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4. FLAVELL,R. B., and J. R. S . FINCHAM.1968. Acetate-nonutilizing mutants of Neurospora crassa 11. Biochemical deficiencies and the roles of certain enzymes. J. Bacteriol. 95: 1063-1068. 5. FLECHTNER, V. R., and R. S. HANSON.1970. Regulation of the tricarboxylic acid cycle in bacteria: A comparison of citrate synthase from different bacteria. Biochim. Biophys. Acta, 222: 253-264. Acknowledgment T.,and H. P. KLEIN.1973. Studies 6. SATYANARAYANA, This research was supported by National on acetyl-coenzyme A synthase of yeast: Inhibition by Science Foundation grant GB 28558X and the long-chain acyl-coenzyme A esters. J. Bacteriol. 115: 600406. Faculty Research Corqmittee, Miami University. 7. SINHA,A. K., and J. K. BHATTACHARJEE. 1970. Control ofa lysine biosynthetic step by two unlinked genes 1. BURAND,J . P., R. DRILLIEN, and J. K . BHATTAof Saccharomyces cerevisiae. Biochem. Biophys. CHARJEE.1975. Citrate synthaseless glutamic acid Res. Commun. 39: 1205-1210. auxotroph of Saccharomyces cerevisiae. Mol. Gen. 8. SRERE,P. A. 1965. Palmityl-coenzyme A inhibition of Genet. 139: 303-309. the citrate condensing enzyme. Biochim. Biophys. 2. COLEMAN, J. S., and J. K. BHATTACHARJEE. 1975. Acta, 106: 445455. Regulation of citrate synthase activity of Saccharo9. WEITZMAN, P. D. J . , and P. DUNMORE. 1969. Citrate myces cerevisiae. Antonie Van Leeuwenhoek; J. Misynthesis: Allosteric regulation and molecular size. crobiol. Serol. 41: 249-256. 3. CROCKER, W. H., JR., and J. K . BHATTACHARJEE. Biochim. Biophys. Acta, 171: 198-200. 10. WIELAND,O., L. WEISS, and I. EGER-NEWFELDT. 1973. Biosynthesis of glutamic acid in Saccharomyces 1964. Inhibition of enzymatic citric acid synthesis by cerevisiae : Accumulation of tricarboxylic acid cycle long-chained acyl-thioesters of coenzyme A. Biointermediates in a glutamate auxotroph. Appl. Michem. Z. 339: 501-513. crobiol. 26: 303-308.
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of acetate to its metabolizable form and the entry of acetate into the tricarboxylic acid cycle, both ACS and citrate synthase may function in tandem for the regulation of energy metabolism and the biosynthesis of glutamic acid.
Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet1 K.-J. CHENGA N D R. HIRONAKA Research Station, Agriculture Canada, Lethbridge, Alberta TlJ4Bl AND
J . W . COSTERTON Department of Biology, Universio of Calgary, Calgary, Alberta E N 1N4 Accepted January 13, 1976
CHENG,K.-J., R. HIRONAKA, and J. W. COSTERTON. 1976. Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet. Can. J. Microbiol. 22: 764-769. Alkaline phosphatase (APase) was present in the bovine rumen in both cell-free and cellassociated states and levels of the enzyme varied with dietary regime. Reaction product deposition showed that the enzyme was associated with the mixed bacterial population. No enzyme was observed to be associated with protozoa. Trace activity of APase was also detected in the saliva. The presence of large amounts of APase in cell-free rumen fluid of cattle fed fine concentrate feed is believed to be due, in part, to the breakage of bacterial cells that occurs in the rumen. CHENG,K.-J., R. HIRONAKA et J. W. COSTERTON.1976. Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet. Can. J. Microbiol. 22: 764-769. Dans le rumen des bovins, la phosphatase alkaline (APase) est a la fois associCe aux cellules et dissociCe de celles-ci et le taux d e I'enzyme varie avec le rCgime alimentaire. La skdimentation 'Received September 8, 1975.
765
NOTES
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des produits d e reaction demontre que I'enzyme est associi B la population bacterienne du milieu. Aucun enzyme s'avere associk aux protozoaires. Des traces d'activite Apase sont aussi detectees dans la salive. L a presence de grandes quantites d'APase dissocikes des cellules dans le suc d u rumen chez les bovins nourris B I'aide de fins concentres est due en partie, semble-t-il, au bris d e s cellules bacteriennes localisees dans le rumen. [Traduit par le journal]
We have suggested (4, 5, 6) that bacterial cell lysis occurs during the onset of "feedlot" bloat in cattle, and that macromolecules released during lysis (7), coupled with bacterial slimes, contribute to the high viscosity of the rumen fluid characteristic of bloat. Alkaline phosphatase (APase: EC 3.1.3.1) localization by reaction product deposition has shown that the common rumen bacteria Bacteroides ruminicola and B. succinogenes are constitutive producers of APase, which is localized in the periplasmic area of its cells (2). Some rough strains of gram-negative bacteria shed APase into the medium during growth (lo), but most bacteria in natural habitats and the rumen of cattle are of the smooth type (9) and retain their periplasmic enzymes while their cell envelopes are intact (2, 3, 8). Thus, APase in cell-free rumen fluid would indicate bacterial cell lysis. In this study, we used extracellular levels of APase to indicate bacterial cellular breakdown in the rumen of cattle fed a feedlot bloatprovoking all-concentrate diet or a non-bloatprovoking hay diet. Four mature, rumen-fistulated cattle (A, B, C, D) were used in this experiment. Cattle A and B were fed 5.4 kg/day of a pelleted, fine, particle size (with a geometric mean particle size of 448 pm), all-concentrate diet in two equal feedings. The all-concentrate diet consisted of 50% barley, 30% oats, 15% dried molasses beet pulp, 1.5% beet molasses, 1.5% rapeseed meal, 1.0% limestone, 0.5% defluorinated rock phosphate, 0.45% salt, and 0.05% vitamin A-10 (5). Cattle C and D received alfalfa hay at 8.2 kg/day in two equal feedings. After 5 weeks on the above diets, all the cattle were fed the hay at 8.2 kg/day in two equal feedings for 5 weeks. Subsequently, cattle A and B continued to receive the hay diet and cattle C and D received fine particle size, all-concentrate diet for 5 weeks. Cattle were changed to the all-concentrate diet gradually as described before (5). Saliva was sampled from all four animals. Mixed saliva was collected as it descended into the rumen through the cardia. Detailed methods
of saliva collection were similar to those described by Bailey and Balch (1). Rumen contents were collected daily 4 h after feeding in the 5th week of each experiment. Rumen contents were filtered through cheesecloth and centrifuged at 48 000 x g for 30 min. The supernatant was checked for cell-free APase. The pellet was suspended in 20 ml of 0.05 M tris(hydroxymethy1)aminomethane (Tris) (pH 8.4) and cells were sonified for APase determination. The whole-cell assay system for APase localization was carried out as follows. Pellets obtained from centrifugation at 48 000 x g were washed three times with 0.01 M Mg" in 0.01 M Tris (pH 7.4). The washed cells were incubated for 30 min in a mixture of the following composition : sodium-P-glycerophosphate, 0.5%; p-nitrophenylphosphate, 0.0001%; Ca(NO,),, 0.01 M; MgCl,, 0.01 M; and Tris buffer, 0.05 M (pH 8.4). As controls, incubation mixtures TABLE1. The levels of alkaline phosphatase (APase) in the cell-free rumen fluid a n d whole cells 4 h after feeding in four cattle receiving a fine all-concentrate diet o r a n all-hay diet (unit per rnillilitre of rurnen fluid) Diet Cow
Hay Extracellular 0.02*k0.01 0.04 kO.01 0.02 k 0 . 0 0 4 0.03 k 0 . 0 0 8
0.35 0.61 0.45 0.67
Concentrate APase 4 . 8 5 k 1.05 4.03k0.49 1.54k0.15 4.34k 1.06
Intracellular APase k0.14 24.64k6.31 kO.11 25.68k8.47 k0.13 26.87k5.43 k0.15 36.38k4.41
'Mean of five daily observations from each cow. Values are mean f standard error. One unit of activity of APase in all cases represents the change in optical density at 420 nm per minute per rnillilitre of rumen fluid using p-nitrophenylphosphate a s substrate at 25 "C(2). Rumen fluid samples (20 ml) were centrifuged and supernatant fluids were assayed for cell-free APase activity. The cell pellets obtained were resuspended in 20 ml of 0.05 M Tris (pH 8.4) and cells were sonified for APase determination.
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CAN. J. MICROBIOL. VOL. 22. 1976
lacking sodium-P-glycerophosphate and p-nitrophenylphosphate were used. Fixation, embedding, staining, and electron microscopy procedures were described before (2). Cattle that were fed hay did not develop the foam and high viscosity of rumen fluid that characterize feedlot bloat. Their rumen fluids showed very low levels of APase (Table 1) and most of it was intracellular. The traces of enzyme detected in the cell-free rumen fluid may have come partially from the cattle's mixed saliva which contained only trace amounts of this enzyme (0.10-0.45 units; one unit of activity of APase is defined as change of optical density at 420 nm per hour per millilitre of saliva using p-nitrophenylphosphate as substrate at 25 "C). No reaction product was observed by electron microscopy to be associated with protozoa in reaction product deposition preparations. Reaction product deposition studies of the APase in the bacteria from the rumens of these cattle showed that the enzyme was present in only 15% of the cells, and that it was entirely periplasmic in its location (Fig. 1, arrows). Control preparations, from which the substrate was deleted, showed no reaction product deposition. Very few cells were lysed in preparations from the hayfed cattle. The cattle that were fed the all-concentrate diet of fine particle size developed foam in the rumen and high viscosity of rumen fluid characteristics associated with feedlot bloat, and showed high levels of both extracellular and intracellular APase (Table 1). Many of the rumen bacteria from these cattle were lysed and APase was bound to many of the damaged bacterial cells (Fig. 2, G) and its reaction product was nonspecifically deposited on the surface of cellular debris (Fig. 2, J). The occasional double-trace profiles (Fig. 2, arrows) seen in this debris indicates that some of it is of cellular
origin. Control preparations, from which the substrate was deleted, showed electron-dense deposits, but this "background" was comparatively light. In this study, we have noted that rumen fluids of cattle fed the all-concentrate diet with fine particle size were characterized by a high level of bacterial APase both in the cell-free fluid and in the cellular pellet. The high extracellular enzyme level may reflect massive breakdown of bacterial cells in the rumen, as was suggested earlier (4, 5, 6, 7), since APase is largely confined to the periplasmic space of intact cells, and is both bound to cellular debris and free in the menstruum surrounding broken cells. The higher overall enzyme levels in broken cells, compared with unbroken cells, appear to be a consequence of greater accessibility of substrates to the enzymes associated with disrupted cell envelopes. Thus, it is clear that the feedlot bloat-provoking diet increases the number of cells in population capable of producing APase and the bacterial cell breakdown, both which cause an apparent increase in the general concentration of APase in the rurnen. More particularly, the level of extracellular APase is sharply increased by this lytic activity and may therefore serve as a n indication of the onset of the condition associated with feedlot bloat. 1. B A I L E YC., B., and C. C. BALCH.1961. Saliva secretion and its relation to feeding in cattle. 11. The cornposition and rate of secretion of mixed saliva in the cow during rest. Br. J. Nutr. 15: 383402. 2. CHENG, K.-J., and J. W. COSTERTON. 1973. Localization of alkaline phosphatase in three gram-negative rurnen bacteria. J. Bacterial. 116: 424440. 3. CHENG,K.-J., and J. W. COSTERTON.1975. Ultrastructure of cell-envelope of bacteria of the bovine rurnen. Appl. Microbial. 29: 841-849. 4. CHENG,K.-J., and R. HIRONAKA. 1972. Influence of feed particle size on feedlot bloat. Proc. West. Sect. Am. Soc. Anirn. Sci. 23: 385-388.
FIG. 1. Electron micrograph of a section of rurnen material from a cow fed an alfalfa hay diet. The preparation had been treated for reaction product deposition by APase and the electron-dense reaction product (arrows) of this enzyme can be seen in the periplasmic area of three of the cells. The cytoplasmic and outer membranes, which define the periplasmic space, are only resolved when they are in near perfect cross section (arrows). Control preparations from which the substrate had been omitted showed no comparable electron-dense deposits. The bar indicates 0.1 pm. FIG.2. Electron micrograph of a section of rumen material from a cow fed a feedlot bloat-provoking diet. Reaction product deposition shows that APase was associated with recognizable remnants of lysed bacterial cells (G) and with the surface of cells and detritus (J). This detritus could be identified as being of cellular origin by the occasional resolution of double-track membrane fragments at favorable sectioning angles (arrows). The bar indicates 0.1 pm.
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NOTES
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768 CAN. J. MICROBIOL. VOL. 22, 1976
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8. COSTERTON, J. W. 1973. The relationship of a wall5. CHENG,K.-J., and R. HIRONAKA. 1973. Influence of associated enzyme with specific layers of the cell wall feed particle size on pH, carbohydrate content, and of a gram-negative bacterium. J. Bacteriol. 114: viscosity of rumen fluid. Can. J. Anim. Sci. 53: 1281-1293. 417-422. J. W., J. M. INGRAM, and K.-J. CHENG. 6. CHENG,K.-J., R. HIRONAKA, G. A. JONES,T . NICAS, 9. COSTERTON, 1974. Structure and function of the cell envelope of and J. W. COSTERTON.1976. Frothy feedlot bloat in gram-negative bacteria. Bacteriol. Rev. 38: 87-1 10. cattle: production of extracellular polysaccharides 10. LINDSAY, S. B., B. WHEELER, K. E. SANDERSON, J. and development of viscosity in cultures of W. COSTERTON, and K.-J. CHENG.1973. The release Sfrepfococcusbovis. Can. J. Microbiol. 22: 45W59. of alkaline phosphatase and of lipopolysaccharide dur7. CHENG,K.-J., R. HIRONAKA, D. W. A. ROBERTS, and ing the growth of rough and smooth strains of 1973. Cytoplasmic glycogen incluJ. W. COSTERTON. Salmonella fyphimurium. Can. J. Microbiol. 19: sions in cells of anaerobic gram-negative rumen bac335-343. teria. Can. J. Microbiol. 19: 1501-1506.
Un nouveau sCrotype de Salmonella isole au Canada: Salmonella quebec (44, ,44,: c:e, n,z A. BASUET S. PETROW Direction des Laborafoires du Minisfere des Affaires Sociales du Que'bec, Chomedey, Ville de Laval, Q u l . ET
S . LE M I N O R , L. LE MINORET E. LE COUEFFIC CenfreNritional de Salmonella, InsfiflcrPasfercr, Paris, France
ET
S. S. KASATIYA~ I.N.R.S. UniversifeduQuebec, Que'bec, QuP. Approuve le 16janvier 1976
BASU,A., S. PETROW,S. LE MINOR,L. L E MINOR, E. L E COUEFFIC et S. S. KASATIYA. 1976. Un nouveau sCrotype de Salmonella isole au Canada: Salmonella qlrebec (44, ,44,:c:e,n,zI5). Can. J. Microbiol..22: 769-770. Un nouveau serotype de Salmonella, Salmonella quebec (44, ,44,:c:e,n,z15) appartenant au sous-genre I de Kauffmann a ete isole par coproculture chez une femme asymptomatique lors d'un examen medical routinier. BASU,A., S. PETROW,S. LE MINOR,L. L E MINOR,E. L E COUEFFIC, and S . S. KASATIYA. 1976. Un nouveau serotype de Salmonella isole au Canada: Salmonella quebec ( M I , 44,:c:e,n,z15). Can. J. Microbiol. 22: 769-770. A new serotype of Salmonella, Salmonella quebec (44,,44,:c:e,n,z15)belonging to Kauffmann's sub-genus I was isolated from the faeces of an asymptomatic female during a routine medical examination.
Un nouveau strotype de Salmonella appartenant au sous-genre I de Kauffmann (1) a ttC isolt par coproculture chez une femme asymptomatique lors d'un examen medical routinier. Cette souche acidifie en 24 h les eaux peptonCes additionntes de: xylose, rhamnose, glucose (avec production de gaz), mannitol, sorbitol, ' R e p le 23 juin 1975. 'Adresse alternative: Direction des Laboratoires du Ministkre des Affaires Sociales du QuCbec, Chomedey, Ville de Laval, P. Q. Canada.
dulcitol, maltose, trthalose, inositol, melibiosy et glyctrol (Stern). Elle n'a aucune action sur l'adonitol, le lactose, le saccharose et la salicine. Elle posskde une lysine et une ornithine dtcarboxylase, une arginine dihydrolase, une nitratase, une tttrathionate rtductase mais ne posdde pas d'urtase, de P-galactosidase et de gtlatinase. Elle produit du H,S et se cultive sur milieu au citrate de Simmons, mais elle ne produit pas d'indole et ne se cultive pas sur KCN. Elle donne une rtaction positive au rouge de mtthyle mais nCgative
CAN. J . MICROBIOL. VOL. 22, 1976
4. FLAVELL,R. B., and J. R. S . FINCHAM.1968. Acetate-nonutilizing mutants of Neurospora crassa 11. Biochemical deficiencies and the roles of certain enzymes. J. Bacteriol. 95: 1063-1068. 5. FLECHTNER, V. R., and R. S. HANSON.1970. Regulation of the tricarboxylic acid cycle in bacteria: A comparison of citrate synthase from different bacteria. Biochim. Biophys. Acta, 222: 253-264. Acknowledgment T.,and H. P. KLEIN.1973. Studies 6. SATYANARAYANA, This research was supported by National on acetyl-coenzyme A synthase of yeast: Inhibition by Science Foundation grant GB 28558X and the long-chain acyl-coenzyme A esters. J. Bacteriol. 115: 600406. Faculty Research Corqmittee, Miami University. 7. SINHA,A. K., and J. K. BHATTACHARJEE. 1970. Control ofa lysine biosynthetic step by two unlinked genes 1. BURAND,J . P., R. DRILLIEN, and J. K . BHATTAof Saccharomyces cerevisiae. Biochem. Biophys. CHARJEE.1975. Citrate synthaseless glutamic acid Res. Commun. 39: 1205-1210. auxotroph of Saccharomyces cerevisiae. Mol. Gen. 8. SRERE,P. A. 1965. Palmityl-coenzyme A inhibition of Genet. 139: 303-309. the citrate condensing enzyme. Biochim. Biophys. 2. COLEMAN, J. S., and J. K. BHATTACHARJEE. 1975. Acta, 106: 445455. Regulation of citrate synthase activity of Saccharo9. WEITZMAN, P. D. J . , and P. DUNMORE. 1969. Citrate myces cerevisiae. Antonie Van Leeuwenhoek; J. Misynthesis: Allosteric regulation and molecular size. crobiol. Serol. 41: 249-256. 3. CROCKER, W. H., JR., and J. K . BHATTACHARJEE. Biochim. Biophys. Acta, 171: 198-200. 10. WIELAND,O., L. WEISS, and I. EGER-NEWFELDT. 1973. Biosynthesis of glutamic acid in Saccharomyces 1964. Inhibition of enzymatic citric acid synthesis by cerevisiae : Accumulation of tricarboxylic acid cycle long-chained acyl-thioesters of coenzyme A. Biointermediates in a glutamate auxotroph. Appl. Michem. Z. 339: 501-513. crobiol. 26: 303-308.
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of acetate to its metabolizable form and the entry of acetate into the tricarboxylic acid cycle, both ACS and citrate synthase may function in tandem for the regulation of energy metabolism and the biosynthesis of glutamic acid.
Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet1 K.-J. CHENGA N D R. HIRONAKA Research Station, Agriculture Canada, Lethbridge, Alberta TlJ4Bl AND
J . W . COSTERTON Department of Biology, Universio of Calgary, Calgary, Alberta E N 1N4 Accepted January 13, 1976
CHENG,K.-J., R. HIRONAKA, and J. W. COSTERTON. 1976. Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet. Can. J. Microbiol. 22: 764-769. Alkaline phosphatase (APase) was present in the bovine rumen in both cell-free and cellassociated states and levels of the enzyme varied with dietary regime. Reaction product deposition showed that the enzyme was associated with the mixed bacterial population. No enzyme was observed to be associated with protozoa. Trace activity of APase was also detected in the saliva. The presence of large amounts of APase in cell-free rumen fluid of cattle fed fine concentrate feed is believed to be due, in part, to the breakage of bacterial cells that occurs in the rumen. CHENG,K.-J., R. HIRONAKA et J. W. COSTERTON.1976. Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet. Can. J. Microbiol. 22: 764-769. Dans le rumen des bovins, la phosphatase alkaline (APase) est a la fois associCe aux cellules et dissociCe de celles-ci et le taux d e I'enzyme varie avec le rCgime alimentaire. La skdimentation 'Received September 8, 1975.
765
NOTES
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des produits d e reaction demontre que I'enzyme est associi B la population bacterienne du milieu. Aucun enzyme s'avere associk aux protozoaires. Des traces d'activite Apase sont aussi detectees dans la salive. L a presence de grandes quantites d'APase dissocikes des cellules dans le suc d u rumen chez les bovins nourris B I'aide de fins concentres est due en partie, semble-t-il, au bris d e s cellules bacteriennes localisees dans le rumen. [Traduit par le journal]
We have suggested (4, 5, 6) that bacterial cell lysis occurs during the onset of "feedlot" bloat in cattle, and that macromolecules released during lysis (7), coupled with bacterial slimes, contribute to the high viscosity of the rumen fluid characteristic of bloat. Alkaline phosphatase (APase: EC 3.1.3.1) localization by reaction product deposition has shown that the common rumen bacteria Bacteroides ruminicola and B. succinogenes are constitutive producers of APase, which is localized in the periplasmic area of its cells (2). Some rough strains of gram-negative bacteria shed APase into the medium during growth (lo), but most bacteria in natural habitats and the rumen of cattle are of the smooth type (9) and retain their periplasmic enzymes while their cell envelopes are intact (2, 3, 8). Thus, APase in cell-free rumen fluid would indicate bacterial cell lysis. In this study, we used extracellular levels of APase to indicate bacterial cellular breakdown in the rumen of cattle fed a feedlot bloatprovoking all-concentrate diet or a non-bloatprovoking hay diet. Four mature, rumen-fistulated cattle (A, B, C, D) were used in this experiment. Cattle A and B were fed 5.4 kg/day of a pelleted, fine, particle size (with a geometric mean particle size of 448 pm), all-concentrate diet in two equal feedings. The all-concentrate diet consisted of 50% barley, 30% oats, 15% dried molasses beet pulp, 1.5% beet molasses, 1.5% rapeseed meal, 1.0% limestone, 0.5% defluorinated rock phosphate, 0.45% salt, and 0.05% vitamin A-10 (5). Cattle C and D received alfalfa hay at 8.2 kg/day in two equal feedings. After 5 weeks on the above diets, all the cattle were fed the hay at 8.2 kg/day in two equal feedings for 5 weeks. Subsequently, cattle A and B continued to receive the hay diet and cattle C and D received fine particle size, all-concentrate diet for 5 weeks. Cattle were changed to the all-concentrate diet gradually as described before (5). Saliva was sampled from all four animals. Mixed saliva was collected as it descended into the rumen through the cardia. Detailed methods
of saliva collection were similar to those described by Bailey and Balch (1). Rumen contents were collected daily 4 h after feeding in the 5th week of each experiment. Rumen contents were filtered through cheesecloth and centrifuged at 48 000 x g for 30 min. The supernatant was checked for cell-free APase. The pellet was suspended in 20 ml of 0.05 M tris(hydroxymethy1)aminomethane (Tris) (pH 8.4) and cells were sonified for APase determination. The whole-cell assay system for APase localization was carried out as follows. Pellets obtained from centrifugation at 48 000 x g were washed three times with 0.01 M Mg" in 0.01 M Tris (pH 7.4). The washed cells were incubated for 30 min in a mixture of the following composition : sodium-P-glycerophosphate, 0.5%; p-nitrophenylphosphate, 0.0001%; Ca(NO,),, 0.01 M; MgCl,, 0.01 M; and Tris buffer, 0.05 M (pH 8.4). As controls, incubation mixtures TABLE1. The levels of alkaline phosphatase (APase) in the cell-free rumen fluid a n d whole cells 4 h after feeding in four cattle receiving a fine all-concentrate diet o r a n all-hay diet (unit per rnillilitre of rurnen fluid) Diet Cow
Hay Extracellular 0.02*k0.01 0.04 kO.01 0.02 k 0 . 0 0 4 0.03 k 0 . 0 0 8
0.35 0.61 0.45 0.67
Concentrate APase 4 . 8 5 k 1.05 4.03k0.49 1.54k0.15 4.34k 1.06
Intracellular APase k0.14 24.64k6.31 kO.11 25.68k8.47 k0.13 26.87k5.43 k0.15 36.38k4.41
'Mean of five daily observations from each cow. Values are mean f standard error. One unit of activity of APase in all cases represents the change in optical density at 420 nm per minute per rnillilitre of rumen fluid using p-nitrophenylphosphate a s substrate at 25 "C(2). Rumen fluid samples (20 ml) were centrifuged and supernatant fluids were assayed for cell-free APase activity. The cell pellets obtained were resuspended in 20 ml of 0.05 M Tris (pH 8.4) and cells were sonified for APase determination.
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CAN. J. MICROBIOL. VOL. 22. 1976
lacking sodium-P-glycerophosphate and p-nitrophenylphosphate were used. Fixation, embedding, staining, and electron microscopy procedures were described before (2). Cattle that were fed hay did not develop the foam and high viscosity of rumen fluid that characterize feedlot bloat. Their rumen fluids showed very low levels of APase (Table 1) and most of it was intracellular. The traces of enzyme detected in the cell-free rumen fluid may have come partially from the cattle's mixed saliva which contained only trace amounts of this enzyme (0.10-0.45 units; one unit of activity of APase is defined as change of optical density at 420 nm per hour per millilitre of saliva using p-nitrophenylphosphate as substrate at 25 "C). No reaction product was observed by electron microscopy to be associated with protozoa in reaction product deposition preparations. Reaction product deposition studies of the APase in the bacteria from the rumens of these cattle showed that the enzyme was present in only 15% of the cells, and that it was entirely periplasmic in its location (Fig. 1, arrows). Control preparations, from which the substrate was deleted, showed no reaction product deposition. Very few cells were lysed in preparations from the hayfed cattle. The cattle that were fed the all-concentrate diet of fine particle size developed foam in the rumen and high viscosity of rumen fluid characteristics associated with feedlot bloat, and showed high levels of both extracellular and intracellular APase (Table 1). Many of the rumen bacteria from these cattle were lysed and APase was bound to many of the damaged bacterial cells (Fig. 2, G) and its reaction product was nonspecifically deposited on the surface of cellular debris (Fig. 2, J). The occasional double-trace profiles (Fig. 2, arrows) seen in this debris indicates that some of it is of cellular
origin. Control preparations, from which the substrate was deleted, showed electron-dense deposits, but this "background" was comparatively light. In this study, we have noted that rumen fluids of cattle fed the all-concentrate diet with fine particle size were characterized by a high level of bacterial APase both in the cell-free fluid and in the cellular pellet. The high extracellular enzyme level may reflect massive breakdown of bacterial cells in the rumen, as was suggested earlier (4, 5, 6, 7), since APase is largely confined to the periplasmic space of intact cells, and is both bound to cellular debris and free in the menstruum surrounding broken cells. The higher overall enzyme levels in broken cells, compared with unbroken cells, appear to be a consequence of greater accessibility of substrates to the enzymes associated with disrupted cell envelopes. Thus, it is clear that the feedlot bloat-provoking diet increases the number of cells in population capable of producing APase and the bacterial cell breakdown, both which cause an apparent increase in the general concentration of APase in the rurnen. More particularly, the level of extracellular APase is sharply increased by this lytic activity and may therefore serve as a n indication of the onset of the condition associated with feedlot bloat. 1. B A I L E YC., B., and C. C. BALCH.1961. Saliva secretion and its relation to feeding in cattle. 11. The cornposition and rate of secretion of mixed saliva in the cow during rest. Br. J. Nutr. 15: 383402. 2. CHENG, K.-J., and J. W. COSTERTON. 1973. Localization of alkaline phosphatase in three gram-negative rurnen bacteria. J. Bacterial. 116: 424440. 3. CHENG,K.-J., and J. W. COSTERTON.1975. Ultrastructure of cell-envelope of bacteria of the bovine rurnen. Appl. Microbial. 29: 841-849. 4. CHENG,K.-J., and R. HIRONAKA. 1972. Influence of feed particle size on feedlot bloat. Proc. West. Sect. Am. Soc. Anirn. Sci. 23: 385-388.
FIG. 1. Electron micrograph of a section of rurnen material from a cow fed an alfalfa hay diet. The preparation had been treated for reaction product deposition by APase and the electron-dense reaction product (arrows) of this enzyme can be seen in the periplasmic area of three of the cells. The cytoplasmic and outer membranes, which define the periplasmic space, are only resolved when they are in near perfect cross section (arrows). Control preparations from which the substrate had been omitted showed no comparable electron-dense deposits. The bar indicates 0.1 pm. FIG.2. Electron micrograph of a section of rumen material from a cow fed a feedlot bloat-provoking diet. Reaction product deposition shows that APase was associated with recognizable remnants of lysed bacterial cells (G) and with the surface of cells and detritus (J). This detritus could be identified as being of cellular origin by the occasional resolution of double-track membrane fragments at favorable sectioning angles (arrows). The bar indicates 0.1 pm.
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8. COSTERTON, J. W. 1973. The relationship of a wall5. CHENG,K.-J., and R. HIRONAKA. 1973. Influence of associated enzyme with specific layers of the cell wall feed particle size on pH, carbohydrate content, and of a gram-negative bacterium. J. Bacteriol. 114: viscosity of rumen fluid. Can. J. Anim. Sci. 53: 1281-1293. 417-422. J. W., J. M. INGRAM, and K.-J. CHENG. 6. CHENG,K.-J., R. HIRONAKA, G. A. JONES,T . NICAS, 9. COSTERTON, 1974. Structure and function of the cell envelope of and J. W. COSTERTON.1976. Frothy feedlot bloat in gram-negative bacteria. Bacteriol. Rev. 38: 87-1 10. cattle: production of extracellular polysaccharides 10. LINDSAY, S. B., B. WHEELER, K. E. SANDERSON, J. and development of viscosity in cultures of W. COSTERTON, and K.-J. CHENG.1973. The release Sfrepfococcusbovis. Can. J. Microbiol. 22: 45W59. of alkaline phosphatase and of lipopolysaccharide dur7. CHENG,K.-J., R. HIRONAKA, D. W. A. ROBERTS, and ing the growth of rough and smooth strains of 1973. Cytoplasmic glycogen incluJ. W. COSTERTON. Salmonella fyphimurium. Can. J. Microbiol. 19: sions in cells of anaerobic gram-negative rumen bac335-343. teria. Can. J. Microbiol. 19: 1501-1506.
Un nouveau sCrotype de Salmonella isole au Canada: Salmonella quebec (44, ,44,: c:e, n,z A. BASUET S. PETROW Direction des Laborafoires du Minisfere des Affaires Sociales du Que'bec, Chomedey, Ville de Laval, Q u l . ET
S . LE M I N O R , L. LE MINORET E. LE COUEFFIC CenfreNritional de Salmonella, InsfiflcrPasfercr, Paris, France
ET
S. S. KASATIYA~ I.N.R.S. UniversifeduQuebec, Que'bec, QuP. Approuve le 16janvier 1976
BASU,A., S. PETROW,S. LE MINOR,L. L E MINOR, E. L E COUEFFIC et S. S. KASATIYA. 1976. Un nouveau sCrotype de Salmonella isole au Canada: Salmonella qlrebec (44, ,44,:c:e,n,zI5). Can. J. Microbiol..22: 769-770. Un nouveau serotype de Salmonella, Salmonella quebec (44, ,44,:c:e,n,z15) appartenant au sous-genre I de Kauffmann a ete isole par coproculture chez une femme asymptomatique lors d'un examen medical routinier. BASU,A., S. PETROW,S. LE MINOR,L. L E MINOR,E. L E COUEFFIC, and S . S. KASATIYA. 1976. Un nouveau serotype de Salmonella isole au Canada: Salmonella quebec ( M I , 44,:c:e,n,z15). Can. J. Microbiol. 22: 769-770. A new serotype of Salmonella, Salmonella quebec (44,,44,:c:e,n,z15)belonging to Kauffmann's sub-genus I was isolated from the faeces of an asymptomatic female during a routine medical examination.
Un nouveau strotype de Salmonella appartenant au sous-genre I de Kauffmann (1) a ttC isolt par coproculture chez une femme asymptomatique lors d'un examen medical routinier. Cette souche acidifie en 24 h les eaux peptonCes additionntes de: xylose, rhamnose, glucose (avec production de gaz), mannitol, sorbitol, ' R e p le 23 juin 1975. 'Adresse alternative: Direction des Laboratoires du Ministkre des Affaires Sociales du QuCbec, Chomedey, Ville de Laval, P. Q. Canada.
dulcitol, maltose, trthalose, inositol, melibiosy et glyctrol (Stern). Elle n'a aucune action sur l'adonitol, le lactose, le saccharose et la salicine. Elle posskde une lysine et une ornithine dtcarboxylase, une arginine dihydrolase, une nitratase, une tttrathionate rtductase mais ne posdde pas d'urtase, de P-galactosidase et de gtlatinase. Elle produit du H,S et se cultive sur milieu au citrate de Simmons, mais elle ne produit pas d'indole et ne se cultive pas sur KCN. Elle donne une rtaction positive au rouge de mtthyle mais nCgative
