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Inhibition of Avian Mycoplasmal Hemagglutination by IgM Type Antibody1
Inhibition of Avian Mycoplasmal Hemagglutination by IgM Type Antibody 1 K. R. RHOADES U.S. Department of Agriculture, ARS National Animal Disease Center, Ames, Iowa 50010 (Received for publication August 29, 1977) ABSTRACT Intravenous exposure of turkeys to Mycoplasma synoviae resulted in development of hemagglutination-inhibition (HI) activity in serums within 4 days. Treatment with 2-mercaptoethanol and gel-filtration separation of serum immunoglobulins indicated that the activity was due to IgM type antibody. Similar early HI activity was demonstrated in serums from turkeys exposed intravenously to Mycoplasma gallisepticum and Mycoplasma meleagridis.
MATERIALS AND METHODS
Information gained from serologic tests is an i m p o r t a n t aid in establishing t h e presence of avian mycoplasmal infections. The serologic tests used most c o m m o n l y for this purpose are t h e agglutination and hemagglutination-inhibition (HI) tests. T h e effectiveness of these t w o types of tests for detecting a n t i b o d y against mycoplasmas is related, at least in part, t o t h e p r e d o m i n a t i n g i m m u n o g l o b u l i n of which t h e specific a n t i b o d y consists. For example, studies of a n t i b o d y response to Mycoplasma meleagridis (Kleven and P o m e r o y , 1971) and Mycoplasma gallisepticum ( R o b e r t s , 1 9 6 9 ) indicated t h a t t h e HI tests d e m o n s t r a t e d IgG b u t n o t IgM, and t h a t plate agglutination tests d e m o n strated IgM b u t were relatively insensitive for d e m o n s t r a t i n g IgG. However, a s t u d y ( R h o a d e s , 1975) of the a n t i b o d y response of t u r k e y s to Mycoplasma synoviae indicated t h a t in addition to the expected agglutinins, low level HI activity was present within a few days after intravenous (IV) e x p o s u r e . This HI activity was sensitive to the action of 2-mercaptoethanol (2ME) suggesting t h a t IgM t y p e a n t i b o d y was involved.
Mycoplasma synoviae, strain WVU 1 8 5 3 ; M. gallisepticum, strain S6; and M. meleagridis, strain 8M92, were used to p r o d u c e antigens for inoculating turkeys and c o n d u c t i n g serologic tests. A h e a r t infusion broth (Kelton and Van Roekel, 1963) with yeast a u t o l y s a t e substituted for yeast e x t r a c t was used to cultivate M. gallisepticum and M. meleagridis. The MF4 m e d i u m described b y Frey et al., ( 1 9 6 8 ) was used to cultivate M. synoviae. T h e mycoplasmas were sedimented by centrifugation at 1 0 0 0 0 G for 45 min, washed 2 times in p h o s p h a t e buffered saline (PBS), p H 7.2, and resuspended according to their intended use. Cells used to inoculate t u r k e y s were resuspended in PBS and adjusted s p e c t r o p h o t o m e t r i c a l l y to an optical density of .18 at 6 0 0 n m . Cells used as antigens for HI tests were resuspended in PBS at a p p r o x i m a t e l y 1/100 t h e original volume, mixed with an equal volume of glycerin, divided i n t o 1-ml aliquots, and frozen and stored at ^ 4 5 C until used.
This r e p o r t describes the study of serums obtained from t u r k e y s 4 days after TV exposure with M. synoviae to d e t e r m i n e whether early HI activity was d u e t o IgM t y p e a n t i b o d y . O t h e r pathogenic avian mycoplasmas, M. meleagridis and M. gallisepticum, also were used to expose t u r k e y s IV t o d e t e r m i n e whether they induced a similar early HI response.
1
No endorsements are implied herein.
1978 Poultry Sci 57:608-610
Mycoplasma-free turkeys were used to prepare antiserums. T w o were inoculated IV with 1 ml of M. gallisepticum antigen, and 2 were similarly inoculated with M. meleagridis antigen. Three t u r k e y s were inoculated IV 4 times at 3- or 4-day intervals with .5 ml of M. synoviae antigen. Serums from blood samples taken before and at various intervals after inoculation were examined for HI a n t i b o d y . Each of t h e serums was also examined after t r e a t m e n t with 2ME. Serum immunoglobulins from a representative t u r k e y inoculated with M. synoviae antigen were separated by gel filtration, and samples were examined for HI activity. The HI tests were c o n d u c t e d as previously
608
Downloaded from http://ps.oxfordjournals.org/ by guest on April 24, 2015
INTRODUCTION
MYCOPLASMAL H E M A G G L U T I N A T I O N INHIBITION
609
effect of 2ME t r e a t m e n t on these titers are indicated in Table 1. T h e results of gel filtration separation of serum proteins and s u b s e q u e n t HI testing of representative samples are indicated in Figure 1. T h e HI titers of serums from t u r k e y s inoculated with M. gallisepticum and M. meleagridis antigens and the effect of 2ME t r e a t m e n t on the titers are presented in Tables 2 and 3, respectively.
DISCUSSION
15 25 35 45 55 65 75 SAMPLE NUMBER
FIG. 1. Optical densities and hemagglutination-inhibition titers of samples obtained by gel-filtration separation of serum proteins from a turkey 4 days after intravenous exposure to Mycoplasma synoviae.
described (Yoder, 1 9 7 5 ) , as was t r e a t m e n t of serums with 2ME (Kleven and P o m e r o y , 1 9 7 1 ) . Immunoglobulin separation by gel filtration was essentially as previously reported (Klevven and P o m e r o y , 1971). Eluted samples were examined for HI activity w i t h o u t c o n c e n t r a t i o n . RESULTS
T h e HI titers of serums obtained from turkeys inoculated with M. synoviae and t h e
In a previous s t u d y (Kleven and P o m e r o y ,
TABLE 1 .—Hemagglutination-inhibition (HI) titers of serums from turkeys inoculated intravenously with Mycoplasma synoviae Days after exposure Turkey serums 2 2 2 (untreated) 222 (2ME treated)*-'
0
4 .a
80^
7
11
14
18
3 20 80
320 160
3 20 160
640 320
226 (untreated) 226 (2ME treated)
40
40 20
160 40
320 160
1280 640
235 (untreated) 2 3 5 (2ME treated)
80
320 80
640 3 20
640 320
640 320
No evidence of HI activity at a 1:10 dilution of serum. Reciprocal of highest dilution of serum causing complete HI. Serum pretreated with .05 M 2-mercaptoethanol.
Downloaded from http://ps.oxfordjournals.org/ by guest on April 24, 2015
5
T h e sensitivity of early HI activity to 2ME t r e a t m e n t and d e m o n s t r a t i o n of this activity in only t h e first protein fraction eluted during gel filtration separation indicates t h a t IgM t y p e a n t i b o d y was responsible. T h e d e m o n s t r a t i o n of HI due to IgM t y p e a n t i b o d y in this s t u d y differs from that in previous studies (Kleven and P o m e r o y , 1 9 7 1 ; R o b e r t s , 1969) p r o b a b l y due to differences in IgM a n t i b o d y titers and possibly related to b o t h the r o u t e of exposure and t h e a m o u n t of antigen administered. Previous reports have indicated that t h e IV r o u t e of exposure results in higher titers than o t h e r routes (Kleven and P o m e r o y , 1 9 7 1 ; M o h a m m e d and Bohl, 1 9 6 8 ) . T h e c o n c e n t r a t i o n of antigens in the present s t u d y was adjusted s p e c t r o p h o t o metrically and n o t according to n u m b e r s of colony-forming units, b u t probably contained several times t h e antigen c o n c e n t r a t i o n of the cultures used to expose birds in t h e previous studies (Kleven and P o m e r o y , 1 9 7 1 ; R o b e r t s , 1969).
RHOADES
610
TABLE 2.—Hemagglutination-inhibition (HI) titers of serums from turkeys inoculated intravenously with Mycoplasma gallisepticum Days after exposure Turkey serums 699 699 728 728
40b
(untreated) (2ME treated) 0 (untreated) (2ME treated)
40
36
60
160 80 160 160
640 320 80 80
No evidence of HI activity at a 1:10 dilution of serum. Reciprocal of highest dilution of serum causing complete HI. Serum pretreated with .05 M 2-mercaptoethanol.
REFERENCES Frey, M. L., R. P. Hanson, and D. P. Anderson, 1968. A medium for the isolation of avian mycoplasmas. Amer. J. Vet. Res. 29:2163-2171. Kelton, W. H., and H. Van Roekel, 1963. Serological studies of mycoplasma (PPLO) of avian origin. Avian Dis. 7:272-286. Kleven, S. H., and B. S. Pomeroy, 1971. Characterization of the antibody response of turkeys to Mycoplasma meleagridis. Avian Dis. 15:291—298. Kuniyasu, C , 1969. Nature of antibody response of chickens to Mycoplasma gallisepticum. Nat. Inst. Anim. Health Quart. 9 : 1 1 9 - 1 2 8 . Mohammed, Y. S., and E. H. Bohl, 1968. Serologic studies on Mycoplasma meleagridis in turkeys. Avian Dis. 12:554-566. Rhoades, K. R., 1975. Antibody responses of turkeys experimentally exposed to Mycoplasma synoviae. Avian Dis. 19:437-442. Roberts, D. H., 1969. Serologic response produced in chickens by three strains of Mycoplasma gallisepticum. J. Appl. Bact. 3 2 : 3 9 5 - 4 0 1 . Yoder, H. W., Jr., 1975. Mycoplasma gallisepticum infection. In Diseases of poultry. 6th ed. M. S. Hofstad, B. W. Calnek, C. F. Hemboldt, W. M. Reid and H. W. Yoder, Jr., Ed. Iowa State University Press, Ames, Iowa, pp. 287—307.
TABLE 3 .—Hemagglutination-inhibition (HI) titers of serums from turkeys intravenously with Mycoplasma meleagridis
inoculated
Days after exposure Turkey serums 290 290 746 746
(untreated) (2ME treated) 0 (untreated) (2ME treated)
12
80b
80
40
40
80
160
40
40
No evidence of HI activity at a 1:10 dilution of serum. Reciprocal of highest dilution of serum causing complete HI. Serum pretreated with .05 M 2-mercaptoethanol.
Downloaded from http://ps.oxfordjournals.org/ by guest on April 24, 2015
1971), early low level HI activity, sensitive to treatment with 2ME, was demonstrated in serums obtained from a turkey a few days after IV exposure with .1 ml of broth culture of M. meleagridis. However, this activity could not be demonstrated in serum fractions separated by gel filtration. In another study (Kuniyasu, 1969), chickens inoculated IV with .5 ml of broth culture of M. gallisepticum developed early HI activity that was sensitive to 2ME treatment and was present in the first protein fraction eluted when serums were fractionated by gel filtration. Although IgM type antibody against mycoplasmas can be detected under certain conditions by HI tests, agglutination tests are certainly much more sensitive. Types of exposure other than IV induce an early antibody response detectable by agglutination tests but not by HI tests, and the early HI antibody titers induced by IV inoculation are lower than antibody titers demonstrated by tube agglutination tests (Kuniyasu, 1969; Kleven and Pomeroy, 1971; and Rhoades, 1975).
MATERIALS AND METHODS
Information gained from serologic tests is an i m p o r t a n t aid in establishing t h e presence of avian mycoplasmal infections. The serologic tests used most c o m m o n l y for this purpose are t h e agglutination and hemagglutination-inhibition (HI) tests. T h e effectiveness of these t w o types of tests for detecting a n t i b o d y against mycoplasmas is related, at least in part, t o t h e p r e d o m i n a t i n g i m m u n o g l o b u l i n of which t h e specific a n t i b o d y consists. For example, studies of a n t i b o d y response to Mycoplasma meleagridis (Kleven and P o m e r o y , 1971) and Mycoplasma gallisepticum ( R o b e r t s , 1 9 6 9 ) indicated t h a t t h e HI tests d e m o n s t r a t e d IgG b u t n o t IgM, and t h a t plate agglutination tests d e m o n strated IgM b u t were relatively insensitive for d e m o n s t r a t i n g IgG. However, a s t u d y ( R h o a d e s , 1975) of the a n t i b o d y response of t u r k e y s to Mycoplasma synoviae indicated t h a t in addition to the expected agglutinins, low level HI activity was present within a few days after intravenous (IV) e x p o s u r e . This HI activity was sensitive to the action of 2-mercaptoethanol (2ME) suggesting t h a t IgM t y p e a n t i b o d y was involved.
Mycoplasma synoviae, strain WVU 1 8 5 3 ; M. gallisepticum, strain S6; and M. meleagridis, strain 8M92, were used to p r o d u c e antigens for inoculating turkeys and c o n d u c t i n g serologic tests. A h e a r t infusion broth (Kelton and Van Roekel, 1963) with yeast a u t o l y s a t e substituted for yeast e x t r a c t was used to cultivate M. gallisepticum and M. meleagridis. The MF4 m e d i u m described b y Frey et al., ( 1 9 6 8 ) was used to cultivate M. synoviae. T h e mycoplasmas were sedimented by centrifugation at 1 0 0 0 0 G for 45 min, washed 2 times in p h o s p h a t e buffered saline (PBS), p H 7.2, and resuspended according to their intended use. Cells used to inoculate t u r k e y s were resuspended in PBS and adjusted s p e c t r o p h o t o m e t r i c a l l y to an optical density of .18 at 6 0 0 n m . Cells used as antigens for HI tests were resuspended in PBS at a p p r o x i m a t e l y 1/100 t h e original volume, mixed with an equal volume of glycerin, divided i n t o 1-ml aliquots, and frozen and stored at ^ 4 5 C until used.
This r e p o r t describes the study of serums obtained from t u r k e y s 4 days after TV exposure with M. synoviae to d e t e r m i n e whether early HI activity was d u e t o IgM t y p e a n t i b o d y . O t h e r pathogenic avian mycoplasmas, M. meleagridis and M. gallisepticum, also were used to expose t u r k e y s IV t o d e t e r m i n e whether they induced a similar early HI response.
1
No endorsements are implied herein.
1978 Poultry Sci 57:608-610
Mycoplasma-free turkeys were used to prepare antiserums. T w o were inoculated IV with 1 ml of M. gallisepticum antigen, and 2 were similarly inoculated with M. meleagridis antigen. Three t u r k e y s were inoculated IV 4 times at 3- or 4-day intervals with .5 ml of M. synoviae antigen. Serums from blood samples taken before and at various intervals after inoculation were examined for HI a n t i b o d y . Each of t h e serums was also examined after t r e a t m e n t with 2ME. Serum immunoglobulins from a representative t u r k e y inoculated with M. synoviae antigen were separated by gel filtration, and samples were examined for HI activity. The HI tests were c o n d u c t e d as previously
608
Downloaded from http://ps.oxfordjournals.org/ by guest on April 24, 2015
INTRODUCTION
MYCOPLASMAL H E M A G G L U T I N A T I O N INHIBITION
609
effect of 2ME t r e a t m e n t on these titers are indicated in Table 1. T h e results of gel filtration separation of serum proteins and s u b s e q u e n t HI testing of representative samples are indicated in Figure 1. T h e HI titers of serums from t u r k e y s inoculated with M. gallisepticum and M. meleagridis antigens and the effect of 2ME t r e a t m e n t on the titers are presented in Tables 2 and 3, respectively.
DISCUSSION
15 25 35 45 55 65 75 SAMPLE NUMBER
FIG. 1. Optical densities and hemagglutination-inhibition titers of samples obtained by gel-filtration separation of serum proteins from a turkey 4 days after intravenous exposure to Mycoplasma synoviae.
described (Yoder, 1 9 7 5 ) , as was t r e a t m e n t of serums with 2ME (Kleven and P o m e r o y , 1 9 7 1 ) . Immunoglobulin separation by gel filtration was essentially as previously reported (Klevven and P o m e r o y , 1971). Eluted samples were examined for HI activity w i t h o u t c o n c e n t r a t i o n . RESULTS
T h e HI titers of serums obtained from turkeys inoculated with M. synoviae and t h e
In a previous s t u d y (Kleven and P o m e r o y ,
TABLE 1 .—Hemagglutination-inhibition (HI) titers of serums from turkeys inoculated intravenously with Mycoplasma synoviae Days after exposure Turkey serums 2 2 2 (untreated) 222 (2ME treated)*-'
0
4 .a
80^
7
11
14
18
3 20 80
320 160
3 20 160
640 320
226 (untreated) 226 (2ME treated)
40
40 20
160 40
320 160
1280 640
235 (untreated) 2 3 5 (2ME treated)
80
320 80
640 3 20
640 320
640 320
No evidence of HI activity at a 1:10 dilution of serum. Reciprocal of highest dilution of serum causing complete HI. Serum pretreated with .05 M 2-mercaptoethanol.
Downloaded from http://ps.oxfordjournals.org/ by guest on April 24, 2015
5
T h e sensitivity of early HI activity to 2ME t r e a t m e n t and d e m o n s t r a t i o n of this activity in only t h e first protein fraction eluted during gel filtration separation indicates t h a t IgM t y p e a n t i b o d y was responsible. T h e d e m o n s t r a t i o n of HI due to IgM t y p e a n t i b o d y in this s t u d y differs from that in previous studies (Kleven and P o m e r o y , 1 9 7 1 ; R o b e r t s , 1969) p r o b a b l y due to differences in IgM a n t i b o d y titers and possibly related to b o t h the r o u t e of exposure and t h e a m o u n t of antigen administered. Previous reports have indicated that t h e IV r o u t e of exposure results in higher titers than o t h e r routes (Kleven and P o m e r o y , 1 9 7 1 ; M o h a m m e d and Bohl, 1 9 6 8 ) . T h e c o n c e n t r a t i o n of antigens in the present s t u d y was adjusted s p e c t r o p h o t o metrically and n o t according to n u m b e r s of colony-forming units, b u t probably contained several times t h e antigen c o n c e n t r a t i o n of the cultures used to expose birds in t h e previous studies (Kleven and P o m e r o y , 1 9 7 1 ; R o b e r t s , 1969).
RHOADES
610
TABLE 2.—Hemagglutination-inhibition (HI) titers of serums from turkeys inoculated intravenously with Mycoplasma gallisepticum Days after exposure Turkey serums 699 699 728 728
40b
(untreated) (2ME treated) 0 (untreated) (2ME treated)
40
36
60
160 80 160 160
640 320 80 80
No evidence of HI activity at a 1:10 dilution of serum. Reciprocal of highest dilution of serum causing complete HI. Serum pretreated with .05 M 2-mercaptoethanol.
REFERENCES Frey, M. L., R. P. Hanson, and D. P. Anderson, 1968. A medium for the isolation of avian mycoplasmas. Amer. J. Vet. Res. 29:2163-2171. Kelton, W. H., and H. Van Roekel, 1963. Serological studies of mycoplasma (PPLO) of avian origin. Avian Dis. 7:272-286. Kleven, S. H., and B. S. Pomeroy, 1971. Characterization of the antibody response of turkeys to Mycoplasma meleagridis. Avian Dis. 15:291—298. Kuniyasu, C , 1969. Nature of antibody response of chickens to Mycoplasma gallisepticum. Nat. Inst. Anim. Health Quart. 9 : 1 1 9 - 1 2 8 . Mohammed, Y. S., and E. H. Bohl, 1968. Serologic studies on Mycoplasma meleagridis in turkeys. Avian Dis. 12:554-566. Rhoades, K. R., 1975. Antibody responses of turkeys experimentally exposed to Mycoplasma synoviae. Avian Dis. 19:437-442. Roberts, D. H., 1969. Serologic response produced in chickens by three strains of Mycoplasma gallisepticum. J. Appl. Bact. 3 2 : 3 9 5 - 4 0 1 . Yoder, H. W., Jr., 1975. Mycoplasma gallisepticum infection. In Diseases of poultry. 6th ed. M. S. Hofstad, B. W. Calnek, C. F. Hemboldt, W. M. Reid and H. W. Yoder, Jr., Ed. Iowa State University Press, Ames, Iowa, pp. 287—307.
TABLE 3 .—Hemagglutination-inhibition (HI) titers of serums from turkeys intravenously with Mycoplasma meleagridis
inoculated
Days after exposure Turkey serums 290 290 746 746
(untreated) (2ME treated) 0 (untreated) (2ME treated)
12
80b
80
40
40
80
160
40
40
No evidence of HI activity at a 1:10 dilution of serum. Reciprocal of highest dilution of serum causing complete HI. Serum pretreated with .05 M 2-mercaptoethanol.
Downloaded from http://ps.oxfordjournals.org/ by guest on April 24, 2015
1971), early low level HI activity, sensitive to treatment with 2ME, was demonstrated in serums obtained from a turkey a few days after IV exposure with .1 ml of broth culture of M. meleagridis. However, this activity could not be demonstrated in serum fractions separated by gel filtration. In another study (Kuniyasu, 1969), chickens inoculated IV with .5 ml of broth culture of M. gallisepticum developed early HI activity that was sensitive to 2ME treatment and was present in the first protein fraction eluted when serums were fractionated by gel filtration. Although IgM type antibody against mycoplasmas can be detected under certain conditions by HI tests, agglutination tests are certainly much more sensitive. Types of exposure other than IV induce an early antibody response detectable by agglutination tests but not by HI tests, and the early HI antibody titers induced by IV inoculation are lower than antibody titers demonstrated by tube agglutination tests (Kuniyasu, 1969; Kleven and Pomeroy, 1971; and Rhoades, 1975).