Veterinary Microbiology, 32 ( 1992 ) 343-350 Elsevier Science Publishers B.V., Amsterdam
343
Aggregation of bovine platelets by Fusobacterium necrophorum Minoru Horose a, Hiroko Kiyoyamaa, Hiroyuki Ogawa b and Toshiharu Shinjo a aDepartment of Veterinary Microbiology, Faculty of Agriculture, Miyazaki University, Gakuenkibanadai, Japan bDepartment of Veterinery Surgery, Faculty of Agriculture, Miyazaki University, Gakuenkibanadai, Japan (Accepted 13 march 1992 )
ABSTRACT Horose, M., Kiyoyama, H., Ogawa, H. and Shinjo, T., 1992. Aggregation of bovine platelets by Fusobacterium necrophorum. Vet. Microbiol., 32: 343-350. Washed cell suspensions ofbiovar A strains ofFusobacterium necrophorum aggregated cattle platelets, but similar suspensions of biovar B strains did not. Platelets were also aggregated by heat-treated bacterial cells or the lipopolysaccharide of biovar A. No platelet aggregation occurred in the presence of the cell-free culture supernatant of biovar A and of all samples prepared from biovar B. Scanning electron microscopy revealed that aggregated platelets were not damaged. Platelet aggregation was inhibited by EDTA, aspirin and quinacrine, and lag time was retarded by these inhibitors, indicating the reaction was a Ca2+-dependent, cyclo-oxygenase sensitive event. Platelet aggregation may be a virulence marker, probably mediated by the lipopolysaccharide ofF. necrophorum biovar A strains.
INTRODUCTION
Fusobacterium necrophorum is divided into two biovars, A and B, by their haemagglutinating activity (Fi6vez, 1963; Shinjo et al., 1981 ). The former has this activity, but the latter does not. Biovar A is mainly isolated from pathological lesions, such as bovine hepatic abscesses or foot-rot, and biovar B is present in the alimentary tract of animals. The two biovars also differ in their pathogenicity for mice (Fievez, 1963; Shinjo et al., 1981 ), adherence to bovine ruminal epithelial cells (Kanoe and Iwaki, 1987) and cultured cell lines (Shinjo et al., 1988), composition of lipopolysaccharide (LPS) (Inoue et al., 1985), haemolytic activity (Emery et al., 1985), production of leucotoxin (Scanlan et al., 1986 ) and hydrophobicity (Shinjo et al., 1987 ). The ability of bacteria to aggregate platelets may be a virulence determiCorrespondence to: Toshiharu Shinjo, Dept of Veterinary Microbiology. Faculty of Agriculture, Miyazaki University, Gakuenkibanadai, Miyazaki 889-21, Japan.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
344
M. HOROSE ET AL.
nant in some infections. Microbial components such as LPS (Morrison et al., 1978, Springer and Adye, 1975 ), tuberculin (Rourka et al., 1979), mannan from Candida (Maisch and Calderone, 1981 ), peptidoglycan (Greenblatt et al., 1978 ) and lipoteichoic acid (Beachy et al., 1977 ) are known to contribute to platelet aggregation. F. necrophorum aggregated human platelets in platelet-rich plasma (PRP) (Forrester et al., 1985 ) and bovine platelets were aggregated by the bacterial haemagglutinin (Kanoe and Yamanaka, 1989). In this study, the authors investigated in detail the ability of F. necrophorum to aggregate bovine platelets. The experiments were carried out to determine whether the reaction is a cyclo-oxygenase-sensitive event. MATERIALS AND METHODS
Bacterial strains and growth conditions Three biovar A strains were used: strain VPI 2891 was supplied by Dr W.E.C. Moore of the Virginia Polytechnic Institute and State University, Blacksburg, VA, U.S.A., strain Fn 43 originated from a bovine hepatic abscess, and strain SPH 1 was supplied by Dr Aalbaek of the Royal Veterinary and Agricultural University, Copenhagen, Denmark. Biovar B strain VPI 6161 was supplied by Dr M. Kanoe of Yamaguchi University, Yamaguchi, Japan and strain SPH 6 was supplied by Dr. Aalbaek. The organisms were cultured at 37°C for 24 h in GAM broth (Nissui Seiyaku, Co., Tokyo), collected by centrifugation at 8000 g for 20 min, washed three times with phosphate buffered saline (PBS, pH 7.2) and resuspended in PBS. Bacterial suspensions containing 105 cfu/ml were used.
Preparations of platelets and washed platelets Nine volumes of bovine blood were drawn in to one volume of 4.6% sodium citrate solution in a plastic syringe. The mixture was transferred to plastic tubes and centrifuged at 150 g for 10 min. The supernatant was collected into plastic tubes and used as PRP. The platelets were washed three times in modified Tyrode solution (0.3% BSA, 0.2 mM EGTA in Tyrode solution, pH 6.8 ) and suspended in Tyrode solution (pH 7.4) and used as washed platelets ( 105 cells/ml) (WP).
Extraction of LPS LPS of F. necrophorum was extracted by the hot phenol water method described by Westphal and Jann (1965 ). The purified LPS was dissolved at 1 mg/ml in distilled water and diluted to 1 pg/ml in distilled water for use in platelet aggregation.
AGGREGATION OF BOVINE PLATELETS BY FUSOBACTERIUM NECROPHORUM
345
Platelet aggregation Aggregation responses were evaluated using an aggregometer (Hematracer, model PAT-2A, NKK, Tokyo ). An aliquot of 200/tl of PRP or WP was placed in a cuvette containing a stir bar. The cuvette was placed in the aggregometer (temperature 37 °C) and 25/tl of 30 m M CaCI2 solution was added. One min later 25/tl of the bacterial or LPS suspension was added, and aggregation was monitored for 3 of 5 min. The number of bacteria was adjusted to be equal to that of platelets using PBS. 25/tl horse Achilles tendon collage (500/zg/ml) was used as the reference aggregating agent. The experiments were carried out in triplicate.
Inhibition of platelet aggregation In the inhibition test, aspirin, quinacrine and EDTA were used. The three drugs were diluted to 10, 5, 2.5, 1.25 and 0.62 mM, and 25/zl of these solutions was preincubated with WP for 10 min at room temperature before being transferred to the 37 °C environment in the aggregometer. When this temperature was achieved, the bacteria were added to initiate the experiment.
Observation of platelets by scanning electron microscope Scanning electron microscope observations of PRP after aggregation by bacteria or LPS were carried out. Bacterial and LPS suspensions and PRP suspensions were mixed and incubated at 37°C for 30 min. A few drops of the mixture were fixed in 6 ml of 1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. Samples were dried in a critical point dryer and examined with a JSM-35C scanning electron microscope (Japan Electron Optics, Tokyo). RESULTS
Platelet aggregation All biovar A strains aggregated bovine PRP, while the two biovar B strains did not (Table 1 ). The kinetics of aggregation by strain VPI 2891 and strain VPI 6161 is recorded in Figs 1 and 2. Strain VPI 2891 aggregated bovine PRP within 10 min. Cell-free culture supernatants of two biovars were unable to aggregate PRP. Heat-treated bacterial cells of biovar A were also able to aggregate PRP. Furthermore, LPS aggregated platelets in proportion to bacterial strains (Fig. 3 ). Bovine platelets aggregated by bacterial cells or LPS were apparently intact when observed by scanning electron microscopy. The biovar A strains aggregated WP but the biovar B strains did not (Fig. 4 ). The kinetics was similar to that for aggregation of PRP.
Inhibition test Platelet aggregation by biovar A strains was inhibited by the three drugs. As shown in Table 2, EDTA at 1.25 mM, aspirin at 2.5 mM and quinacrine
346
M. HOROSE ET AL.
TABLE 1 Platelet aggregation ofF. necrophorum Biovar
Strain
Percent aggregation
A
VPI 2891 Fn 43 SPH 1 VP1 6161 SPH 6
86 68 70 7 3
B
C ollage..nn 80
4o
I
I
5 TIME(min)
I
10
Fig. 1. Aggregation of platelets by F. necrophorum b i o v a r A (strain VPI 2 8 91 ).
at 1.25 m M inhibited aggregation completely, and EDTA at 1.25 mM, aspirin at 2.5 mM and quinacrine at 0.62 m M produced 82%, 90% and 85% inhibition, respectively. In addition, the lag time was retarded from 0.5 rain to 1.5 or 2.0 min by these inhibitors (Table 2 ).
AGGREGATION OF BOVINE PLATELETS BY FUSOBACTERIUM NECROPHORUM
347
Collagen..
i"l ~'°-t
t
Washed ba [~P
-
-
="--
--- J l - f,--'e
I
culture
ria/cells fluid
I
I
5 TIM E(min)
10
Fig. 2. Aggregation of platelets by F. necrophorum biovar B (strain VPI 6161 ). Biovar A LPS 80Biovar A heat treated
bacterial cells i
z om p¢¢ c3 4 0 14. n-1 c~ Biovar B LPS
-
Biovar B heat t r e a t e d
I
i
5
t
10
T I M E (min) Fig. 3. Aggregation of platelets by LPS and heat-treated bacterial cells ofF. necrophorum biovar A (strain VPI 2891 ) and biovar B (strain VPI 6161 ).
348
M. H O R O S E ET AL.
80--
Biovar A Z
O
--
040-ill rr
O O
---
I
I
I
5 TIME (min)
10
Fig. 4. Aggregation of washed platelets by/~: necrophorum b i o v a r A ( s t r a i n V P I 2891 ) and biovar B (strain V P I 6161 ). TABLE 2
Inhibition ofF. necrophorum VPI 2891 platelet aggregation Inhibitor
Concentration
Lag time
Aggregation at
(mM)
(min)
10 m i n (%)
0.5
100 0 18 0 10 0 15
None EDTA EDTA
Aspirin Aspirin Quinacrine Quinacrine
10.0 1.25 10.0 2.5 1.25 0.62
1.5 2.0 2.0
DISCUSSION
The present study demonstrated that bacterial cells, heat-treated bacterial cells and the LPS ofF. necrophorumbiovar A strains aggregated bovine platelets, whereas cell-free culture supernatant was unable to do so. These results indicate that active bacterial metabolism was not responsible for the aggregation, and that heat-resistant surface components of bacteria induced platelet aggregation. Scanning electron microscope observations revealed that no
AGGREGATION OF BOVINE PLATELETS BY FUSOBACTERIUM NECROPHORUM
349
platelet lysis occurred, as has been observed with Listeria monocytgenes (Czuprynski and Balish, 1981 ) and Streptococcus pyogenes (Sullam et al., 1987). Immunoglobulin (Sullam et al., 1988), complement (Maish and Calderone, 1981 ) and fibrinogen (Kurpiewsky et al., 1983; Sullam et al., 1987) are necessary for platelet aggregation in some bacteria. Forrester et al. (1985) reported that F, necrophorum needed fibrinogen for platelet aggregation. In our experiments platelet aggregation by F. necrophorum occurred without the addition of fibrinogen. However, they used gel-filtered human platelets and we used washed bovine platelets; either of them may be a species difference, or the differing methods used may explain the different result. The inhibition of platelet aggregation by EDTA suggests that the reaction is calcium-dependent. Aggregation may be associated with cyclo-oxygenation of arachidonic acid, since aspirin and quinacrine, both cyclo-oxygenase inhibitors, inhibited aggregation. Endotoxin may induce thrombus formation in the hepatic sinusoids with subsequent hepatic necrosis (Ruiter et al., 1981; Maier and Hafnel, 1984; Shibayama, 1987 ). Endotoxin of F. necrophorum biovar A might directly affect the platelet membrane, inducing platelet activation in thrombus formation. Platelet aggregation might thus be associated with the virulence of F. necrophorum, probably mediated by LPS.
REFERENCES Beachy, E.H., Chiang, T.M., Ofek, I. and Kang, A.H., 1977. Interaction of lipoteichoic acid of group A streptococci with human platelets. Infect. Immun., 16: 649-654. Czuprynski, C.J. and Balish, E., 1981. Interaction of rat platelets with Listeria monocytogenes. Infect. Immun., 33: 103-108. Emery, D.L., Vaughan, J.A., Clark, B.L., Dufly, J.H. and Stewart, D.J., 1985. Cultural characteristics and virulence of strains of Fusobacterium necrophorurn isolated from the feet of cattle and sheep. Aust. Vet. J., 62: 43-46. Fi6vez, L., 1963. Etude compar6e des souches de Sphaerophorus necrophorus Isol6es chez l'Homme et chez l'Animal. Presses Academiques Europ6ennes, Brussels. Forrester, L.J., Campbell, B.J. and Berg, J.N. and Barrett, J.T., 1985. Aggregation of platelets by Fusobacterium necrophorum. J. Clin. Microbiol., 22: 245-249. Greenblatt, J., Boackle, R.J. and Schwab, J.H., 1978. Activation of the alternate complement pathway by peptidoglycan from streptococcal cell wall. Infect. Immun., 19: 269-303. Inoue, T., Kanoe, M., Goto, N., Matusumura, K. and Nakano, K., 1985. Chemical and biological properties oflipopolysaccharides from Fusobacterium necrophorum biovar A and biovar B strains. Jpn. J. Vet. Sci., 47: 639-645. Kanoe, M. and lwaki, K., 1987. Adherence of Fusobacterium necrophorum to bovine ruminal cells. J. Med. Microbiol., 23: 69-73. Kanoe, M. and Yamanaka, M., 1989. Bovine platelet aggregation by Fusobacterium necrophorum. J. Med. Microbiol., 29: 13-17.
350
M. HOROSE Eq- AL.
Kurpiewsky, G.E., Forrester, L.J., Campbell, B.J. and Barrett, J.T., 1983. Platelet aggregation by Streptococcus pyogenes. Infect. Immun., 39: 704-708. Maier, R.V. and Hahnel, G.B., 1984. Potential for endotoxin-activated Kupffer's cells to induce microvascular thrombosis. Arch. Surg., 119: 62-67. Maisch, P.A. and Calderone, R.A., 1981. Role of surface mannan in the adherence of Candida albicans to fibrin-platelet clots formed in vitro. Infect. Immun., 32: 92-97. Morrison, D.C., Kline, L.F., Oades, Z.G. and Henson, P.M., 1978. Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: Alternative complement pathway dependence of the lyric response. Infect. Immun., 20:744-751. Rourke, F.J., Fan, S.S. and Wilder, M.S., 1979. Anticomplementary activity of tuberculin: Relationship to platelet aggregation and lytic response. Infect. Immun., 23:160-167. Ruiter, D.J., Van der Meulen, J., Brouwer, A., Hummel, M.J.R., Mauw, B.J., Van der Ploeg, J.C.M. and Wisse, E., 1981. Uptake by liver cells of enterotoxin following its intravenous injection. Lab. Invest., 45: 38-45. Scanlan, C.M., Berg, J.N. and Campbell, F.F., 1986. Biochemical characterization of the leukotoxins of three bovine strains ofFusobacterium necrophorum. Am. J. Vet. Res., 47: 14221425. Shibayama, Y., 1987. Sinusoidal circulatory disturbance by microthrombosis as a cause of endotoxin-induced hepatic injury. J. Pathol., 151:315-321. Shinjo, T., Miyazato, S., Kaneuchi, C. and Mitsuoka. T., 1981. Physiological and biochemical characteristics of Fusobacterium necrophorum biovar A and biovar B strains and their deoxyribonucleic acid homology. Jpn. J. Vet. Sci., 43: 233-241. Shinjo, T., Hazu, H. and Kiyoyama, H., 1987. Hydrophobicity ofFusobacterium necrophoru~ biovars A and B. FEMS Microbiol. Lett., 48: 243-247. Shinjo, T., Miyazato, S. and Kiyoyama, H., 1988. Adherence of Fusobacterium necrophorum biovar A and B strains to erythrocytes and tissue culture cells. Ann. Inst. Pasteur, Microbiol., 139: 456-460. Springer, G.F. and Adye, J.C., 1975. Endotoxin-binding substances from human leukocytes and platelets. Infect. Immun., 12: 978-986. Sullam, P.M., Valone, F.H. and Mills, J., 1987. Mechanisms of platelet aggregation by viridans group streptococci. Infect. Immun., 55:1743-1750. Sullam, P.M., Jarvis, G.A. and Valone, F.H., 1988. Role ofimmunoglobulin G in platelet aggregation by viridans group streptococci. Infect. Immun., 56:2907-2911. Westphal, O. and Jann, K., 1965. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem., 5:83-91.
343
Aggregation of bovine platelets by Fusobacterium necrophorum Minoru Horose a, Hiroko Kiyoyamaa, Hiroyuki Ogawa b and Toshiharu Shinjo a aDepartment of Veterinary Microbiology, Faculty of Agriculture, Miyazaki University, Gakuenkibanadai, Japan bDepartment of Veterinery Surgery, Faculty of Agriculture, Miyazaki University, Gakuenkibanadai, Japan (Accepted 13 march 1992 )
ABSTRACT Horose, M., Kiyoyama, H., Ogawa, H. and Shinjo, T., 1992. Aggregation of bovine platelets by Fusobacterium necrophorum. Vet. Microbiol., 32: 343-350. Washed cell suspensions ofbiovar A strains ofFusobacterium necrophorum aggregated cattle platelets, but similar suspensions of biovar B strains did not. Platelets were also aggregated by heat-treated bacterial cells or the lipopolysaccharide of biovar A. No platelet aggregation occurred in the presence of the cell-free culture supernatant of biovar A and of all samples prepared from biovar B. Scanning electron microscopy revealed that aggregated platelets were not damaged. Platelet aggregation was inhibited by EDTA, aspirin and quinacrine, and lag time was retarded by these inhibitors, indicating the reaction was a Ca2+-dependent, cyclo-oxygenase sensitive event. Platelet aggregation may be a virulence marker, probably mediated by the lipopolysaccharide ofF. necrophorum biovar A strains.
INTRODUCTION
Fusobacterium necrophorum is divided into two biovars, A and B, by their haemagglutinating activity (Fi6vez, 1963; Shinjo et al., 1981 ). The former has this activity, but the latter does not. Biovar A is mainly isolated from pathological lesions, such as bovine hepatic abscesses or foot-rot, and biovar B is present in the alimentary tract of animals. The two biovars also differ in their pathogenicity for mice (Fievez, 1963; Shinjo et al., 1981 ), adherence to bovine ruminal epithelial cells (Kanoe and Iwaki, 1987) and cultured cell lines (Shinjo et al., 1988), composition of lipopolysaccharide (LPS) (Inoue et al., 1985), haemolytic activity (Emery et al., 1985), production of leucotoxin (Scanlan et al., 1986 ) and hydrophobicity (Shinjo et al., 1987 ). The ability of bacteria to aggregate platelets may be a virulence determiCorrespondence to: Toshiharu Shinjo, Dept of Veterinary Microbiology. Faculty of Agriculture, Miyazaki University, Gakuenkibanadai, Miyazaki 889-21, Japan.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
344
M. HOROSE ET AL.
nant in some infections. Microbial components such as LPS (Morrison et al., 1978, Springer and Adye, 1975 ), tuberculin (Rourka et al., 1979), mannan from Candida (Maisch and Calderone, 1981 ), peptidoglycan (Greenblatt et al., 1978 ) and lipoteichoic acid (Beachy et al., 1977 ) are known to contribute to platelet aggregation. F. necrophorum aggregated human platelets in platelet-rich plasma (PRP) (Forrester et al., 1985 ) and bovine platelets were aggregated by the bacterial haemagglutinin (Kanoe and Yamanaka, 1989). In this study, the authors investigated in detail the ability of F. necrophorum to aggregate bovine platelets. The experiments were carried out to determine whether the reaction is a cyclo-oxygenase-sensitive event. MATERIALS AND METHODS
Bacterial strains and growth conditions Three biovar A strains were used: strain VPI 2891 was supplied by Dr W.E.C. Moore of the Virginia Polytechnic Institute and State University, Blacksburg, VA, U.S.A., strain Fn 43 originated from a bovine hepatic abscess, and strain SPH 1 was supplied by Dr Aalbaek of the Royal Veterinary and Agricultural University, Copenhagen, Denmark. Biovar B strain VPI 6161 was supplied by Dr M. Kanoe of Yamaguchi University, Yamaguchi, Japan and strain SPH 6 was supplied by Dr. Aalbaek. The organisms were cultured at 37°C for 24 h in GAM broth (Nissui Seiyaku, Co., Tokyo), collected by centrifugation at 8000 g for 20 min, washed three times with phosphate buffered saline (PBS, pH 7.2) and resuspended in PBS. Bacterial suspensions containing 105 cfu/ml were used.
Preparations of platelets and washed platelets Nine volumes of bovine blood were drawn in to one volume of 4.6% sodium citrate solution in a plastic syringe. The mixture was transferred to plastic tubes and centrifuged at 150 g for 10 min. The supernatant was collected into plastic tubes and used as PRP. The platelets were washed three times in modified Tyrode solution (0.3% BSA, 0.2 mM EGTA in Tyrode solution, pH 6.8 ) and suspended in Tyrode solution (pH 7.4) and used as washed platelets ( 105 cells/ml) (WP).
Extraction of LPS LPS of F. necrophorum was extracted by the hot phenol water method described by Westphal and Jann (1965 ). The purified LPS was dissolved at 1 mg/ml in distilled water and diluted to 1 pg/ml in distilled water for use in platelet aggregation.
AGGREGATION OF BOVINE PLATELETS BY FUSOBACTERIUM NECROPHORUM
345
Platelet aggregation Aggregation responses were evaluated using an aggregometer (Hematracer, model PAT-2A, NKK, Tokyo ). An aliquot of 200/tl of PRP or WP was placed in a cuvette containing a stir bar. The cuvette was placed in the aggregometer (temperature 37 °C) and 25/tl of 30 m M CaCI2 solution was added. One min later 25/tl of the bacterial or LPS suspension was added, and aggregation was monitored for 3 of 5 min. The number of bacteria was adjusted to be equal to that of platelets using PBS. 25/tl horse Achilles tendon collage (500/zg/ml) was used as the reference aggregating agent. The experiments were carried out in triplicate.
Inhibition of platelet aggregation In the inhibition test, aspirin, quinacrine and EDTA were used. The three drugs were diluted to 10, 5, 2.5, 1.25 and 0.62 mM, and 25/zl of these solutions was preincubated with WP for 10 min at room temperature before being transferred to the 37 °C environment in the aggregometer. When this temperature was achieved, the bacteria were added to initiate the experiment.
Observation of platelets by scanning electron microscope Scanning electron microscope observations of PRP after aggregation by bacteria or LPS were carried out. Bacterial and LPS suspensions and PRP suspensions were mixed and incubated at 37°C for 30 min. A few drops of the mixture were fixed in 6 ml of 1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. Samples were dried in a critical point dryer and examined with a JSM-35C scanning electron microscope (Japan Electron Optics, Tokyo). RESULTS
Platelet aggregation All biovar A strains aggregated bovine PRP, while the two biovar B strains did not (Table 1 ). The kinetics of aggregation by strain VPI 2891 and strain VPI 6161 is recorded in Figs 1 and 2. Strain VPI 2891 aggregated bovine PRP within 10 min. Cell-free culture supernatants of two biovars were unable to aggregate PRP. Heat-treated bacterial cells of biovar A were also able to aggregate PRP. Furthermore, LPS aggregated platelets in proportion to bacterial strains (Fig. 3 ). Bovine platelets aggregated by bacterial cells or LPS were apparently intact when observed by scanning electron microscopy. The biovar A strains aggregated WP but the biovar B strains did not (Fig. 4 ). The kinetics was similar to that for aggregation of PRP.
Inhibition test Platelet aggregation by biovar A strains was inhibited by the three drugs. As shown in Table 2, EDTA at 1.25 mM, aspirin at 2.5 mM and quinacrine
346
M. HOROSE ET AL.
TABLE 1 Platelet aggregation ofF. necrophorum Biovar
Strain
Percent aggregation
A
VPI 2891 Fn 43 SPH 1 VP1 6161 SPH 6
86 68 70 7 3
B
C ollage..nn 80
4o
I
I
5 TIME(min)
I
10
Fig. 1. Aggregation of platelets by F. necrophorum b i o v a r A (strain VPI 2 8 91 ).
at 1.25 m M inhibited aggregation completely, and EDTA at 1.25 mM, aspirin at 2.5 mM and quinacrine at 0.62 m M produced 82%, 90% and 85% inhibition, respectively. In addition, the lag time was retarded from 0.5 rain to 1.5 or 2.0 min by these inhibitors (Table 2 ).
AGGREGATION OF BOVINE PLATELETS BY FUSOBACTERIUM NECROPHORUM
347
Collagen..
i"l ~'°-t
t
Washed ba [~P
-
-
="--
--- J l - f,--'e
I
culture
ria/cells fluid
I
I
5 TIM E(min)
10
Fig. 2. Aggregation of platelets by F. necrophorum biovar B (strain VPI 6161 ). Biovar A LPS 80Biovar A heat treated
bacterial cells i
z om p¢¢ c3 4 0 14. n-1 c~ Biovar B LPS
-
Biovar B heat t r e a t e d
I
i
5
t
10
T I M E (min) Fig. 3. Aggregation of platelets by LPS and heat-treated bacterial cells ofF. necrophorum biovar A (strain VPI 2891 ) and biovar B (strain VPI 6161 ).
348
M. H O R O S E ET AL.
80--
Biovar A Z
O
--
040-ill rr
O O
---
I
I
I
5 TIME (min)
10
Fig. 4. Aggregation of washed platelets by/~: necrophorum b i o v a r A ( s t r a i n V P I 2891 ) and biovar B (strain V P I 6161 ). TABLE 2
Inhibition ofF. necrophorum VPI 2891 platelet aggregation Inhibitor
Concentration
Lag time
Aggregation at
(mM)
(min)
10 m i n (%)
0.5
100 0 18 0 10 0 15
None EDTA EDTA
Aspirin Aspirin Quinacrine Quinacrine
10.0 1.25 10.0 2.5 1.25 0.62
1.5 2.0 2.0
DISCUSSION
The present study demonstrated that bacterial cells, heat-treated bacterial cells and the LPS ofF. necrophorumbiovar A strains aggregated bovine platelets, whereas cell-free culture supernatant was unable to do so. These results indicate that active bacterial metabolism was not responsible for the aggregation, and that heat-resistant surface components of bacteria induced platelet aggregation. Scanning electron microscope observations revealed that no
AGGREGATION OF BOVINE PLATELETS BY FUSOBACTERIUM NECROPHORUM
349
platelet lysis occurred, as has been observed with Listeria monocytgenes (Czuprynski and Balish, 1981 ) and Streptococcus pyogenes (Sullam et al., 1987). Immunoglobulin (Sullam et al., 1988), complement (Maish and Calderone, 1981 ) and fibrinogen (Kurpiewsky et al., 1983; Sullam et al., 1987) are necessary for platelet aggregation in some bacteria. Forrester et al. (1985) reported that F, necrophorum needed fibrinogen for platelet aggregation. In our experiments platelet aggregation by F. necrophorum occurred without the addition of fibrinogen. However, they used gel-filtered human platelets and we used washed bovine platelets; either of them may be a species difference, or the differing methods used may explain the different result. The inhibition of platelet aggregation by EDTA suggests that the reaction is calcium-dependent. Aggregation may be associated with cyclo-oxygenation of arachidonic acid, since aspirin and quinacrine, both cyclo-oxygenase inhibitors, inhibited aggregation. Endotoxin may induce thrombus formation in the hepatic sinusoids with subsequent hepatic necrosis (Ruiter et al., 1981; Maier and Hafnel, 1984; Shibayama, 1987 ). Endotoxin of F. necrophorum biovar A might directly affect the platelet membrane, inducing platelet activation in thrombus formation. Platelet aggregation might thus be associated with the virulence of F. necrophorum, probably mediated by LPS.
REFERENCES Beachy, E.H., Chiang, T.M., Ofek, I. and Kang, A.H., 1977. Interaction of lipoteichoic acid of group A streptococci with human platelets. Infect. Immun., 16: 649-654. Czuprynski, C.J. and Balish, E., 1981. Interaction of rat platelets with Listeria monocytogenes. Infect. Immun., 33: 103-108. Emery, D.L., Vaughan, J.A., Clark, B.L., Dufly, J.H. and Stewart, D.J., 1985. Cultural characteristics and virulence of strains of Fusobacterium necrophorurn isolated from the feet of cattle and sheep. Aust. Vet. J., 62: 43-46. Fi6vez, L., 1963. Etude compar6e des souches de Sphaerophorus necrophorus Isol6es chez l'Homme et chez l'Animal. Presses Academiques Europ6ennes, Brussels. Forrester, L.J., Campbell, B.J. and Berg, J.N. and Barrett, J.T., 1985. Aggregation of platelets by Fusobacterium necrophorum. J. Clin. Microbiol., 22: 245-249. Greenblatt, J., Boackle, R.J. and Schwab, J.H., 1978. Activation of the alternate complement pathway by peptidoglycan from streptococcal cell wall. Infect. Immun., 19: 269-303. Inoue, T., Kanoe, M., Goto, N., Matusumura, K. and Nakano, K., 1985. Chemical and biological properties oflipopolysaccharides from Fusobacterium necrophorum biovar A and biovar B strains. Jpn. J. Vet. Sci., 47: 639-645. Kanoe, M. and lwaki, K., 1987. Adherence of Fusobacterium necrophorum to bovine ruminal cells. J. Med. Microbiol., 23: 69-73. Kanoe, M. and Yamanaka, M., 1989. Bovine platelet aggregation by Fusobacterium necrophorum. J. Med. Microbiol., 29: 13-17.
350
M. HOROSE Eq- AL.
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