4-linked trisaccharide of glucose was the preferred substrate of Epi3, as determined by analysis with the p-nitrophenyl form of the substrate. To our knowledge, this is the first report of the isolation of an endoglucanase gene from a rumen protozoan.     

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.     

Rumen Ciliates of Musk-Oxen (Ovibos moschatus Z.) from the Canadian Arctic1

Samples of rumen contents were obtained from 13 musk-oxen living on Banks Island, N.W.T., Canada. Total protozoan numbers ranged from 38.5 to 124.6 times 10⁴ per ml of rumen contents with a mean generic distribution of 52.4%Entodinium, 45.9%Diplodinium, and 1.7%Epidinium. A total of 18 protozoan species were found, six of which have not previously been observed in this host, i.e. D. (Diplodinium) costatum, D. (Ostracodinium) gracile, D. (Ostracodinium) trivesiculatum. D. (Eudiplodinium) medium, Epidinium quadricaudatum, and Epidinium parvicaudatum. Two new species of protozoa are described, Entodinium ovibos n. sp. and Diplodinium (Eudiplodinium) banksi n. sp.     

Rumen Ciliate Fauna of Some Brazilian Cattle: Occurrence of Several Ciliates New to the Rumen,Including the Cycloposthid Parentodinium africanum1

ABSTRACT. Parentodinium africanum was observed in rumen contents of four Brazilian cattle and constituted 4.4, 0.9, > 0.1, and 10.0% of the total ciliates. This appears to be the first observation of the family Cycloposthiidae in the rumen habitat. Blepharoconus krugerensis, previously observed in the intestines of the elephant, and an unknown species of ciliate with many characteristics similar to the family Paraisotrichidae were each observed in a single animal. A new subspecies, Diplodinium flabellum laterospinatum n. subsp., is also described. Total number of ciliates per ml of rumen contents ranged from 9.0 to 51.2 × 10⁴ and included an overall total of 55 species and 4 subspecies.     

Rumen Anaerobic Fungi of Cattle and Sheep

A system for the large-scale production and purification of mouse mammary tumor virus in the absence of detectable endogenous murine leukemia virus is described. By utilizing the Mm5mt/c1 cell line established from an adenocarcinoma of a C3H mouse, the continuous production of over 25,000 liters of mouse mammary tumor virus-containing tissue culture fluids has been achieved. By the strict adherence to well-defined tissue culture conditions, mammary tumor virus production was accomplished without the expression of murine leukemia virus. Various biochemical and immunological systems were established for the rapid and precise detection of the endogenous leukemia virus, the expression of which could be enhanced under conditions of culture stress.     

Glycogen Phosphorylase and Synthase Activities of Rumen Ciliates of the Genus Entodinium

Glycogen phosphorylase and synthase activities were detected in the sonic lysate of rumen ciliates of the genus Entodinium. The ciliate phosphorylase had the following properties. The pH optimum was narrow and centered at pH 5.9. The activity was maximum at 30°C; above 40°C a rapid inactivation occurred. The K_m value for glucose-1-phosphate (G-1-P) and for glycogen was 15 mM and 0.069% (w/v), respectively. NaF and ethylenediamine tetraacetic acid had no stimulative effect on the enzyme activity, though adenosine 3′,5′-monophosphate and theophylline activated it. NaHSO₃ inhibited the enzyme activity at a concentration of 1 mM. The inhibition of glucose was noncompetitive for G-1-P. Glycolytic intermediates and nucleotides had a minor effect on phosphorylase activity. Glycogen synthase existed in two forms, glucose-6-phosphate dependent and independent forms: the proportion of the latter form increased with the decrease of reserve polysaccharide levels in the ciliates. Correlations between glycolytic enzyme activities included phosphorylase and synthase activities and reserve polysaccharide contents in the ciliates were determined, and a possible regulatory mechanism of polysaccharide synthesis and degradation was discussed.     

Concentrations of Free Sugars in Sheep Rumen Fluids

A convenient method was proposed for the estimation of glucosamine content in koji. In this method, the time of acid hydrolysis of the mold sample was markedly shortened modifying the previously reported method as follows; the mold sample was immersed in 60% of sulfuric acid for 24 hr at 25°C, and then the mixture was diluted with water to make the concentration of sulforis acid 1 n and autoclaved under the pressure at 1 kg/cm² for 1 hr. The liberated glucosamine was assayed by Blix’s method. The glucosamine contents of mycelia obtained from the submerged and surface cultures were increased about 2 times of initial stages in prolonged incubation. The significant differences of the glucosamine contents in various kojis were seen depending on the raw materials used; the values were 18~26mg/g in wheat bran koji and 6~16 mg/g in rice and defatted soybean meal koji.     

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°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°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.