Aspects of selenium metabolism in sheep and selected rumen bacteria

High rates of application of superphosphate lead to lower glutathione peroxidase levels in sheep and higher incidence of nutritional myopathy. There is no convincing evidence that sulphate and selenium interact to the detriment of selenium metabolism in sheep. In this work the possible interaction of selenium and phosphorous is investigated. Sheep fed a low selenium, low phosphate diet and intraruminally infused with either sodium phosphate or sodium chloride developed skeletal or cardiac muscle lesions. The severity of the skeletal muscle lesions was dramatically increased by the intraruminal infusion of sodium phosphate. There was no evidence of skeletal or cardiac muscle lesions in sheep fed diets adequate in selenium and infused with either sodium phosphate or sodium chloride. Glutathione peroxidase activity in the whole blood declined more rapidly in phosphate infused sheep as compared to chloride infused sheep, suggesting that erythrocytes produced subsequent to phosphate infusion contained very small amounts of this seleno-enzyme. Elevated plasma creatine phosphokinase activity was associated with the skeletal muscle lesions in the sheep fed a low selenium, low phosphate diet and intraruminally infused with sodium phosphate. The concentration of selenium in the livers of the sodium phosphate infused sheep was significantly lower than that of the sodium chloride infusea sheep on the same diet. The selenium concentrations of kidney, blood and cardiac and skeletal muscle of sheep fed a low selenium, low phosphate diet and subsequently intraruminally infused with either sodium phosphate or sodium chloride were similar. The glutathione peroxidase activities and selenium concentrations in the whole blood of sheep with nutritional myopathy showed that low levels of these two parameters were not necessarily associated with nutritional myopathy nor reliable indicators of animals at risk of developing nutritional myopathy. A study of the bioavailability of selenium incorporated into rumen bacteria showed that there were no significant differences in the tissue metabolism of 75 [SeJ associated with bacteria (derived from 75 [SeJselenite) , or from inorganic 75 [Se ] selenite or 75 [SeJselenate. The results indicated that there were differences in the metabolism of selenate and selenite by mixed populations of rumen bacteria. 75 [SeJ incorporated into bacterial cells, or that derived from inorganic selenite and selenate did not behave in the same manner after infusion into the rumen. The rate of entry into blood and plasma of 75 [Se] incorporated into bacterial cells "in vitro" was more rapid than 75 [Se] derived from inorganic selenite or selenate. Other isotopic data showed that 75 [Sejselenium infused into the rumen was excreted predominantly in the faeces. Selenium associated with whole blood was predominantly in the plasma TCA precipitate and erythrocytes. Very little 75 [Sejselenium was present in the TCA precipitated plasma supernatant. The majority of selenium in the blood was associated with the plasma and selenium was incorporated more rapidly into the plasma than into the erythrocyte. In older sheep (>G months old) selenium incorporated into the erythrocyte was not associated with an increase in whole blood glutathione peroxidase activity at least in the short term (over 4 weeks). Sheep fed the selenium deficient, low phosphate diet and intraruminally infused with sodium chloride developed osteoporosis, whereas there was no evidence of osteoporosis in sheep fed the same diet and infused with sodium phosphate. A dietary insufficiency of phosphate was implicated in the development of osteoporosis and the urinary excretion of calcium was elevated in the osteoporotic sheep. There are two possible sites for this interaction of phosphorous and selenium — either within the rumen or within the animal. In the first instance, the metabolism of selenium was studied in mixed populations of rumen bacteria and then the study was continued with pure cultures of three selected rumen isolates. A study of the incorporation of selenium into mixed rumen bacteria showed that 75 [Sejselenite was metabolised "in vitro" by mixed populations of rumen bacteria and that seleno-amino acids could be identified in the bacterial extracts. 75 [Sejselenate was not metabolised by rumen bacteria under the same conditions. Selenite uptake and incorporation in Selenomonas ruminantium, Butyrivibrio fibrisolvens, and Bacteroides ruminicola were by constitutive systems. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit uptake, nor could sulphate or selenate uptake be demonstrated in the three species. Selenite uptake had an apparent Km of 1.28 mM, 1.82 mM and 1.71 mM in S. ruminantium, B. fibrisolvens and B. ruminicola respectively. The K values were associated 0.11 and 1.5 /xg Se min 1 (mg protein) 1 respectively in the three species. 75 [Sejselenite by S. ruminantium, B. fibrisolvens and B. ruminicola was sensitive to inhibition m with Vmax values of 0.15, Uptake of by 2,4-dinitrophenol (2,4,DNP), iodoacetic acid (IAA) and N-ethyImaleimide but not chlorpromazine (CPZ) or N,N1-dicyclohexy1-carbodiimide (DCCD). Azide partially inhibited selenite uptake in cells of S. ruminantium and B. fibrisolvens but not in B. ruminicola. Arsenate partially inhibited selente uptake by cells of B. fibrisolvens as did carbonyl cyanide m-chloropheny1 hydrazone (CCCP) in S. ruminantium and fluoride in cells of B. ruminicola. These compounds were not inhibitory to the other species. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate or selenate in cells of S. ruminantium, B. fibrisolvens and B. ruminicola. S. ruminantium and B. fibrisolvens were capable of converting 75 [Se]selenite into seleno-amino acids whereas there was no conversion of selenite into seleno-amino acids by B. ruminicola. B. ruminicola converted selenite into red elemental selenium. S. ruminantium and B. fibrisolvens also produced some red elemental selenium. Clearly these isolated bacteria metabolise selenate and selenite in a manner different to the whole animal. Similarly phosphorous does not affect selenium metabolism in these isolated bacteria. This suggests that although phosphorous does affect selenium metabolism leading to nutritional myopathy, the rumen does not appear to be the primary site of this interaction. Therefore there is a need to look at the effect of phosphorous loading on selenium metabolism in animal tissues. One possibility is to use non-ruminant animals as a model for this effect

Glucose-induced morphological variation in Selenomonas ruminantium

Selenomonas ruminantium (strain I10) isolated from the ovine rumen showed considerable morphological variation and lack of motility when cultured in a phosphate-limited chemostat in the presence of high levels of glucose (55.5 mM). Transmission electron microscopy showed that some of these variants were capable of producing daughter cells with a typical selenomonad morphology but lacking flagella. The reduction of the levels of glucose (27.8 mM) in the media caused the numbers of cells exhibiting variation to decrease, with a corresponding increase in motile cells possessing a typical selenomonad morphology. The removal of trypticase from the media had no effect on the morphology or motility of the cells. During the initial stages of changeover to reduced glucose levels variants could be found in the chemostat which were flagellate. The flagellae were consistently attached to a concave section of the cells

Selenium uptake by Butyrivibrio fibrisolvens and Bacteroides ruminicola

The uptake and incorporation of 75[Se]selenite by Butyrivibrio fibrisolvens and Bacteroides ruminicola were by constitutive systems. Rates of uptake were higher in chemostat culture than in batch culture and there may be some inducible component. Uptake of [75Se]selenite was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit selenite uptake, nor could sulphate or selenate uptake be demonstrated in these organisms. Selenite uptake in B. fibrisolvens had and apparent Km of 1.74 mM and a Vmax of 109 ng Se · min−1· (mg protein)−1. An apparent Km of 1.76 mM and Vmax of 1.5 μg Se · min−1· (mg protein)−1 was obtained for B. ruminicola. [75Se]Selenite uptake by both organisms was partially sensitive to inhibition by 2,4‐DNP. Uptake by B. fibrisolvens was also partially inhibited by azide and arsenate and in B. ruminicola it was partially inhibited by fluoride. CCCP, CPZ, DCCD or quinine did not inhibit uptake in either B. fibrisolvens or B. ruminicola. Selenite transport by both organisms was sensitive to IAA and NEM and was strongly inhibited by sulphite and nitrite. [75Se]Selenite was converted to selenocystine, selenohomocystine and selenomethionine by B. fibrisolvens. B. ruminicola did not incorporate [75Se]selenite into organic compounds, but did reduce it to red elemental selenium

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up 14C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue α-methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K m for α-methyl glucose uptake was 2.9×10-4 M

Selenite uptake and incorporation by Selenomonas ruminatium

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhbit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent Km of 1.28 mM and a Vmax of 148 ng Se min-1 mg-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chloropromazine (CPZ), N,N′-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating 75[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. 75[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium

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 (φSb02, φSb03 and φSb04) were specific to the strain of Strep. bovis from which they were isolated, while phages 2BV and φSb01 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