Zbl. Vet. Med. B, 26, 340-343 (1979) @ 1979 Verlag Paul Parey, Berlin und Hamburg ISSN 0514-7166lASTM-Coden: ZVRBA2
M A F F , Central Veterinary Laboratory, N e w H a w , Weybridge, England
Immunoglobulins Detected by the Serum Agglutination Test for Salmonella dublin Infection of Cattle BY
C. WRAY,J. A. MORRIS and W. J. SOJKA W i t h one table
(Received for publication September 2 1 , 1978)
Introduction The classes of immunoglobulins produced against salmonella have been investigated in various species of animals, but there appears to have been no investigation in cattle. Because the nature of the antibodies formed during salmonella infection in cattle is not known it is possible that characterisation of these antibodies may improve the diagnostic specificity of tests for detection of infection in animals, especially carriers. For instance, in bovine brucellosis, use has been made of 2-mercaptoethanol to destroy IgM so as to 1976). increase the specificity of tests (ANCZYKOWSKI,
Material and Methods Serum Samples Serial serum samples were obtained from 12 experimentally infected cattle, infected either intravenously or orally, and from two naturally infected cows, a constant S. dublin excreter and a convalescent animal. In addition single serum samples were obtained from 36 cows with a history of S. dublirz infection. Samples were also obtained from 6 calves and 4 adults immunised with a live S. dublin vaccine’) and from one calf immunised with boiled S. dublin in Freund’s complete antigen. Serological Tests Serum agglutination tests and disulphide bond reduction using dithiothreitol (D’TT) 1977). More samples were performed as described previously (WRAY,SOJKAand CALLOW, contained flagellar agglutinins than somatic agglutinins. Density gradient ultracentrifugation and ion exchange chromatography were used for serum fractionation and the immunoglobulin
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341
Immunoglobulins Detected by the Serum Agglutination Test
classes were identified by gel diffusion and immunoelectrophoresis using rabbit antiserum to whole bovine globulin and class-specific antiglobulins (MORRIS and HUSSAINI, 1974).
Results Disulphide Bond Reduction Somatic agglutinins were detected at low titre in three (two from experimentally infected animals and one from an abortion case) of the 230 samples examined after DTT treatment (Tab. 1). In the case of the flagellar agglutination test, 134 of the 282 samples retained agglutinating activity, often at high titre, after DTT treatment. Table 1 Number of samples showing S. dublin agglutinins belonging to the IgG class of immunoglobulins Method of immunoglobulin identification Somatic Agglutinins
Method of infection and no. of animals
Vaccination Calves l 3 ) * C 3 months Calves I31 > 3 months Adults 14) Calf ( I ) * *
1 Experimental
infection (121
Ultracentrifugation
Disulphide Bond Reduction
Ultracentrifugation
ND
ND ND 01 2 ND
21 1 5 1 45 I 5 2 13 I 3 2 ND
ND ND 3 1 I ND
ND 01 4 01 4
15 1 3 6 20 I 33 12 1 1 6
ND 3 1 4 41 4
0 I 42 0 I 32 0 I 18
Natural infection Abortion cases (36) Excreter 11) Convalescent animal (1)
1 I 36
0 I 15 0 1 16
I
Flagellar Agglutinins
Disulphide Bond Reduct ion
2 I 71
I
I
0 I 11 ~
2 I62 ~
I
7 I 11
I
~~~~
Numerator = No. of samples showing serological activity in the IgG class. Denominator = Total No. of samples examined (all these samples showed serological activity in the IgM class). ’>= Figure in parenthesis shows number of animals or samples. = Vaccinated with somatic antigen. ND = Not done
Density Gradient Ultracentrifugation Flagellar agglutinins were detected in the fast (shown to contain IgM) and slow (shown to contain IgG) sedimenting fractions in most of the samples examined (Tab. 1). Somatic agglutinins were not detected in the IgG containing fractions. Ion-exchange Chromatography Sixteen serum samples from 4 calves (including one immunised with the somatic antigen) were collected a t varying periods after vaccination and after a booster vaccination and fractionated by ion exchange chromatography. Somatic agglutinins were detected only in the peak containing IgM. Flagellar agglutinins, however, were detected in the fractions containing IgG,, IgG,, and IgM. Discussion In the case of the somatic agglutinins all three methods showed that their activity was predominantly of the IgM class. Some investigators (LOSPALLUTO et al., 1962; BAUERet al., 1963) have suggested that only the IgM class of
342
WRAY,MORRIS and SOJKA
immunoglobulins is produced against the somatic antigen of Gram-negative bacteria. Other workers detected some specific IgG response but only with large doses of antigen and repeated inoculations (MUHOLLAND et al., 1965). DUFFUS and ALLEN(1968) found that S. gallinarum infection in poultry produced IgM followed by IgG, which often appeared to be incomplete antibody because it was detected only by the antiglobulin test. In pigs, however, (1964) showed that S. typhimurium “0”antigen induces ROWLEY and TURNER selective synthesis of IgM antibodies. Thus the failure to demonstrate somatic agglutinins of the IgG class may relate to the species of animals used, the serological tests employed and the method of challenge. It should be pointed out that the present study utilised both animals naturally infected and animals experimentally infected by various routes with different S. dublin antigens. Thus it is unlikely that either the method of challenge or the somatic antigen preparations used influenced the class of immunoglobulins produced. In contrast, both IgG and IgM were produced in calves and adults in response to the flagellar antigen. This is perhaps not surprising since protein antigens usually elicit an IgM response which is followed by IgG, while the polysaccharide somatic antigens of Gram-negative bacteria usually induce a 1967). predominant and persistent IgM response (PIKE, It is also apparent that the classes of immunoglobulins detected by the SAT in the constant excreter and in samples from the field did not differ from those found in animals immunised by other methods and it was not possible to differentiate the excreter. However, a study of the immunoglobulins detected by other serological tests may be of value and provide additional information about immunity against salmonella infection in cattle.
Summary The classes of immunoglobulins detected by the serum agglutination test were investigated in Salmonella dublin infected cattle. Somatic agglutinins were predominantly of the IgM class, whereas flagellar agglutinins were found in both the IgM and IgG classes. Zusammenfassung Nachweis von Immunglobulinen durch den Serumagglutinationstest bei mit Salmonella dublin infizierten Kzlbern Mit dem Serumagglutinationstest wurden die Immunglobulinklassen bei mit Salmonella dublin infizierten Kalbern untersucht. Die somatischen Agglutinine gehorten uberwiegend der IgM-Klasse an, wahrend das Geiflelagglutinin sowohl in der IgM- als auch in der IgG-Klasse gefunden wurde. References ANCZYKOWSKI, F., 1976: Diagnostic specificity of the mercaptoethanol test in the diagnosis of bovine brucellosis in cattle. 111. Effect of mercaptoethanol on the agglutination activity of sera of cattle pestered with brucellosis. Bull. vet. Inst. Pulawy. 20, 17-21. BAUER,D. C., M. J. MATHIES, and A. B. STAVITSKY, 1963: Sequence of synthesis of gamma-1 macroglobulin and gamma-2 globulin antibodies during primary and secondary responses to proteins, Salmonella antigens and phage. J. exp. Med. 217, 889-907. DUFFUS,W. P. H., and D. ALLAN,1968: A study on the immunological response in chidcens to the somatic antigen of Salmonella gallinarum. Immunology 15, 653-660. LOSPALLUTO, J., W. MILLER,B. DORWARD, and C. W. FINK, 1962: Formation of macroglobulin antibodies. I. Studies on adult humans. J. Clin. Invest. 41, 1415-1421. MORRIS,J. A,, and S. N. HUSSAINI, 1974: Characterization of the antibodies detected by the microscopic agglutination test for bovine leptospirosis. J. Hyg. 73, 425-432.
Immunoglobulins Detected by the Serum Agglutination Test
343
MULHOLLAND, J. H., S. M. WOLFF, A. L. JACKSON, and M. LANDY,1965: Quantitative studies of febrile tolerance and levels of specific antibody evoked by bacterial endotoxin. J. d i n . Invest. 44, 920-928. PIKE,R. M., 1967: Antibody heterogeneity and serological reactions. Bact. Rev. 31, 157-174. 1964: Increase in macroglobulin antibodies of mouse m d ROWLEY, D., and K. J. TURNER, pig following injection of bacterial lipopolysaccharide. Immunology 7, 394-402. WRAY,C., W. J. SOJKA,and R. J. CALLOW, 1977: Serological response in cattle to salmonella infection. Br. vet. J. 133, 25-32. Authors’ address: Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, KT 15 3NB, England.
M A F F , Central Veterinary Laboratory, N e w H a w , Weybridge, England
Immunoglobulins Detected by the Serum Agglutination Test for Salmonella dublin Infection of Cattle BY
C. WRAY,J. A. MORRIS and W. J. SOJKA W i t h one table
(Received for publication September 2 1 , 1978)
Introduction The classes of immunoglobulins produced against salmonella have been investigated in various species of animals, but there appears to have been no investigation in cattle. Because the nature of the antibodies formed during salmonella infection in cattle is not known it is possible that characterisation of these antibodies may improve the diagnostic specificity of tests for detection of infection in animals, especially carriers. For instance, in bovine brucellosis, use has been made of 2-mercaptoethanol to destroy IgM so as to 1976). increase the specificity of tests (ANCZYKOWSKI,
Material and Methods Serum Samples Serial serum samples were obtained from 12 experimentally infected cattle, infected either intravenously or orally, and from two naturally infected cows, a constant S. dublin excreter and a convalescent animal. In addition single serum samples were obtained from 36 cows with a history of S. dublirz infection. Samples were also obtained from 6 calves and 4 adults immunised with a live S. dublin vaccine’) and from one calf immunised with boiled S. dublin in Freund’s complete antigen. Serological Tests Serum agglutination tests and disulphide bond reduction using dithiothreitol (D’TT) 1977). More samples were performed as described previously (WRAY,SOJKAand CALLOW, contained flagellar agglutinins than somatic agglutinins. Density gradient ultracentrifugation and ion exchange chromatography were used for serum fractionation and the immunoglobulin
‘* Mellavax - Burroughs Wellcome.
u. s. Copyright Clearance Center
Code Statement:
0514-7166/79/2604-0340$02.50/0
341
Immunoglobulins Detected by the Serum Agglutination Test
classes were identified by gel diffusion and immunoelectrophoresis using rabbit antiserum to whole bovine globulin and class-specific antiglobulins (MORRIS and HUSSAINI, 1974).
Results Disulphide Bond Reduction Somatic agglutinins were detected at low titre in three (two from experimentally infected animals and one from an abortion case) of the 230 samples examined after DTT treatment (Tab. 1). In the case of the flagellar agglutination test, 134 of the 282 samples retained agglutinating activity, often at high titre, after DTT treatment. Table 1 Number of samples showing S. dublin agglutinins belonging to the IgG class of immunoglobulins Method of immunoglobulin identification Somatic Agglutinins
Method of infection and no. of animals
Vaccination Calves l 3 ) * C 3 months Calves I31 > 3 months Adults 14) Calf ( I ) * *
1 Experimental
infection (121
Ultracentrifugation
Disulphide Bond Reduction
Ultracentrifugation
ND
ND ND 01 2 ND
21 1 5 1 45 I 5 2 13 I 3 2 ND
ND ND 3 1 I ND
ND 01 4 01 4
15 1 3 6 20 I 33 12 1 1 6
ND 3 1 4 41 4
0 I 42 0 I 32 0 I 18
Natural infection Abortion cases (36) Excreter 11) Convalescent animal (1)
1 I 36
0 I 15 0 1 16
I
Flagellar Agglutinins
Disulphide Bond Reduct ion
2 I 71
I
I
0 I 11 ~
2 I62 ~
I
7 I 11
I
~~~~
Numerator = No. of samples showing serological activity in the IgG class. Denominator = Total No. of samples examined (all these samples showed serological activity in the IgM class). ’>= Figure in parenthesis shows number of animals or samples. = Vaccinated with somatic antigen. ND = Not done
Density Gradient Ultracentrifugation Flagellar agglutinins were detected in the fast (shown to contain IgM) and slow (shown to contain IgG) sedimenting fractions in most of the samples examined (Tab. 1). Somatic agglutinins were not detected in the IgG containing fractions. Ion-exchange Chromatography Sixteen serum samples from 4 calves (including one immunised with the somatic antigen) were collected a t varying periods after vaccination and after a booster vaccination and fractionated by ion exchange chromatography. Somatic agglutinins were detected only in the peak containing IgM. Flagellar agglutinins, however, were detected in the fractions containing IgG,, IgG,, and IgM. Discussion In the case of the somatic agglutinins all three methods showed that their activity was predominantly of the IgM class. Some investigators (LOSPALLUTO et al., 1962; BAUERet al., 1963) have suggested that only the IgM class of
342
WRAY,MORRIS and SOJKA
immunoglobulins is produced against the somatic antigen of Gram-negative bacteria. Other workers detected some specific IgG response but only with large doses of antigen and repeated inoculations (MUHOLLAND et al., 1965). DUFFUS and ALLEN(1968) found that S. gallinarum infection in poultry produced IgM followed by IgG, which often appeared to be incomplete antibody because it was detected only by the antiglobulin test. In pigs, however, (1964) showed that S. typhimurium “0”antigen induces ROWLEY and TURNER selective synthesis of IgM antibodies. Thus the failure to demonstrate somatic agglutinins of the IgG class may relate to the species of animals used, the serological tests employed and the method of challenge. It should be pointed out that the present study utilised both animals naturally infected and animals experimentally infected by various routes with different S. dublin antigens. Thus it is unlikely that either the method of challenge or the somatic antigen preparations used influenced the class of immunoglobulins produced. In contrast, both IgG and IgM were produced in calves and adults in response to the flagellar antigen. This is perhaps not surprising since protein antigens usually elicit an IgM response which is followed by IgG, while the polysaccharide somatic antigens of Gram-negative bacteria usually induce a 1967). predominant and persistent IgM response (PIKE, It is also apparent that the classes of immunoglobulins detected by the SAT in the constant excreter and in samples from the field did not differ from those found in animals immunised by other methods and it was not possible to differentiate the excreter. However, a study of the immunoglobulins detected by other serological tests may be of value and provide additional information about immunity against salmonella infection in cattle.
Summary The classes of immunoglobulins detected by the serum agglutination test were investigated in Salmonella dublin infected cattle. Somatic agglutinins were predominantly of the IgM class, whereas flagellar agglutinins were found in both the IgM and IgG classes. Zusammenfassung Nachweis von Immunglobulinen durch den Serumagglutinationstest bei mit Salmonella dublin infizierten Kzlbern Mit dem Serumagglutinationstest wurden die Immunglobulinklassen bei mit Salmonella dublin infizierten Kalbern untersucht. Die somatischen Agglutinine gehorten uberwiegend der IgM-Klasse an, wahrend das Geiflelagglutinin sowohl in der IgM- als auch in der IgG-Klasse gefunden wurde. References ANCZYKOWSKI, F., 1976: Diagnostic specificity of the mercaptoethanol test in the diagnosis of bovine brucellosis in cattle. 111. Effect of mercaptoethanol on the agglutination activity of sera of cattle pestered with brucellosis. Bull. vet. Inst. Pulawy. 20, 17-21. BAUER,D. C., M. J. MATHIES, and A. B. STAVITSKY, 1963: Sequence of synthesis of gamma-1 macroglobulin and gamma-2 globulin antibodies during primary and secondary responses to proteins, Salmonella antigens and phage. J. exp. Med. 217, 889-907. DUFFUS,W. P. H., and D. ALLAN,1968: A study on the immunological response in chidcens to the somatic antigen of Salmonella gallinarum. Immunology 15, 653-660. LOSPALLUTO, J., W. MILLER,B. DORWARD, and C. W. FINK, 1962: Formation of macroglobulin antibodies. I. Studies on adult humans. J. Clin. Invest. 41, 1415-1421. MORRIS,J. A,, and S. N. HUSSAINI, 1974: Characterization of the antibodies detected by the microscopic agglutination test for bovine leptospirosis. J. Hyg. 73, 425-432.
Immunoglobulins Detected by the Serum Agglutination Test
343
MULHOLLAND, J. H., S. M. WOLFF, A. L. JACKSON, and M. LANDY,1965: Quantitative studies of febrile tolerance and levels of specific antibody evoked by bacterial endotoxin. J. d i n . Invest. 44, 920-928. PIKE,R. M., 1967: Antibody heterogeneity and serological reactions. Bact. Rev. 31, 157-174. 1964: Increase in macroglobulin antibodies of mouse m d ROWLEY, D., and K. J. TURNER, pig following injection of bacterial lipopolysaccharide. Immunology 7, 394-402. WRAY,C., W. J. SOJKA,and R. J. CALLOW, 1977: Serological response in cattle to salmonella infection. Br. vet. J. 133, 25-32. Authors’ address: Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, KT 15 3NB, England.
