The diversity of methanogens in the rumen of sheep fed three different diets was examined using denaturing gradient gel electrophoresis (DGGE). In addition, half of the sheep had pot scrubbers added to their rumen to increase flow rate. The methanogens were separated by a series of washing steps into three populations: free-living, ecto-symbiotic and endo-symbiotic.Preliminary DGGE banding patterns demonstrated considerable differences between populations, treatment groups, and within groups. This suggests that the diversity of methanogens is influenced by diet, flow rates of digesta and the niche they occupy in relation to the protozoa.

Ruminant tissues and products contain conjugated linoleic acids (CLA) due to biohydrogenation in the rumen.We hypothesize that kangaroos would have higher concentrations of CLA compared to lambs due to incomplete biohydrogenation of fatty acids in the kangaroo foregut.Fatty acid composition of adipose tissue (including cis 9, trans 11 CLA) from lambs and kangaroos were signifi cantly different.The concentrations of CLA and its precursor trans vaccenic acid (TVA) in the adipose tissue of kangaroos were approximately four and fi ve times that of lambs.Kangaroo fat was signifi cantly less saturated and had a lower melting point.

This work was conducted to determine if methane emissions from sheep immunized with an anti-methanogen vaccine were significantly lower than methane emissions from non-immunized sheep, to test the effectiveness of two different vaccine formulations (VF) on methane abatement, and to compare methane emissions measured using a closed-circuit respiration chamber and the sulphur-hexafluoride (SF6) tracer technique. Thirty mature wether sheep were randomly allocated to three treatment groups (n = 10). One group received an immunization of adjuvant only on days 0 and 153 (control), a second group received an immunization with a 3-methanogen mix on days 0 and 153 (VF3 + 3), and a third group received an immunization of a 7-methanogen mix on day 0 followed by a 3-methanogen mix on day 153 (VF7 + 3). Four weeks post-secondary immunization, there was a significant 7.7% reduction in methane production per kg dry matter intake in the VF7 + 3 group compared to the controls (P = 0.051). However, methane emissions from sheep immunized with VF7 + 3 were not significantly different when compared to the sheep in the control group (P = 0.883). The average IgG and IgA antibody titres in both plasma and saliva of the VF3 + 3 immunized sheep were four to nine times higher than those immunized with VF7 + 3 (P< 0.001) at both 3 and 6 weeks post-secondary immunization. Data also revealed that SF6 methane estimates were consistently higher than the respiration chamber estimates and that there was no significant correlation between the SF6 methane estimates and the respiration chamber methane estimates (R2 = 0.11).

ABSTRACT 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 relationships within the kinetoplastid flagellates were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included 5 new gene sequences, Trypanosoma fallisi (2,239 bp), Trypanosoma chattoni (2,180 bp), Trypanosoma mega (2,211 bp), Trypanosoma neveulemairei (2,197 bp), and Trypanosoma ranarum (2,203 bp). Trees produced using maximum-parsimony and distance-matrix methods (least-squares, neighbor-joining, and maximum-likelihood), supported by strong bootstrap and quartet-puzzle analyses, indicated that the trypanosomes are a monophyletic group that divides into 2 major lineages, the salivarian trypanosomes and the nonsalivarian trypanosomes. The nonsalivarian trypanosomes further divide into 2 lineages, 1 containing trypanosomes of birds, mammals, and reptiles and the other containing trypanosomes of fish, reptiles, and anurans. Among the giant trypanosomes, T. chattoni is clearly shown to be distantly related to all the other anuran trypanosome species. Trypanosoma mega is closely associated with T. fallisi and T. ranarum, whereas T. neveulemairei and Trypanosoma rotatorium are sister taxa. The branching order of the anuran trypanosomes suggests that some toad trypanosomes may have evolved by host switching from frogs to toads.

Cryptocaryon irritans, a ciliated protozoan, is one of the most devastating parasites of marine fish cultured in temperate and tropical seas. Based on the remarkable affinities with its "freshwater counterpart" Ichthyophthirius multifiliis, C, irritans has traditionally been included in the Ichthyophthiriidae, although cytological studies failed to show ultrastructural features to warrant its inclusion in this family. We sequenced the complete 18S rRNA gene (1,765 nucleotides) from a C, irritans isolate from the Red Sea. Our phylogenetic analyses unequivocally show that C. irritans is taxonomically distinct from I. multifiliis (15.4% divergence) and justify C, irritans' inclusion into the order Prorodontida within the Class Prostomatea. Cryptocaryonidae is proposed as a new family name.

The knowledge of what happens to the diversity of methanogens in the rumen of sheep when the fermentation pattern changed is limited. Many studies have shown that changing rumen fermentation leads to a change in the amount of methane produced (Baker 1999). Monensin is an ionophore that has a wide range of effects on rumen fermentation (Bergen and Bates 1984). The aim of this experiment was to examine whether monensin changes the diversity of methanogens in the rumen of sheep.

The genus Paramecium includes species that are well known and very common in freshwater environments. Species of Paramecium are morphologically divided into two distinct groups: the "bursaria" subgroup (foot-shaped) and the "aurelia" subgroup (cigar-shaped). Their placement within the class Oligohymenophorea has been supported by the analysis of the small subunit rRNA gene sequence of P. tetraurelia. To confirm the stability of this placement and to resolve relationships within the genus, small subunit rRNA gene sequences of P. bursaria, P. calkinsi, P. duboscqui, P. jenningsi, P. nephridiatum, P. primaurelia, and P. polycaryum were determined and aligned. Trees constructed using distance-matrix, maximum-likelihood, and maximum-parsimony methods all depicted the genus as a monophyletic group, clustering with the other oligohymenophorean taxa. Within the Paramecium clade, P. bursaria branches basal to the other species, although the remaining species of the morphologically defined "bursaria" subgroup do not group with P. bursaria, nor do they form a monophyletic subgroup. However, the species of the "aurelia" subgroup are closely related and strongly supported as a monophyletic group.

Peniculine ciliates have been recognized as a distinct higher taxon of ciliates for almost 50 years. However, phylogenetic relationships within the Subclass Peniculia are still unsettled. To contribute to our understanding of their phylogeny and provide evidence for the position of Urocentrum turbo, we sequenced its small subunit (SS) rRNA gene and the SSrRNA genes from Lembadion bullinum, Frontonia sp., Paramecium caudatum, Paramecium multimicronucleatum, Paramecium putrinum, and Paramecium woodruffi. Urocentrum turbo was the only one of these species not to exhibit a shortened Helix E10_1, which we conclude characterizes the "higher" peniculines. Except for U. turbo, the peniculines are strongly supported as a monophyletic clade with Lembadion, Frontonia, and Paramecium species forming separate and strongly supported clades by bootstrap analysis using distance matrix, maximum parsimony, and maximum likelihood methods. Urocentrum turbo is associated with different lineages, depending upon the analysis used. The Paramecium species form at least four clades with the Paramecium aurelia subgroup being the most derived. We conclude that the Subclass Peniculia should be divided into two orders, the Order Urocentrida and Order Peniculida, with the latter order having two suborders, the Suborder Frontoniina and Peniculina. We place U. turbo with the peniculines because of shared morphological and stomatogenetic features.

The small subunit rRNA (SSrRNA) genes were sequenced for Protocruzia sp2, Phacodinium metchnicoffi, Holosticha multistylata, and Halteria grandinella. All four genera are placed within the Class Spirotrichea with strong bootstrap support in both distance matrix and parsimony tree construction methods and by maximum likelihood analysis using quartet puzzling. Protocruzia sp2 groups with Protocruzia sp1 with 100% bootstrap support, and the 5.6% genetic difference between them strongly argues that they are different species although they are morphologically quite similar. The Protocruzia species branch first in the spirotrich clade at a deep level, supporting their recognition as a Subclass Protocruziidia. Phacodinium branches after Euplotes at a deep level, confirming the conclusion that others have reached that this genus is related to the hypotrich and stichotrich spirotrichs. Phacodinium is assigned to its own family and order, and we conclude that the deep branching within the spirotrichs argues for its own subclass, the Subclass Phacodiniidia. Consistent with the partial SSrRNA sequences and with the sequence of a polymerase gene, Halteria groups within the stichotrich clade, supporting the argument that the oligotrichs are not monophyletic as currently conceived. Finally, Holosticha, which has been assigned to the Order Urostylida, groups outside the stichotrichs with parsimony analysis, which is consistent with this ordinal assignment. However, it is associated with Halteria and Oxytricha granulifera in the other analyses. Additional stichotrich sequences obviously are required before we can confidently begin revision of the Subclass Stichotrichia.

An approximately 10 yr-old male Dalmation was admitted to the Exeter Animal Hospital presenting symptoms of continuous urination, polyuria/polydypsia, and regurgitation. Urinalysis showed glucosuria, pyuria, proteinuria, casts, and white blood cells. Microscopic examination of urine revealed considerable numbers of a ciliated protozoan. The ciliate was isolated and aseptically cultured in protease peptone medium. Cytological staining of cells with the Chatton-Lwoff silver nitrate and silver proteinate procedures demonstrated that the ciliate was a species of the genus Tetrahymena, measuring about 50 x 25 mum, placing it within the "pyriformis" species complex. Polymerase chain reaction amplification of the small subunit rRNA (SSrRNA) gene followed by DNA sequence analysis confirmed this identification. Analysis of the complete SSrRNA gene demonstrated significant differences in primary sequence from all other members of the "pyriformis" species complex and justified the designation of a new species, Tetrahymena farleyi sp. n.

The internally transcribed spacer regions 1 and 2 (ITS-1 and ITS-2) and the 5.8S ribosomal RNA gene of Isotricha prostoma were examined for intraspecific sequence variation. There were no differences in the ITS-1/5.8S/ITS-2 region among cattle and sheep isolates of I. prostoma from Australia, Canada, and the United States, indicating that this region is 100% conserved among eight isolates from two continents.

Phylogenetic relationships within the kinetoplastid flagellates were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included three new gene sequences from Cryptobia bullocki, (2091 bp), Cryptobia catostomi (2090 bp), and Cryptobia salmositica (2091 bp). Trees produced using maximum parsimony and distance-matrix methods (least squares and neighbor-joining) demonstrated with strong bootstrap support, that the kinetoplastids are a monophyletic group divided into two major lineages consistent with the two suborders, Trypanosomatina and Bodonina. Within the trypanosomatid clade, the genus Trypanosoma is a monophyletic group that divides into two groups, the salivarian trypanosomes and the stercorarian trypanosomes. Dimastigella and Rhynchobodo, currently classified in the Bodonina, are basal to the trypanosomatid-bodonid clade, suggesting that the suborder Bodonina is paraphyletic. Further, Trypanoplasma borreli grouped within the Cryptobia clade, and was more closely related to C. salmositica than to either C. bullocki or C. catostomi. This new molecular evidence, coupled with morphological similarities of the two genera, again calls into question the validity of the genus Trypanoplasma.

We estimated the rate of nucleotide substitution for the obligate freshwater fish ectoparasite, Ichthyophthirius, and its closest free-living relative, Ophryoglena, using an independently-timed event — the origin of freshwater fish in the fossil record. Based on this information, the rate of nucleotide substitution per site, per year, per lineage is 1.25 to 1.4 × 10−8 or 1% divergence per 72 to 80 million years (My). Using this rate, we determined that the origin of the ciliates (i.e. crown eukaryotes) is much older than previously speculated, dating back to the Paleoproterozoic some 1980 to 2200 million years ago (Ma). We also determined that the wellestablished lineages recognized as classes today (e.g. Spirotrichea, Oligohymenophorea, Nassophorea, Colpodea, Heterotrichea, Karyorelictea, and Litostomatea) diverged within 600-My of the ciliate-like ancestor diverging from the main eukaryotic line.

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.

Phylogenetic relationships within the largest family of entodiniomorphid rumen ciliates, the Ophryoscolecidae, were inferred from comparisons of small-subunit ribosomal RNA gene sequences. These included three new sequences from Diplodinium dentatum (1638 base pairs (bp)), Eudiplodinium maggii (1637 bp), and Ophryoscolex purkynjei (1636 bp). Using morphological characters, Lubinsky constructed a cladogram of the Ophryoscolecidae, and on the basis of his analysis, he divided the family into three subfamilies (Entodiniinae, Diplodiniinae, Ophryoscolecinae) to reflect his "natural" groupings (G. Lubinsky. 1957. Can. J. Zool. 35: 141 – 159). Our cladistic analysis, based on the limited morphological and ultrastructural data available, indicates that there are no synapomorphies supporting the Diplodiniinae sensu Lubinsky. However, based upon the six 18S sequences for the Ophryoscolecidae, the rumen ciliates are monophyletic and fall into three distinct groups corresponding to Lubinsky's subfamilial division of the family. Our molecular analysis shows Entodinium to be the earliest branching rumen ciliate (subfamily Entodiniinae) and Eudiplodinium, not Diplodiium, branching first among the diplodiniines.

Known spectroscopic and kinetic data are used to formulate pathways of the physiological and transfer reactions and the substrate inhibition of phenol sulfotransferase. Kinetic mechanisms indicate that release of PAP from enzyme complex is required for the physiological reaction but not for the transfer reaction. The pathways explain rate difference between the physiological and transfer reactions since the release of PAP is the rate-limiting step of the former reaction. Two enzyme species of phenol sulfotransferase which distinguish the physiological and transfer reaction were found to involve the binding of PAP. Differences between two forms of phenol sulfotransferase, α and β, indicate that they assemble through different folding process. It is demonstrated that only α enzyme renatures in the presence of PAP and β enzyme renatures only in the absence of PAP in vitro. In the over-expressed system, formation of α and β phenol sulfotransferase is also dependent on the availability of PAP in Escherichia coli. It is concluded that folding of phenol sulfotransferase is assisted by PAP to form α enzyme. In the absence of PAP, β form of phenol sulfotransferase is produced.

Phylogenetic relationships within the subclass Hymenostomatia were inferred from the comparisons of three new SSrRNA gene sequences from Ichthyophthirius multifiliis (1,751 bp), Ophryoglena catenula (1,748 bp), and Tetrahymena corlissi (1,753 bp). Using maximum-parsimony and distance-matrix methods, Ichthyophthirius and Ophryoglena were consistently paired and formed a sister group to the tetrahymenines, consistent with their placement in the Ophryoglenina. Tetrahymenids formed a monophyletic group that was divided into main lineages: T. corlissi diverged from the base of the lineage that included T. thermophila.