Condensed tannins in forage legumes improve the nutrition of sheep by reducing ruminal degradation of plant protein and increasing crude protein flow to the intestine. However, the effects of condensed tannins in forage legumes on rumen bacterial populations in vivo are poorly understood. The aim of this study was to investigate the specific effects of condensed tannins from Lotus corniculatus on four proteolytic rumen bacteria in sheep during and after transition from a ryegrass (Lolium perenne)-white clover (Trifolium repens) diet (i.e., low condensed tannins) to a Lotus corniculatus diet (i.e., higher condensed tannins). The bacterial populations were quantified using a competitive polymerase chain reaction. Lotus corniculatus was fed with or without ruminal infusions of polyethylene glycol (PEG), which binds to and inactivates condensed tannins, enabling the effect of condensed tannins on bacterial populations to be examined. When sheep fed on ryegrass-white clover, populations of Clostridium proteoclasticum B316T, Butyrivibrio fibrisolvens C211a, Eubacterium sp. C12b, and Streptococcus bovis B315 were 1.5 x 10(8), 1.1 x 10(6), 4.6 x 10(8), and 7.1 x 10(6) mL(-1), respectively. When the diet was changed to Lotus corniculatus, the average populations (after 8-120 h) of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis decreased (P < 0.001) to 2.4 x 10(7), 1.1 x 10(5), 1.1 x 10(8), and 2.5 x 10(5) mL(-1), respectively. When PEG was infused into the rumen of sheep fed Lotus corniculatus, the populations of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis were higher (P < 0.01-0.001) than in sheep fed Lotus corniculatus without the PEG infusion, with average populations (after 8-120 h) of 4.9 x 10(7), 3.8 x 10(5), 1.9 x 10(8), and 1.0 x 10(6), respectively. Sheep fed the Lotus corniculatus diet had lower rumen proteinase activity, ammonia, and soluble nitrogen (P < 0.05-0.001) than sheep that were fed Lotus corniculatus plus PEG. The Lotus corniculatus diet reduced rumen nitrogen digestibility (P < 0.05) and ammonia pool size and increased the flow of undegraded feed nitrogen to the abomasum. The nitrogen intake, rumen non-ammonia nitrogen pool size, rumen microbial non-ammonia nitrogen pool size, and abomasal microbial non-ammonia nitrogen fluxes were similar both in sheep fed only Lotus corniculatus and in sheep fed Lotus corniculatus plus PEG, but nonmicrobial non-ammonia nitrogen flux to the abomasum was higher (P < 0.01) for the sheep fed only Lotus corniculatus. Although condensed tannins in Lotus corniculatus reduced the populations of some proteolytic bacteria, total ruminal microbial protein and microbial protein outflow to the abomasum were unchanged, suggesting a species-specific effect of condensed tannins on bacteria in the rumen.

A novel proteolytic bacterium was isolated from rumen contents of New Zealand cattle grazing fresh forage and was designated strain B316T (T = type strain). Strain B316T cells were straight to slightly curved rods (width, 0.4 to 0.6 pm; length, 1.3 to 3.0 pm) that were gram-positive and possessed a single subterminal flagellum. This isolate did not produce catalase, indole, ammonia, lipase, or lecithinase or reduce nitrate, but it did produce a curd reaction with milk. Strain B316T was proteolytic, hydrolyzing casein and fraction I leaf protein. The crude proteinase was predominantly the serine type, but some cysteine proteinase and metalloproteinase activities were also detected. The DNA base composition of strain B316T was 28 mol% G+C. A 16s ribosomal DNA sequence analysis of strain B316T indicated that it was most closely related to a member of clostridial cluster XIVa, viz., Clostridium aminophilum, an amino acid-fermenting organism isolated from the rumen; the similarity value was 92.2%. The results of the phenotypic characterization analysis, G+C content analysis, and phylogenetic analysis of the 16s ribosomal DNA sequence set strain B3MT apart from all of the members of cluster m a . We propose that strain B316T should be designated a new species of the genus Clostridium, Clostridiumproteochticum. Strain B316 is the type strain and has been deposited in the American Type Culture Collection as strain ATCC 51982.In New Zealand ruminants, the breakdown of plant protein in the rumen can lead to the loss of up to 50% of the available protein (14) and therefore to the inefficient use of pasture nitrogen. Bacteria are the most active proteolytic organisms in the rumen (2, 18), and their populations are influenced by the diet of the animal and the physical form of the feed. Many rumen bacteria are able to degrade protein, and members of the genera Prevotella, Ruminobacter, Selenomonas, Butyn'vibrio, and Streptococcus are commonly identified as being proteolytic. More recently, members of the genera Peptostreptococcus and Clostridium have been identified as being important in peptide and amino acid fermentation in the rumen (6, 7, 20). In a recent study of the proteolytic bacteria present in New Zealand cattle, species belonging to the genera Streptococcus, Eubacterium, and Butyrivibrio were identified as important members of the proteolytic flora (1). One highly proteolytic strain, strain B3MT (T = type strain), was initially identified as a Butyrivibrio-like organism. However, further phenotypic characterization and a phylogenetic analysis of the 16s ribosomal DNA (rDNA) sequence of this strain identified it as a member of a new species of the genus Clostridium. We propose that this organism should be named Clostridiurn proteoclasticum sp. nov. MATERLALS AND METHODSIsolation and characterization of strain B316T. Strain B316T was isolated from a rumen sample from a cow grazing in a fresh pasture as described previously (1). Unless otherwise indicated, all procedures were carried out anaerobically in Hungate tubes...

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To investigate the role of mannose-binding lectin-A (MBL-A) in protection against infectious disease, MBL-A−/−-deficient mice were generated. Using a well-characterized mouse model of human filarial nematode infection, nematode survival and protective immune responses were tested in vivo. Blood-borne Brugia malayi microfilariae survived for significantly longer time periods in MBL-A−/− than in wild-type (WT) mice. However, no differences in either splenic cytokine responses or induction of leukocytes in the blood were observed. A profound abrogation of Ag-specific IgM levels was measured in B. malayi-infected MBL-A−/− mice, and some IgG isotypes were higher than those observed in WT animals. To establish whether there was a defect in Ab responses per se in MBL-A−/− mice or the effect was specific to filarial infection, we immunized these mice with OVA or a carbohydrate-free protein. Significantly, Ag-specific IgM responses were defective to both of these Ags, and Ag-specific IgG responses were largely unaffected. Furthermore, in naive mice, total IgM levels did not differ between MBL-A−/− and WT mice. This article describes the first demonstration that MBL-A may function independently of MBL-C and suggests that MBL-A, like other C-type lectins and members of the complement cascade, is intimately involved in the priming of the humoral Ab response.

The protease activities of 212 strains of rumen bacteria isolated from New Zealand cattle grazing pasture were measured. Thirty-seven per cent of strains had activity greater than or equal to the proteolytic rumen bacterium Prevotella ruminicola and 43 of these isolates were identified by morphology, carbon source utilization, Gram stain, biochemical tests and fermentation end-product analysis. Hierarchical Cluster Analysis showed that the strains formed four clusters: cluster A contained 26 strains and clustered with a reference strain of Streptococcus bovis; cluster C contained three strains and clustered with a reference strain of Butyrivibrio fibrisolvens, while clusters B (10 strains) and D (three strains) did not cluster with any of the remaining rumen bacterial type strains. Further tests identified strains of cluster B as Eubacterium budayi, while cluster D strains most closely resembled B. fibrisolvens and were described as B. fibrisolvens-like. An unclustered strain, C21a, was identified as P. ruminicola. The significance of these proteolytic bacterial populations is discussed in relation to protein breakdown in New Zealand ruminants.