Veterinary Microbiology, 32 (1992) 135-148 Elsevier Science Publishers B.V., Amsterdam
135
Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec K.R. Mittal, R. Higgins, S. Larivi~re and M. Nadeau Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, St. Hyacinthe, Que, Canada (Accepted 10 January 1992)
ABSTRACT Mittal, K.R., Higgins, R., Larivibre, S. and Nadeau, M., 1992. Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec. Vet. Microbiol., 32:135-148. A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains ofA. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.
INTRODUCTION
Actinobacillus pleuropeumoniae is the causative agent of porcine pleurop n e u m o n i a characterized by acute fibrino-haemorrhagic or chronic localized necrotizing p n e u m o n i a with pleuritis. The disease is spread all over the world and causes substantial economic losses. Morbidity and mortality rates may Correspondence to: K.R. Mittal, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, P.O. Box 5000, St. Hyacinthe, Quebec, J2S 7C6, Canada.
0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
136
K.R. MITTAL ETAL.
exceed 50% during acute outbreaks and further losses occur due to poor feed conversion by chronically infected pigs and high cost of medication (Nicolet, 1986). The agent of porcine pleuropneumonia has been transferred from the genus Haemophilus to Actinobacillus on the basis of phenotypic and deoxyribonucleic acid relatedness (Pohl et al., 1983). A. pleuropneumoniae strains have been subdivided as: biovar I (NAD-dependent) and biovar II (NAD-independent) (Nicolet, 1988 ). Serological diagnosis and vaccination programs have been used in an attempt to control the disease. Both these programs require an extensive knowledge of the different serotypes existing in a particular region, as this organism is known to be antigenically heterogenous with different serotypes showing different geographical distributions (Table 1 ). Nicolet ( 1971 ) defined 3 serotypes based on capsule associated type-specific antigens. This classification scheme was extended by Gunnarsson et al., ( 1977 ) who described serotypes 4 and 5, and by Nielsen ( 1982 ), and Rosendal and Boyd ( 1982 ) who proposed the existence of serotypes 6 and 7 respectively. Serotypes 8, 9,10 and 12 were added by Nielsen and O'Connor ( 1984 ) and Nielsen (1985a,b and 1987) and serotype 11 by Kamp et al., (1987). Thus, to date, 12 serotypes, variably distributed throughout the world have been proposed. Tests to determine the serotypes include tube agglutination (Gunnarsson et al., 1977 ), immunofluorescence (Nicolet, 1971 ), immunodiffusion (Nicolet, 1971 ) slide agglutination (Mittal et al., ( 1982 ), 2-mercaptoethanol tube agglutination test (Mittal et al., 1984, Mittal and Bourdon, 1991 ), ring precipitation (Mittal et al., 1982), indirect hemagglutination (Mittal et al., 1983a), Coagglutination (Mittal et al., 1983b), counterimmunoelectrophoresis (Piffer et al., 1986) complement fixation (Lombin et al., 1985), paper chromatography (Utrera et al., 1988) and slide precipitation (Hommez et al., 1990). Each procedure has merits but none has been free from problems mainly due to dissociation of colonies or cross-reactivity. Monoclonal antibodies directed to specific epitopes have been evaluated to conduct a more accurate serological classification (Korvuo, et al., 1988; Lida et al., 1990; Nakai et al., 1990). The purpose of this paper was to provide a review of epidemiological information regarding porcine pleuropneumonia in Quebec by ascertaining which serotypes ofA. pleuropneumoniae are associated with acute and chronic forms of disease. Data covering the prevalence of different serotypes in the world as well as evolution of serotyping methodology at St. Hyacinthe (Quebec) during last 10 years are also presented. MATERIALS AND METHODS
Bacterial strains Reference strains representing serotypes 1 through 12 ofA. pleuropneurnoniae were shope 4074, S-1536, S1421, M62, K17, L-20, Femo, WF83, 405,
SEROLOGICALCHARACTERIZATIONOF ACTINOBACILLUS PLEUROPLEUMONIAE
137
TABLE 1 Geographic distribution ofActinobacillus pleuropneumoniae serotypes in the world Country
Prevalent serotypes
Dominant serotype (s)
References
Argentina Australia Belgium Brazil Canada
1, 2, 1, 2, 2, 3, 1, 3, 1, 2,
1 1 3 5 1,5
Chile
1, 5
1,5
Czechoslovakia Denmark
2 2
France
1, 2, 7 1, 2, 3, 5, 6, 7, 8, 10, 11, 12 2, 3, 7, 8, 9
Vena et al. (1988) Eaves and Blackall, ( 1988 ) Hommez et al. ( 1988, 1990) Piffer et al. (1986, 1987) Rosendal et al. ( 1981 ); Mittal et al. (1982) Olivares and Morgado (1988) Skollova and Gois ( 1987 ) Nielsen ( 1982, 1987)
Germany
2, 3, 4, 5, 6, 7, 9, 10
9,2,7
Hungary
1, 2, 3, 5, 6, 7, 10, 11, 12
1,2
Italy
1, 2, 3, 4, 5, 7
5
Ireland Japan
3, UT l, 2, 3, 5, 6, 7, 8, 9, 12
3 2
Korea Mexico
2, 3, 5, 7
5, 2
1, 2, 3, 4, 5, 6, 7, 8, 9
1
Netherlands Norway Poland
1, 2, 3, 5, 7, 8, 9, 11 2 1, 2, 5, 9
2,9,11 2 1
Spain Sweden Switzerland United Kingdom
1, 2, 2, 3, 2, 3, 1, 2,
4 2 2 3
United States
1, 3, 5, 7, 8, 9
3, 3, 6, 4, 3,
5 7, 7, 5, 5,
UT 8, 9, 11, U T UT 6, 7, 8, 10, 12
3, 4, 5, 6, 7, 8, 9, l0 4 7, 9 3, 5, 6, 7, 8, 10
9
1,5
Kobisch (1990) (personal communication ) Schimmel and Hass ( 1983); Muller et al. (1986) Fodor et al. ( 1989); Molnar ( 1990 ) Manzat et al. ( 1987); Sidoli et al. (1987) Power et al. (1983) Chan et al. (1978; Kume et al. ( 1986); Fukuyasu et al. ( 1991 ) Yeh (1990) Diazi et al. ( 1988); Ciprian et al. (1988) Kamp etal. (1987) Falk et al. ( 1991 ) Molenda ( 1988); Tarasiuk Ferri et al. (1990) Gunnarsson (1979) Nicolet ( 1988 ) Hunter et al. (1983); Brandreth and Smith ( 1985 ) Schultz et al. ( 1983); Rapp et al. ( 1985); Hoffman et al. ( 1985); Fales et al. (1989)
CVI 1326 l, D 13039, 56153 and 8329. Most of the field isolates originated from pulmonary tissues of pigs which died of acute pleuropneumonia. Some isolates were also recovered from tonsils or nasal cavities of pigs from chronically infected herds. Identification of isolates was based on cultural and biochemical characteristics. The isolates were presumptively identified as A.
138
K.R. MITTAL ET AL.
pleuropneumonia if they were Gram-negative, V-factor dependent but X-factor independent and positive for CAMP and urease reactions. Further characterization and differentiation from Haernophilus parasuis and other Haemophilus sp. were carried out as described by Biberstein et al., (1977) and Kilian et al., ( 1978 ), using additional biochemical tests such as dextrose, nitrate, lactose, mannitol, xylose, and indole.
Preparation of antisera in rabbits Antisera were prepared in rabbits by intravenous inoculations of formalinized whole cell suspension (F-WC) of 6-8 hour-old culture on pleuropneumonia like organism (PPLO) agar medium. Two young adult New Zealand male rabbits weighing about 3 kg were injected twice a week with an antigen preparation of each reference strain of 12 different serotypes. A total of seven injections were given in increasing doses. The details of the method were given earlier (Mittal et al., 1982). The rabbits were bled 7 days after the last injection. Sera were harvested and stored at - 20 ° C until used.
Serotyping of A. pleuropneumoniae A. pleuropneumoniae field isolates recovered during the period 1980-1982 were serotyped by slide agglutination (SA), tube agglutination (TA) and ring precipitation (RP) tests using rabbit hyperimmune sera against F-WC of reference strains of serotypes 1 through 5 (Mittal et al., 1982). Antisera against serotypes 6 and 7 were added to the serotyping scheme by 1983, against serotypes 8, 9 and 10 by 1985 and against serotypes 11 and 12 by 1987. During the period from 1982 to date, all the isolates were serotyped by coagglutination (CoA) test (Mittal et al., 1 983b). Immunodiffusion (ID) (Mittal et al., 1988b ) and counterimmunoelectrophoresis (CIE) (Piffer et al., 1986 ) tests were used mainly to serotype isolates which showed either positive reactions for more than one serotype (polyagglutinating strains) or negative reactions for all the serotypes (non-agglutinating strains) in CoA test. Scheme for serotyping A. pleuropneumoniae isolates used in our laboratory is shown in Table 2. The isolates were serotyped by using antisera against all the known serotypes and positive reaction in one of the antisera and negative in all the remaining antisera was considered as the serotype specific reaction. Isolates showing positive reactions for serotypes 3, 6 and 8 in CoA test were grouped together and differentiated further. Isolates belonging to serotype 5 were further differentiated into two subtypes ( 5a and 5b ) by using indirect hemagglutination ( I H A ) t e s t (Nielsen, 1986). Quantitation of serotype and group specific antigens by CoA a n d / o r ID and CIE tests was carried out in order to differentiate cross-reacting isolates of serotypes 3, 6 and 8, (Mittal et al., 1988a), 1 and 9 (Mittal 1990) and 9 and 11 (Mittal et al., 1991 ). IHA test was used mainly to differentiate strains
SEROLOGICALCHARACTERIZATIONOF ACT1NOBACILLUS PLEUROPLEUMONIAE
139
TABLE 2 Scheme for serotyping ofActinobacillus pleuropneumoniae currently used at the college of Veterinary Medicine of the University of Montreal, St. Hyacinthe, Quebec Strains
Antigens
Tests used
Mucoid or smooth Auto-agglutinating Non-agglutinating Poly-agglutinating Cross-reacting strains (Serotypes 3, 6 and 8 ) (Serotypes 1, 9 and 11 ) (Serotypes 4 and 7 ) Subtyping of serotype 5 strains (5a and 5b)
F-WC or F-WC-SE F-WC-SE B-WC; B-WC-SE B-WC; B-WC-SE F-WC; B-WC; F-WC-SE; B-WC-SE
CoA, ID CoA, ID CoA, ID, CIE CoA, ID, CIE Quantitation of type and group specific antigens by CoA, ID and CIE and antibodies by IHA IHA
F-WC-SE; B-WC-SE
F-WC: formalinized whole cell-suspension F-WC-SE: F-WC-saline extract B-WC: F-WC boiled for one hour in a water bath B-WC-SE: B-WC-saline extract CoA: coagglutination ID: immunodiffusion CIF: Counterimmunoelectrophoresis IHA: indirect hemagglutination
of serotypes 3, 6 and 8 (Mittal et al., 1988c,d, 1989) serotypes 1 and 9. (Mittal, 1990) and serotypes 4 and 7 (Mittal and Bourdon, 1991 ).
Antigens used in various serological tests for serotyping Formalinized whole cell suspensions (F-WC) of 18 hour-old culture on chocolate blood agar were used for agglutination test. For auto agglutinating isolates, clear supernatant (cell-free soluble antigen) obtained after centrifugation of F-WC was used in RP test (Mittal et al., 1982). During the period from 1983 to date, chocolate blood agar medium was replaced with PPLO agar medium. In the case of auto-agglutinating isolates on PPLO agar, 6-8 hour-old culture was used in place of 18 hour-old culture. F-WC or its saline extract (SE) was used for serotyping by CoA test. Details of the preparation of CoA reagents and antigens are given earlier (Mittal et al., 1983b). For serotyping isolates which were polyagglutinating or non-agglutinating in CoA test, boiled whole cell suspensions (B-WC) or their saline extracts (SE) were used in CoA test (Mittal et al., 1987a). Some isolates still showing some ambiguous reactions in CoA test were tested by ID and/or CIE tests using B-WC-SE as antigen. Saline extracts of F-WC or B-WC were used to sensitize sheep red blood cells in IHA test. F-WC or B-WC or their saline extracts were used for quantitation of serotype and group specific antigens by CoA test and B-WC-SE by ID test (Mittal et al., 1988a).
140
K.R. MITTAL ET AL.
RESULTS
Prevalence of different serotypes isolated from pigs that died due to acute pleuropneumonia in Quebec during the period from 1980 to 1990 is shown in Table 3. A total of 3306 isolates were serotyped and results clearly indicate that during the last l0 years, serotypes 1 and 5 have continued to remain dominant in Quebec followed by serotypes 7, 2 and other serotypes which were in small numbers. Both subtypes of serotypes 5 (5a and 5b) were presTABLE 3 D i s t r i b u t i o n ofActinobacilluspleuropneumoniae serotypes in Q u e b e c during the period f r o m 1980 to 1990 isolated f r o m pigs that died d u e to acute p l e u r o p n e u m o n i a Year
Total number
1
2
3/6/8
4
5
7
295 169 466 390 300 367 402 444 473
87 69 84 71 60 53 55 61 60
2 2 2 1 2 2 2 0 1
0
135
Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec K.R. Mittal, R. Higgins, S. Larivi~re and M. Nadeau Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, St. Hyacinthe, Que, Canada (Accepted 10 January 1992)
ABSTRACT Mittal, K.R., Higgins, R., Larivibre, S. and Nadeau, M., 1992. Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec. Vet. Microbiol., 32:135-148. A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains ofA. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.
INTRODUCTION
Actinobacillus pleuropeumoniae is the causative agent of porcine pleurop n e u m o n i a characterized by acute fibrino-haemorrhagic or chronic localized necrotizing p n e u m o n i a with pleuritis. The disease is spread all over the world and causes substantial economic losses. Morbidity and mortality rates may Correspondence to: K.R. Mittal, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, P.O. Box 5000, St. Hyacinthe, Quebec, J2S 7C6, Canada.
0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
136
K.R. MITTAL ETAL.
exceed 50% during acute outbreaks and further losses occur due to poor feed conversion by chronically infected pigs and high cost of medication (Nicolet, 1986). The agent of porcine pleuropneumonia has been transferred from the genus Haemophilus to Actinobacillus on the basis of phenotypic and deoxyribonucleic acid relatedness (Pohl et al., 1983). A. pleuropneumoniae strains have been subdivided as: biovar I (NAD-dependent) and biovar II (NAD-independent) (Nicolet, 1988 ). Serological diagnosis and vaccination programs have been used in an attempt to control the disease. Both these programs require an extensive knowledge of the different serotypes existing in a particular region, as this organism is known to be antigenically heterogenous with different serotypes showing different geographical distributions (Table 1 ). Nicolet ( 1971 ) defined 3 serotypes based on capsule associated type-specific antigens. This classification scheme was extended by Gunnarsson et al., ( 1977 ) who described serotypes 4 and 5, and by Nielsen ( 1982 ), and Rosendal and Boyd ( 1982 ) who proposed the existence of serotypes 6 and 7 respectively. Serotypes 8, 9,10 and 12 were added by Nielsen and O'Connor ( 1984 ) and Nielsen (1985a,b and 1987) and serotype 11 by Kamp et al., (1987). Thus, to date, 12 serotypes, variably distributed throughout the world have been proposed. Tests to determine the serotypes include tube agglutination (Gunnarsson et al., 1977 ), immunofluorescence (Nicolet, 1971 ), immunodiffusion (Nicolet, 1971 ) slide agglutination (Mittal et al., ( 1982 ), 2-mercaptoethanol tube agglutination test (Mittal et al., 1984, Mittal and Bourdon, 1991 ), ring precipitation (Mittal et al., 1982), indirect hemagglutination (Mittal et al., 1983a), Coagglutination (Mittal et al., 1983b), counterimmunoelectrophoresis (Piffer et al., 1986) complement fixation (Lombin et al., 1985), paper chromatography (Utrera et al., 1988) and slide precipitation (Hommez et al., 1990). Each procedure has merits but none has been free from problems mainly due to dissociation of colonies or cross-reactivity. Monoclonal antibodies directed to specific epitopes have been evaluated to conduct a more accurate serological classification (Korvuo, et al., 1988; Lida et al., 1990; Nakai et al., 1990). The purpose of this paper was to provide a review of epidemiological information regarding porcine pleuropneumonia in Quebec by ascertaining which serotypes ofA. pleuropneumoniae are associated with acute and chronic forms of disease. Data covering the prevalence of different serotypes in the world as well as evolution of serotyping methodology at St. Hyacinthe (Quebec) during last 10 years are also presented. MATERIALS AND METHODS
Bacterial strains Reference strains representing serotypes 1 through 12 ofA. pleuropneurnoniae were shope 4074, S-1536, S1421, M62, K17, L-20, Femo, WF83, 405,
SEROLOGICALCHARACTERIZATIONOF ACTINOBACILLUS PLEUROPLEUMONIAE
137
TABLE 1 Geographic distribution ofActinobacillus pleuropneumoniae serotypes in the world Country
Prevalent serotypes
Dominant serotype (s)
References
Argentina Australia Belgium Brazil Canada
1, 2, 1, 2, 2, 3, 1, 3, 1, 2,
1 1 3 5 1,5
Chile
1, 5
1,5
Czechoslovakia Denmark
2 2
France
1, 2, 7 1, 2, 3, 5, 6, 7, 8, 10, 11, 12 2, 3, 7, 8, 9
Vena et al. (1988) Eaves and Blackall, ( 1988 ) Hommez et al. ( 1988, 1990) Piffer et al. (1986, 1987) Rosendal et al. ( 1981 ); Mittal et al. (1982) Olivares and Morgado (1988) Skollova and Gois ( 1987 ) Nielsen ( 1982, 1987)
Germany
2, 3, 4, 5, 6, 7, 9, 10
9,2,7
Hungary
1, 2, 3, 5, 6, 7, 10, 11, 12
1,2
Italy
1, 2, 3, 4, 5, 7
5
Ireland Japan
3, UT l, 2, 3, 5, 6, 7, 8, 9, 12
3 2
Korea Mexico
2, 3, 5, 7
5, 2
1, 2, 3, 4, 5, 6, 7, 8, 9
1
Netherlands Norway Poland
1, 2, 3, 5, 7, 8, 9, 11 2 1, 2, 5, 9
2,9,11 2 1
Spain Sweden Switzerland United Kingdom
1, 2, 2, 3, 2, 3, 1, 2,
4 2 2 3
United States
1, 3, 5, 7, 8, 9
3, 3, 6, 4, 3,
5 7, 7, 5, 5,
UT 8, 9, 11, U T UT 6, 7, 8, 10, 12
3, 4, 5, 6, 7, 8, 9, l0 4 7, 9 3, 5, 6, 7, 8, 10
9
1,5
Kobisch (1990) (personal communication ) Schimmel and Hass ( 1983); Muller et al. (1986) Fodor et al. ( 1989); Molnar ( 1990 ) Manzat et al. ( 1987); Sidoli et al. (1987) Power et al. (1983) Chan et al. (1978; Kume et al. ( 1986); Fukuyasu et al. ( 1991 ) Yeh (1990) Diazi et al. ( 1988); Ciprian et al. (1988) Kamp etal. (1987) Falk et al. ( 1991 ) Molenda ( 1988); Tarasiuk Ferri et al. (1990) Gunnarsson (1979) Nicolet ( 1988 ) Hunter et al. (1983); Brandreth and Smith ( 1985 ) Schultz et al. ( 1983); Rapp et al. ( 1985); Hoffman et al. ( 1985); Fales et al. (1989)
CVI 1326 l, D 13039, 56153 and 8329. Most of the field isolates originated from pulmonary tissues of pigs which died of acute pleuropneumonia. Some isolates were also recovered from tonsils or nasal cavities of pigs from chronically infected herds. Identification of isolates was based on cultural and biochemical characteristics. The isolates were presumptively identified as A.
138
K.R. MITTAL ET AL.
pleuropneumonia if they were Gram-negative, V-factor dependent but X-factor independent and positive for CAMP and urease reactions. Further characterization and differentiation from Haernophilus parasuis and other Haemophilus sp. were carried out as described by Biberstein et al., (1977) and Kilian et al., ( 1978 ), using additional biochemical tests such as dextrose, nitrate, lactose, mannitol, xylose, and indole.
Preparation of antisera in rabbits Antisera were prepared in rabbits by intravenous inoculations of formalinized whole cell suspension (F-WC) of 6-8 hour-old culture on pleuropneumonia like organism (PPLO) agar medium. Two young adult New Zealand male rabbits weighing about 3 kg were injected twice a week with an antigen preparation of each reference strain of 12 different serotypes. A total of seven injections were given in increasing doses. The details of the method were given earlier (Mittal et al., 1982). The rabbits were bled 7 days after the last injection. Sera were harvested and stored at - 20 ° C until used.
Serotyping of A. pleuropneumoniae A. pleuropneumoniae field isolates recovered during the period 1980-1982 were serotyped by slide agglutination (SA), tube agglutination (TA) and ring precipitation (RP) tests using rabbit hyperimmune sera against F-WC of reference strains of serotypes 1 through 5 (Mittal et al., 1982). Antisera against serotypes 6 and 7 were added to the serotyping scheme by 1983, against serotypes 8, 9 and 10 by 1985 and against serotypes 11 and 12 by 1987. During the period from 1982 to date, all the isolates were serotyped by coagglutination (CoA) test (Mittal et al., 1 983b). Immunodiffusion (ID) (Mittal et al., 1988b ) and counterimmunoelectrophoresis (CIE) (Piffer et al., 1986 ) tests were used mainly to serotype isolates which showed either positive reactions for more than one serotype (polyagglutinating strains) or negative reactions for all the serotypes (non-agglutinating strains) in CoA test. Scheme for serotyping A. pleuropneumoniae isolates used in our laboratory is shown in Table 2. The isolates were serotyped by using antisera against all the known serotypes and positive reaction in one of the antisera and negative in all the remaining antisera was considered as the serotype specific reaction. Isolates showing positive reactions for serotypes 3, 6 and 8 in CoA test were grouped together and differentiated further. Isolates belonging to serotype 5 were further differentiated into two subtypes ( 5a and 5b ) by using indirect hemagglutination ( I H A ) t e s t (Nielsen, 1986). Quantitation of serotype and group specific antigens by CoA a n d / o r ID and CIE tests was carried out in order to differentiate cross-reacting isolates of serotypes 3, 6 and 8, (Mittal et al., 1988a), 1 and 9 (Mittal 1990) and 9 and 11 (Mittal et al., 1991 ). IHA test was used mainly to differentiate strains
SEROLOGICALCHARACTERIZATIONOF ACT1NOBACILLUS PLEUROPLEUMONIAE
139
TABLE 2 Scheme for serotyping ofActinobacillus pleuropneumoniae currently used at the college of Veterinary Medicine of the University of Montreal, St. Hyacinthe, Quebec Strains
Antigens
Tests used
Mucoid or smooth Auto-agglutinating Non-agglutinating Poly-agglutinating Cross-reacting strains (Serotypes 3, 6 and 8 ) (Serotypes 1, 9 and 11 ) (Serotypes 4 and 7 ) Subtyping of serotype 5 strains (5a and 5b)
F-WC or F-WC-SE F-WC-SE B-WC; B-WC-SE B-WC; B-WC-SE F-WC; B-WC; F-WC-SE; B-WC-SE
CoA, ID CoA, ID CoA, ID, CIE CoA, ID, CIE Quantitation of type and group specific antigens by CoA, ID and CIE and antibodies by IHA IHA
F-WC-SE; B-WC-SE
F-WC: formalinized whole cell-suspension F-WC-SE: F-WC-saline extract B-WC: F-WC boiled for one hour in a water bath B-WC-SE: B-WC-saline extract CoA: coagglutination ID: immunodiffusion CIF: Counterimmunoelectrophoresis IHA: indirect hemagglutination
of serotypes 3, 6 and 8 (Mittal et al., 1988c,d, 1989) serotypes 1 and 9. (Mittal, 1990) and serotypes 4 and 7 (Mittal and Bourdon, 1991 ).
Antigens used in various serological tests for serotyping Formalinized whole cell suspensions (F-WC) of 18 hour-old culture on chocolate blood agar were used for agglutination test. For auto agglutinating isolates, clear supernatant (cell-free soluble antigen) obtained after centrifugation of F-WC was used in RP test (Mittal et al., 1982). During the period from 1983 to date, chocolate blood agar medium was replaced with PPLO agar medium. In the case of auto-agglutinating isolates on PPLO agar, 6-8 hour-old culture was used in place of 18 hour-old culture. F-WC or its saline extract (SE) was used for serotyping by CoA test. Details of the preparation of CoA reagents and antigens are given earlier (Mittal et al., 1983b). For serotyping isolates which were polyagglutinating or non-agglutinating in CoA test, boiled whole cell suspensions (B-WC) or their saline extracts (SE) were used in CoA test (Mittal et al., 1987a). Some isolates still showing some ambiguous reactions in CoA test were tested by ID and/or CIE tests using B-WC-SE as antigen. Saline extracts of F-WC or B-WC were used to sensitize sheep red blood cells in IHA test. F-WC or B-WC or their saline extracts were used for quantitation of serotype and group specific antigens by CoA test and B-WC-SE by ID test (Mittal et al., 1988a).
140
K.R. MITTAL ET AL.
RESULTS
Prevalence of different serotypes isolated from pigs that died due to acute pleuropneumonia in Quebec during the period from 1980 to 1990 is shown in Table 3. A total of 3306 isolates were serotyped and results clearly indicate that during the last l0 years, serotypes 1 and 5 have continued to remain dominant in Quebec followed by serotypes 7, 2 and other serotypes which were in small numbers. Both subtypes of serotypes 5 (5a and 5b) were presTABLE 3 D i s t r i b u t i o n ofActinobacilluspleuropneumoniae serotypes in Q u e b e c during the period f r o m 1980 to 1990 isolated f r o m pigs that died d u e to acute p l e u r o p n e u m o n i a Year
Total number
1
2
3/6/8
4
5
7
295 169 466 390 300 367 402 444 473
87 69 84 71 60 53 55 61 60
2 2 2 1 2 2 2 0 1
0
