Strains Of Streptococcus Bovis Research Articles

Immunological cross-reactivity between the vaccine and other isolates of Streptococcus bovis and Lactobacillus.

Recent studies have showed that immunisation with Streptococcus bovis (Sb-5) and Lactobacillus (LB-27) may confer protection against lactic acidosis in sheep and cattle. The present study was designed to determine the degree of immunological cross-reactivity between Sb-5 and eight other strains of Streptococcus bovis; and between LB-27 and four other isolates of Lactobacillus. The cross-reactivity index (CRI, a low CRI indicates a high degree of immunological cross-reactivity) ranged from 7.3 to 56.1% between the strains of S. bovis (the encapsulated strains with CRIs ranging from 7.3 to 12.4%). For isolates of Lactobacillus the CRIs ranged from 11.5 to 72.2%. The results indicate that all the isolates tested have a certain degree of immunological homology with Sb-5 and LB-27, and suggest that the vaccine may cross-react with a large number of strains of S. bovis and Lactobacillus which may cause lactic acidosis. As most of the S. bovis strains in the rumen are encapsulated, the high degree of homology between Sb-5 and encapsulated S. bovis strains further suggests that the vaccine containing Sb-5 may be effective against a wide range of strains of S. bovis in sheep and cattle.

Genetic homogeneity and phage susceptibility of ruminal strains of Streptococcus bovis isolated in Australia.

The genetic homogeneity of 37 strains of ruminal streptococci was investigated by comparing DNA fragment profiles on agarose gels following restriction endonuclease digestion with Hae III, Cfo I and Msp I. Thirty strains were indistinguishable from Streptococcus bovis strains, 2B, H24 and AR3. The remaining three strains were similar but not identical to a ruminal strain of Strep. intermedius (AR36). In addition, the susceptibility of these strains to infection by five bacteriophages was examined. Three of the phages (phi Sb02, phi Sb03 and phi Sb04) were specific to the strain of Strep. bovis from which they were isolated, while phages 2BV and phi Sb01 infected one and two strains, respectively, in addition to their primary host. It was concluded that although Strep. bovis is relatively homogeneous genetically, broad host range phages appear to be uncommon with this bacterial species.

Hydration of linoleic acid by bacteria isolated from ruminants.

Two strains of Enterococcus faecalis isolated from the ovine rumen and known to hydrate oleic acid were shown to transform linoleic acid by hydration into two products. The products, identified as 10-hydroxy-12-octadecenoic acid and 13-hydroxy-9-octadecenoic acid, were formed during stationary phase in yields of 13% and 6% respectively. Yields increased to 22% and 14% when culture conditions were optimised. To our knowledge, this is the first report of 13-hydroxy-9-octadecenoic acid production by bacteria. During a search for further linoleic-acid-hydrating bacteria, a strain of Streptococcus bovis isolated from bovine faeces and the ruminal strain S. bovis JB1 were found to hydrate linoleic acid. Both strains formed only one product and the most rapid appearance occurred during exponential growth. The S. bovis product, identified as 13-hydroxy-9-octadecenoic acid, formed in a yield of 28%. This study provides the first information on linoleic acid hydration by ruminal bacteria.

A bacteriocin-mediated antagonism by Enterococcus faecium BC25 against ruminal Streptococcus bovis

A bacteriocin-like activity produced by Enterococcus faecium BC25, isolated from the rumen of a cow, was partially purified and characterized. The active substance was prepared by ammonium sulfate and chloroform/methanol precipitation of culture supernatant. The bacteriocin was a protein of molecular mass 17.5 kDa. Activity was inactivated by trypsin and proteinase K. The bacteriocin BC25 inhibited growth of amylolytic ruminal strains of Streptococcus bovis, including S. bovis AO 24185. Results showed that bacteriocine substance BC25 has a bacteriostatic effect when present in concentrations exceeding 125 AU/ml. Agar overlays and batch culture growth experiments proved that E. faecium BC25 was producing bacteriocin that inhibited the growth of S. bovis.

Phenotypic and phylogenetic characterization of ruminal tannin-tolerant bacteria.

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.

The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis.

Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 +/- 0.12 h-1) then than on glucose (1.67 +/- 0.12 h-1), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less intracellular fructose 1,6-bisphosphate (Frul,6P2) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Frul,6P2. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h-1) of S. bovis.

Bile-Esculin Test for Presumptive Identification of Enterococci and Streptococci: Effects of Bile Concentration, Inoculation Technique, and Incubation Time

The bile-esculin test is used to differentiate enterococci and group D streptococci from non-group D viridans group streptococci. The effects on test performance of the concentration of bile salts, inoculum, and duration of incubation were examined with 110 strains of enterococci, 30 strains of Streptococcus bovis, and 110 strains of non-group D viridans group streptococci. Optimal sensitivity (> 99%) and specificity (97%) of the bile-esculin test can be obtained with a bile concentration of 40%, a standardized inoculum of 10(6) CFU, and incubation for 24 h.

Lack of surface receptors not restriction-modification system determines F4 phage resistance in Streptococcus bovis II/1.

The resistance of Streptococcus bovis strain II/1, the producer of SbvI restriction endonuclease, to F4 phage infection was demonstrated by the double-agar-layer method. Despite the presence of restriction endonuclease SbvI which can cleave F4 phage DNA to numerous fragments in vitro, the evidence that adsorption inhibition is the most important defence mechanism in phage resistance of S. bovis II/1 strain was obtained by adhesion experiments in vivo. Electron microscopy of phage-host mixtures showed many phage particles on the bacterial surface of phage-sensitive S. bovis 47/3 control strain in comparison with no phage particles seen on S. bovis II/1 (phage-resistant) strain surface.

Extracellular proteins and virulence in Streptococcus bovis isolates from pigeons

The association between virulence and the occurrence of the extracellular proteins A, T1, T2 and T3 in the culture supernatant of pigeon Streptococcus bovis strains, was examined in experimental infection studies. Fourteen groups of 10–17 pigeons were inoculated intravenously with 1 × 10 9 CFU of S. bovis strains that belonged to the phenotypes A +T1, A −T1, A +T2, A −T2, A +T3 and A −T3, respectively. The overall postinoculation morbidity in the phenotype groups was 85%, 87%, 70%, 5%, 100% and 37%, respectively. These results indicate that strains producing A or T1 are of high virulence, those producing T3 only are of moderate virulence and those producing T2 are of low virulence. Virulence of S. bovis for pigeons was more clearly correlated with supernatant-phenotype than with serotype.

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

Lactate dehydrogenase (LDH) was purified from three strains of Streptococcus bovis, and the gene ldh was cloned and sequenced. The ldh of S. bovis from a goat (TH1) was different from the ldhs of two other strains from cattle (TH2, JB1) in that Asp(220) was substituted for Glu. Northern blot analysis revealed that the LDH-mRNA of S. bovis was approximately 1.0 kbp, which was transcribed in a monocistronic fashion. When cells were grown at pH 6.9 in a batch culture, the level of ldh transcript decreased as the growth phase changed; from exponential growth to the cessation of growth. The level of ldh transcript was higher in cells grown at pH 4.5 than at pH 6.9. This observation was consistent with the amounts of LDH in cells and the percentages of lactate produced. These results support the hypothesis that S. bovis regulates LDH synthesis at the transcriptional level probably in response to intracellular pH.

Secreted antigens as virulence associated markers in Streptococcus bovis strains from pigeons

SDS-PAGE and Western blot analysis were performed on the culture supernatant of 7 pigeon S. bovis reference strains belonging to the serotypes 1, 2, 3 and 5. The culture supernatant of highly virulent serotype 1, 2 and 5 strains contained four antigens that were absent in low virulent serotype 3 strains, notably a 185 kDa minor protein band (A) and a triplet (T1) of 70 kDa. The less virulent serotype 3 strains on the other hand contained protein triplets, that had a molecular mass of either 68 kDa (T2) or 74 kDa (T3). The prevalence of A, T1, T2 and T3 was examined in 68 S. bovis strains isolated from healthy pigeons and in 68 S. bovis strains isolated from pigeons that died of S. bovis septicaemia. Six supernatant phenotypes were identified: A − T1 (32 strains), A − T2 (10 strains), A − T3 (7 strains), A + T1 (84 strains), A + T2 (1 strain) and A + T3 (2 strains). Ninety-four percent of the strains lacking the A and T1 proteins were isolated from healthy pigeons, and only 6% were isolated from septicaemia. Strains expressing A and/or T1, however, were isolated from septicaemia in 57% of the cases. These observations may indicate that the A and/or T1 proteins are associated with virulence.

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle.

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.

Effects of a strain of Saccharomyces cerevisiae (Levucell SC1), a microbial additive for ruminants, on lactate metabolism in vitro.

The effect of Levucell SC, a strain of Saccharomyces cerevisiae marked as a feed additive for ruminants, was investigated in vitro on lactate metabolism by the ruminal bacteria Streptococcus bovis and Megasphaera elsdenii. The coculture between 10(7) live cells x mL(-1) of SC and a Streptococcus bovis strain in the presence of glucose reduced lactate production by the bacterial strain. Live yeast cells were able to compete with Streptococcus bovis for glucose utilization in strictly anaerobic conditions, so less glucose was available for the bacterium. SC also stimulated L-lactate utilization by a strain of M. elsdenii. The effect depended on the concentration of yeast cells added. Bacterial growth and fermentation end-product concentrations were also increased in the presence of SC. Some amino acids and vitamins, but not dicarboxylic acids, stimulated the bacterial specific activity of L-lactate uptake. SC was able to provide amino acids to M. elsdenii. In a coculture of Streptococcus bovis and M. elsdenii on glucose, the reduction of lactate concentration was improved by SC, the same trend being observed when maltose or soluble starch were used as carbon and energy source. These results indicate that SC can be a very useful tool to reduce lactate accumulation in vitro during fermentation of soluble sugars.

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes.

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains (K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4-38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.

Intracellular survival and multiplication of virulent and less virulent strains of Streptococcus bovis in pigeon macrophages

The intracellular fate of pigeons S. bovis strains ingested by macrophages was studied in vivo and in vitro. During in vivo experiments, histological and electron microscopical examinations demonstrated numerous cocci, which appeared to be actively multiplying, within splenic macrophages of pigeons experimentally inoculated with a highly virulent S. bovis serotype 1 strain. In pigeons inoculated with a low virulence serotype 3 strain, intracellular cocci were only occasionally observed. For in vitro experiments, pigeon peritoneal macrophages were inoculated with a S. bovis serotype 1 or serotype 3 strain and incubated. Following an initial decrease, an increase in the number of intracellular bacteria was observed in tests performed with the S. bovis serotype 1 strain, demonstrating intracellular multiplication. Macrophages in these experiments had all died after 7 h of incubation, possibly indicating that the intracellular replication of S. bovis resulted in the release of substances toxic for macrophages. In experiments performed with the S. bovis serotype 3 strain, the number of intracellular bacteria continuously decreased, reflecting killing of organisms. Significant changes in the number of adhering macrophages in S. bovis serotype 3 inoculated cultures were not observed. These results indicate S. bovis in pigeons is a facultative intracellular bacterium and intracellular multiplication may be involved in virulence.

Identification of proteolytic rumen bacteria isolated from New Zealand cattle.

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.

Location of the alpha-amylase gene in rumen Streptococcus bovis strains distinguished by unstable amylase activity.

Genetic stability of amylase activity after serial subcultivation experiments with amylolytic ruminal Streptococcus bovis strains was investigated. Two strains Amy+ and Amy- were obtained. Loss of amylase activity connected with the loss of plasmid DNA was not found in these strains. The presence of the gene responsible for the amylase activity in the chromosome of these strains was revealed by hybridization of the alpha-amylase gene on pJK108 against chromosomal DNA of S. bovis and Bacillus subtilis after a complete restriction with EcoRI.

The adherence of three Streptococcus bovis strains to cells of rumen epithelium primoculture under various conditions.

Three Streptococcus bovis strains were tested in biotype assay and examined for the adherence to cells of rumen epithelium primoculture. The adherence pattern of ruminal streptococci in phosphate buffered saline at pH values ranging from 4.1 to 8.5 was determined. Our isolates of Streptococcus bovis strains adhered best at pH 7.0-7.3. To characterize the adhesive determinants, the bacterial cells were exposed to various treatments. Protease treatment dramatically decreased the adherence of all Streptococcus bovis strains, thus suggesting that the determinants responsible for the adherence are largely proteinaceous. Carbohydrates could be also significantly involved in the active sites of bacterial surface because metaperiodate-treated cells adhered much more poorly than control, sodium iodate-treated cells. Addition of carbohydrates (lactose, maltose and saccharose) had no significant effect on the adherence of Streptococcus bovis strains although a slight decrease in the adhesion was detected.

Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).

Use of transposon Tn916 as a genetic marker in the rumen

Streptococcus bovis strain SB3 was genetically marked by conjugal transfer of the tetracycline-resistant transposon, Tn916, from Enterococcus faecalis to Strep. bovis. The transposon was stable in the Strep. bovis chromosome in the presence or absence of tetracycline. Streptococcus bovis: Tn916 was introduced into the rumen of experimental sheep and was maintained for at least 76 d. The population was stable in the presence of a grain-based ration but rapidly declined when sheep were transferred to pasture. On return to the grain-based diet, the Strep. bovis: Tn916 population reappeared. These data demonstrate the potential of this technique in studies of microbial interactions in the rumen.