Letters in Applied Microbioloyy 1991, 13, 55-57
ADONIS 026682549 100083V
Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies C .GIL-TURNE &SJ . A . G . A L E I X OUniversidade Federal de Pelotas, Centro de Biotecnologiu, 96100 Pelotas, RS, Brazil MSJ207: received 8 April 1991 and accepted I0 April 1991 GIL-TURNES, C . & A L E I X O , J . A . G . 1991. Quantification of Moraxella hovis haemagglutinating adhesins with monoclonal antibodies. Letters in Applied Microbiology 13, 55-57. Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.
Moraxella-bouis is the cause o f infectious bovine keratoconjunctivitis, the most important ocular disease of cattle, which affects European breeds throughout the world (Baptista 1979). Fimbriae and probably other adhesins mediate the adherence of the bacteria to target cells (Gil-Turnes 1983; Annuar & Wilcox 1985). Vaccines produced with adherent M . bouis are more efficient than those prepared with nonadherent strains (Gil-Turnes ef a / . 1986a). The diversity of somatic antigens (Gil-Turnes & Araujo 1982) and adhesins (Gil-Turnes et al. 1986b; Lepper & Hermans, 1986) of M . houis has been demonstrated by double immunodiffusion and ELISA. Lepper & Hermans (1986) detected six antigenically different groups of fimbriae by ELISA with rabbit antisera, while Moore & Rutter (198’7) also with rabbit antisera detected seven group!j of fimhriae with a seroagglutination test . Monoclonal antibodies (MAbs) were used in the present work to detect and quantify haemagglutinins of 16 strains of M . houis. Material and Methods STRAINS
Sixteen M . hocis strains recovered from cattle with infectious bovine keratoconjunctivitis were
used. They were identified biochemically (Fraser & Gilmour 1979) and lyophilized. All haemagglutinated chicken and sheep erythrocytes. Strains R1 to R7 are clones obtained from the same animal; 2439-1, 2419-3 and 2358-1 were isolated in Uruguay; JUR, JUR 1, JUR 3, JUR 5 and JUR 7 were recovered from different animals of the same herd in successive years; GF 9 was recovered from an outbreak in Rio Grande d o Sul, Brazil, and FLA 64 was sent by Dr G.W. Pugh, NADL,Iowa, USA. MONOCLONAL ANTIBODIES
A panel of six MAbs produced against M . bouis GF 9 were used (Aleixo, Cardozo & Gil-Turnes, unpublished). The hybridomas were obtained according to established procedures. Briefly, spleen cells from immunized mice were fused with SP 2/0-AG 14 myeloma cells and culture fluids from the resulting hybridomas screened by ELISA. Cloning was carried out by the limiting dillution technique and selected clones were injected into pristane-primed Balb/c mice for production of ascites. MAbs were partially purified from ascites fluids by precipitation with 50% (w/v) ammonium sulphate, dialysed against PBS and redissolved to the original volume with PBS. Protein concentrations of MAbs were determined from absorbance at 280nm ( A , , , = 1.35%). For use in the experiments the protein
C. Gil-Turnes and J . A . G. Aleixo
56
concentration of each MAb was adjusted to 5 mg/ml with PBS. QUANTIFICATION OF E P I T O P E S
The epitopes were quantified to determine the amount of MAb used to inhibit one haemagglutinating unit (HAu) of each strain. One HAu was the least amount of bacteria contained in 50 pl of suspension that agglutinated an equal volume of 1% sheep red blood cells. To estimate the amount of MAb used, 50 pl of doubled dilutions of each MAb were incubated with one HAu of the strain at 37°C for 30 min. Fifty p1 of 1% sheep red blood cells in saline were then added, mixed and placed at 4"C, and the results read 1 h and 24 h later. The maximal dilution that inhibited haemagglutination was considered the haemagglutination inhibition titer. For comparisons, the titers of strain G F 9 were considered loo%, and those obtained with the other strains were expressed as percentages of those of strain G F 9. Results and Discussion
The use of a panel of six MAbs allowed detection of differences between isolates. Four of these, that express the six antigens detectable by our panel, may be considered dominant strains. Even though they express the same antigens, the quantity of each one varied between strains. Only three of the strains tested did not react with the panel of MAbs: one recovered from the same animal from which two dominant strains were isolated, one from Uruguay and the third from an outbreak in Rio Crande do Sui, Brazil. Some epitopes are expressed by more strains than others. The epitope recognized by MAb 36E was detected in 11 out of 16 strains; nine
strains expressed the epitopes recognized by MAbs 47D, 410D, 35F and 311B, while four were recognized by MAb 52E. This antigen was expressed only by the dominant strains. The amount of MAb used to neutralize one HAu of the strains varied between 400% and 0.7% of those used by strain G F 9 (Table 1). For example, strain R4 used four times the amount of MAb 47D consumed by strain G F 9, suggesting that the former has four times more epitopes, while strain 2419-3 used only 0.007 times, indicating that this epitope is in a much lower concentration in this strain. Quantitative methods such as haemagglutination inhibition with MAbs are able to differentiate strains presumed to be identical by nonquantitative methods. Four strains tested were used previously to study antigenic relationships by a double immunodiffusion test with seven polyclonal antifimbrial sera (GilTurnes et al. 1986b). Strains 2358-1 and 2419-3 were identical by that technique, but when studied by haemagglutination inhibition with MAbs, the former did not react with any of these sera while the other reacted with all of them. Strain 2439-1 reacted with three polyclonal sera and with four MAbs, and strain G F 9, used to prepare the MAbs, reacted with four polyclonal sera and with the six MAbs. The study of somatic (Gil-Turnes & Araujo 1982) and fimbrial antigens (Gil-Turnes et al. 1986b; Lepper & Hermans 1986; Moore & Rutter 1987) of strains isolated from diseased animals, showed that M. bouis is antigenically heterogeneous and that the prevalent serogroups vary in different regions. It was also shown that some strains express more antigens than others, indicating the existence of dominant strains that could be useful to prepare vaccines with wide protection.
Table 1. Relative quantities of nonoclonal antibodies necessary to neutralize one hemagglutinating unit of Moraxella boois ~
~
~~~~~~~~~~~
~
~
Strains of Moraxella bouis
Monoclonal antibody GF-9 R-1 R-2 R-3 R-4 R-7 2358-1 2439-1 2419-3 JUR-I JUR-3 JUR-5 JUR-7 JUR FLA-64 CTMT 47D 410D 35F 36E 311B 52E
100 200 1 0 0 200 100 25 100 25 100 10 100 100 -
- 400
58
- 50 - - 10 50 6 - - 100 - 50 -
- ,absence of inhibition.
-
-
6
50 10 50
-
87 6 10
-
50 10
10 100 200 100
50
-
-
100
-
25
200
50
200
100 100
10 25
6 10
25 25
100
100
200
-
58 -
-
-
-
-
-
-
Quantfication of Moraxella bovis haemagglutinating adhesins More work is needed to detect and quantify epitopes involved in the adherence of M. bouis. However, our results are encouraging, because they demonstrate that strains of this organism differ not only serologically but also in their capability to produce these epitopes. References ANNUAK, B.O. & WILCOX,G.E. 1985 Adherence of Moraxella bouis to cell cultures of bovine origin. Research in Veterinary Science 39, 241-246. BAPTISTA, P.J.H.P. 1979 Infectious bovine keratoconjunctivitis: a review. British Veterinary Journal 135, 225-242. N.J.L. 1979 The identification FRASER. J. & GILMOUR. of Moraxella houis and Neisseria ouis from the eyes of cattle and sheep. Research in Veterinary Science 27, 127-128. GIL-TLJRNES, C. 1983 Hemagglutination, autoagglutination and pathogenicity of Moraxella bouis strains. Canadian Journal of Comparative Medicine 47, 503504.
57
GIL-TURNES,C. & ARAUJO,F.L. 1982 Serological characterization of strains of Moraxella bovis using double immunodiffusion. Canadian Journal of Comparative Medicine 46, 16S168. GIL-TURNES,C., SOUZA, R.S.M., ARAUIO, F.L. & REYES,J.C.S. 1986a Evaluation of a vaccine prepared with adherent Moraxella bovis for the control of Infectious Bovine Keratoconjunctivitis. Proceedings X I V World Congress of Diseases of Cattle pp. 1233-1235. World Association for Buiatrics, Dublin. C., RIBEIRO,G.A., ALBUQUERQUE,I.M.B. GIL-TURNES, & CHAGAS,P.R.S. 1986b Serological characterization of adhesins of Moraxella boois. Proceedings X I V International Congress of Microbiology p. 281. International Union of Microbiological Societies, Manchester. LEPPER,A.W.D. & HERMANS,L.R. 1986 Characterization and quantification of pilus antigens of Moraxella bonk by ELISA. Australian Veterinary Journal 6 3 , 4 0 1 4 5 . MOORE,L.S. & RUTTER,J.M. 1987 Antigenic analysis of fimbrial proteins from Moraxella bouis. Journal of Clinical Microbiology 25,2063-2070.
ADONIS 026682549 100083V
Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies C .GIL-TURNE &SJ . A . G . A L E I X OUniversidade Federal de Pelotas, Centro de Biotecnologiu, 96100 Pelotas, RS, Brazil MSJ207: received 8 April 1991 and accepted I0 April 1991 GIL-TURNES, C . & A L E I X O , J . A . G . 1991. Quantification of Moraxella hovis haemagglutinating adhesins with monoclonal antibodies. Letters in Applied Microbiology 13, 55-57. Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.
Moraxella-bouis is the cause o f infectious bovine keratoconjunctivitis, the most important ocular disease of cattle, which affects European breeds throughout the world (Baptista 1979). Fimbriae and probably other adhesins mediate the adherence of the bacteria to target cells (Gil-Turnes 1983; Annuar & Wilcox 1985). Vaccines produced with adherent M . bouis are more efficient than those prepared with nonadherent strains (Gil-Turnes ef a / . 1986a). The diversity of somatic antigens (Gil-Turnes & Araujo 1982) and adhesins (Gil-Turnes et al. 1986b; Lepper & Hermans, 1986) of M . houis has been demonstrated by double immunodiffusion and ELISA. Lepper & Hermans (1986) detected six antigenically different groups of fimbriae by ELISA with rabbit antisera, while Moore & Rutter (198’7) also with rabbit antisera detected seven group!j of fimhriae with a seroagglutination test . Monoclonal antibodies (MAbs) were used in the present work to detect and quantify haemagglutinins of 16 strains of M . houis. Material and Methods STRAINS
Sixteen M . hocis strains recovered from cattle with infectious bovine keratoconjunctivitis were
used. They were identified biochemically (Fraser & Gilmour 1979) and lyophilized. All haemagglutinated chicken and sheep erythrocytes. Strains R1 to R7 are clones obtained from the same animal; 2439-1, 2419-3 and 2358-1 were isolated in Uruguay; JUR, JUR 1, JUR 3, JUR 5 and JUR 7 were recovered from different animals of the same herd in successive years; GF 9 was recovered from an outbreak in Rio Grande d o Sul, Brazil, and FLA 64 was sent by Dr G.W. Pugh, NADL,Iowa, USA. MONOCLONAL ANTIBODIES
A panel of six MAbs produced against M . bouis GF 9 were used (Aleixo, Cardozo & Gil-Turnes, unpublished). The hybridomas were obtained according to established procedures. Briefly, spleen cells from immunized mice were fused with SP 2/0-AG 14 myeloma cells and culture fluids from the resulting hybridomas screened by ELISA. Cloning was carried out by the limiting dillution technique and selected clones were injected into pristane-primed Balb/c mice for production of ascites. MAbs were partially purified from ascites fluids by precipitation with 50% (w/v) ammonium sulphate, dialysed against PBS and redissolved to the original volume with PBS. Protein concentrations of MAbs were determined from absorbance at 280nm ( A , , , = 1.35%). For use in the experiments the protein
C. Gil-Turnes and J . A . G. Aleixo
56
concentration of each MAb was adjusted to 5 mg/ml with PBS. QUANTIFICATION OF E P I T O P E S
The epitopes were quantified to determine the amount of MAb used to inhibit one haemagglutinating unit (HAu) of each strain. One HAu was the least amount of bacteria contained in 50 pl of suspension that agglutinated an equal volume of 1% sheep red blood cells. To estimate the amount of MAb used, 50 pl of doubled dilutions of each MAb were incubated with one HAu of the strain at 37°C for 30 min. Fifty p1 of 1% sheep red blood cells in saline were then added, mixed and placed at 4"C, and the results read 1 h and 24 h later. The maximal dilution that inhibited haemagglutination was considered the haemagglutination inhibition titer. For comparisons, the titers of strain G F 9 were considered loo%, and those obtained with the other strains were expressed as percentages of those of strain G F 9. Results and Discussion
The use of a panel of six MAbs allowed detection of differences between isolates. Four of these, that express the six antigens detectable by our panel, may be considered dominant strains. Even though they express the same antigens, the quantity of each one varied between strains. Only three of the strains tested did not react with the panel of MAbs: one recovered from the same animal from which two dominant strains were isolated, one from Uruguay and the third from an outbreak in Rio Crande do Sui, Brazil. Some epitopes are expressed by more strains than others. The epitope recognized by MAb 36E was detected in 11 out of 16 strains; nine
strains expressed the epitopes recognized by MAbs 47D, 410D, 35F and 311B, while four were recognized by MAb 52E. This antigen was expressed only by the dominant strains. The amount of MAb used to neutralize one HAu of the strains varied between 400% and 0.7% of those used by strain G F 9 (Table 1). For example, strain R4 used four times the amount of MAb 47D consumed by strain G F 9, suggesting that the former has four times more epitopes, while strain 2419-3 used only 0.007 times, indicating that this epitope is in a much lower concentration in this strain. Quantitative methods such as haemagglutination inhibition with MAbs are able to differentiate strains presumed to be identical by nonquantitative methods. Four strains tested were used previously to study antigenic relationships by a double immunodiffusion test with seven polyclonal antifimbrial sera (GilTurnes et al. 1986b). Strains 2358-1 and 2419-3 were identical by that technique, but when studied by haemagglutination inhibition with MAbs, the former did not react with any of these sera while the other reacted with all of them. Strain 2439-1 reacted with three polyclonal sera and with four MAbs, and strain G F 9, used to prepare the MAbs, reacted with four polyclonal sera and with the six MAbs. The study of somatic (Gil-Turnes & Araujo 1982) and fimbrial antigens (Gil-Turnes et al. 1986b; Lepper & Hermans 1986; Moore & Rutter 1987) of strains isolated from diseased animals, showed that M. bouis is antigenically heterogeneous and that the prevalent serogroups vary in different regions. It was also shown that some strains express more antigens than others, indicating the existence of dominant strains that could be useful to prepare vaccines with wide protection.
Table 1. Relative quantities of nonoclonal antibodies necessary to neutralize one hemagglutinating unit of Moraxella boois ~
~
~~~~~~~~~~~
~
~
Strains of Moraxella bouis
Monoclonal antibody GF-9 R-1 R-2 R-3 R-4 R-7 2358-1 2439-1 2419-3 JUR-I JUR-3 JUR-5 JUR-7 JUR FLA-64 CTMT 47D 410D 35F 36E 311B 52E
100 200 1 0 0 200 100 25 100 25 100 10 100 100 -
- 400
58
- 50 - - 10 50 6 - - 100 - 50 -
- ,absence of inhibition.
-
-
6
50 10 50
-
87 6 10
-
50 10
10 100 200 100
50
-
-
100
-
25
200
50
200
100 100
10 25
6 10
25 25
100
100
200
-
58 -
-
-
-
-
-
-
Quantfication of Moraxella bovis haemagglutinating adhesins More work is needed to detect and quantify epitopes involved in the adherence of M. bouis. However, our results are encouraging, because they demonstrate that strains of this organism differ not only serologically but also in their capability to produce these epitopes. References ANNUAK, B.O. & WILCOX,G.E. 1985 Adherence of Moraxella bouis to cell cultures of bovine origin. Research in Veterinary Science 39, 241-246. BAPTISTA, P.J.H.P. 1979 Infectious bovine keratoconjunctivitis: a review. British Veterinary Journal 135, 225-242. N.J.L. 1979 The identification FRASER. J. & GILMOUR. of Moraxella houis and Neisseria ouis from the eyes of cattle and sheep. Research in Veterinary Science 27, 127-128. GIL-TLJRNES, C. 1983 Hemagglutination, autoagglutination and pathogenicity of Moraxella bouis strains. Canadian Journal of Comparative Medicine 47, 503504.
57
GIL-TURNES,C. & ARAUJO,F.L. 1982 Serological characterization of strains of Moraxella bovis using double immunodiffusion. Canadian Journal of Comparative Medicine 46, 16S168. GIL-TURNES,C., SOUZA, R.S.M., ARAUIO, F.L. & REYES,J.C.S. 1986a Evaluation of a vaccine prepared with adherent Moraxella bovis for the control of Infectious Bovine Keratoconjunctivitis. Proceedings X I V World Congress of Diseases of Cattle pp. 1233-1235. World Association for Buiatrics, Dublin. C., RIBEIRO,G.A., ALBUQUERQUE,I.M.B. GIL-TURNES, & CHAGAS,P.R.S. 1986b Serological characterization of adhesins of Moraxella boois. Proceedings X I V International Congress of Microbiology p. 281. International Union of Microbiological Societies, Manchester. LEPPER,A.W.D. & HERMANS,L.R. 1986 Characterization and quantification of pilus antigens of Moraxella bonk by ELISA. Australian Veterinary Journal 6 3 , 4 0 1 4 5 . MOORE,L.S. & RUTTER,J.M. 1987 Antigenic analysis of fimbrial proteins from Moraxella bouis. Journal of Clinical Microbiology 25,2063-2070.
