Veterinary Microbiology, 24 ( 1 9 9 0 ) 155-169 Elsevier Science Publishers B.V., A m s t e r d a m
155
The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting
A l a n P. A v a k i a n * a n d S t a n l e y H . K l e v e n
Poultry Disease Research Center, Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30606 (U.S.A.) (Accepted 2 November 1989 )
ABSTRACT
Avakian, A.P. and Kleven, S.H., 1990. The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting. Vet. Microbiol., 24:155-169. The humoral immune response over time of White Leghorn chickens experimentally infected with
Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization ofM. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gaUisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53 ) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein ofM. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests. *Present address: D e p a r t m e n t o f Food A n i m a l a n d Equine Medicine, College of Veterinary Medicine, N o r t h Carolina State University, 4700 Hillsborough St., Raleigh, N C 27606, U.S.A.
0378-1135/90/$03.50
© 1990 1 Elsevier Science Publishers B.V.
156
A.P. AVAKIAN AND S.H. KLEVEN
INTRODUCTION
Mycoplasma gallisepticum and M. synoviae can cause respiratory disease in chickens, turkeys, and other species of birds (Jordan, 1979; Olson, 1984). Poultry breeders and producers need to maintain their flocks free of pathogenic mycoplasmas. Thus, most monitor their flocks for infection by serology and culture. When M. gallisepticum infection in a breeder flock is suspected but unconfirmed, it can cost the breeder company significant economic loss each day the uncertainty exists. Poultry industries worldwide need a sensitive and specific serodiagnostic test which can rapidly differentiate between M. gallisepticum and M. synoviae. Presently, flocks are screened for antibodies to M. gallisepticum or M. synoviae by the serum plate agglutination (SPA) test, which primarily measures IgM (Roberts, 1969). The SPA test is rapid and sensitive, but false positives are common (Boyer et al., 1960; Bradbury and Jordan, 1971; Avakian et al., 1988; Avakian and Kleven, 1990). Positive SPA results are confirmed by the hemagglutination-inhibition (HI) test, which primarily measures IgG (Roberts, 1969). The HI test is specific, but lacks sensitivity (Kleven, 1975; Kleven et al., 1988). Enzyme-linked immunosorbent assays (ELISA) have been developed for M. gallisepticum (Ansari et al., 1983; Patten et al., 1984; Talkington et al., 1985; Avakian et al., 1988; Avakian and Kleven, 1990) and M. synoviae (Patten et al., 1984). However, these ELISA tests suffer from poor specificity and/or sensitivity primarily in the acute phase of the infection. These serological tests are often combined with tracheal culturing of birds to produce a final diagnosis. Using these procedures, it takes 1 to 3 weeks for experienced laboratory diagnosticians to determine which if any species of Mycoplasma is infecting a flock. Recent studies employing immunoblotting have shown an antigenic relationship between M. gallisepticum and M. synoviae (Bradley et al., 1988; Yogev et al., 1989; Avakian and Kleven, 1990). It has been suggested that purified antigens would be necessary to develop specific serological tests (Avakian and Kleven, 1990). In this report, immunogenic species-specific proteins of M. gallisepticum and M. synoviae are identified. The humoral immune response of chickens inoculated with viable M. gallisepticum or M. synoviae have recently been documented by immunoblotting (Bradley et al., 1988; Avakian and Kleven, 1990). The authors felt that chickens infected by a contact exposure would respond more like birds naturally infected in the field, and that this information would be necessary to design serodiagnostic tests for these bacteria. Thus, the humoral immune response of chickens to M. gallisepticum or M. synoviae initiated by an aerosol-exposure or contact-exposure were compared by immunoblotting. Antisera from natural field outbreaks of mycoplasmosis were also analyzed by immunoblotting.
HUMORAL IMMUNE RESPONSE OF CHICKENS TO M. GALLISEPTICUM AND M. SYNOVIAE
| 57
MATERIALS
Experimental design Specific-pathogen-free (SPF) White Leghorn chickens were reared in Horsfall units and determined to be free of Mycoplasma species by tracheal culture (Jordan, 1983) and serology (Avakian et al., 1988) prior to experimental infection. At 8 weeks of age, 12 birds were moved to an isolation house with battery cages. Six of the birds were exposed using a nebulizer to a 5-rain aerosol inoculation (Kleven et al., 1972 ) with 109 color-changing units (CCU) per ml of viable M. gallisepticum strain R (22 m e d i u m passages) grown in Frey's m e d i u m (Frey et al., 1968 ) with liposomes substituting for swine serum ( A h m a d et al., 1988 ). Four hours later, six contact birds were introduced so that each of two cages contained three aerosol inoculated birds and three contact birds. Antisera were collected from all birds at various intervals starting at day 0 and continuing until the end of the trial on day 116. In a second trial, ten White Leghorn chickens determined to be free of Mycoplasma species were exposed at 20 weeks of age to a 5-min aerosol inoculation (109 C C U / ml) with M. synoviae isolate F 10-2AS ( 13 m e d i u m passages) grown in Frey's m e d i u m with 12% swine serum (FMS). Aerosol-inoculated chickens were placed in individual cages in an isolation room with a layer cage battery system. Nine uninoculated chickens free of mycoplasmas were introduced 4 h later, and were individually caged adjacent to two aerosol-inoculated birds. Antisera were collected at various intervals starting on day 0 and continuing until the end of the trial on day 70. In both trials, the three aerosol-inoculated chickens were chosen at random for i m m u n o b l o t analysis. Uninoculated control birds were maintained in separate facilities during both trials. At the end of the trials, all birds were examined for the presence of Mycoplasma species by tracheal culture (Jordan, 1983 ). Isolated mycoplasmas were identified to species by direct immunofluorescence (Baas and Jasper, 1972 ), and only the Mycoplasma species used for inoculation was recovered. Contact birds were tracheal-cultured periodically to determine whether and approximately when contact infection initiated. In both trials the contact-exposed chickens used for i m m u n o b l o t analysis were chosen because they became colonized early in the trials with either M. gallisepticum or M. synoviae Chicken antisera from commercial poultry growers sent to our laboratory or to the Georgia Poultry Laboratory (Oakwood, GA) for serodiagnosis were assayed by i m m u n o b l o t analysis. Field sera were used only ifM. gallisepticum or M. synoviae were isolated and identified by direct immunofluorescence (Baas and Jasper, 1972). The SPA (Anonymous, 1972) and the HI (Vardaman and Yoder, 1970) tests were also used to analyze these sera. Antigen for these two tests were made using strain A5969 of M. gallisepticum and strain WVU 1853 ofM. synoviae.
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Antigen preparation The R strain of M. gallisepticum and the F10-2AS isolate of M. synoviae were filter-cloned three times through a 450/zm filter (Tully, 1983) and assayed for purity by immunofluorescence (Baas and Jasper, 1972). Filter cloned mycoplasmas were grown in FMS to log phase (until culture m e d i u m turned orange) at 37 °C, then they were collected and washed three times in 150 m M phosphate buffered saline solution (pH 7.2) by centrifugation at 18 0 0 0 X g for 30 min at 4°C. Washed mycoplasma cells were solubilized in 10 m M Tris containing 0.2% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 1.0% (w/v) Triton X-100, 10 m M EDTA, and 1 m M phenylmethylsulfonyl fluoride (pH 7.8) for 30 min at 37°C (Krause and Baseman, 1983 ) and stored at - 20°C until used.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Solubilized mycoplasma proteins were assayed for protein content (Bradford, 1976), mixed with lysis buffer consisting of 125 m M Tris, 4% (w/v) SDS, 10.0% (v/v) 2-beta mercaptoethanol, and 20.0% (v/v) glycerol, pH 6.8, and heated to 100°C for 5 min. Samples (30/zg/well) were applied to 0.75 m m thick, 4% stacking/10% resolving SDS-PAGE gels and run overnight at 4 mA constant current/gel using the buffer system of Laemmli (1970). The reported molecular masses of proteins were determined by the method of Weber and Osborn ( 1969 ) in 10% resolving gels using low range molecular size standards (Stock # SDS-7, Sigma Chemicals, St. Louis, MO and catalog # 161 0304, Bio-Rad Laboratories, Richmond, CA). The standard size markers indicated in the left margin of Figs. 1-6 are those of Bio-Rad Laboratories.
Immunoblot analysis Proteins were transferred to 0.45 p m nitrocellulose paper (Bio-Rad) at 50 V constant voltage for 2 h (Towbin et al., 1979). Total transferred protein was visualized using 0.1% amido black stain. Nitrocellulose (NC) strips with transferred mycoplasma proteins were blocked by incubation for 3 h at room temperature (RT) or overnight at 4°C in Tris buffered saline (TBS; 20 m M Tris, 500 m M NaC1, pH 7.5) containing 4% calf serum. Primary antiserum was diluted 1:80 in TBS containing 1% swine serum and 1% calf serum (TBSsera). The diluted antisera was allowed to stand at RT for 15 min before incubating with NC strips for 4 h at RT and then held overnight at 4°C. To increase sensitivity, experimental antisera used in Figs. 1 and 4 were incubated with NC strips for 6 h at RT, and then held overnight at 4 ° C. This was followed by four 8-min washes in TBS containing 0.05% Tween 20. Affinity purified rabbit anti-chicken IgG ( H + L chains )-horseradish peroxidase (Zymed Laboratories, So. San Francisco, CA) was diluted 1 : 1000 in TBSsera and incubated with NC strips for 4 h at RT. This was followed by four
HUMORAL IMMUNE RESPONSE OF CHICKENS TO M. GALLISEPTICUM AND M. SYNOVIAE
159
washes as above and then a 3-min wash in TBS. Immunoblots were developed for 5 min using H202 and 4-chloro-l-naphthol (Bio-Rad) as directed by the manufacturer.
Purification of proteins bands Specific protein bands of M. gallisepticum and M. synoviae were purified in the following manner. SDS-PAGE gels (10%) with 15 lanes/gel were loaded with 60 #g protein/lane of either M. gallisepticum strain R or M. synoviae isolate F 10-2AS. Electrophoresis occurred overnight at 8 mA constant current/gel. Gels were passively stained for 5 to 6 min in 0.2% Coomassie blue and then destained for 30 min in destaining solution (50% methanol, 10% glacial acetic acid, 40% distilled water). Gels were further destained in 25% m e t h a n o l / 7 5 % distilled water until individual protein bands could be easily distinguished. Proteins of interest were cut from gels with a razor blade and destained completely in destaining solution (8 to 20 h). Protein was electroeluted from the cut gels using a model 422 electro-eluter (Bio-Rad) as instructed by the manufacturer. Eluted protein was stored at - 80 °C until used. RESULTS
Species-specific proteins of M. gallisepticum are shown in Fig. 1. Proteins of 82 (p82), 65-63 (p64), and 35 (p35) kDa were shown to be species-specific and were assigned molecular masses of 85, 67, and 29 kDa, respectively, in an earlier report (Avakian and Kleven, 1990). The molecular masses given in this report are considered more accurate. Antisera from chickens infected with M. synoviae by aerosol or contact exposure did not react with p82, p64, or p35. In addition, three highly immunogenic species-specific proteins of approximately 56 (p56), 26 (p26) and 24 (p24) kDa were identified. All three M. gallisepticum aerosol-inoculated chickens (Fig. 2 ) produced a strong antibody response by day 11 that lasted throughout the 116-day trial. Although the response was delayed, the M. gallisepticum contact-exposed chickens (Fig. 3 ) responded as strongly to infection as the aerosol-inoculated birds. Day 0 for contact-exposed chickens (Figs. 3 and 6 ) was the day they were placed with the aerosol-inoculated birds. M. gallisepticum contact-exposed chickens were negative by tracheal culture on day 6. M. gallisepticum was first isolated from birds one and three on day 11, and on day 16 from bird two (bird numbers correspond to bird numbers in Fig. 3 ). The proteins p64, p56, and p26 were among the first to be recognized by chickens in both the aerosol and contact groups. The response to p64, p56, and p26 persisted throughout the trial. The occurrence of antibodies recognizing p82, p35, and p24 was more variable in individual birds with respect to onset and duration. Two contact-exposed chickens in the M. gallisepticum trial never became positive by tracheal culture or by the SPA test. Day 33 and 64 antisera from these two contact birds were analyzed by immunoblotting. A weak response to two
160
A.P. AVAK1AN AND S.H. KLEVEN
82 64 56
35 26 24
1
2
3
4
5
6
7
8
Fig. 1. Immunoblots of cell proteins of Mycoplasma gall&epticum (Mg) strain R (lanes 1-8) reacted with chicken antisera (diluted 1:80) to M. synoviae (Ms; lanes 1-7) or Mg (lane 8). Lane 1: bird 29, 28 days post Ms contact exposure; lane 2: bird 3l, 28 days post Ms contact exposure; lane 3: bird 33, 41 days post Ms contact exposure; lane 4: bird 34, 20 days post Ms aerosol inoculation; lane 5: bird 38, 20 days post Ms aerosol inoculation; lane 6: bird 40, 20 days post Ms aerosol inoculation; lane 7: bird 36, 20 days post Ms aerosol infection; lane 8: bird 89, 99 days post Mg contact exposure. Mg immunogenic species-specific proteins of approximately 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kDa are shown on the right. Relative size markers in kDa indicated on the left.
M. gallisepticum proteins was observed in one of the birds at day 64 (data not shown). Fig. 4 shows the identification of two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa. Also shown is a highly immunogenic, but not species-specific, protein of 41 kDa (p41). In Fig. 4 it can be seen that antisera from chickens infected with M. gallisepticum by contact (lanes l and 2 ) or by aerosolization (lanes 3, 4, and 5 ) responded with a number of antibodies that cross-reacted with proteins of M. synoviae. Antisera from chickens infected with M. synoviae by contact exposure (lane 6 ) and by aerosolization (lane 7 ) reacted with p53, p41, and p22, as well as with a number of other proteins. Chickens infected by aerosolization of viable M. synoviae (Fig. 5) responded to more M. synoviae proteins and with greater intensity than birds infected by contact exposure (Fig. 6). M. synoviae contact-exposed chickens were negative by tracheal culture on day 6. M. synoviae was first isolated from birds one and two on day 10 and from bird three on day 14 (bird numbers
161
HUMORAL IMMUNE RESPONSE OF CHICKENS TO M. G A L L I S E P ~ C U M AND M. SYNOVIAE
0
123
11
21
33
48
1 2 3 1 2 3 1 2 3 1 2 3
77
116
1 2 3 1 2 3
Fig. 2. Immunoblots of cell proteins of M. gallisepticum (Mg) strain R with Mg-positive antiserum collected over time from aerosol-inoculated chickens. Days post aerosol inoculation with Mg strain R are indicated at the top. The same three birds ( 1, 2, and 3) sampled at various times post inoculation are indicated at the bottom. The locations of Mg species-specific proteins are indicated on the right. Relative size markers in kDa indicated on the left.
0
11
21
33
48
77
116
F
97.4
-
66.2 45.0 .....
-
155
The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting
A l a n P. A v a k i a n * a n d S t a n l e y H . K l e v e n
Poultry Disease Research Center, Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30606 (U.S.A.) (Accepted 2 November 1989 )
ABSTRACT
Avakian, A.P. and Kleven, S.H., 1990. The humoral immune response of chickens to Mycoplasma gallisepticum and Mycoplasma synoviae studied by immunoblotting. Vet. Microbiol., 24:155-169. The humoral immune response over time of White Leghorn chickens experimentally infected with
Mycoplasma gallisepticum or M. synoviae by an aerosol inoculation or a contact exposure were compared by immunoblotting. The response of chickens infected with M. gallisepticum were similar with respect to proteins recognized and intensity of response, regardless of mode of infection. On the other hand, chickens infected by aerosolization ofM. synoviae responded to more proteins and with greater intensity than did M. synoviae contact-exposed birds. Chickens infected with M. gallisepticum responded with antibodies to over 20 proteins, while chickens infected with M. synoviae responded with antibodies to 12 proteins. Field sera from chickens naturally infected on commercial poultry farms with M. gallisepticum or M. synoviae were analyzed by immunoblotting and were found to react with a number of mycoplasma proteins. However, no correlation was seen when comparing intensity of immunoblot staining and hemagglutination-inhibition titer of the field sera. The experimental antisera were used to identify species-specific proteins of M. gallisepticum and M. synoviae. Six immunogenic species-specific proteins of M. gaUisepticum with relative molecular masses of 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kilodaltons (kDa) were identified. Two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53 ) and 22 (p22) kDa were identified. Additionally, a highly immunogenic 41 (p41) kDa protein of M. synoviae was identified. Species-specific proteins identified in these mycoplasmas and the 41 kDa protein ofM. synoviae were purified by preparative SDS-PAGE in amounts sufficient for further characterization and for use in serodiagnostic tests. *Present address: D e p a r t m e n t o f Food A n i m a l a n d Equine Medicine, College of Veterinary Medicine, N o r t h Carolina State University, 4700 Hillsborough St., Raleigh, N C 27606, U.S.A.
0378-1135/90/$03.50
© 1990 1 Elsevier Science Publishers B.V.
156
A.P. AVAKIAN AND S.H. KLEVEN
INTRODUCTION
Mycoplasma gallisepticum and M. synoviae can cause respiratory disease in chickens, turkeys, and other species of birds (Jordan, 1979; Olson, 1984). Poultry breeders and producers need to maintain their flocks free of pathogenic mycoplasmas. Thus, most monitor their flocks for infection by serology and culture. When M. gallisepticum infection in a breeder flock is suspected but unconfirmed, it can cost the breeder company significant economic loss each day the uncertainty exists. Poultry industries worldwide need a sensitive and specific serodiagnostic test which can rapidly differentiate between M. gallisepticum and M. synoviae. Presently, flocks are screened for antibodies to M. gallisepticum or M. synoviae by the serum plate agglutination (SPA) test, which primarily measures IgM (Roberts, 1969). The SPA test is rapid and sensitive, but false positives are common (Boyer et al., 1960; Bradbury and Jordan, 1971; Avakian et al., 1988; Avakian and Kleven, 1990). Positive SPA results are confirmed by the hemagglutination-inhibition (HI) test, which primarily measures IgG (Roberts, 1969). The HI test is specific, but lacks sensitivity (Kleven, 1975; Kleven et al., 1988). Enzyme-linked immunosorbent assays (ELISA) have been developed for M. gallisepticum (Ansari et al., 1983; Patten et al., 1984; Talkington et al., 1985; Avakian et al., 1988; Avakian and Kleven, 1990) and M. synoviae (Patten et al., 1984). However, these ELISA tests suffer from poor specificity and/or sensitivity primarily in the acute phase of the infection. These serological tests are often combined with tracheal culturing of birds to produce a final diagnosis. Using these procedures, it takes 1 to 3 weeks for experienced laboratory diagnosticians to determine which if any species of Mycoplasma is infecting a flock. Recent studies employing immunoblotting have shown an antigenic relationship between M. gallisepticum and M. synoviae (Bradley et al., 1988; Yogev et al., 1989; Avakian and Kleven, 1990). It has been suggested that purified antigens would be necessary to develop specific serological tests (Avakian and Kleven, 1990). In this report, immunogenic species-specific proteins of M. gallisepticum and M. synoviae are identified. The humoral immune response of chickens inoculated with viable M. gallisepticum or M. synoviae have recently been documented by immunoblotting (Bradley et al., 1988; Avakian and Kleven, 1990). The authors felt that chickens infected by a contact exposure would respond more like birds naturally infected in the field, and that this information would be necessary to design serodiagnostic tests for these bacteria. Thus, the humoral immune response of chickens to M. gallisepticum or M. synoviae initiated by an aerosol-exposure or contact-exposure were compared by immunoblotting. Antisera from natural field outbreaks of mycoplasmosis were also analyzed by immunoblotting.
HUMORAL IMMUNE RESPONSE OF CHICKENS TO M. GALLISEPTICUM AND M. SYNOVIAE
| 57
MATERIALS
Experimental design Specific-pathogen-free (SPF) White Leghorn chickens were reared in Horsfall units and determined to be free of Mycoplasma species by tracheal culture (Jordan, 1983) and serology (Avakian et al., 1988) prior to experimental infection. At 8 weeks of age, 12 birds were moved to an isolation house with battery cages. Six of the birds were exposed using a nebulizer to a 5-rain aerosol inoculation (Kleven et al., 1972 ) with 109 color-changing units (CCU) per ml of viable M. gallisepticum strain R (22 m e d i u m passages) grown in Frey's m e d i u m (Frey et al., 1968 ) with liposomes substituting for swine serum ( A h m a d et al., 1988 ). Four hours later, six contact birds were introduced so that each of two cages contained three aerosol inoculated birds and three contact birds. Antisera were collected from all birds at various intervals starting at day 0 and continuing until the end of the trial on day 116. In a second trial, ten White Leghorn chickens determined to be free of Mycoplasma species were exposed at 20 weeks of age to a 5-min aerosol inoculation (109 C C U / ml) with M. synoviae isolate F 10-2AS ( 13 m e d i u m passages) grown in Frey's m e d i u m with 12% swine serum (FMS). Aerosol-inoculated chickens were placed in individual cages in an isolation room with a layer cage battery system. Nine uninoculated chickens free of mycoplasmas were introduced 4 h later, and were individually caged adjacent to two aerosol-inoculated birds. Antisera were collected at various intervals starting on day 0 and continuing until the end of the trial on day 70. In both trials, the three aerosol-inoculated chickens were chosen at random for i m m u n o b l o t analysis. Uninoculated control birds were maintained in separate facilities during both trials. At the end of the trials, all birds were examined for the presence of Mycoplasma species by tracheal culture (Jordan, 1983 ). Isolated mycoplasmas were identified to species by direct immunofluorescence (Baas and Jasper, 1972 ), and only the Mycoplasma species used for inoculation was recovered. Contact birds were tracheal-cultured periodically to determine whether and approximately when contact infection initiated. In both trials the contact-exposed chickens used for i m m u n o b l o t analysis were chosen because they became colonized early in the trials with either M. gallisepticum or M. synoviae Chicken antisera from commercial poultry growers sent to our laboratory or to the Georgia Poultry Laboratory (Oakwood, GA) for serodiagnosis were assayed by i m m u n o b l o t analysis. Field sera were used only ifM. gallisepticum or M. synoviae were isolated and identified by direct immunofluorescence (Baas and Jasper, 1972). The SPA (Anonymous, 1972) and the HI (Vardaman and Yoder, 1970) tests were also used to analyze these sera. Antigen for these two tests were made using strain A5969 of M. gallisepticum and strain WVU 1853 ofM. synoviae.
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A.P. AVAKIAN AND S.H. KLEVEN
Antigen preparation The R strain of M. gallisepticum and the F10-2AS isolate of M. synoviae were filter-cloned three times through a 450/zm filter (Tully, 1983) and assayed for purity by immunofluorescence (Baas and Jasper, 1972). Filter cloned mycoplasmas were grown in FMS to log phase (until culture m e d i u m turned orange) at 37 °C, then they were collected and washed three times in 150 m M phosphate buffered saline solution (pH 7.2) by centrifugation at 18 0 0 0 X g for 30 min at 4°C. Washed mycoplasma cells were solubilized in 10 m M Tris containing 0.2% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, 1.0% (w/v) Triton X-100, 10 m M EDTA, and 1 m M phenylmethylsulfonyl fluoride (pH 7.8) for 30 min at 37°C (Krause and Baseman, 1983 ) and stored at - 20°C until used.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Solubilized mycoplasma proteins were assayed for protein content (Bradford, 1976), mixed with lysis buffer consisting of 125 m M Tris, 4% (w/v) SDS, 10.0% (v/v) 2-beta mercaptoethanol, and 20.0% (v/v) glycerol, pH 6.8, and heated to 100°C for 5 min. Samples (30/zg/well) were applied to 0.75 m m thick, 4% stacking/10% resolving SDS-PAGE gels and run overnight at 4 mA constant current/gel using the buffer system of Laemmli (1970). The reported molecular masses of proteins were determined by the method of Weber and Osborn ( 1969 ) in 10% resolving gels using low range molecular size standards (Stock # SDS-7, Sigma Chemicals, St. Louis, MO and catalog # 161 0304, Bio-Rad Laboratories, Richmond, CA). The standard size markers indicated in the left margin of Figs. 1-6 are those of Bio-Rad Laboratories.
Immunoblot analysis Proteins were transferred to 0.45 p m nitrocellulose paper (Bio-Rad) at 50 V constant voltage for 2 h (Towbin et al., 1979). Total transferred protein was visualized using 0.1% amido black stain. Nitrocellulose (NC) strips with transferred mycoplasma proteins were blocked by incubation for 3 h at room temperature (RT) or overnight at 4°C in Tris buffered saline (TBS; 20 m M Tris, 500 m M NaC1, pH 7.5) containing 4% calf serum. Primary antiserum was diluted 1:80 in TBS containing 1% swine serum and 1% calf serum (TBSsera). The diluted antisera was allowed to stand at RT for 15 min before incubating with NC strips for 4 h at RT and then held overnight at 4°C. To increase sensitivity, experimental antisera used in Figs. 1 and 4 were incubated with NC strips for 6 h at RT, and then held overnight at 4 ° C. This was followed by four 8-min washes in TBS containing 0.05% Tween 20. Affinity purified rabbit anti-chicken IgG ( H + L chains )-horseradish peroxidase (Zymed Laboratories, So. San Francisco, CA) was diluted 1 : 1000 in TBSsera and incubated with NC strips for 4 h at RT. This was followed by four
HUMORAL IMMUNE RESPONSE OF CHICKENS TO M. GALLISEPTICUM AND M. SYNOVIAE
159
washes as above and then a 3-min wash in TBS. Immunoblots were developed for 5 min using H202 and 4-chloro-l-naphthol (Bio-Rad) as directed by the manufacturer.
Purification of proteins bands Specific protein bands of M. gallisepticum and M. synoviae were purified in the following manner. SDS-PAGE gels (10%) with 15 lanes/gel were loaded with 60 #g protein/lane of either M. gallisepticum strain R or M. synoviae isolate F 10-2AS. Electrophoresis occurred overnight at 8 mA constant current/gel. Gels were passively stained for 5 to 6 min in 0.2% Coomassie blue and then destained for 30 min in destaining solution (50% methanol, 10% glacial acetic acid, 40% distilled water). Gels were further destained in 25% m e t h a n o l / 7 5 % distilled water until individual protein bands could be easily distinguished. Proteins of interest were cut from gels with a razor blade and destained completely in destaining solution (8 to 20 h). Protein was electroeluted from the cut gels using a model 422 electro-eluter (Bio-Rad) as instructed by the manufacturer. Eluted protein was stored at - 80 °C until used. RESULTS
Species-specific proteins of M. gallisepticum are shown in Fig. 1. Proteins of 82 (p82), 65-63 (p64), and 35 (p35) kDa were shown to be species-specific and were assigned molecular masses of 85, 67, and 29 kDa, respectively, in an earlier report (Avakian and Kleven, 1990). The molecular masses given in this report are considered more accurate. Antisera from chickens infected with M. synoviae by aerosol or contact exposure did not react with p82, p64, or p35. In addition, three highly immunogenic species-specific proteins of approximately 56 (p56), 26 (p26) and 24 (p24) kDa were identified. All three M. gallisepticum aerosol-inoculated chickens (Fig. 2 ) produced a strong antibody response by day 11 that lasted throughout the 116-day trial. Although the response was delayed, the M. gallisepticum contact-exposed chickens (Fig. 3 ) responded as strongly to infection as the aerosol-inoculated birds. Day 0 for contact-exposed chickens (Figs. 3 and 6 ) was the day they were placed with the aerosol-inoculated birds. M. gallisepticum contact-exposed chickens were negative by tracheal culture on day 6. M. gallisepticum was first isolated from birds one and three on day 11, and on day 16 from bird two (bird numbers correspond to bird numbers in Fig. 3 ). The proteins p64, p56, and p26 were among the first to be recognized by chickens in both the aerosol and contact groups. The response to p64, p56, and p26 persisted throughout the trial. The occurrence of antibodies recognizing p82, p35, and p24 was more variable in individual birds with respect to onset and duration. Two contact-exposed chickens in the M. gallisepticum trial never became positive by tracheal culture or by the SPA test. Day 33 and 64 antisera from these two contact birds were analyzed by immunoblotting. A weak response to two
160
A.P. AVAK1AN AND S.H. KLEVEN
82 64 56
35 26 24
1
2
3
4
5
6
7
8
Fig. 1. Immunoblots of cell proteins of Mycoplasma gall&epticum (Mg) strain R (lanes 1-8) reacted with chicken antisera (diluted 1:80) to M. synoviae (Ms; lanes 1-7) or Mg (lane 8). Lane 1: bird 29, 28 days post Ms contact exposure; lane 2: bird 3l, 28 days post Ms contact exposure; lane 3: bird 33, 41 days post Ms contact exposure; lane 4: bird 34, 20 days post Ms aerosol inoculation; lane 5: bird 38, 20 days post Ms aerosol inoculation; lane 6: bird 40, 20 days post Ms aerosol inoculation; lane 7: bird 36, 20 days post Ms aerosol infection; lane 8: bird 89, 99 days post Mg contact exposure. Mg immunogenic species-specific proteins of approximately 82 (p82), 65-63 (p64), 56 (p56), 35 (p35), 26 (p26), and 24 (p24) kDa are shown on the right. Relative size markers in kDa indicated on the left.
M. gallisepticum proteins was observed in one of the birds at day 64 (data not shown). Fig. 4 shows the identification of two species-specific proteins of M. synoviae with relative molecular masses of 53 (p53) and 22 (p22) kDa. Also shown is a highly immunogenic, but not species-specific, protein of 41 kDa (p41). In Fig. 4 it can be seen that antisera from chickens infected with M. gallisepticum by contact (lanes l and 2 ) or by aerosolization (lanes 3, 4, and 5 ) responded with a number of antibodies that cross-reacted with proteins of M. synoviae. Antisera from chickens infected with M. synoviae by contact exposure (lane 6 ) and by aerosolization (lane 7 ) reacted with p53, p41, and p22, as well as with a number of other proteins. Chickens infected by aerosolization of viable M. synoviae (Fig. 5) responded to more M. synoviae proteins and with greater intensity than birds infected by contact exposure (Fig. 6). M. synoviae contact-exposed chickens were negative by tracheal culture on day 6. M. synoviae was first isolated from birds one and two on day 10 and from bird three on day 14 (bird numbers
161
HUMORAL IMMUNE RESPONSE OF CHICKENS TO M. G A L L I S E P ~ C U M AND M. SYNOVIAE
0
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1 2 3 1 2 3 1 2 3 1 2 3
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1 2 3 1 2 3
Fig. 2. Immunoblots of cell proteins of M. gallisepticum (Mg) strain R with Mg-positive antiserum collected over time from aerosol-inoculated chickens. Days post aerosol inoculation with Mg strain R are indicated at the top. The same three birds ( 1, 2, and 3) sampled at various times post inoculation are indicated at the bottom. The locations of Mg species-specific proteins are indicated on the right. Relative size markers in kDa indicated on the left.
0
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F
97.4
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66.2 45.0 .....
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