Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains

Veterinary Microbiology 78 (2001) 49±60

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains M. Tadjine, K.R. Mittal* DeÂpartement de pathologie et microbiologie, Faculte de meÂdecine veÂteÂrinaire, Universite de MontreÂal, 3200, Sicotte, C.P. 5000, Saint-Hyacinthe, QueÂbec, Canada J2S 7C6 Received 28 January 2000; received in revised form 13 July 2000; accepted 13 July 2000

Abstract Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two ®eld strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identi®ed as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a con®rmatory test to identify serotype-speci®c capsular and somatic antigens. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Actinobacillus pleuropneumoniae; Serotyping; Cross-reactivities

1. Introduction Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae is one of major problems in the modern swine industry all over the world. The organism may cause an acute respiratory infection, resulting in high morbidity and mortality, or a chronic persistent infection, resulting in severe economic loss (Nicolet, 1992). Twelve serotypes

*

Corresponding author. Tel.: ‡1-514-345-8521/ext. 8296; fax: ‡1-450-778-8113.

0378-1135/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 9 2 - 3

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M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

of A. pleuropneumoniae biotype I, based on capsular antigens, have been recognized (Nielsen, 1986). Serotypes 1, 5 and 7 are the most prevalent in North America and an increasing number of serotype 7 infection has been observed during the last few years, especially in high health status herds (Mittal et al., 1998). Serological diagnosis and vaccination programs have been used in an attempt to control the disease. Both these programs require an extensive knowledge of different serotypes existing in a particular region, as this organism is antigenically heterogeneous with different serotypes showing different geographical distributions. Serotype-speci®c antigens are mainly capsular polysaccharide epitopes, whereas cell wall lipopolysaccharide may be shared between different serotypes (Mittal et al., 1988b; Mittal, 1990; Perry et al., 1990). Strains of serotype 7 are known to be antigenically heterogeneous and share common antigens with several other serotypes. Strong cross-reactivities between strains of serotypes 4 and 7 as well as between strains of serotype 7 and those of other serotypes may cause a great deal of problems in both serotyping and serodiagnosis (Mittal and Bourdon, 1991; Gottschalk et al., 2000). The purpose of the present study was to examine the antigenic heterogeneity among different strains of serotype 7 and their cross-reactivities with other serotypes using a battery of serological tests and rabbit polyclonal hyperimmune sera. 2. Materials and methods 2.1. Bacterial strains and antigen preparation Reference strains representing serotypes 1 through 12 of A. pleuropneumoniae were 4074, ATCC 27088 (1A), ISU158 (1B), S1536 (2), 1421 (3), M62 (4), K-17 (5), Fem1 (6), WF83 (7), 405 (8), CVJ-13261 (9), D13039 (10), 56153 (11) and 8329 (12). One hundred and twenty two ®eld isolates of serotype 7 originating from pulmonary tissues of pigs that had died of acute pleuropneumonia, or from tonsils of chronically infected pigs, were received from different diagnostic laboratories in Canada. The cultural and biochemical characterization of the bacteria was done according to the method of Biberstein et al. (1977). Reference and ®eld strains of A. pleuropneumoniae were grown on enriched pleuropneumonia-like organism (PPLO) agar medium overnight at 378C. Each plate was washed off gently in 3 ml of phosphate-buffered saline solution (PBS; pH 7.0) containing 0.5% formalin, and pooled. The pooled whole-cell suspension of each strain was divided into two portions. One portion was kept at room temperature for 4 h and a second portion was kept at 1008C in a water bath for 1 h; two antigen preparations were referred to as WC and BC, respectively. BC antigens were centrifuged at 800  g for 30 min and supernatants were collected and referred to as BC-SE. 2.2. Preparation of hyperimmune sera in rabbits Antisera against all the reference strains of serotypes 1±12 and two atypical ®eld strains of serotype 7 (90-3182 and 86-1411) were prepared in rabbits by intravenous

M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

51

inoculations of WC antigens. Young adult rabbits were injected twice a week with an antigen preparation of each strain. In total, seven injections were given in an increasing dose. The method used has been described previously by Mittal et al. (1982). The rabbits were bled 7 days after the last injection; the sera were separated and stored at ÿ208C until used. 2.3. Absorption of rabbit antisera Rabbit hyperimmune sera against the WC antigens of reference (WF83) and two atypical ®eld strains (90-3182 and 86-1411) were absorbed with an equal volume of 10% suspensions of WC antigens of homologous and heterologous strains in PBS. The mixtures were kept at 378C for 2 h and then centrifuged at 800  g for 30 min. The second absorption was done in the same way. The sera were tested before and after absorption for antibodies against homologous and heterologous strains by ID test. 2.4. Serological tests Coagglutination (COA), indirect haemagglutination (IHA), immunodiffusion (ID) and counter immunoelectophoresis (CIE) tests were used as described earlier Mittal et al., 1983a,b, 1988a; 1993). Rapid dot-ELISA (RDE) was used as described by Achacha and Mittal (1995). BC was used in COA and RDE tests, and BC-SE was used in ID, IHA and CIE tests. Colonial morphology in serum-soft agar (SSA) was determined according to the method of Finkelstein and Sulkin (1958). Conversion of diffuse type colonies to compact type in the presence of type-speci®c antiserum was considered as positive. Growth agglutination (GA) was carried out as described by Mittal and Tizard (1979). Live bacterial suspension were used for both SSA and GA tests. 2.5. Puri®cation of capsular polysaccharide Capsular material was extracted as described by Inzana et al. (1988). 2.6. Treatment of antigens with proteinase-K WC antigens were treated with 100 mg/ml of proteinase-K (Sigma) in PBS at 558C for 1 h as described by Hamel et al. (1987). 2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting WC antigens of ®eld and reference strains of serotype 7 were separated by SDS-PAGE (Laemmli, 1970). Gels were either stained with silver nitrate as described by Tsai and Frasch (1982) or transferred to nitrocellulose membranes (0.2 mm; Bio-Rad) for Western blotting (Towbin et al., 1979).

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M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

2.8. Tricine-SDS-PAGE (TSDS-PAGE) TSDS-PAGE was carried out as described by Jacques et al. (1996). 3. Results 3.1. Serological reactivity of A. pleuropneumoniae reference and ®eld strains of serotype 7 using rabbit hyperimmune sera against reference strains of all the known 12 serotypes of APP A total of 122 ®eld isolates of serotype 7 were tested for their reactivities with rabbit hyperimmune sera against all the known 12 serotypes. Reference strain of serotype 7 shared cross-reactivity only with serotype 1B; whereas ®eld strains of serotypes 7 shared cross-reactivities with serotypes 1A, 1B, 4, 9, 10 and 11 (Table 1). Seventeen percent of strains did not show any cross-reactivities with any other serotype, whereas, 58% of the ®eld isolates reacted with antiserum against serotype 1. Among these isolates, majority of strains reacted only with antiserum against serotype 1B in all the serological tests except IHA and CIE. Thirteen percent of strains reacted with antiserum against serotype 4, and 7% of stains reacted with antiserum against serotype 10. 3.2. Effect of absorption of rabbit hyperimmune sera against reference and ®eld strains of A. pleuropneumoniae serotype 7 with homologous and heterologous strains of serotype 7 as detected by ID test Results shown in Table 2 clearly demonstrate the existence of antigenic heterogeneity within serotype 7 strains as shown by the ID test. Absorption of anti-WF83 serum with ®eld strain 86-1411 did not remove antibodies against WF83. Similarly, absorption of anti-861411 serum with reference strain WF83 failed to remove antibodies against ®eld strain 86-1411. Most of the strains tested in ID test using puri®ed capsular extract and anti-WF83 hyperimmune serum gave a precipitation line completely identical with strain WF83, and only a few strains were found to give a line of partial identity with WF83 (Table 3). 3.3. SDS-PAGE and Western blot analysis of whole-cell preparations of serotype 7 strains probed with rabbit hyperimmune serum against serotype 7 reference strain SDS-PAGE and Western blot analysis did not show signi®cant differences in the pro®le of major outer membrane proteins of reference and ®eld strains of serotype 7 (Fig. 1). 3.4. Western blot analysis of proteinase-K treated whole cell preparations of A. pleuropneumoniae serotypes 1, 4 and 7 strains probed with rabbit hyperimmune serum against serotype 7 reference strain The results shown in Fig. 2 present the evidence of cross-reactivities between capsular polysaccharide antigens of reference serotype 1B and 7 strains. Strains 86-1411 and

Table 1 Serological reactivity of Actinobacillus pleuropneumoniae reference and ®eld strains of serotype 7 representing various groups using rabbit hyperimmune sera against all the known 12 serotypes of A. pleuropneumoniaea Tests used

WF83

COA ID IHA CIE RDE SSA GA

98-1102

Rabbit hyperimmune sera against reference strains of serotypes 1A

NRS Controlb

2

3

4

5

6

7

8

9

10

11

12

ÿ ÿ 0 ÿ ÿ ÿ ÿ

2‡ ‡ 0 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

4‡ ‡ 1600 ‡ ‡ ‡ ‡

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ ÿ ÿ ÿ ÿ ÿ

COA ID IHA CIE RDE SSA GA

2‡ ÿ 0 ÿ ÿ ÿ ÿ

2‡ ÿ 0 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

2‡ ‡ 0 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

3‡ ‡ 400 ‡ ‡ ‡ ‡

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ ÿ ÿ ÿ ÿ ÿ

96-2462

COA ID IHA CIE RDE SSA GA

ÿ ÿ 0 ÿ ÿ ÿ ÿ

3‡ ‡ 0 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

3‡ ‡ 0 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

4‡ ‡ 400 ‡ ‡ ‡ ‡

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

97-2629

COA ID IHA CIE

ÿ ÿ 0 ÿ

2‡ ÿ 0 ÿ

ÿ ÿ 0 ÿ

ÿ ÿ 0 ÿ

2‡ ÿ 0 ÿ

ÿ ÿ 0 ÿ

ÿ ÿ 0 ÿ

4‡ ‡ 400 ‡

ÿ ÿ 0 ÿ

ÿ ÿ 0 ÿ

3‡ ÿ 0 ÿ

ÿ ÿ 0 ÿ

ÿ ÿ 0 ÿ

ÿ ÿ 0 ÿ

53

1B

M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

Strains of serotype 7

54

Strains of serotype 7

90-3182

Tests used

Rabbit hyperimmune sera against reference strains of serotypes 1A

1B

2

3

4

5

6

7

8

9

10

11

12

NRS Controlb

RDE SSA GA

ÿ ÿ ÿ

‡ ÿ ÿ

ÿ ÿ ÿ

ÿ ÿ ÿ

‡ ÿ ÿ

ÿ ÿ ÿ

ÿ ÿ ÿ

‡ ‡ ‡

ÿ ÿ ÿ

ÿ ÿ ÿ

ÿ ÿ ÿ

ÿ ÿ ÿ

ÿ ÿ ÿ

ÿ ÿ ÿ

COA ID IHA CIE RDE SSA GA

3‡ ‡ 800 ‡ ‡ ‡ ‡

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ‡ 0 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

3‡ ‡ 800 ‡ ‡ ‡ ‡

ÿ ÿ 0 ÿ ÿ ÿ ÿ

‡ ‡ 200 ÿ ÿ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

3‡ ÿ 100 ÿ ‡ ÿ ÿ

ÿ ÿ 0 ÿ ÿ ÿ ÿ

ÿ ÿ ÿ ÿ ÿ ÿ ÿ

a 1‡, 2‡, 3‡ and 4‡ indicate the intensity of the reaction in the COA test, ‡ indicates positive reaction in ID, CIE, RDE, SSA and GA tests. 0 and ÿ indicate negative reaction. b Normal rabbit serum control.

M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

Table 1 (Continued )

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Table 2 Effect of absorption of rabbit hyperimmune sera against reference and ®eld strains of A. pleuropneumoniae serotype 7 with homologous and heterologous strains of serotype 7 as detected by immunodiffusion test Strain

WF83 86-1411

Anti-WF83 serum absorbed with

Anti-86-1411 serum absorbed with

Nil

WF83

86-1411

Nil

WF83

86-1411

‡ ‡

ÿ ÿ

‡ ÿ

‡ ‡

ÿ ‡

ÿ ÿ

WF83 showed an identical pro®le of both capsular and lipopolysaccharidic antigens. However, strain 90-3182 showed a similar pattern as shown by the reference serotype 1A (4074) strain with anti-WF83 serum. The serotype 4 strain (M62) showed one-way capsular polysaccharidic cross-reaction with anti-WF83 serum (results not shown). Strain 90-3182 shared capsular polysaccharide with reference strain WF83, but shared neither capsular nor somatic antigens with strain 86-1411 (results not shown). Results of the Western blot analysis shown in Fig. 3 presented evidence of an identical pro®le of both capsular and somatic antigens of serotype 1A (4074) and ®eld strain 90-3182 with antiserum against 90-3182. Strain 86-1411 showed strong reactions with rabbit antiserum against serotype 7 reference strain WF83 in both SSA and GA tests, but not with antisera against other serotypes, whereas strain 90-3182 reacted equally well in both SSA and GA tests with antisera against reference strains of both serotypes 1B and 7. Antiserum against strain 903182 also reacted in both SSA and GA tests with reference strains of serotypes 1A, 1B and 7 (results not shown). These observations prompted us to further study this strain which showed two-way cross-reactions in various tests. The results of both Western blot and Tricine SDS-PAGE clearly demonstrated that strain 90-3182 belonged to serotype 1A and not to serotype 7 as identi®ed earlier by conventional serological tests, whereas, strain 86-1411 belonged to serotype 7 as identi®ed by other serological tests. Tricine SDS-PAGE was used to further characterize the serotype identity of strain 903182 by determining its core type. Strain 90-3182 was found to possess the same core Table 3 Results of the immunodiffusion test using puri®ed capsular antigen and rabbit hyperimmune sera against reference and two selected ®eld strains of Actinobacillus pleuropneumoniae serotype 7 Rabbit antisera against strains

Capsular antigen of straina WF83

WF83 90-3182 86-1411 a

b

‡ ‡c ‡

90-3182 ‡ ‡c ÿ

Negative is indicated by symbol (ÿ). The positive symbol (‡) here indicates identical precipitation line. c The positive symbol (‡) here indicates identical precipitation line. b

b

86-1411 ÿ ÿ ‡

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M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

Fig. 1. SDS-PAGE (A) and Western blot (B) analysis of whole-cell preparations of reference and ®eld strains A. pleuropneumoniae serotype 7 probed with rabbit hyoerimmune serum against serotype 7 reference strain. WF83 (lane 1), 90-3182 (lane 2), 86-1411 (lane 3), 98-827 (lane 4), 97-A714 (lane 5), 95-4590 (lane 6), 97-1061 (lane 7).

type (type I) as possessed by the serotype 1A reference strain. Whereas, strain 86-1411 possessed the core type II as that possessed by the serotype 7 reference strain. The reference serotype 1 strains (1A and 1B) did not show any reactivity with antiWF83 serum in the CIE, IHA, RDE, SSA and GA assays (results not shown).

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Fig. 2. Western blot analysis of proteinase-K-treated whole-cell preparations of reference and ®eld strain of A. pleuropneumoniae serotype 1, 4 and 7 probed with rabbit hyperimmune serum against serotype 7 reference strain. Atypical strain 90-3182 (serotype 7) (lane 1), WF83 (serotype 7) (lane 2), M62 (serotype 4) (lane 3), SHY85-77 (serotype 1) (lane 4), 86-1411 (serotype 7) (lane 5), ISU158 (serotype 1B) (lane 6), 4074 (serotype 1A) (lane 7).

Fig. 3. Western blot analysis of proteinase-K-treated whole-cell preparations of A. pleuropneumoniae probed with rabbit hyperimmune serum against serotype 7 ®eld strain 90-3182. SHY85-77 (serotype 1) (lane 1), 861411 (serotype 7) (lane 2), 90-3182 (serotype 7) (lane 3), WF83 (serotype 7) (lane 4), M62 (serotype 4) (lane 5), ISU158 (serotype 1B) (lane 6), 4074 (serotype 1A) (lane 7).

4. Discussion Various techniques used for serotyping have their own limitations. Although all of these methods may be reliable, some tests may show problems of cross-reactivity. Both GA and SSA methods were found to be suitable serological tests to characterize the most super®cially located surface antigens because of the use of live bacterial cells instead of

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M. Tadjine, K.R. Mittal / Veterinary Microbiology 78 (2001) 49±60

killed cell suspensions or cell extractions routinely used in conventional serological tests such as COA, ID, IHA and CIE. Both GA and SSA method have been used earlier by various workers to study the surface antigens of various microbes. However, these two tests have not been used earlier for surface characterization of A. pleuropneumoniae. Precise identi®cation of serotype 7 becomes extremely important especially in view of the recently observed strong cross-reactions between serotypes 7 and 1 strains (Mittal and Bourdon, 1991; Gottschalk et al., 2000). In view of the fact that serotype 1 is recognized as the most virulent serotype involved in acute swine pleuropneumonia and that serotype 7 isolates are the second most prevalent in North America, such cross-reactions may cause a great deal of problems in both serotyping and serodiagnosis. It is not uncommon to encounter serotype 7 strains which give confusing and contradictory results in serotyping because of the presence of strong cross-reactions with other serotypes. Perry et al. (1990) suggested that serotypes of A. pleuropneumoniae could be more rigorously de®ned by speci®cations of both their capsular polysaccharide and cell wall LPS antigenic characteristics. Nielsen et al. (1996) demonstrated the presence of identical smooth LPS ladder pattern between a ®eld strain (7317) of serotype 2 and WF83 reference strain of serotype 7 and they proposed the strain 7317 to be designated as K2:O7. Using serotype-speci®c monoclonal antibodies (Mabs) in dot-ELISA and ELISAinhibition test, Gottschalk et al. (2000) studied three isolates of A. pleureuropneumoniae (1B, 27E and 26B). The results obtained indicated that all three isolates possessed serotype 1-related CPS and serotype 7-related O-chain LPS antigens. In addition, isolate 26B also possessed serotype 4-speci®c O-chain LPS epitopes. Strains 1B and 27E were proposed to be designated as K1:O7 and strain 26E as K1:O7/4. Results shown in Table 1 indicate the existence of several common antigens between serotype 7 ®eld strains and those of other serotypes. Until recently serotype 1 was considered as the major cause of swine pleuropneumonia in North America (Mittal et al., 1992; Mittal and Higgins, 1994). Results of this study are suggestive of the fact that pigs infected with some strains of serotype 1 could give false positive results in serology for serotype 7. The IHA test was found to be the most reliable serological test to characterize serotype-speci®c antigens (Mittal et al., 1983b, 1993; Gutierrez et al., 1991). The implications of the heterogeneity of the strains described above for serodiagnosis and vaccination would require further studies. Jacques et al. (1996) suggested the presence of two core types in A. pleuropneumoniae based on separation of lipopolysaccharides by Tricine SDS-polyacrylamide gel electrophoresis, which has been shown to improve resolution of low molecular mass migrating bands. Strains representing 12 serotypes of A. pleureupneumoniae were divided into two types according to the gel mobility of the core lipid A region of their LPS. The ®rst electrophoretic core type (type I) was found in serotypes 1, 6, 9 and 11. The second electrophoretic core type (type II) was found in serotypes 2, 3, 4, 5, 7, 8, 10 and 12. Strain 90-3182 possessed the same core (type I) as that possessed by serotype 1 and strain 861411 possessed a core type II as that possessed by serotype 7 (results not shown). These results further con®rmed the results obtained by Western blot assay. Observed apparent serological cross-reactions between A. pleuropneumoniae serotypes 4 and 7 may be due to the presence of antibodies to their LPS components since their LPS O-chains are both structurally similar being composed of repeating tetrasaccharide units

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with identical trisaccharide residues forming a common backbone (Perry et al., 1990). Previous investigations have attributed apparent serological cross-reactivity between different serotypes to common LPS antigens (Mittal, 1990). Gottschalk et al. (1998) used monoclonal antibodies to demonstrate the existence of commons epitopes to both serotypes 4 and 7 of A. pleuropneumoniae located on O-chain LPS. One- or two-way cross-reactions between serotypes 4 and 7 and those between serotypes 7 and 1, 9, 10 or 11 may affect the reliability of serotyping and serodiagnostic results. Although in most cases, it is not dif®cult to identify the serotype-speci®c antigens, because of their stronger antigenic activity compared with cross-reacting common antigens, which are only minor in nature. Usually, a combination of two or more serological tests is able to identify serotype-speci®c antigens in strains showing strong cross-reactivities with other serotypes. Currently used serological tests are speci®c and sensitive to identify type-speci®c antigens but may give confusing results for serotyping some atypical strains (Mittal et al., 1987). In such cases, it is recommended to use Western blot assay as a con®rmatory test to identify serotype-speci®c capsular and somatic antigens. Identi®cation and characterization of the type-speci®c and cross-reacting common antigens may help in the precise and accurate serotyping of APP as well as in the correct serodiagnosis of swine pleuropneumonia. Acknowledgements We gratefully thank Suzanne Bourdon for her help in serotyping. References Achacha, M., Mittal, K.R., 1995. Identi®cation and characterization of genus-speci®c epitopes of Serpulina species using monoclonal antibodies. J. Clin. Microbiol. 33, 2519±2521. Biberstein, E.L., Gunnarsson, A., Hurvell, B., 1977. Cultural and biochemical criteria for the identi®cation of Haemophilus spp. from swine. Am. J. Vet. Res. 38, 7±11. Finkelstein, R.A., Sulkin, S.E., 1958. Characteristics of coagulase positive and coagulase negative Staphylococcus in serum-soft agar. J. Bacteriol. 75, 339±344. Gottschalk, M., Lebrun, A., Lacouture, S., CoÃteÂ, D., Mittal, K.R., 1998. Identi®cation of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii. Vet. Microbiol. 65, 182±271. Gottschalk, M., Lebrun, A., Lacouture, S., Harel, J., Forget, C., Mittal, K.R., 2000. Atypical Actinobacillus pleuropneumoniae isolates which share antigenic determinants with both serotypes 1 and 7. J. Vet. Diagn. Invest. 12, 444±449. Gutierrez, C.B., Tascon, R.I., Vazquez, J.A., Rodriguez Ferri, E.F., 1991. Cross-reactivity between Actinobacillus pleuropneumoniae serotypes comparing different antigens and serological tests. Res. Vet. Sci. 50, 308±310. Hamel, J., Brodeur, B.R., Larose, Y., Tsang, P.S., Belmaaza, A., Montplaisir, S., 1987. Monoclonal antibody directed against a serotype-speci®c, outer-membrane protein of Haemophilus in¯uenzae type b. J. Med. Microbiol. 23, 163±170. Inzana, T.J., Workman, Ma.T., Gogolewski, R.P., Anderson, P., 1988. Virulence properties and protective ef®cacy of the capsular polymer of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5. Infect. Immun. 56, 1880±1889.

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