Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.     

Smooth muscle cells in the rat testicular capsule: a developmental study.

This study of the postnatal development (from 1 to 60 days) of smooth muscle elements in the rat testicular capsule has demonstrated that while such elements are identifiable by light microscopy at 30 days, myocytes are present at birth as seen by electron microscopy. The differentiation of smooth muscle from birth to 30 days has been described, by which time it is of adult morphology and content. Perhaps significantly, it is at 30 days that the testis achieves a scrotal position, although sexual maturity does not occur until about 60 days. Presumably, at 30 days the testicular capsule of the rat is capable of the spontaneous contractions which are known to occur in the adult and which are assumed to aid the transport of non-motile spermatozoa from the testis to the spididymis. The presence of occasional striated muscle fibers in the rat testicular capsule as reported previously has not been confirmed by this investigation, although their possible origin is discussed.     

A 4 year observation of gastrointestinal nematode egg counts,nemabiomes and the benzimidazole resistance genotypes of Teladorsagia circumcincta on a Scottish sheep farm

Anthelmintic resistance threatens the sustainability of sheep production globally. Advice regarding strategies to reduce the development of anthelmintic resistance incorporates the outcomes of modelling exercises. Further understanding of gastrointestinal nematode species diversity, and population dynamics and genetics (which may vary between species) is required to refine these models; and field studies combining faecal egg outputs, species composition and resistance genetics are needed to calibrate them. In this study, faecal samples were taken from ewes and lambs on a commercial farm in south-eastern Scotland at approximately 3 t-4 week intervals between spring and autumn over a period of 4 years. Faecal egg counts were performed on these samples, and L₃ were collected from pooled coprocultures. Deep amplicon sequencing was used to determine both the species composition of these L₃ and the proportions of benzimidazole-resistant single nucleotide polymorphisms in the isotype-1 β-tubulin locus of the predominant species, Teladorsagia circumcincta L₃. Despite consistent management throughout the study, the results show variation in gastrointestinal nematode species composition with time and between age groups, that was potentially associated with weather conditions. The F200Y benzimidazole resistance mutation is close to genetic fixation in the T. circumcincta population on this farm. There was no evidence of variation in isotype-1 β-tubulin single nucleotide polymorphisms frequency between age groups, and no genetic evidence of reversion to benzimidazole susceptibility, despite targeted benzimidazole usage. This study highlights the need to include speciation when investigating gastrointestinal nematode epidemiology and anthelmintic resistance, and serves as an example of how genetic data may be analysed alongside species diversity and faecal egg counts, when markers for other anthelmintic classes are identified.     

A proton and fluorine-19 nuclear magnetic resonance and fluorescence study of the binding of some natural and synthetic thyromimetics to prealbumin (transthyretin)

Interactions between prealbumin and several thyromimetic compounds have been examined by proton magnetic resonance spectroscopy. One equivalent of thyroxine (T4) or reverse triiodothyronine (rT3) selectively broadens a number of protein signals, while addition of a second equivalent induces much less widespread changes. One equivalent of triiodothyronine (T3), however, produces much less dramatic changes, and effects comparable with T4 and rT3 are only apparent when a second equivalent binds. The broadening is ascribed to immobilization of flexible residues. The non-halogenated analogue 3,5-dimethyl-3'-isopropylthyronine induces qualitatively different changes suggesting incomplete entry into the thyromimetic binding channel. The fluorinated analogue SK&F 95049 (3,5-bis-thiotrifluoromethyl-3'-isopropylthyronine) induces very similar changes to T3. A fluorine-19 NMR signal with a half-height line width of approximately 150 Hz can be observed from the bound ligand. Finally, a spin-labeled T4 analogue, with a nitroxyl on the alanyl moiety, induces changes identical to those induced by T4 itself, and additionally broadens some signals corresponding to residues at the opening of the ligand binding channel. The natural tryptophan fluorescence of the protein is shown to be a sensitive indicator of binding. The possible influence of the dynamic restrictions induced by binding the first molecule of T4 or rT3 on the protein's affinity for a second hormone is discussed. It is suggested that the first interaction confers rigidity on the second site and reduces its ability to flex open and accommodate a second thyromimetic, which results in the marked negative co-operativity associated with the occupancy of this site.     

Plasmalemma structure in freezing tolerant unicellular algae

Summary Electron microscopy of several freezing tolerant species of the algal generaChlamydomonas andChloromonas revealed plasmalemma invaginations which are absent from freezing sensitiveChlamydomonas species. The morphology of these invaginations in freezing tolerant strains is described and compared with similar structures in the yeastSaccharomyces cerevisiae.     

The taste of polycose in hamsters

Hamsters show a preference for Polycose, a mixture of starch-derived glucose polymers, that is as strong as their preference for sucrose. However, in the hamster, taste aversions to Polycose may be less easily acquired than taste aversions to sucrose and the qualitative aspects of Polycose are unknown in this species. In order to examine the taste of Polycose in the hamster, we utilized a taste-aversion protocol with two conditioning trials. Animals were trained to avoid one of three different conditioning stimuli: 50 mM sucrose, 100 mM Polycose and a mixture of 50 mM sucrose with 100 mM Polycose. Control animals were conditioned with deionized water. After the second conditioning trial, generalization testing began for the three conditioning stimuli plus 3 mM citric acid, 300 mM KCI and 30 mM NaCl. The results showed that aversions to Polycose, sucrose or the Polycose/sucrose mixture cross- generalized, demonstrating that Polycose and sucrose share a common taste percept in the hamster. None of the aversions generalized to NaCl, citric acid or KCI. In addition, comparisons among the patterns of taste generalizations indicated that the tastes of Polycose and sucrose also had distinct qualitative components. Finally, although the taste of 100 mM Polycose was more salient than the taste of 50 mM sucrose, the taste of sucrose could still be detected in a mixture with Polycose.      

The effects of farrowing nest size and width on sow and piglet behaviour and piglet survival

The ‘Werribee farrowing pen' (WFP) was developed as a loose housing alternative to the farrowing crate. The WFP occupies about twice the space of a crate and comprises two compartments, a ‘nest' and a ‘non-nest' area. In this experiment, we investigated the effects of reducing total pen space by modifying the dimensions of the ‘nest'. The hypothesis was that modifying ‘nest' size and width would not negatively affect piglet survival. A reduction in total floor space in the WFP may increase attractiveness for adoption of the system by pig producers. The experiment had a 2×2 factorial design with nine replicates and a total of 72 primiparous sows (Large White×Landrace) and their litters. All subjects were included to day 4 of lactation, but production data to weaning (day 23) was restricted to 36 litters. We examined the effects of ‘nest' size (large: L vs. small: S) and width (wide: W vs. narrow: N) on sow and piglet behaviours and piglet survival. The W, as compared to N treatment sows, had longer mean bouts of standing in the ‘nest' during 16–8 h pre-farrowing (3.7 vs. 2.0 min, P<0.01), suggesting that pre-farrowing (‘nesting') behaviour may have been adversely affected by narrow nest width. On the first 2 days of lactation, sow suckling grunts occurred more in the W than N treatment (14.1 and 8.0% of observations, P<0.01). However, this was due to an approximately 50% lower incidence in the SN treatment, as compared to the other treatments combined (5.9 and 12.7%). Piglets spent more (P<0.01) time at the udder in L vs. S (41 and 30%) and W vs. N nests (40 and 31%) and performed more (P<0.05) sucking behaviour (i.e., drinking milk from a teat) in L vs. S nests (2.5 and 1.2%). These differences were due to SN treatment litters sucking less than other treatments combined (0.9 vs. 2.2%). Of the 680 piglets born (n=72 litters), 3.8% were stillborn and 6.8% died between birth and day 4 of lactation. From day 5 to weaning (n=36 litters), 3.5% of piglets died, all due to Escherichia coli infection. While there were no effects of treatment on piglet survival, liveborn piglets were less likely (P<0.05) to die from E. coli in the L vs. S treatments (9/24 vs. 23/33 piglets). In conclusion, our results provide evidence that the size and width of the farrowing ‘nest' in a loose farrowing system may effect sow and piglet behaviours that may be relevant to piglet survival.     

D-arabitol metabolism in Candida albicans: construction and analysis of mutants lacking D-arabitol dehydrogenase.

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol + NAD <=> D-ribulose + NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol or D-arabinose medium. Thus, ArDH catalyzes the first step in D-arabitol utilization and a necessary intermediate step in D-arabinose utilization. Unexpectedly, the ard null mutant synthesized D-arabitol from glucose. Moreover, 13C nuclear magnetic resonance studies showed that the ard null mutant and its wild-type parent synthesized D-arabitol via the same pathway. These results imply that C. albicans synthesizes and utilizes D-arabitol via separate metabolic pathways, which was not previously suspected for fungi.