Exocellular Carbohydrase Formation by Rumen Holotrich Ciliates

SYNOPSIS Exocellular carbohydrase activity was detected, in the absence of cell lysis, in cell-free culture supernatant fluids of rumen holotrich ciliates after incubations in buffer systems of varying tonicity, from cell suspensions that were isolated by various technics, and in which bacterial activity had been suppressed by antibiotics. The kinetic characteristics of the holotrich invertases and β–glucosidase from Dasytricha , although having interspecies variations, were the same for the intra- and extracellular form of the enzyme. The properties of the invertase activity present in cell-free rumen contents resembled those of the exocellular enzymes formed by the holotrichs. Invertase activity in the in vitro culture supernatant fluid of Dasytricha ruminantium increased throughout the incubation period and was influenced by the initial pH, temperature, sucrose concentration, and inoculum size. Exocellular holotrich carbohydrase activity was increased when the incubation substrate was not readily utilized by the protozoa. Intracellular carbohydrase activity was also influenced by the carbohydrate substrate.     

Molecular Diversity of Rumen Methanogens from Sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobacteriales. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

Phylogenetic Analysis of Methanogens in the Pig Feces

In order to assess methanogen diversity in feces of pigs, archaeal 16S rRNA gene clone libraries were constructed from feces of the pig. After the amplification by PCR using primers Met86F and Met1340R, equal quantities of PCR products from each of the five pigs were mixed together and used to construct the library. Sequence analysis showed that the 74 clones were divided into ten phylotypes as defined by RFLP analysis. Phylogenetic analysis showed that three phylotypes were most closely affiliated with the genus Methanobrevibacter (46% of clones). The library comprised 55.4% unidentified euryarchaeal clones. Three phylotypes (LMG4, LMG6, LMG8) were not closely related to any known Euryarchaeota sequences. The phylogenetic analysis indicated that the archaea found in the libraries were all clustered into the Euryarchaeota. The data from the phylogenetic tree showed that those sequences belonged to three monophyletic groups. Phylotypes LGM2 and LGM7 grouped within the genus Methanobrevibacter. Phylotypes LGM4, LGM6, LGM8 and LGM9 grouped within the genus Methanosphaera. Other phylotypes grouped together, and formed a distantly related sister group to Aciduliprofundum boonei and species of the Thermoplasmatales including Thermoplasma volcanium and Thermoplasm acidophilum. Our results showed that methanogens belonging to the genus Methanobrevibacter were predominant in pig feces, and that many unique unknown archaea sequences were also found in the library. Nevertheless, whether these unique sequences represent new taxonomic groups and their role in the pig gut need further investigation.     

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.     

Rumen Ciliates with Description of Two New Species from Three African Reedbuck Species

Thirteen bohor reedbuck Redunca redunca wardi (Thomas, 1900) from Kenya and Tanzania, ten common reedbuck Redunca arundinum arundinum (Boddaert, 1785) from Zimbabwe and 34 mountain reedbuck Redunca fulvorufula fulvorufula (Afzelius, 1915) from South Africa were sampled. Twenty-six protozoan species have been identified, 18 in the bohor reedbuck, which included seven entoninia, two new Diplodinium species, but no holotrichs. In the southern reedbuck 13 species were recorded, which included two entodinia, and 11 diplodinia including the two new species; holotrichs were also absent. The mountain reedbuck which is a mixed feeder as opposed to the grazers had 22 species including two holotrichs.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

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.     

Scanning Electron Microscope Study of Bacteria Associated with the Rumen Epithelium of Sheep

Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed.     

The Effect of Some Factors on the Cell Volume of Rumen Ciliates

The effect of diluting rumen samples with water and that of formol fixation, on the volume of ophryoscolecid rumen ciliates, was studied. The dilution with water caused within 2 hrs. the ciliates to swell to an average of 60 % (15.4–107.3). During 15 days of formol fixation, the shrinkage was on an average 10 % (0.0–19.8) of the original volume. When the thickness of the cells was conceived equal to the width, volumes 35–77 % too large were obtained. The influence of these sources of error on calculations of rumen fauna volumes is discussed.     

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.     

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.     

西藏三种苔藓栖生纤毛虫的形态和系统发育分析

本研究利用活体观察和蛋白银染色方法对采自西藏日喀则和那曲的3种苔藓栖生纤毛虫,鬃异源棘尾虫(Tetmemena pustulata)、棘毛虫未定种(Sterkiella sp.)和殖口虫未定种(Gonostomum sp.)进行了形态学研究,描述了活体、核器以及纤毛图式等形态学特征.同时,测定了这3种纤毛虫的SSU r...     

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.     

Light Microscopic Observations of Infraciliature and Morphogenesis in Six Species of Rumen Ostracodinium Ciliates

Infraciliature and morphogenesis of six rumen ciliates, Ostracodinium mammosum, O. munham, O. dilobum, O. rugoloricatum, O. iwawoi, and O. tiete are described from pyridinated silver carbonate-impregnated specimens. These six Ostracodinium have a similar polybrachykinety arrangement, distinct from that of other ophryoscolecid ciliates and intermediate between those of O. gracile and 0. damaliscus. Buccal infraciliature is composed of three polybrachykineties, a kinety loop, and paralabial kineties. Small dorso-adoral polybrachykinety, slender vestibular polybrachykinety, and kinety loop are characteristic. Dorsal infraciliature consists of the dorsal polybrachykinety that extends laterally along the dorsal side of the body. Morphogenesis is different from that of O. gracile, because the right end of ventral primordium extends dorsally to grow into a right primordium without separation.     

采用未培养技术对荷斯坦奶牛瘤胃细菌多样性进行初步分析

采用未培养(Culture independent)技术直接从荷斯坦奶牛瘤胃液中提取瘤胃细菌微生物混合DNA(也叫元基因组DNA),利用细菌16SrDNA通用引物27F与1492R,扩增瘤胃混合微生物的16SrDNA,根据16SrDNA序列对瘤胃细菌多样性进行初步分析。通过16SrDNA序列同源性分析,发现有多于一半以上的序列与可培养的菌株的同源性小于90%,属于不可培养的菌株。选用45条测得序列与已知序列构建系统发育树,分析结果表明,它们分属于两大类LGCGPB(the lowG CGram positivebac teria)和CFB(Cytophaga_Flexibacter $CBacteroides group),剩下的克隆尚难确定其分类地位,可能是代表新属和种的序列,这些序列已向GenBank提交并得到序列号(AY986777_AY986791)。     

Zooxanthellae Harvested by Ciliates Associated with Brown Band Syndrome of Corals Remain Photosynthetically Competent

Brown band syndrome is a new coral affliction characterized by a local accumulation of yet-unidentified ciliates migrating as a band along the branches of coral colonies. In the current study, morphologically intact zooxanthellae (= Symbiodinium) were observed in great numbers inside the ciliates (>50 dinoflagellates per ciliate). Microscale oxygen measurements and variable chlorophyll a fluorescence analysis along with microscopic observations demonstrated that zooxanthellae within the ciliates are photosynthetically competent and do not become compromised during the progression of the brown band zone. Zooxanthellae showed similar trends in light acclimation in a comparison of rapid light curve and steady-state light curve measures of variable chlorophyll a fluorescence. Extended light exposure of steady-state light curves resulted in higher quantum yields of photosystem II. The brown band tissue exhibited higher photosynthetically active radiation absorptivity, indicating more efficient light absorption due to a higher density of zooxanthellae in the ciliate-dominated zone. This caused relatively higher gross photosynthesis rates in the zone with zooxanthella-containing ciliates compared to healthy coral tissue. The observation of photosynthetically active intracellular zooxanthellae in the ciliates suggests that the latter can benefit from photosynthates produced by ingested zooxanthellae and from photosynthetic oxygen production that alleviates diffusion limitation of oxic respiration in the densely populated brown band tissue. It remains to be shown whether the zooxanthellae form a stable symbiotic association with the ciliate or are engulfed incidentally during grazing on coral tissue and then maintained as active inside the ciliate for a period before being digested and replaced by new zooxanthellae.     

The Cytology of Sheep Rumen Ciliates. I. Ultrastructure of Epidinium caudatum Crawley1

Epidinium caudatum has an anterior vestibulum containing the adoral zone syncilia (AZS) on an extrusible peristome. The cytopharyngeal structures include a funnel-shaped arrangement of nematodesmata, longitudinal and transversely oriented microtubular ribbons all of which are located in the peristome, a structure which also contains filamentous phagoplasm. The origins of the microtubular ribbons indicate affinities to the rhabdos type of cytopharynx. The peristomal base is continuous with the tubular esophagus, the region connecting the two being ensheathed by a fibrous layer and low density cytoplasm. The esophagus has a microtubular/microfilamentous wall. A distinct cytoproct with associated myonemal structures occurs posteriorly. The skeletal plates consist of a large number of interconnected, variably shaped platelets and may have dual skeletal and storage functions. The endoplasm is more vesicular than the ectoplasm, the two separated by a fibrous boundary layer. The five-layered cortex has an external glycocalyx, a plasma membrane with two subtending membranes, homogeneous, microtubular, and microfilamentous layers. The syncilia of the AZS are mounted in a U-shaped band on the peristome with transversely oriented kinetics consisting of kinetosomes linked by a sub-kinetosomal rod. There is a bifurcated kinetodesma, dense support material forming a lateral spur with associated transverse microtubules, and postciliary, interkinetal, and occasional basal microtubules, nematodesmata, and a subciliary reticulum. A barren, possibly vestigial, somatic infraciliature consists of non-ciliated kinetosomes and a basal striated fiber with associated basal and perpendicular (cortical) microtubules.     

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.