Veterinary Research Communications, 16 (1992) 97-105 Copyright C) Kluwer Academic Publishers bv - Printed in the Netherlands
PASSIVE PROTECTION OF MICE WITH ANTISERUM TO NEURAMINIDASE FROM PASTEURELLA MUL TOCIDA SEROTYPE A:3 F.I. IFEANYI* AND W.E. BAILIE Department of Laboratory Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA *Current address: Department of Biological Sciences, Grambling State University, Grambling, LA 71245, USA
ABSTRACT Ifeanyi, F.I. and Bailie, W.E., 1992. Passive protection of mice with antiserum to neuraminidase from Pasteurella multocida serotype A:3. Veterinary Research Communications, 16 (2), 97-105 A n t i s e r u m to a partially purified neuraminidase from Pasteurella multocida, type A:3, was adsorbed with p r o t e a s e - d i g e s t e d P. multocida type 3 lipopolysaccharide (LPS) to r e m o v e LPS i m m u n o r e a c t i v i t y . T h e L P S - a d s o r b e d a n t i n e u r a m i n i d a s e caused a 77% r e d u c t i o n in the neuraminidase activity of homologous P. multocida in an in vitro enzyme neutralization test. All 14 mice passively immunized with the adsorbed antineuraminidase were protected against challenge infection with homologous P. multocida in a m o u s e protection test. Ten out of 14 mice in one group that received antisera containing antibodies to both neuraminidase and LPS were protected. In contrast, only 1 out of 14 mice that were immunized with pre-immune serum survived the challenge. These results suggest that antiserum to P. multocida neuraminidase was, at least partly, responsible for the protection observed in this study. Neuraminidase may be one of the immunogenic protective proteins present in aqueous extracts ofPasteurella multocida.
Keywords: antineuraminidase, mouse, protection, Pasteurella multocida
INTRODUCTION Pasteurella species cause a variety of diseases including pneumonia in sheep and cattle
and acute septicaemia in turkeys and chickens. Formalized bacterins and various types of live products have been developed for protecting animals against pasteurellosis. However, the efficacy of these vaccines has been questioned (Lu and Pakes, 1981; Bennet, 1982). Streptomycin-dependent (Chengappa et aL, 1980; Kadel et aL, 1985), chemically altered (Kucera et al., 1983) and live vaccines (Confer et aL, 1985) have been developed. These vaccines have not produced uniformly good results. Various extracts of P. multocida have been used as immunogens to protect animals against experimental infection with the organism (Rebers and Heddleston, 1974; Kodama et aL, 1981). Because these components conferred better protection to animals than intact P. multocida cells (Syuto and Matsumoto, 1982), it is of interest to purify and characterize the different antigenic fractions ofP. multocida extracts. Neuraminidase (N-acetylneuraminyl hydrolase, EC 3.2.1.18) is produced by many microorganisms including P. multocida (Muller, 1974). This enzyme removes sialic acid from glycoprotein and glycolipid compounds. Neuraminidase has been implicated as a virulence factor in certain bacterial species, particularly those that inhabit
98 mucosal surfaces (Moriyama and Barksdale, 1%7; Hayano and Tanaka, 1%9). This report describes the purification of a saline extract of P. multocida neuraminidase, the immunogenicity of neuraminidase-LPS complex in rabbits and the protective capability of antineuraminidase in mice.
MATERIALS AND METHODS
Bacterial strain, media and growth conditions Pasteurella multocida type A:3 (strain KSU 50859), originally isolated from bovine respiratory tract, was obtained from the culture collection of the Department of Laboratory Medicine, Kansas State University. All bacterial growth media were purchased from Difco Laboratories, Detroit, MI, USA. The culture was grown for 18 h at 37°C in brain heart infusion broth and stored frozen in 1-ml aliquots at -70°C. The organism was subcultured on tryptic soy agar base, supplemented with 5% bovine blood, and grown for 24 h at 37°C. The bacterial growth was suspended in brain heart infusion broth and lawns were made on dextrose starch agar plates which were incubated for 18 h at 37°C. Bacterial cells were harvested and suspended in 0.1 mol/L sodium phosphate buffer, pH 6.5, containing 0.04% sodium azide.
Extraction and purification of P. multocida neuraminidase Crude neuraminidase was extracted from the cell suspension by the method of Drzeniek et al. (1972). Briefly, NaC1 was added to the cell suspension to a final concentration of 0.4 mol/L. Following 30 min incubation in a 37°C water bath, the ceils were pelleted by centrifugation at 15,000 g for 20 min. The supernatant was pooled as crude neuraminidase extract. The pelleted cells were resuspended in the phosphate buffer and re-extracted as described above.
Hydrophobic interaction chromatography (HIC). Crude neuraminidase was sonicated in an ice bath for 5 min and ammonium sulphate was added to the sonicated material to achieve 25% saturation. The enzyme preparation was applied to a 12 cm x 1 cm internal diameter column of Octyl Sepharose CL-4B (Sigma, St Louis, MO, USA) previously equilibrated with 25% ammonium sulphate saturated distilled water. The column was thoroughly washed with the equilibrating solution and eluted with distilled water containing 0.04% sodium azide. Neuraminidase assay. Fractions containing neuraminidase were identified by neuraminidase assay. For this, 0.1 ml of each fraction was added to 0.9 ml of substrate solution (2 mg/ml fetuin type III) and the mixture was incubated for 30 min in a 37°C waterbath. To stop the enzyme action, 1 ml of 5% phosphotungstic acid in 2.5 mol/L HCI was added and the enzyme-substrate mixture was centrifuged at 8000 g for 5 min. Then 0.2 ml of the supernatant was assayed for released sialic acid by the thiobarbituric acid assay (Warren, 1959). Protein was determined by the method of Bradford (1976) using bovine gamma globulin as the standard.
99
DEAE ion-exchange chromatography. Neuraminidase-containing fractions from the HIC column were pooled and exhaustively dialysed against 0.05 mol/L Bistris buffer, pH 6.0, containing 0.04% sodium azide (BIS). The dialysed material was applied to a 6.0 cm x 2.5 cm internal diameter column of DEAE Bio-gel A, which was washed with BIS and eluted with 0.3 mol/L sodium sulphate in BIS. High-performance liquid chromatography. The material pooled from the preceding step was subjected to further ion-exchange chromatography on a 75 mm x 7.5 mm internal diameter Bio-Sil DEAE-TSK 3-SW HPLC column (Bio-Rad Chemical Division, Richmond, CA, USA) equilibrated with BIS (solvent A). The eluting buffer (solvent B) was 1 mol/L sodium sulphate in BIS. The gradient was generated with a dual pump ISCO Model 350 and the absorbance at 280 nm was monitored with a V4 absorbance detector. The flow rate was 0.8 ml/min. Fractions containing neuraminidase were pooled and again re-applied to the HPLC column to obtain the final enzyme preparation, designated purified enzyme (PE). The purification was monitored at each stage by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). Gels were silver-stained as described by Oakley et aL (1980).
Preparation of Pasteurella multocida type 3 smooth lipopolysaccharide Pasteurella multocida type 3 lipopolysaccharide (LPS) was extracted with phenol-water by the method of Westphal and Jann (1965). LPS (10 mg) was further purified by digestion with protease type XXV (Sigma, St Louis). Protease-digested LPS was dissolved in 0.03 mol/L Tris-HCl buffer, pH 7.5, and the protein content was determined according to Bradford (1976), to ensure that essentially all the protein had been removed from the digested LPS.
Preparation of antisera Pasteurella-free New Zealand white rabbits were bled I week prior to immunization to obtain pre-immune serum. The rabbits were then injected subcutaneously with either 2 mg of PE or 200 mg of undigested P. multocida type 3 LPS in complete Freund's adjuvant. Two weeks later, the rabbits were test bled and then given a third dose of the antigens. Two months after the last injection, all the rabbits were killed. The serum was harvested and stored frozen in 1 ml aliquots at -70°C.
Counterimmunoelectrophoresis (CIE) Anti-PE was examined for LPS immunoreactivity using a modification of CIE described by Crowle (1980). The wells near the anode end of the gel were filled with either anti-PE or pre-immune serum, while those near the cathode end were filled with either PE or undigested type 3 P. multocida LPS. Electrophoresis was done for i h at a constant current of 3 mA/cm. The gel was pressed, dried and stained in a solution of Coomassie brilliant blue G in methanol-acetic acid-distilled water (5 : 1 : 5, by volume) and de-stained in the same solvent.
lOO Serum adsorption
Anti-PE was adsorbed with protease-digested LPS essentially as described by Fick et al. (1980). The antiserum was recycled continuously for 24 h at room temperature on an immunoadsorbent column prepared by binding protease-digested P. multocida type 3 LPS to Detoxigel (Pierce Chemical Co., Rockford, IL, USA). Samples of antiserum before and after adsorption were reacted with protease-digested and undigested P. multocida type 3 LPS in an immunodiffusion test to verify removal of anti-LPS from the adsorbed serum.
Enzyme neutralization
The LPS-adsorbed serum (anti-neuraminidase) was examined for the presence of neuraminidase-neutralizing antibody by the method of Brown and Straus (1987). Briefly, 200/zl of antineuraminidase, pre-immune serum or buffer was mixed with an equal volume of PE. The mixture was incubated at 37°C for 30 min. Thereafter, 100 /zl of the mixture in each of these primary tubes was transferred to each of two secondary tubes containing 0.9 ml of 2 mg/ml fetuin type III. The reaction in one of the secondary tubes was stopped immediately (time 0) by the addition of 1 ml of 5% phosphotungstic acid in 2.5 mol/L HC1, while the other secondary tube was further incubated for 30 min before the reaction was stopped as described above. The tubes were centrifuged at 8000 g for 5 min and 200/zl of the supernatant was assayed for released sialic acid according to Warren (1959). The enzyme incubated in pre-immune serum and buffer served as controls. The ability of antineuraminidase to neutralize neuraminidase activity was determined by comparing the enzymatic activity of neuraminidase incubated with substrate in the presence of antineuraminidase, with the enzymatic activity of neuraminidase incubated with substrate in the presence of pre-immune serum or buffer.
Mouse protection test
Seventy Swiss white mice each weighing approximately 20 g were injected intraperitoneally with 0.5 ml of either pre-immune serum, sterile normal saline, unadsorbed anti-PE or LPS-adsorbed anti-PE (antineuraminidase). Uninjected mice served as an additional control group. Twenty-four hours after pre-treatment, all the mice were injected intraperitoneally with 1 x 103 colony-forming units (cfu) in 0.5 ml of an 18-h broth culture of homologous P. multocida. This challenge dose was approximately 30 times the mean LDs0 for unprotected control groups of mice as determined in a preliminary study. Deaths were recorded daily and all surviving mice were killed 7 days after challenge.
Statistics
The Mann-Whitney U-test was used to analyse the difference in protection between the treatment group and the control groups that received pre-immune serum.
101
RESULTS The neuraminidase preparation obtained by chromatography on the HIC and the DEAE Bio-gel A columns contained type 3 P. multocida LPS as determined by SDS-PAGE. The subsequent purification on the HPLC column was an attempt to eliminate the LPS. The enzyme activity of neuraminidase determined at the end of each stage of purification was as follows: crude extract, 0.269 units/ml; hydrophobic interaction chromatography, 0.285 units/ml; DEAE ion-exchange chromatography, 0.170 units/ml; and, high-performance liquid chromatography, 0.245 units/ml. The unit of activity for neuramidase is the amount that liberates 1/zmole of sialic acid from fetrin type III in 1 min at 37°, pH 6.5. The elution profile from the last HPLC run is shown in Figure 1. Fractions collected under peak 2 (Figure 1) were pooled and designated purified enzyme (PE). When PE was subjected to SDS-PAGE, LPS could still be detected but in a considerably reduced amount (Figure 2, lane c). As a result, the antiserum raised against PE showed anti-LPS immunoreactivity as determined by CIE in which anti-PE was reacted against undigested type 3 P. multocida LPS. To remove the LPS immunoreactivity, the anti-PE was adsorbed with protease-digested LPS. The immunodiffusion test with anti-PE before and after adsorption indicated that LPS immunoreactivity had been removed from the adsorbed serum (Figure 3). To ensure that the adsorbed serum (antineuraminidase) still retained enzyme-neutralizing activity, an in vitro enzyme neutralization test was done. Antineuraminidase reduced the neuraminidase activity of homologous P. multocida by 77% (Table I). The results of the mouse protection test are presented in Table II. All mice passively immunized with adsorbed anti-PE (antineuraminidase) were protected against challenge with homologous P. multocida, while 78% of the mice immunized with unadsorbed anti-PE were protected. Only 7% of mice that received pre-immune rabbit serum survived the challenge. l00
t
9O % 8O 7O S 0 6O L V 50 E
2
L
40
50 20
I
r ............... I
G4 &,'2 16 20 24 I/
°-"°-
I0
".,___r "-. ~"
2'8 12 3J6 4'0 4'4 4'8 5'2 5'6 6'0 Time (Min)
Figure 1. Chromatography of P. multocida neuraminidase extract on a Bio-SU DEAE-TSK 3-SW HPLC column. The gradient (broken line) was 0-5% solvent B for 1 min, 5% B for 10 min, 5-20% B for 20 min followed by solvent A for 10 min. Fractions pooled under peak 2 were designated purified enzyme (PE)
102
Figure 2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified neuraminidase. Lanes a and f" molecular weight markers. Lane b" P. multocida LPS. Lane c: purified neuraminidase pooled from peak 2. Lane d: neuraminidase pooled from peak 1. Lane e: crude neuraminidase extract. The single band migrating at about 35 kDa (lane c) was neuraminidase
Figure 3. Immunodiffusion test with anti-PE serum before and after adsorption with protease-digested P. multocida type 3 LPS. Centre wells A and B contained anti-PE before and after adsorption, respectively. Wells 1A and 1B contained undigested LPS, while wells 2A and 2B contained protease-digested LPS
103 TABLE I Neutralization of neuraminidase activity of homologous Pasteurella multocida with adsorbed anti-PE (antineuraminidase)
Neuraminidase activity (units/ml) a Reaction mixture
Time 0
After 30 min
Enzyme/anti-PE
0.024 _+ 0.005
0.073 _ 0.006
Enzyme/pre-immune serum
0.056 -+ 0.011
0.269 _+ 0.007
Enzyme/buffer
0.000 _+ 0.000
0.175 -+ 0.007
aMean - SD of three separate determinations. The determination at time 0 served as the baseline value estimating the amount of sialic acid released from serum glycoproteins by neuraminidase. These values were subtracted from the corresponding values determined after 30 min incubation of enzyme/sera mixtures with fetuin. The resulting values represented the activity of unneutralized neuraminidase, thereby indirectly quantitating the neuraminidase neutralizing activity of the antiserum.
TABLE II Mouse protection test with antiserum to P. multocida neuraminidase
Treatment
None Normal saline Pre-immune serum Anti-PE (unadsorbed) a Anti-PE (adsorbed) a
Dead
Alive
13 12 13 3 0
1 2 1 11 14
Per cent protected
7.1 14.2 7.1 78.5 100.0
aSigniticant p-value determined by Mann-Whitney U-test relative to pre-immune serum was p < 0.01. Unadsorbed anti-PE contained antibodies to both neuraminidase and LPS, while adsorbed anti-PE contained only antineuraminidase as determined by immunodiffusion and enzyme neutralization tests
104 DISCUSSION Despite repeated chromatographic resolution of the neuraminidase extract, LPS could not be completely eliminated without denaturing the enzyme. Other investigators have had problems dissociating LPS from endotoxin-protein complexes of many Gramnegative bacteria (Kurtz and Berman, 1986). To circumvent this problem, anti-PE that contained both antineuraminidase and anti-LPS was adsorbed with proteasedigested type 3 P. multocida LPS. Protease digestion removed almost all the protein present in the LPS as determined by protein assay of digested LPS. Adsorption of anti-PE with the protease-digested LPS removed LPS immunoreactivity without interfering with the neuraminidase-neutralizing antibody as demonstrated by the enzyme neutralization test (Table I). The immunodiffusion test did not detect LPS immunoreactivity in the adsorbed anti-PE. Before adsorption, anti-PE reacted with both undigested and protease-digested LPS (Figure 3A), indicating the presence of anti-LPS in the unadsorbed antiserum. Following adsorption, the adsorbed antiserum did not react with the undigested LPS or protease-digested LPS (Figure 3B), indicating the removal of LPS immunoreactivity. Although it is possible that the immunodiffusion test might not have detected non-precipitating antibodies, we believe that essentially all anti-LPS was removed by the adsorption process. The presence of neuraminidase-neutralizing antibody in the adsorbed serum was demonstrated by the 77% reduction in the neuraminidase activity of homologous P. multocida (Table I). The major finding in this study was that all the mice passively immunized with rabbit antiserum to P. multocida neuraminidase (adsorbed anti-PE) were protected against subsequent homologous challenge infection. Mice that were immunized with antiserum containing antibodies to both neuraminidase and LPS (unadsorbed anti-PE) were partially protected. However, there was no significant difference in protection between the mice treated with unadsorbed sera and the mice treated with LPS adsorbed sera. Srivastava and Foster (1977) reported that an LPS fraction obtained from a P. multocida LPS-protein complex had low immunogenicity in mice compared to the protein fraction. Rebers et al. (1980) demonstrated antibody to P. multocida type 1 LPS in mice, but the mice were not protected against a homologous challenge infection. It is therefore probable that the partial protection provided by unadsorbed anti-PE was due to the antineuraminidase rather than the anti-LPS contained in the antiserum. The high degree of mouse protection observed in this study was possibly due to in vivo neutralization of the adverse effects of neuraminidase by antineuraminidase. Further experiments will be conducted using immunoblotting techniques and an attempt will be made to obtain P. multocida neuraminidase free of LPS contamination. It should then be possible to adsorb anti-PE with the neuraminidase preparation in order to determine whether the neuraminidase-adsorbed antiserum will fail to protect mice against challenge infection with homologous P. multocida. ACKNOWLEDGEMENTS This work was supported by grants from Kansas Agricultural Experiment Station, of Kansas State University, to W.E. Bailie. This is Contribution No. 89-56-J.
105 REFERENCES Bennet, B.W., 1982. Efficacy of Pasteurella bacterins for yearling feedlot cattle. Bovine Practitioner, 3, 26-30 Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 249-254 Brown, J. and Straus, D., 1987. Characterization of neuraminidase produced by various serotypes of group B streptococci. Infection and Immunity, 55, 1-6 Chengappa, M.M., Myers, R.C. and Carter, G.R., 1980. A streptomycin dependent live Pasteurella rnultocida vaccine for prevention of fowl cholera in turkeys. Avian Diseases, 91, 57-61 Confer, A.W., Panciera, R.J., Fulton, R.W., Gentry, M.J. and Rummage, J.A., 1985. Effect of vaccination with live or killed Pasteurella hoemolytica on resistance to experimental bovine pneumonic pasteurellosis. American Journal of Veterinary Research, 46, 342-347 Crowle, A.J., 1980. Precipitin and microprecipitin reactions in fluid mediums and in gels. In: N.R. Rose and H. Friedman (eds), Manual of Clinical Immunology, 2nd edn, (American Society for Microbiology, Washington DC), 3-14 Drzneik, IL, Scharman, W. and Blake, E., 1972. Neuraminidase and N-acetylneuraminyl pyruvate lyase of Pasteurella multocida. Journal of General Microbiology, 22, 352-368 Fick, R.B., Nagel, G.P. and Reynolds, H.Y., 1980. Use of Pseudomonas aeruginosa lipopolysaccharide immunoadsorbents to prepare high potency monospec,.'fic antibodies. Journal of Immunological Methods, 38, 103-116 Hayano, S. and Tanaka, A., 1969. Sialidase-like enzymes produced by groups A, B, C, G and L streptococci and Streptococcus sanguis. Journal of Bacteriology, 97, 1328-1333 Kadel, W.L., Chengappa, M.M. and Herren, C.E., 1985. Field trial evaluation of a Pasteurella vaccine in preconditioned and nonpreconditioned light weight calves. American Journal of Veterinary Research, 46, 1944-1948 Kodama, H., Matsumoto, M. and Snow, L.M., 1981. Immunogenicity of capsular antigens of Pasteurella multocida in turkeys. American Journal of Veterinary Research, 42, 1838-1841 Kucera, C.J., Wong, J.C.S. and Feldner, T.J., 1983. Challenge exposure of cattle vaccinated with a chemically-altered strain of Pasteurella haemolytica. American Journal of Veterinary Research, 44, 1848-1852 Kurtz, R.S. and Berman, D.J., 1986. Influence of endotoxin-protein complex in immunoglobin G isotypes of mice to Brucella abortus lipopolysaccharide. Infection and Immunity, 54, 728-734 Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature, 227, 680-685 Lu, Y.S. and Pakes, S.P., 1981. Protection of rabbits against experimental pasteurellosis by a streptomycin-dependent Pasteurella multocida serotype 3:A live mutant vaccine. Infection and Immunity, 34, 1018-1024 Moriyama, T. and Barksdale, L., 1967. Neuraminidase of Corynebacteriurn dipthen'ae. Journal of Bacteriology, 94, 1565-1581 Muller, H.E., 1974. Neuraminidase of mycoplasmas, bacteria and protozoa and their pathogenic role. Behn'ng Institute Mitteilungen, 8, 34-56 Oakley, B.R., Donald, R.K. and Ronald Morris, N., 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry, 105, 361-363 Rebers, P.A. and Heddleston, K.A., 1974. Immunogenic comparison of Westphal-type lipopolysaccharide and free endotoxins from encapsulated avian strain of Pasteurella multocida. American Journal of Veterinary Research, 35, 556-560 Rebers, P.A., Phillips, M., Rimler, IL, Boykins, tLA., Keith, B.S. and Rhodes, R., 1980. Immunizing properties of Westphal lipopolysaccharides from avian strain of Pasteurella multocida. American Journal of Veterinary Research, 41, 1650-1654 Srivastava, K.IC and Foster, J.W., 1977. Characterization of immunogenic fraction of Pasteurella multocida culture filtrates. Canadian Journal of Microbiology, 23, 197-201 Syuto, B. and Matsumoto, M., 1982. Purification of protective antigen from a saline extract of Pasteurella multocida. Infection and Immunity, 37, 1218-1226 Warren, L., 1959. The thiobarbituric acid assay of sialic acids. Journal of Biological Chemistry, 234, 1971-1975 Westphal, O. and Jann, K., 1965. Bacterial lipopolysaccharides. In: ILL. Whistler and M.L. Worform (eds), Methods in Carbohydrate Chemistry, vol. 5, (Academic Press, New York), 83-91 (Accepted: 27 January 1992)
PASSIVE PROTECTION OF MICE WITH ANTISERUM TO NEURAMINIDASE FROM PASTEURELLA MUL TOCIDA SEROTYPE A:3 F.I. IFEANYI* AND W.E. BAILIE Department of Laboratory Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA *Current address: Department of Biological Sciences, Grambling State University, Grambling, LA 71245, USA
ABSTRACT Ifeanyi, F.I. and Bailie, W.E., 1992. Passive protection of mice with antiserum to neuraminidase from Pasteurella multocida serotype A:3. Veterinary Research Communications, 16 (2), 97-105 A n t i s e r u m to a partially purified neuraminidase from Pasteurella multocida, type A:3, was adsorbed with p r o t e a s e - d i g e s t e d P. multocida type 3 lipopolysaccharide (LPS) to r e m o v e LPS i m m u n o r e a c t i v i t y . T h e L P S - a d s o r b e d a n t i n e u r a m i n i d a s e caused a 77% r e d u c t i o n in the neuraminidase activity of homologous P. multocida in an in vitro enzyme neutralization test. All 14 mice passively immunized with the adsorbed antineuraminidase were protected against challenge infection with homologous P. multocida in a m o u s e protection test. Ten out of 14 mice in one group that received antisera containing antibodies to both neuraminidase and LPS were protected. In contrast, only 1 out of 14 mice that were immunized with pre-immune serum survived the challenge. These results suggest that antiserum to P. multocida neuraminidase was, at least partly, responsible for the protection observed in this study. Neuraminidase may be one of the immunogenic protective proteins present in aqueous extracts ofPasteurella multocida.
Keywords: antineuraminidase, mouse, protection, Pasteurella multocida
INTRODUCTION Pasteurella species cause a variety of diseases including pneumonia in sheep and cattle
and acute septicaemia in turkeys and chickens. Formalized bacterins and various types of live products have been developed for protecting animals against pasteurellosis. However, the efficacy of these vaccines has been questioned (Lu and Pakes, 1981; Bennet, 1982). Streptomycin-dependent (Chengappa et aL, 1980; Kadel et aL, 1985), chemically altered (Kucera et al., 1983) and live vaccines (Confer et aL, 1985) have been developed. These vaccines have not produced uniformly good results. Various extracts of P. multocida have been used as immunogens to protect animals against experimental infection with the organism (Rebers and Heddleston, 1974; Kodama et aL, 1981). Because these components conferred better protection to animals than intact P. multocida cells (Syuto and Matsumoto, 1982), it is of interest to purify and characterize the different antigenic fractions ofP. multocida extracts. Neuraminidase (N-acetylneuraminyl hydrolase, EC 3.2.1.18) is produced by many microorganisms including P. multocida (Muller, 1974). This enzyme removes sialic acid from glycoprotein and glycolipid compounds. Neuraminidase has been implicated as a virulence factor in certain bacterial species, particularly those that inhabit
98 mucosal surfaces (Moriyama and Barksdale, 1%7; Hayano and Tanaka, 1%9). This report describes the purification of a saline extract of P. multocida neuraminidase, the immunogenicity of neuraminidase-LPS complex in rabbits and the protective capability of antineuraminidase in mice.
MATERIALS AND METHODS
Bacterial strain, media and growth conditions Pasteurella multocida type A:3 (strain KSU 50859), originally isolated from bovine respiratory tract, was obtained from the culture collection of the Department of Laboratory Medicine, Kansas State University. All bacterial growth media were purchased from Difco Laboratories, Detroit, MI, USA. The culture was grown for 18 h at 37°C in brain heart infusion broth and stored frozen in 1-ml aliquots at -70°C. The organism was subcultured on tryptic soy agar base, supplemented with 5% bovine blood, and grown for 24 h at 37°C. The bacterial growth was suspended in brain heart infusion broth and lawns were made on dextrose starch agar plates which were incubated for 18 h at 37°C. Bacterial cells were harvested and suspended in 0.1 mol/L sodium phosphate buffer, pH 6.5, containing 0.04% sodium azide.
Extraction and purification of P. multocida neuraminidase Crude neuraminidase was extracted from the cell suspension by the method of Drzeniek et al. (1972). Briefly, NaC1 was added to the cell suspension to a final concentration of 0.4 mol/L. Following 30 min incubation in a 37°C water bath, the ceils were pelleted by centrifugation at 15,000 g for 20 min. The supernatant was pooled as crude neuraminidase extract. The pelleted cells were resuspended in the phosphate buffer and re-extracted as described above.
Hydrophobic interaction chromatography (HIC). Crude neuraminidase was sonicated in an ice bath for 5 min and ammonium sulphate was added to the sonicated material to achieve 25% saturation. The enzyme preparation was applied to a 12 cm x 1 cm internal diameter column of Octyl Sepharose CL-4B (Sigma, St Louis, MO, USA) previously equilibrated with 25% ammonium sulphate saturated distilled water. The column was thoroughly washed with the equilibrating solution and eluted with distilled water containing 0.04% sodium azide. Neuraminidase assay. Fractions containing neuraminidase were identified by neuraminidase assay. For this, 0.1 ml of each fraction was added to 0.9 ml of substrate solution (2 mg/ml fetuin type III) and the mixture was incubated for 30 min in a 37°C waterbath. To stop the enzyme action, 1 ml of 5% phosphotungstic acid in 2.5 mol/L HCI was added and the enzyme-substrate mixture was centrifuged at 8000 g for 5 min. Then 0.2 ml of the supernatant was assayed for released sialic acid by the thiobarbituric acid assay (Warren, 1959). Protein was determined by the method of Bradford (1976) using bovine gamma globulin as the standard.
99
DEAE ion-exchange chromatography. Neuraminidase-containing fractions from the HIC column were pooled and exhaustively dialysed against 0.05 mol/L Bistris buffer, pH 6.0, containing 0.04% sodium azide (BIS). The dialysed material was applied to a 6.0 cm x 2.5 cm internal diameter column of DEAE Bio-gel A, which was washed with BIS and eluted with 0.3 mol/L sodium sulphate in BIS. High-performance liquid chromatography. The material pooled from the preceding step was subjected to further ion-exchange chromatography on a 75 mm x 7.5 mm internal diameter Bio-Sil DEAE-TSK 3-SW HPLC column (Bio-Rad Chemical Division, Richmond, CA, USA) equilibrated with BIS (solvent A). The eluting buffer (solvent B) was 1 mol/L sodium sulphate in BIS. The gradient was generated with a dual pump ISCO Model 350 and the absorbance at 280 nm was monitored with a V4 absorbance detector. The flow rate was 0.8 ml/min. Fractions containing neuraminidase were pooled and again re-applied to the HPLC column to obtain the final enzyme preparation, designated purified enzyme (PE). The purification was monitored at each stage by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). Gels were silver-stained as described by Oakley et aL (1980).
Preparation of Pasteurella multocida type 3 smooth lipopolysaccharide Pasteurella multocida type 3 lipopolysaccharide (LPS) was extracted with phenol-water by the method of Westphal and Jann (1965). LPS (10 mg) was further purified by digestion with protease type XXV (Sigma, St Louis). Protease-digested LPS was dissolved in 0.03 mol/L Tris-HCl buffer, pH 7.5, and the protein content was determined according to Bradford (1976), to ensure that essentially all the protein had been removed from the digested LPS.
Preparation of antisera Pasteurella-free New Zealand white rabbits were bled I week prior to immunization to obtain pre-immune serum. The rabbits were then injected subcutaneously with either 2 mg of PE or 200 mg of undigested P. multocida type 3 LPS in complete Freund's adjuvant. Two weeks later, the rabbits were test bled and then given a third dose of the antigens. Two months after the last injection, all the rabbits were killed. The serum was harvested and stored frozen in 1 ml aliquots at -70°C.
Counterimmunoelectrophoresis (CIE) Anti-PE was examined for LPS immunoreactivity using a modification of CIE described by Crowle (1980). The wells near the anode end of the gel were filled with either anti-PE or pre-immune serum, while those near the cathode end were filled with either PE or undigested type 3 P. multocida LPS. Electrophoresis was done for i h at a constant current of 3 mA/cm. The gel was pressed, dried and stained in a solution of Coomassie brilliant blue G in methanol-acetic acid-distilled water (5 : 1 : 5, by volume) and de-stained in the same solvent.
lOO Serum adsorption
Anti-PE was adsorbed with protease-digested LPS essentially as described by Fick et al. (1980). The antiserum was recycled continuously for 24 h at room temperature on an immunoadsorbent column prepared by binding protease-digested P. multocida type 3 LPS to Detoxigel (Pierce Chemical Co., Rockford, IL, USA). Samples of antiserum before and after adsorption were reacted with protease-digested and undigested P. multocida type 3 LPS in an immunodiffusion test to verify removal of anti-LPS from the adsorbed serum.
Enzyme neutralization
The LPS-adsorbed serum (anti-neuraminidase) was examined for the presence of neuraminidase-neutralizing antibody by the method of Brown and Straus (1987). Briefly, 200/zl of antineuraminidase, pre-immune serum or buffer was mixed with an equal volume of PE. The mixture was incubated at 37°C for 30 min. Thereafter, 100 /zl of the mixture in each of these primary tubes was transferred to each of two secondary tubes containing 0.9 ml of 2 mg/ml fetuin type III. The reaction in one of the secondary tubes was stopped immediately (time 0) by the addition of 1 ml of 5% phosphotungstic acid in 2.5 mol/L HC1, while the other secondary tube was further incubated for 30 min before the reaction was stopped as described above. The tubes were centrifuged at 8000 g for 5 min and 200/zl of the supernatant was assayed for released sialic acid according to Warren (1959). The enzyme incubated in pre-immune serum and buffer served as controls. The ability of antineuraminidase to neutralize neuraminidase activity was determined by comparing the enzymatic activity of neuraminidase incubated with substrate in the presence of antineuraminidase, with the enzymatic activity of neuraminidase incubated with substrate in the presence of pre-immune serum or buffer.
Mouse protection test
Seventy Swiss white mice each weighing approximately 20 g were injected intraperitoneally with 0.5 ml of either pre-immune serum, sterile normal saline, unadsorbed anti-PE or LPS-adsorbed anti-PE (antineuraminidase). Uninjected mice served as an additional control group. Twenty-four hours after pre-treatment, all the mice were injected intraperitoneally with 1 x 103 colony-forming units (cfu) in 0.5 ml of an 18-h broth culture of homologous P. multocida. This challenge dose was approximately 30 times the mean LDs0 for unprotected control groups of mice as determined in a preliminary study. Deaths were recorded daily and all surviving mice were killed 7 days after challenge.
Statistics
The Mann-Whitney U-test was used to analyse the difference in protection between the treatment group and the control groups that received pre-immune serum.
101
RESULTS The neuraminidase preparation obtained by chromatography on the HIC and the DEAE Bio-gel A columns contained type 3 P. multocida LPS as determined by SDS-PAGE. The subsequent purification on the HPLC column was an attempt to eliminate the LPS. The enzyme activity of neuraminidase determined at the end of each stage of purification was as follows: crude extract, 0.269 units/ml; hydrophobic interaction chromatography, 0.285 units/ml; DEAE ion-exchange chromatography, 0.170 units/ml; and, high-performance liquid chromatography, 0.245 units/ml. The unit of activity for neuramidase is the amount that liberates 1/zmole of sialic acid from fetrin type III in 1 min at 37°, pH 6.5. The elution profile from the last HPLC run is shown in Figure 1. Fractions collected under peak 2 (Figure 1) were pooled and designated purified enzyme (PE). When PE was subjected to SDS-PAGE, LPS could still be detected but in a considerably reduced amount (Figure 2, lane c). As a result, the antiserum raised against PE showed anti-LPS immunoreactivity as determined by CIE in which anti-PE was reacted against undigested type 3 P. multocida LPS. To remove the LPS immunoreactivity, the anti-PE was adsorbed with protease-digested LPS. The immunodiffusion test with anti-PE before and after adsorption indicated that LPS immunoreactivity had been removed from the adsorbed serum (Figure 3). To ensure that the adsorbed serum (antineuraminidase) still retained enzyme-neutralizing activity, an in vitro enzyme neutralization test was done. Antineuraminidase reduced the neuraminidase activity of homologous P. multocida by 77% (Table I). The results of the mouse protection test are presented in Table II. All mice passively immunized with adsorbed anti-PE (antineuraminidase) were protected against challenge with homologous P. multocida, while 78% of the mice immunized with unadsorbed anti-PE were protected. Only 7% of mice that received pre-immune rabbit serum survived the challenge. l00
t
9O % 8O 7O S 0 6O L V 50 E
2
L
40
50 20
I
r ............... I
G4 &,'2 16 20 24 I/
°-"°-
I0
".,___r "-. ~"
2'8 12 3J6 4'0 4'4 4'8 5'2 5'6 6'0 Time (Min)
Figure 1. Chromatography of P. multocida neuraminidase extract on a Bio-SU DEAE-TSK 3-SW HPLC column. The gradient (broken line) was 0-5% solvent B for 1 min, 5% B for 10 min, 5-20% B for 20 min followed by solvent A for 10 min. Fractions pooled under peak 2 were designated purified enzyme (PE)
102
Figure 2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified neuraminidase. Lanes a and f" molecular weight markers. Lane b" P. multocida LPS. Lane c: purified neuraminidase pooled from peak 2. Lane d: neuraminidase pooled from peak 1. Lane e: crude neuraminidase extract. The single band migrating at about 35 kDa (lane c) was neuraminidase
Figure 3. Immunodiffusion test with anti-PE serum before and after adsorption with protease-digested P. multocida type 3 LPS. Centre wells A and B contained anti-PE before and after adsorption, respectively. Wells 1A and 1B contained undigested LPS, while wells 2A and 2B contained protease-digested LPS
103 TABLE I Neutralization of neuraminidase activity of homologous Pasteurella multocida with adsorbed anti-PE (antineuraminidase)
Neuraminidase activity (units/ml) a Reaction mixture
Time 0
After 30 min
Enzyme/anti-PE
0.024 _+ 0.005
0.073 _ 0.006
Enzyme/pre-immune serum
0.056 -+ 0.011
0.269 _+ 0.007
Enzyme/buffer
0.000 _+ 0.000
0.175 -+ 0.007
aMean - SD of three separate determinations. The determination at time 0 served as the baseline value estimating the amount of sialic acid released from serum glycoproteins by neuraminidase. These values were subtracted from the corresponding values determined after 30 min incubation of enzyme/sera mixtures with fetuin. The resulting values represented the activity of unneutralized neuraminidase, thereby indirectly quantitating the neuraminidase neutralizing activity of the antiserum.
TABLE II Mouse protection test with antiserum to P. multocida neuraminidase
Treatment
None Normal saline Pre-immune serum Anti-PE (unadsorbed) a Anti-PE (adsorbed) a
Dead
Alive
13 12 13 3 0
1 2 1 11 14
Per cent protected
7.1 14.2 7.1 78.5 100.0
aSigniticant p-value determined by Mann-Whitney U-test relative to pre-immune serum was p < 0.01. Unadsorbed anti-PE contained antibodies to both neuraminidase and LPS, while adsorbed anti-PE contained only antineuraminidase as determined by immunodiffusion and enzyme neutralization tests
104 DISCUSSION Despite repeated chromatographic resolution of the neuraminidase extract, LPS could not be completely eliminated without denaturing the enzyme. Other investigators have had problems dissociating LPS from endotoxin-protein complexes of many Gramnegative bacteria (Kurtz and Berman, 1986). To circumvent this problem, anti-PE that contained both antineuraminidase and anti-LPS was adsorbed with proteasedigested type 3 P. multocida LPS. Protease digestion removed almost all the protein present in the LPS as determined by protein assay of digested LPS. Adsorption of anti-PE with the protease-digested LPS removed LPS immunoreactivity without interfering with the neuraminidase-neutralizing antibody as demonstrated by the enzyme neutralization test (Table I). The immunodiffusion test did not detect LPS immunoreactivity in the adsorbed anti-PE. Before adsorption, anti-PE reacted with both undigested and protease-digested LPS (Figure 3A), indicating the presence of anti-LPS in the unadsorbed antiserum. Following adsorption, the adsorbed antiserum did not react with the undigested LPS or protease-digested LPS (Figure 3B), indicating the removal of LPS immunoreactivity. Although it is possible that the immunodiffusion test might not have detected non-precipitating antibodies, we believe that essentially all anti-LPS was removed by the adsorption process. The presence of neuraminidase-neutralizing antibody in the adsorbed serum was demonstrated by the 77% reduction in the neuraminidase activity of homologous P. multocida (Table I). The major finding in this study was that all the mice passively immunized with rabbit antiserum to P. multocida neuraminidase (adsorbed anti-PE) were protected against subsequent homologous challenge infection. Mice that were immunized with antiserum containing antibodies to both neuraminidase and LPS (unadsorbed anti-PE) were partially protected. However, there was no significant difference in protection between the mice treated with unadsorbed sera and the mice treated with LPS adsorbed sera. Srivastava and Foster (1977) reported that an LPS fraction obtained from a P. multocida LPS-protein complex had low immunogenicity in mice compared to the protein fraction. Rebers et al. (1980) demonstrated antibody to P. multocida type 1 LPS in mice, but the mice were not protected against a homologous challenge infection. It is therefore probable that the partial protection provided by unadsorbed anti-PE was due to the antineuraminidase rather than the anti-LPS contained in the antiserum. The high degree of mouse protection observed in this study was possibly due to in vivo neutralization of the adverse effects of neuraminidase by antineuraminidase. Further experiments will be conducted using immunoblotting techniques and an attempt will be made to obtain P. multocida neuraminidase free of LPS contamination. It should then be possible to adsorb anti-PE with the neuraminidase preparation in order to determine whether the neuraminidase-adsorbed antiserum will fail to protect mice against challenge infection with homologous P. multocida. ACKNOWLEDGEMENTS This work was supported by grants from Kansas Agricultural Experiment Station, of Kansas State University, to W.E. Bailie. This is Contribution No. 89-56-J.
105 REFERENCES Bennet, B.W., 1982. Efficacy of Pasteurella bacterins for yearling feedlot cattle. Bovine Practitioner, 3, 26-30 Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 249-254 Brown, J. and Straus, D., 1987. Characterization of neuraminidase produced by various serotypes of group B streptococci. Infection and Immunity, 55, 1-6 Chengappa, M.M., Myers, R.C. and Carter, G.R., 1980. A streptomycin dependent live Pasteurella rnultocida vaccine for prevention of fowl cholera in turkeys. Avian Diseases, 91, 57-61 Confer, A.W., Panciera, R.J., Fulton, R.W., Gentry, M.J. and Rummage, J.A., 1985. Effect of vaccination with live or killed Pasteurella hoemolytica on resistance to experimental bovine pneumonic pasteurellosis. American Journal of Veterinary Research, 46, 342-347 Crowle, A.J., 1980. Precipitin and microprecipitin reactions in fluid mediums and in gels. In: N.R. Rose and H. Friedman (eds), Manual of Clinical Immunology, 2nd edn, (American Society for Microbiology, Washington DC), 3-14 Drzneik, IL, Scharman, W. and Blake, E., 1972. Neuraminidase and N-acetylneuraminyl pyruvate lyase of Pasteurella multocida. Journal of General Microbiology, 22, 352-368 Fick, R.B., Nagel, G.P. and Reynolds, H.Y., 1980. Use of Pseudomonas aeruginosa lipopolysaccharide immunoadsorbents to prepare high potency monospec,.'fic antibodies. Journal of Immunological Methods, 38, 103-116 Hayano, S. and Tanaka, A., 1969. Sialidase-like enzymes produced by groups A, B, C, G and L streptococci and Streptococcus sanguis. Journal of Bacteriology, 97, 1328-1333 Kadel, W.L., Chengappa, M.M. and Herren, C.E., 1985. Field trial evaluation of a Pasteurella vaccine in preconditioned and nonpreconditioned light weight calves. American Journal of Veterinary Research, 46, 1944-1948 Kodama, H., Matsumoto, M. and Snow, L.M., 1981. Immunogenicity of capsular antigens of Pasteurella multocida in turkeys. American Journal of Veterinary Research, 42, 1838-1841 Kucera, C.J., Wong, J.C.S. and Feldner, T.J., 1983. Challenge exposure of cattle vaccinated with a chemically-altered strain of Pasteurella haemolytica. American Journal of Veterinary Research, 44, 1848-1852 Kurtz, R.S. and Berman, D.J., 1986. Influence of endotoxin-protein complex in immunoglobin G isotypes of mice to Brucella abortus lipopolysaccharide. Infection and Immunity, 54, 728-734 Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature, 227, 680-685 Lu, Y.S. and Pakes, S.P., 1981. Protection of rabbits against experimental pasteurellosis by a streptomycin-dependent Pasteurella multocida serotype 3:A live mutant vaccine. Infection and Immunity, 34, 1018-1024 Moriyama, T. and Barksdale, L., 1967. Neuraminidase of Corynebacteriurn dipthen'ae. Journal of Bacteriology, 94, 1565-1581 Muller, H.E., 1974. Neuraminidase of mycoplasmas, bacteria and protozoa and their pathogenic role. Behn'ng Institute Mitteilungen, 8, 34-56 Oakley, B.R., Donald, R.K. and Ronald Morris, N., 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry, 105, 361-363 Rebers, P.A. and Heddleston, K.A., 1974. Immunogenic comparison of Westphal-type lipopolysaccharide and free endotoxins from encapsulated avian strain of Pasteurella multocida. American Journal of Veterinary Research, 35, 556-560 Rebers, P.A., Phillips, M., Rimler, IL, Boykins, tLA., Keith, B.S. and Rhodes, R., 1980. Immunizing properties of Westphal lipopolysaccharides from avian strain of Pasteurella multocida. American Journal of Veterinary Research, 41, 1650-1654 Srivastava, K.IC and Foster, J.W., 1977. Characterization of immunogenic fraction of Pasteurella multocida culture filtrates. Canadian Journal of Microbiology, 23, 197-201 Syuto, B. and Matsumoto, M., 1982. Purification of protective antigen from a saline extract of Pasteurella multocida. Infection and Immunity, 37, 1218-1226 Warren, L., 1959. The thiobarbituric acid assay of sialic acids. Journal of Biological Chemistry, 234, 1971-1975 Westphal, O. and Jann, K., 1965. Bacterial lipopolysaccharides. In: ILL. Whistler and M.L. Worform (eds), Methods in Carbohydrate Chemistry, vol. 5, (Academic Press, New York), 83-91 (Accepted: 27 January 1992)
