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Comparison of Mycoplasma gallisepticum subunit and whole organism vaccines containing different adjuvants by Western immunoblotting
Veterinary Immunology and Immunopathology, 22 (1989) 135-144 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
135
Comparison of Mycoplasma gallisepticum Subunit and Whole Organism Vaccines Containing Different Adjuvants by Western Immunoblotting ELIE K. BARBOUR 1and JOHN A. NEWMAN 2
1Brinton Laboratories, Inc., Department of Research and Development, Willmar, MN 56201 (U.S.A.) 2CoUegeof Veterinary Medicine, University of Minnesota, St. Paul, MN 55108 (U.S.A.) (Accepted 16 March 1989)
ABSTRACT Barbour, E.K. and Newman, J.A., 1989. Comparison of Mycoplasma gaUisepticum subunit and whole organism vaccines containing different adjuvants by western immunoblotting. Vet. Immunol. Immunopathol., 22: 135-144. Chickens were vaccinated with subunit (adhesin protein) or whole organisms of Mycoplasma gaUisepticum (MG) adjuvanted with multilamellar positively charged liposomes or oil-emulsion. Sera were collected before and following the first ( 13 weeks of age ) and second ( 17 weeks of age ) vaccination. The chicken sera were used in western immunoblottingagainst whole MG polypeptides. Vaccination with the subunit (MG-adhesin) bacterin containing positively charged liposomes resulted in antibody response specific to adhesin band (75 kD ) at 3 weeks post the first and second vaccination; however, crossreactions of the same antibodies occurred to MG proteins of 85 kD (3 weeks after the first vaccination) and 56 kD (3 weeks after the second vaccination ). Vaccination with whole MG proteins containing positively charged liposomes resulted in significant immunopotentiation of antibodies against low molecular weight polypeptides of MG ( < 48.0 kD). The addition of Salmonella typhimurium cell wall proteins mitogens (STP) to the different bacterins suppressed the antibody responses to some MG polypeptides.
INTRODUCTION
The need to develop bacterins for eliminating mucosal infections of chickens caused by MycoplasmagaUisepticum (MG) was reported previously (Levisohn and Kleven, 1985). The hope in veterinary biologics research was mainly put on Freund's adjuvant (Chedid et al., 1984). However, the use of an oil-emulsion MG bacterin in chickens resulted in incomplete protection against a large challenge dose (Yoder, 1979; Glisson and Kleven, 1984; Kleven et al., 1984). Using a low challenge dose of 1500 colony-forming units of MG per layer, vac0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
136 cinated with two doses of oil-emulsion MG bacterin resulted in only 40% protection against tracheal MG colonization (Talkington and Kleven, 1985). The first screening report on using MG liposomal adjuvants with microspheres of different charges and sizes, for immunizing chickens by the subcutaneous route, was published recently (Barbour et al., 1987). This work showed that positively and negatively charged liposomes used in MG bacterins were able to induce a positive correlation between total systemic immunoglobulin response and protection against tracheal colonization by MG in chickens. A recent development of a rapid computerized method for quantitation of cellular-mediated immunity (CMI) (Barbour et al., 1988a ) helped in detecting the significant CMI response in chicken layers given the positively charged liposomal bacterin of MG (Barbour et al., 1988b). The same liposomal preparation resulted in a high local IgA level specific for MG in respiratory system washings (Barbour et al., 1988b) as estimated by indirect enzyme linked-immunosorbant assay, using the avidin-biotin system (Barbour et al., 1988c). Lately, adhesin of MG reported and characterized by Kahane et al. (1984) was isolated by electroelution technique (Barbour et al., 1989). This simple technique of protein isolation gave the chance to incorporate the eluted adhesin in the liposome vesicles. The purpose of this study is to use the positively charged liposomes as an adjuvant with whole MG proteins or with the isolated adhesin. The immunopotentiating effect of the different adjuvants (liposomal and oil-emulsion) on the antibody response to different MG polypeptides will be compared by western immunoblotting. The second part of the study was to observe the immunopotentiating effect of the cell wall proteins of Salmonella typhimurium incorporated in the different MG bacterins.
MATERIALAND METHODS
MG strain The MG strain used for preparing the different bacterins was the R-strain. The strain was grown in Frey's medium (Frey et al., 1968) for 72 h and pelleted by centrifugation at 4 ° C. Pellets were washed for three times with haemagglutination-inhibition (HI) buffer (5 m M KH2PO4, 7.5 m M NaOH, 145 m M NaC1, pH 7.2 ). The MG pellets were resuspended in HI buffer containing 0.03% formalin to give a light transmission of 68% at a wavelength of 540 nm; this stock was used to prepare the total protein-MG bacterins. Another part of the pellets was suspended in saline giving a protein concentration equivalent to 2 ttg//tl; this stock was used for isolation of adhesin protein, used in preparing a subunit bacterin of MG.
137
Adhesin Adhesin was isolated as reported previously (Barbour et al., 1989); however, more gels were used to obtain more quantities of the electroeluted protein. Briefly, in preparing for sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) run, twelve gels (each of 1.3 m m thickness) containing 12% acrylamide were set. One large channel was made in each stacking gel containing 4% acrylamide. MG R-strain stock pellets solubilized in equal volumes of sample buffer containing 1.6% SDS resulting in protein equivalence of 1500 ]~g was loaded on each gel and run at 60 mA. Two h later the amperage was changed to 120 mA and kept for an additional 8 h. Coomassie brilliant blue (0.125% w/v in 50% methanol: 10% glacial acetic acid) was used for staining the gels. The gels were destained for 17 h with four changes of 50% methanol: 10% glacial acetic acid. Appropriate cuts were made in the gel to obtain the adhesin band (75 kD). The gel slabs obtained were washed three times using SDS-tank buffer (0.025 M Tris-HC1, 0.192 M glycine, 0.1% SDS, pH 8.3 ). ISCO chamber (ISCO Inc., Lincoln, NE) filled with the same buffer was used for electroelution of the adhesin from slab gels. The elution time was for 1 h at 150 V and 50 mA. The eluted polypeptides were rinsed from the collecting compartment with a total SDS-tank buffer of 28 ml. The eluted adhesin present in SDS-PAGE tank buffer was dialyzed for 5 days at 4°C using a 12 000 molecular weight cut-off dialysis bag. The buffer used for dialysis was HI buffer ÷ 0.03% formalin, and the quantity used was 2 1 per day. A sterility test of the isolated-dialyzed adhesin was performed on blood agar plates that were incubated both aerobically and anaerobically. The protein level in the eluted adhesin was estimated using DU-50 spectrophotometer (Beckman Instruments Inc., Palo Alto, CA). Briefly, adhesin was applied in different volumes on SDS-PAGE gel of 0.5 mm thickness and containing 12% acrylamide (Fig. 1 ). At the same time, bovine serum albumin (BSA) standard was applied in different concentrations using the same characteristic gel. The electroeluted adhesin-protein estimation, based on peaks recorded by DU-50 spectrophotometer corresponding to BSA, was 23.3 ~g adhesin/ml. Bacterins Six different bacterins were prepared (Table 1). The liposomal bacterins included small positively charged multilamellar vesicles, prepared as previously described (Barbour et al., 1987 ). Briefly, the molar ratio of phosphatidyl choline to cholesterol was 7:3 with a total lipid concentration of 11.01-12.98 mg/ml. Stearylamine moeity was used to create the positive charge on liposome microspheres surfaces (Barbour et al., 1987). The total protein estimation of nonviable MG pellets added to the dried lipid film was 169.2 #g/ml as determined by using the dye reagent produced by Bio-Rad (Bio-Rad Laboratories, Richmond, CA). The adhesin subunit bacterin contained 23.3 ~g adhesin protein/ml. The positively charged liposomal preparations were soni-
138 TABLE 1 Chicken treatments with different MycoplasmagaUisepticum (MG) bacterins a Chicken treatments
MG proteins
Adjuvant
Immunostimulant b
Proteins c (ttg/ml)
1 2 3 4 5 6 7 8
Adhesin Adhesin Whole Whole Whole Whole Control Control
Liposome Liposome Oil-emulsion Oil-emulsion Liposome Liposome NA d NA
no yes no yes no yes NA NA
23.3 23.3 169.2 169.2 169.2 169.2 NA NA
aFirst dose (0.5 ml) was given at 13 weeks of age and the second dose (0.5 ml) given 1 month later. hSTP = Salmonella typhimurium cell wall proteins (50 #g/dose of 0.5 ml). CMG protein (s) level. dNA = not applicable.
cated for 15 min, with a noncontinuous pulse, a 50% duty cycle, and a 20-kHz ultrasonic vibration. This procedure resulted in mean vesicle size of liposomes equivalent to 0.03 ttm 3, as identified previously by electron microscopy (Barbour et al., 1988b). The oil-emulsion MG bacterin was prepared from the same stock of nonviable MG pellets, as previously reported (Sasipreeyajan et al., 1985). Other bacterins prepared contained Salmonella typhimurium cell wall proteins mitogens (STP) (Ribi Immunochem Research Inc., Hamilton, MT) in addition to liposomal or oil-emulsion adjuvants. The S T P was added to the total MG protein stock and to adhesin stock, before incorporating the MG protein(s) with adjuvants (liposomes or oil). The final concentration of S T P in the bacterin was 100 ttg/ml.
Chickens Tracheal swabs of 42 White Leghorn pullets were found free of avian mycoplasmas, and their sera were negative by the serum-plate agglutination test (Yoder, 1980). Chickens were divided into eight groups (Table 1 ). Each of the first seven groups consisted of five layers, while seven layers were included in the eighth group.
Vaccination The nature of MG bacterins given to each group of chickens is defined in Table 1. The first MG bacterin dose was given at 13 weeks of age and second dose 1 month later. The bacterins were administered subcutaneously in the
139 anterior cervical region. The volume of each vaccine dose received by each bird was 0.5 ml.
Sera and swabs Bleeding and tracheal swabbing were done prior to and 3 weeks after the second vaccination. No mycoplasma organisms were isolated from the tracheal swab cultures during the study. Sera collected from each group of chickens were pooled using 100 #l of each chicken. Pooled sera were frozen at - 20 ° C for assessing the systemic antibodies immunopotentiated by the different bacterins against different MG polypeptides antigens. SDS-PAGE and immunoblotting SDS-PAGE was performed using the discontinuous buffer system (Laemmli, 1970). The acrylamide levels in the resolving gel and stacking gel were 12% and 4%, respectively. The dimensions of the resolving gel were 120 × 160 × 0.5 mm. The width of the sample loading slots was 6.5 mm. The weight of R-strain proteins applied in each lane was 30 ]tg. Molecular mass standards (Bio-Rad Laboratories, Richmond, CA) were included in the run. The amperage used in the run was 50 mA for 1 h followed by 30 mA for 7 h. The polypeptides of MG resolved on SDS-PAGE gels were electrophoretically transferred onto a nitrocellulose sheet for performing the western immunoblotting (Towbin et al., 1979). Briefly, the active sites on nitrocellulose were blocked with 3% gelatin. Pooled chicken sera were diluted 1/500 before adding to the MG polypeptides present on nitrocellulose strips. The nitrocellulose strips were incubated with the pooled sera for 20 h at 37 ° C. Horseradishperoxidase-labeled antichicken IgG (Nordic Immunological Laboratories, Tilburg, The Netherlands) was diluted 1/100 and applied to the strips for a period of 1 h at 37°C. The color development reagent 4, chloro-l-naphtol {Bio-Rad Laboratories) was used with H202 as substrate to obtain bands of purple color on the nitrocellulose strips. Strips were dried and photographed. RESULTS SDS-PAGE on the purified adhesin protein of MG showed the presence of a single band (Fig. 1 ). The intensity of adhesin band increased with an increasing volume of loaded sample. Western immunoblots of pooled sera collected from different chicken groups before vaccination did not show any antibodies to polypeptides of MG. The antibody responses were prominent in sera of vaccinated chickens. The samples were collected 3 weeks after the first dose (Fig. 2 ) and again 3 weeks after the second vaccination (Fig. 3). The subunit bacterin given to chickens of group 1 resulted in a clear antibody response to adhesin protein (75 kD) 3 weeks following vaccinations 1 and 2. However, there was a clear significant
140
200.0 116.2 92.5
..‘ ._.__^ 2 1
3
- I- I- - -I 4 56
MW
Fig. 1. Characterization of Mycoplasmagallisepticum-adhesin purity by SDS -PAGE. Applied adhesin volumes in lanes 1-6 were 100,75,50,25,12.5 and 6.25 ~1 respectively.
Fig. 2. Western immunoblots of chicken sera against Mycoplasmn gallisepticum (3 weeks post first vaccination). Arabic numerals correspond to defined chicken groups in Table 1. MW is the lane for molecular weight markers.
141
200. 116 92
66
45.
31.
MW
1
2
3
4
5
6
7
8
Fig. 3. Western immunoblotsof chicken sera against MycoplasmagaUisepticum (3 weekspost secondvaccination).Arabic numeralscorrespondto definedchickengroups in Table 1. MW is the lane for molecularweightmarkers. crossreaction of this antibody to the 85 kD protein (3 weeks post the first vaccination) and to the 56 kD (3 weeks post the second vaccination); moreover, minor crossreactions to other bands around the adhesin protein were detected (Fig. 3, lane 1 ). In general, the intensity of bands was stronger for the different antibody reactions 3 weeks following the second vaccination (Fig. 3 ) when compared to the reactions observed 3 weeks after the initial vaccination (Fig. 2 ). Additional antibodies recognized more MG polypeptides after the second vaccination was given (Fig. 3). All bacterins not containing STP were able to create antibodies against the adhesin protein (75 kD) including the subunit, oil-emulsion and liposomal MG proteins-bacterins. The liposomal bacterins (lanes 5 and 6 of Figs. 2 and 3) were able to immunopotentiate better antibody response to MG polypeptides of low molecular weights ( < 48.0 kD). The addition of STP to the MG bacterins (lanes 2, 4 and 6 of Figs. 2 and 3)
142
did create the disappearance of some bands (lanes 2 and 4, Fig. 2 ) or fading of others (lanes 2 and 4, Fig. 3). The control unvaccinated chickens did not show any antibodies to any polypeptides in the period following vaccination of experimental birds (lanes 7 and 8 of Figs. 2 and 3). DISCUSSION
The separation and purification of adhesin protein by electroelution of SDSPAGE gel slabs was characterized and confirmed by the run done on the eluted protein (Fig. 1). This resulted in one band at the 75 kD position. The same protein was isolated before by using column chromatography (Kahane et al., 1984). An application of different volumes of the eluted adhesin (6.25-100 ]~l) (Fig. 1 ) allowed the comparison with the bovine serum albumin standard bands (0.25-2.50 ]~g). This enabled the estimation of adhesin-protein quantity as read by the DU-50 spectrophotometer. Before vaccination, the tracheal swab cultures collected were negative for MG, and so was the rapid plate test done on the chicken sera. These results were confirmed with the highly sensitive western immunoblotting technique showing no antibody formation in the chicken sera against any MG polypeptide. All liposomal and oil-emulsion MG bacterins not containing S T P showed an emergence of antibody response against one or more MG polypeptides; this was demonstrated in sera collected 3 weeks following the first dose (Fig. 2) and 3 weeks following the second dose (Fig. 3). The antibodies formed against adhesin-subunit bacterin recognized the adhesin (lane 1, Figs. 2 and 3) with significant crossreaction to the 85 kD polypeptide (3 weeks post first vaccination, Fig. 2 ) and other significant crossreaction to the 56 kD polypeptide (3 weeks post second vaccination, Fig. 3 ). This raises a question about some common antigenic determinants between the adhesin protein and other polypeptides in MG cells. It is worth mentioning that the purity of the adhesin preparation was demonstrated by obtaining a single band in SDS-PAGE (Fig. 1 ). The observation of bands with deeper color intensity in the immunoblot performed on sera collected 3 weeks post second vaccination in comparison to less intense bands shown for sera collected 3 weeks after the initial vaccination, is a possible indication of higher antibody levels subsequent to the booster dose. Moreover, there was the emergence of antibodies which recognized more polypeptides in MG following the booster; examples will be the new bands formed by reacting sera of group 3 (given the oil-emulsion bacterin) above the 31.0 kD band (lane 3, Fig. 3). The same lane in Fig. 3 showed more bands above adhesin that did not appear in lane 3 of Fig. 2. The same situation appeared in sera of chickens given liposomal bacterins incorporating the total MG proteins (compare lanes 5 and 6 of Fig. 2 to lanes 5 and 6 of Fig. 3). All bacterins not containing S T P were able to create antibodies against the
143 adhesin (75 kD ) 3 weeks following the initial dose and 3 weeks following the second dose. This indicates the highly immunogenic nature of adhesin resulting in early and late immune responses. These antibodies could be of protective nature against adhesion and colonization of MG on mucosal layers. The confirmation of their protection capacity is the subject of our current investigation. The antibody response to a wide range of MG polypeptides in chickens given the positively charged liposomes incorporating the total MG proteins (lane 5, Figs. 2 and 3) included responses against the low molecular weight polypeptides (less than 48.0 kD); this indicates a high adjuvanicity of these positively charged vesicles used in MG bacterins. The addition of S T P to the bacterins (lanes 2, 4 and 6 of Figs. 2 and 3) reduced the immune responses to MG antigens, resulting in vanishing of antibodies to adhesin (lane 2, Fig. 2 ) and fading of other bands (lanes 2 and 4 of Fig. 3 ). The explanation to this could be the incompetetive nature of the chicken immune system where immune responses to a challenging protein (like adhesin) could be switched off or weakened in the presence of numerous proteins challenging the bird immune system at the same time. If this was the case, then the research direction towards developing subunit bacterins could be the solution to avoid burdening the bird immune system with excess antigenic determinants. Another explanation could be the induction by S T P of a shift from humoral immunity to cell-mediated immunity; this possibility needs further investigation. Future emphasis will be directed towards the study of the protection in layers against infection with MG using these same preparations that showed significant differences in immunoblot patterns. These studies could result in increasing our understanding of the protective nature of antibodies to adhesin in comparison to those raised against other MG polypeptides.
REFERENCES Barbour, E.K., Newman,J.A., Sivanandan, V., Halvorson,D.A. and Sasipreeyajan,J., 1987. Protection and immunity in commercialchicken layers administered Mycoplasmagallisepticum liposomalbacterins. AvianDis., 31: 723-729. Barbour, E.K., Newman, J.A. and Sasipreeyajan, J., 1988a. A rapid computerized method for quantitation of cellularmediated immunity in biologicalsystems.Int. J. Biomed.Comput., 22: 225-231. Barbour, E.K., Newman, J.A., Halvorson, D.A., Sivanandan, V. and Sasipreeyajan, J., 1988b. Evaluation of MycoplasmagaUisepticumliposomalbacterins administered by different routes in chicken layers.J. Am. Vet. Med. Assoc., 192: 1783. Barbour, E.K., Newman, J.A., Sivanandan, V. and Sasipreeyajan,J., 1988c. New biotin-conjugated antisera for quantitation of Mycoplasma gallisepticum-specific immunoglobulinA in chicken. Avian Dis., 32: 416-420. Barbour, E.K., Newman,J.A., Sasipreeyajan,J., Caputa, A.C. and Muneer, M.A., 1989. Identification of the antigenic componentsof the virulent MycoplasmagaUisepticum (R) in chickens:
144 their role in differentiation from the vaccine strain (F). Vet. Immunol. Immunopathol., 21: 197-206. Chedid, L., Carelli, C. and Audibert, F., 1984. Use of adjuvants, antigens, and carriers in synthetic veterinary vaccins. Advances in Carriers and Adjuvants for Veterinary Biologics. Iowa State University Press, Ames, IA, pp. 51-59. Frey, M.L., Hanson, R.P. and Anderson, D.P., 1968. A medium for the isolation of avian mycoplasmas. Am. J. Vet. Res., 20: 2163-2171. Glisson, J.R. and Kleven, S.H., 1984. MycoplasmagaUisepticum vaccination effect on egg transmission and egg production. Avian Dis., 28: 406-415. Kahane, I., Graneck, J. and Reisch-Saada, A., 1984. The adhesin of Mycoplasma gallisepticum and M. pneumoniae. Ann. Microbiol., 135A: 25-32. Kleven, S.H., Glisson, J.R., Lin, M.Y and Talkington, F.D., 1984. Bacterins and vaccines for the control of MycoplasmagaUisepticum. Isr. J. Med. Sci., 20: 989-991. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Levisohn, S. and Kleven, S.H., 1985. Vaccination of chickens against Mycoplasmagallisepticum. United States-Israel Binational Agricultural Research and Development Fund, Bet Dagan, Israel, pp. 1-131. Sasipreeyajan, J., Halvorson, D.A. and Newman, J.A., 1985. Bacterin to control the vertical transmission of MycoplasmagaUisepticum in chickens. Avian Dis., 29: 1256-1259. Talkington, F.D. and Kleven, S.H., 1985. Evaluation of protection against colonization of the chicken trachea following administration of Mycoplasma gaUisepticum bacterin. Avian Dis., 29: 998-1003. Towbin, H., Stachelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Yoder, H.W., Jr., 1979. Serological responses of chickens vaccinated with inactivated preparations of MycoplasmagaUisepticum. Avian Dis., 23: 493-506. Yoder, H.W., Jr., 1980. Mycoplasmosis. In: S.B. Hitchner, C.S. Domermuth, H.G. Purchase and J.E. Williams (Editors), Isolation and Identification of Avian Pathogens, 2nd edn. American Association of Avian Pathologists, College Station, TX, pp. 40-42.
135
Comparison of Mycoplasma gallisepticum Subunit and Whole Organism Vaccines Containing Different Adjuvants by Western Immunoblotting ELIE K. BARBOUR 1and JOHN A. NEWMAN 2
1Brinton Laboratories, Inc., Department of Research and Development, Willmar, MN 56201 (U.S.A.) 2CoUegeof Veterinary Medicine, University of Minnesota, St. Paul, MN 55108 (U.S.A.) (Accepted 16 March 1989)
ABSTRACT Barbour, E.K. and Newman, J.A., 1989. Comparison of Mycoplasma gaUisepticum subunit and whole organism vaccines containing different adjuvants by western immunoblotting. Vet. Immunol. Immunopathol., 22: 135-144. Chickens were vaccinated with subunit (adhesin protein) or whole organisms of Mycoplasma gaUisepticum (MG) adjuvanted with multilamellar positively charged liposomes or oil-emulsion. Sera were collected before and following the first ( 13 weeks of age ) and second ( 17 weeks of age ) vaccination. The chicken sera were used in western immunoblottingagainst whole MG polypeptides. Vaccination with the subunit (MG-adhesin) bacterin containing positively charged liposomes resulted in antibody response specific to adhesin band (75 kD ) at 3 weeks post the first and second vaccination; however, crossreactions of the same antibodies occurred to MG proteins of 85 kD (3 weeks after the first vaccination) and 56 kD (3 weeks after the second vaccination ). Vaccination with whole MG proteins containing positively charged liposomes resulted in significant immunopotentiation of antibodies against low molecular weight polypeptides of MG ( < 48.0 kD). The addition of Salmonella typhimurium cell wall proteins mitogens (STP) to the different bacterins suppressed the antibody responses to some MG polypeptides.
INTRODUCTION
The need to develop bacterins for eliminating mucosal infections of chickens caused by MycoplasmagaUisepticum (MG) was reported previously (Levisohn and Kleven, 1985). The hope in veterinary biologics research was mainly put on Freund's adjuvant (Chedid et al., 1984). However, the use of an oil-emulsion MG bacterin in chickens resulted in incomplete protection against a large challenge dose (Yoder, 1979; Glisson and Kleven, 1984; Kleven et al., 1984). Using a low challenge dose of 1500 colony-forming units of MG per layer, vac0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
136 cinated with two doses of oil-emulsion MG bacterin resulted in only 40% protection against tracheal MG colonization (Talkington and Kleven, 1985). The first screening report on using MG liposomal adjuvants with microspheres of different charges and sizes, for immunizing chickens by the subcutaneous route, was published recently (Barbour et al., 1987). This work showed that positively and negatively charged liposomes used in MG bacterins were able to induce a positive correlation between total systemic immunoglobulin response and protection against tracheal colonization by MG in chickens. A recent development of a rapid computerized method for quantitation of cellular-mediated immunity (CMI) (Barbour et al., 1988a ) helped in detecting the significant CMI response in chicken layers given the positively charged liposomal bacterin of MG (Barbour et al., 1988b). The same liposomal preparation resulted in a high local IgA level specific for MG in respiratory system washings (Barbour et al., 1988b) as estimated by indirect enzyme linked-immunosorbant assay, using the avidin-biotin system (Barbour et al., 1988c). Lately, adhesin of MG reported and characterized by Kahane et al. (1984) was isolated by electroelution technique (Barbour et al., 1989). This simple technique of protein isolation gave the chance to incorporate the eluted adhesin in the liposome vesicles. The purpose of this study is to use the positively charged liposomes as an adjuvant with whole MG proteins or with the isolated adhesin. The immunopotentiating effect of the different adjuvants (liposomal and oil-emulsion) on the antibody response to different MG polypeptides will be compared by western immunoblotting. The second part of the study was to observe the immunopotentiating effect of the cell wall proteins of Salmonella typhimurium incorporated in the different MG bacterins.
MATERIALAND METHODS
MG strain The MG strain used for preparing the different bacterins was the R-strain. The strain was grown in Frey's medium (Frey et al., 1968) for 72 h and pelleted by centrifugation at 4 ° C. Pellets were washed for three times with haemagglutination-inhibition (HI) buffer (5 m M KH2PO4, 7.5 m M NaOH, 145 m M NaC1, pH 7.2 ). The MG pellets were resuspended in HI buffer containing 0.03% formalin to give a light transmission of 68% at a wavelength of 540 nm; this stock was used to prepare the total protein-MG bacterins. Another part of the pellets was suspended in saline giving a protein concentration equivalent to 2 ttg//tl; this stock was used for isolation of adhesin protein, used in preparing a subunit bacterin of MG.
137
Adhesin Adhesin was isolated as reported previously (Barbour et al., 1989); however, more gels were used to obtain more quantities of the electroeluted protein. Briefly, in preparing for sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) run, twelve gels (each of 1.3 m m thickness) containing 12% acrylamide were set. One large channel was made in each stacking gel containing 4% acrylamide. MG R-strain stock pellets solubilized in equal volumes of sample buffer containing 1.6% SDS resulting in protein equivalence of 1500 ]~g was loaded on each gel and run at 60 mA. Two h later the amperage was changed to 120 mA and kept for an additional 8 h. Coomassie brilliant blue (0.125% w/v in 50% methanol: 10% glacial acetic acid) was used for staining the gels. The gels were destained for 17 h with four changes of 50% methanol: 10% glacial acetic acid. Appropriate cuts were made in the gel to obtain the adhesin band (75 kD). The gel slabs obtained were washed three times using SDS-tank buffer (0.025 M Tris-HC1, 0.192 M glycine, 0.1% SDS, pH 8.3 ). ISCO chamber (ISCO Inc., Lincoln, NE) filled with the same buffer was used for electroelution of the adhesin from slab gels. The elution time was for 1 h at 150 V and 50 mA. The eluted polypeptides were rinsed from the collecting compartment with a total SDS-tank buffer of 28 ml. The eluted adhesin present in SDS-PAGE tank buffer was dialyzed for 5 days at 4°C using a 12 000 molecular weight cut-off dialysis bag. The buffer used for dialysis was HI buffer ÷ 0.03% formalin, and the quantity used was 2 1 per day. A sterility test of the isolated-dialyzed adhesin was performed on blood agar plates that were incubated both aerobically and anaerobically. The protein level in the eluted adhesin was estimated using DU-50 spectrophotometer (Beckman Instruments Inc., Palo Alto, CA). Briefly, adhesin was applied in different volumes on SDS-PAGE gel of 0.5 mm thickness and containing 12% acrylamide (Fig. 1 ). At the same time, bovine serum albumin (BSA) standard was applied in different concentrations using the same characteristic gel. The electroeluted adhesin-protein estimation, based on peaks recorded by DU-50 spectrophotometer corresponding to BSA, was 23.3 ~g adhesin/ml. Bacterins Six different bacterins were prepared (Table 1). The liposomal bacterins included small positively charged multilamellar vesicles, prepared as previously described (Barbour et al., 1987 ). Briefly, the molar ratio of phosphatidyl choline to cholesterol was 7:3 with a total lipid concentration of 11.01-12.98 mg/ml. Stearylamine moeity was used to create the positive charge on liposome microspheres surfaces (Barbour et al., 1987). The total protein estimation of nonviable MG pellets added to the dried lipid film was 169.2 #g/ml as determined by using the dye reagent produced by Bio-Rad (Bio-Rad Laboratories, Richmond, CA). The adhesin subunit bacterin contained 23.3 ~g adhesin protein/ml. The positively charged liposomal preparations were soni-
138 TABLE 1 Chicken treatments with different MycoplasmagaUisepticum (MG) bacterins a Chicken treatments
MG proteins
Adjuvant
Immunostimulant b
Proteins c (ttg/ml)
1 2 3 4 5 6 7 8
Adhesin Adhesin Whole Whole Whole Whole Control Control
Liposome Liposome Oil-emulsion Oil-emulsion Liposome Liposome NA d NA
no yes no yes no yes NA NA
23.3 23.3 169.2 169.2 169.2 169.2 NA NA
aFirst dose (0.5 ml) was given at 13 weeks of age and the second dose (0.5 ml) given 1 month later. hSTP = Salmonella typhimurium cell wall proteins (50 #g/dose of 0.5 ml). CMG protein (s) level. dNA = not applicable.
cated for 15 min, with a noncontinuous pulse, a 50% duty cycle, and a 20-kHz ultrasonic vibration. This procedure resulted in mean vesicle size of liposomes equivalent to 0.03 ttm 3, as identified previously by electron microscopy (Barbour et al., 1988b). The oil-emulsion MG bacterin was prepared from the same stock of nonviable MG pellets, as previously reported (Sasipreeyajan et al., 1985). Other bacterins prepared contained Salmonella typhimurium cell wall proteins mitogens (STP) (Ribi Immunochem Research Inc., Hamilton, MT) in addition to liposomal or oil-emulsion adjuvants. The S T P was added to the total MG protein stock and to adhesin stock, before incorporating the MG protein(s) with adjuvants (liposomes or oil). The final concentration of S T P in the bacterin was 100 ttg/ml.
Chickens Tracheal swabs of 42 White Leghorn pullets were found free of avian mycoplasmas, and their sera were negative by the serum-plate agglutination test (Yoder, 1980). Chickens were divided into eight groups (Table 1 ). Each of the first seven groups consisted of five layers, while seven layers were included in the eighth group.
Vaccination The nature of MG bacterins given to each group of chickens is defined in Table 1. The first MG bacterin dose was given at 13 weeks of age and second dose 1 month later. The bacterins were administered subcutaneously in the
139 anterior cervical region. The volume of each vaccine dose received by each bird was 0.5 ml.
Sera and swabs Bleeding and tracheal swabbing were done prior to and 3 weeks after the second vaccination. No mycoplasma organisms were isolated from the tracheal swab cultures during the study. Sera collected from each group of chickens were pooled using 100 #l of each chicken. Pooled sera were frozen at - 20 ° C for assessing the systemic antibodies immunopotentiated by the different bacterins against different MG polypeptides antigens. SDS-PAGE and immunoblotting SDS-PAGE was performed using the discontinuous buffer system (Laemmli, 1970). The acrylamide levels in the resolving gel and stacking gel were 12% and 4%, respectively. The dimensions of the resolving gel were 120 × 160 × 0.5 mm. The width of the sample loading slots was 6.5 mm. The weight of R-strain proteins applied in each lane was 30 ]tg. Molecular mass standards (Bio-Rad Laboratories, Richmond, CA) were included in the run. The amperage used in the run was 50 mA for 1 h followed by 30 mA for 7 h. The polypeptides of MG resolved on SDS-PAGE gels were electrophoretically transferred onto a nitrocellulose sheet for performing the western immunoblotting (Towbin et al., 1979). Briefly, the active sites on nitrocellulose were blocked with 3% gelatin. Pooled chicken sera were diluted 1/500 before adding to the MG polypeptides present on nitrocellulose strips. The nitrocellulose strips were incubated with the pooled sera for 20 h at 37 ° C. Horseradishperoxidase-labeled antichicken IgG (Nordic Immunological Laboratories, Tilburg, The Netherlands) was diluted 1/100 and applied to the strips for a period of 1 h at 37°C. The color development reagent 4, chloro-l-naphtol {Bio-Rad Laboratories) was used with H202 as substrate to obtain bands of purple color on the nitrocellulose strips. Strips were dried and photographed. RESULTS SDS-PAGE on the purified adhesin protein of MG showed the presence of a single band (Fig. 1 ). The intensity of adhesin band increased with an increasing volume of loaded sample. Western immunoblots of pooled sera collected from different chicken groups before vaccination did not show any antibodies to polypeptides of MG. The antibody responses were prominent in sera of vaccinated chickens. The samples were collected 3 weeks after the first dose (Fig. 2 ) and again 3 weeks after the second vaccination (Fig. 3). The subunit bacterin given to chickens of group 1 resulted in a clear antibody response to adhesin protein (75 kD) 3 weeks following vaccinations 1 and 2. However, there was a clear significant
140
200.0 116.2 92.5
..‘ ._.__^ 2 1
3
- I- I- - -I 4 56
MW
Fig. 1. Characterization of Mycoplasmagallisepticum-adhesin purity by SDS -PAGE. Applied adhesin volumes in lanes 1-6 were 100,75,50,25,12.5 and 6.25 ~1 respectively.
Fig. 2. Western immunoblots of chicken sera against Mycoplasmn gallisepticum (3 weeks post first vaccination). Arabic numerals correspond to defined chicken groups in Table 1. MW is the lane for molecular weight markers.
141
200. 116 92
66
45.
31.
MW
1
2
3
4
5
6
7
8
Fig. 3. Western immunoblotsof chicken sera against MycoplasmagaUisepticum (3 weekspost secondvaccination).Arabic numeralscorrespondto definedchickengroups in Table 1. MW is the lane for molecularweightmarkers. crossreaction of this antibody to the 85 kD protein (3 weeks post the first vaccination) and to the 56 kD (3 weeks post the second vaccination); moreover, minor crossreactions to other bands around the adhesin protein were detected (Fig. 3, lane 1 ). In general, the intensity of bands was stronger for the different antibody reactions 3 weeks following the second vaccination (Fig. 3 ) when compared to the reactions observed 3 weeks after the initial vaccination (Fig. 2 ). Additional antibodies recognized more MG polypeptides after the second vaccination was given (Fig. 3). All bacterins not containing STP were able to create antibodies against the adhesin protein (75 kD) including the subunit, oil-emulsion and liposomal MG proteins-bacterins. The liposomal bacterins (lanes 5 and 6 of Figs. 2 and 3) were able to immunopotentiate better antibody response to MG polypeptides of low molecular weights ( < 48.0 kD). The addition of STP to the MG bacterins (lanes 2, 4 and 6 of Figs. 2 and 3)
142
did create the disappearance of some bands (lanes 2 and 4, Fig. 2 ) or fading of others (lanes 2 and 4, Fig. 3). The control unvaccinated chickens did not show any antibodies to any polypeptides in the period following vaccination of experimental birds (lanes 7 and 8 of Figs. 2 and 3). DISCUSSION
The separation and purification of adhesin protein by electroelution of SDSPAGE gel slabs was characterized and confirmed by the run done on the eluted protein (Fig. 1). This resulted in one band at the 75 kD position. The same protein was isolated before by using column chromatography (Kahane et al., 1984). An application of different volumes of the eluted adhesin (6.25-100 ]~l) (Fig. 1 ) allowed the comparison with the bovine serum albumin standard bands (0.25-2.50 ]~g). This enabled the estimation of adhesin-protein quantity as read by the DU-50 spectrophotometer. Before vaccination, the tracheal swab cultures collected were negative for MG, and so was the rapid plate test done on the chicken sera. These results were confirmed with the highly sensitive western immunoblotting technique showing no antibody formation in the chicken sera against any MG polypeptide. All liposomal and oil-emulsion MG bacterins not containing S T P showed an emergence of antibody response against one or more MG polypeptides; this was demonstrated in sera collected 3 weeks following the first dose (Fig. 2) and 3 weeks following the second dose (Fig. 3). The antibodies formed against adhesin-subunit bacterin recognized the adhesin (lane 1, Figs. 2 and 3) with significant crossreaction to the 85 kD polypeptide (3 weeks post first vaccination, Fig. 2 ) and other significant crossreaction to the 56 kD polypeptide (3 weeks post second vaccination, Fig. 3 ). This raises a question about some common antigenic determinants between the adhesin protein and other polypeptides in MG cells. It is worth mentioning that the purity of the adhesin preparation was demonstrated by obtaining a single band in SDS-PAGE (Fig. 1 ). The observation of bands with deeper color intensity in the immunoblot performed on sera collected 3 weeks post second vaccination in comparison to less intense bands shown for sera collected 3 weeks after the initial vaccination, is a possible indication of higher antibody levels subsequent to the booster dose. Moreover, there was the emergence of antibodies which recognized more polypeptides in MG following the booster; examples will be the new bands formed by reacting sera of group 3 (given the oil-emulsion bacterin) above the 31.0 kD band (lane 3, Fig. 3). The same lane in Fig. 3 showed more bands above adhesin that did not appear in lane 3 of Fig. 2. The same situation appeared in sera of chickens given liposomal bacterins incorporating the total MG proteins (compare lanes 5 and 6 of Fig. 2 to lanes 5 and 6 of Fig. 3). All bacterins not containing S T P were able to create antibodies against the
143 adhesin (75 kD ) 3 weeks following the initial dose and 3 weeks following the second dose. This indicates the highly immunogenic nature of adhesin resulting in early and late immune responses. These antibodies could be of protective nature against adhesion and colonization of MG on mucosal layers. The confirmation of their protection capacity is the subject of our current investigation. The antibody response to a wide range of MG polypeptides in chickens given the positively charged liposomes incorporating the total MG proteins (lane 5, Figs. 2 and 3) included responses against the low molecular weight polypeptides (less than 48.0 kD); this indicates a high adjuvanicity of these positively charged vesicles used in MG bacterins. The addition of S T P to the bacterins (lanes 2, 4 and 6 of Figs. 2 and 3) reduced the immune responses to MG antigens, resulting in vanishing of antibodies to adhesin (lane 2, Fig. 2 ) and fading of other bands (lanes 2 and 4 of Fig. 3 ). The explanation to this could be the incompetetive nature of the chicken immune system where immune responses to a challenging protein (like adhesin) could be switched off or weakened in the presence of numerous proteins challenging the bird immune system at the same time. If this was the case, then the research direction towards developing subunit bacterins could be the solution to avoid burdening the bird immune system with excess antigenic determinants. Another explanation could be the induction by S T P of a shift from humoral immunity to cell-mediated immunity; this possibility needs further investigation. Future emphasis will be directed towards the study of the protection in layers against infection with MG using these same preparations that showed significant differences in immunoblot patterns. These studies could result in increasing our understanding of the protective nature of antibodies to adhesin in comparison to those raised against other MG polypeptides.
REFERENCES Barbour, E.K., Newman,J.A., Sivanandan, V., Halvorson,D.A. and Sasipreeyajan,J., 1987. Protection and immunity in commercialchicken layers administered Mycoplasmagallisepticum liposomalbacterins. AvianDis., 31: 723-729. Barbour, E.K., Newman, J.A. and Sasipreeyajan, J., 1988a. A rapid computerized method for quantitation of cellularmediated immunity in biologicalsystems.Int. J. Biomed.Comput., 22: 225-231. Barbour, E.K., Newman, J.A., Halvorson, D.A., Sivanandan, V. and Sasipreeyajan, J., 1988b. Evaluation of MycoplasmagaUisepticumliposomalbacterins administered by different routes in chicken layers.J. Am. Vet. Med. Assoc., 192: 1783. Barbour, E.K., Newman, J.A., Sivanandan, V. and Sasipreeyajan,J., 1988c. New biotin-conjugated antisera for quantitation of Mycoplasma gallisepticum-specific immunoglobulinA in chicken. Avian Dis., 32: 416-420. Barbour, E.K., Newman,J.A., Sasipreeyajan,J., Caputa, A.C. and Muneer, M.A., 1989. Identification of the antigenic componentsof the virulent MycoplasmagaUisepticum (R) in chickens:
144 their role in differentiation from the vaccine strain (F). Vet. Immunol. Immunopathol., 21: 197-206. Chedid, L., Carelli, C. and Audibert, F., 1984. Use of adjuvants, antigens, and carriers in synthetic veterinary vaccins. Advances in Carriers and Adjuvants for Veterinary Biologics. Iowa State University Press, Ames, IA, pp. 51-59. Frey, M.L., Hanson, R.P. and Anderson, D.P., 1968. A medium for the isolation of avian mycoplasmas. Am. J. Vet. Res., 20: 2163-2171. Glisson, J.R. and Kleven, S.H., 1984. MycoplasmagaUisepticum vaccination effect on egg transmission and egg production. Avian Dis., 28: 406-415. Kahane, I., Graneck, J. and Reisch-Saada, A., 1984. The adhesin of Mycoplasma gallisepticum and M. pneumoniae. Ann. Microbiol., 135A: 25-32. Kleven, S.H., Glisson, J.R., Lin, M.Y and Talkington, F.D., 1984. Bacterins and vaccines for the control of MycoplasmagaUisepticum. Isr. J. Med. Sci., 20: 989-991. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Levisohn, S. and Kleven, S.H., 1985. Vaccination of chickens against Mycoplasmagallisepticum. United States-Israel Binational Agricultural Research and Development Fund, Bet Dagan, Israel, pp. 1-131. Sasipreeyajan, J., Halvorson, D.A. and Newman, J.A., 1985. Bacterin to control the vertical transmission of MycoplasmagaUisepticum in chickens. Avian Dis., 29: 1256-1259. Talkington, F.D. and Kleven, S.H., 1985. Evaluation of protection against colonization of the chicken trachea following administration of Mycoplasma gaUisepticum bacterin. Avian Dis., 29: 998-1003. Towbin, H., Stachelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76: 4350-4354. Yoder, H.W., Jr., 1979. Serological responses of chickens vaccinated with inactivated preparations of MycoplasmagaUisepticum. Avian Dis., 23: 493-506. Yoder, H.W., Jr., 1980. Mycoplasmosis. In: S.B. Hitchner, C.S. Domermuth, H.G. Purchase and J.E. Williams (Editors), Isolation and Identification of Avian Pathogens, 2nd edn. American Association of Avian Pathologists, College Station, TX, pp. 40-42.