methode de RAPD, celle du champ pulse a permis de distinguer entre elles des souches de serotype 1 et aussi de serotype 5. Toutes les souches a l'exception ...
Genomic Relatedness among Actinobacillus pleuropneumoniae Field Strains of Serotypes 1 and 5 Isolated from Healthy and Diseased Pigs Sonia Chatellier, Josee Harel, Dominique Dugourd, Bruno Chevallier, Marylene Kobisch, and Marcelo Gottschalk
ABSTRACT
Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the
molecular epidemiology of swine pleuropneumonia.
de reference de serotype 1 ou 5. Ces resultats indiquent que la me'thode de RAPD est un outil de typage rapide pour le sous-typage en rouRESUME tine des souches d' A. pleuropneumoniae de serotype 1. Pour les Quarante-quatre souches d'Acti- souches de serotype 5, une associanobacillus pleuropneumoniae isolees tion de plusieurs methodes telles le chez le porc, soient de porteurs champ pulse et le typage des genes sains ou d'animaux malades, ont ete codant pour les toxines Apx est caracterisees par amplification necessaire pour obtenir des inforgenomique arbitraire (technique de mations pertinentes sur le'pideRAPD), par analyse en champ pulse miologie moleculaire de la pleuro(PFGE), et par typage des genes pneumonie porcine. codant pour les toxines Apx. Neuf (Traduit par les auteurs) types RAPD et 14 proflls PFGE ont ete identifie's. Aucun profll commun entre les souches de serotype 1 et INTRODUCTION celles de serotype 5 n'a ete observe. L'analyse par RAPD a indique que Actinobacillus pleuropneumoniae les 15 souches de serotype 1 isolees is the etiological agent of swine pleude porcs malades etaient distribuees ropneumonia, a severe contagious disdans 4 types RAPD avec 66% de ces ease causing important economic souches caracterisees par le meme losses to the swine industry (1,2). The type. Au contraire, les 5 souches de infection is often acute and fatal. Aniserotype 1 obtenues 'a partir de por- mals that survive an A. pleuropneuteurs sains ont ete dispersees dans moniae infection often suffer from 4 types RAPD. Ces resultats sug- chronic lesions and may become cargerent que la diversite genetique riers by which the infection can be des souches de serotype 1 isolees transmitted (1,3). Similarly, animals chez des porcs malades pourrait from subclinically infected herds also etre plus faible que celle des harbor A. pleuropneumoniae in their souches isolees chez des porteurs tonsils. So far, 14 serotypes of sains. En outre, toutes les souches A. pleuropneumoniae have been idende se'rotype 5 ont presente le meme tified based on capsular antigens profil RAPD. Contrairement a la (4-7). The prevalence of serotypes methode de RAPD, celle du champ varies according to countries. pulse a permis de distinguer entre Serotypes 1 and 5 constitute the most elles des souches de serotype 1 et commonly isolated serotypes from aussi de serotype 5. Toutes les clinical cases of pleuropneumonia in souches a l'exception de trois ont North America (8). Various degrees montre le genotype apx des souches of virulence have been reported
Groupe de Recherche sur les Maladies Infectieuses du Porc, D6partement de pathologie et microbiologie, Faculte de medecine vet6rinaire, Universite de Montreal, C.P. 5000, St-Hyacinthe (Quebec) J2S 7C6 (Chatellier, Harel, Dugourd, Gottschalk); CNEVA-Ploufragan UMB, Les Croix, CP 53, 22440 Ploufragan, France (Chevallier, Kobisch). Address correspondence and reprint requests to Dr. Marcelo Gottschalk; telephone: (450) 773-8521 ext. 8374; fax: (514) 778-8108; e-mail: gottschm @ medvet.umontreal.ca. Present address for S. Chatellier: Veterans Affairs Medical Center, Research Service, Memphis, Tennessee 38104 USA. Present address for B. Chevallier: Adiagene, 22000 Saint-Brieuc, France. Received January 14, 1999.
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Can J Vet Res 1999; 63: 170-176
according to the serotype of the isolate (1,9). Several virulence factors such as the capsule polysaccharide (10), the lipopolysaccharide (11,12), apx toxins (13) and certain outer membrane proteins (14-16) have been described to play a role in the pathogenesis of porcine pleuropneumonia. Current vaccines based on these virulence factors fail to prevent the subclinical state of infection (17-21). The standard methods for diagnosis of the infection are culture and serotyping. However, these methods do not give any information regarding to the genetic relatedness of strains. Therefore, other molecular typing methods such as multilocus enzyme electrophoresis (22,23), PCR typing (24-27), detection of apx toxin genes by PCR (28), restriction enzyme analysis (29-31), random amplified polymorphic DNA analysis (RAPD) (32), pulsed field gel electrophoresis (PFGE) (33), and ribotyping (34,35) have been developed for identifying A. pleuropneumoniae for epidemiological investigations and tracking of epidemic strains. These studies allowed the recognition of A. pleuropneumoniae species, and in some cases, the distinction of strains of different serotypes. However, the presence of a given serotype can be noticed in diseased pigs as well as in healthy pigs, making more difficult the decision on prevention and eradication of the virulent types of the pathogen. Therefore, we decided to estimate the relationship between A. pleuropneumoniae isolates of a given serotype isolated from both healthy and diseased swine. We report a genomic characterization of A. pleuropneumoniae isolates serotypes 1 and 5 recovered from both lungs of diseased pigs and tonsils of asymptomatic carrier pigs. The methods used were RAPD, PFGE, species-specific PCR assay, and apx toxin gene typing. The aim of the study was to better characterize A. pleuropneumoniae populations potentially responsible for severe infections in pigs. MATERIALS AND METHODS BACTERIAL STRAINS AND GROWTH CONDITIONS
A total of 44 strains were included in the present study. The reference
strains of A. pleuropneumoniae to a microcomputer (AlphaEase, serotypes 1 and 5 were Shope 4074 Alpha Innotech Corp., San Leandro, (ATCC 27088) and K17 (ATCC California, USA). The RAPD patterns 33377). Field strains of serotype 1 were analyzed by using the Taxotron (19 strains) and serotype 5 (23 strains) package (Taxolab, Institut Pasteur, were isolated from 35 different herds Paris, France). This package is comin Quebec. The strains identified in posed of RestrictoScan, Restrictothe same herd were collected at differ- Typer, Adanson, and Dendrograf proent times. Twenty-nine isolates were grams. The digitized images, in recovered from lungs of clinically tagged image file format, were transaffected pigs, and 13 isolates from ferred to a Macintosh microcomputer, tonsils of asymptomatic animals com- and the band migration distances for ing from herds subclinically infected each lane were determined with the with A. pleuropneumoniae. The RestrictoScan program. The molecustrains were grown at 37°C on PPLO lar size of each fragment was generselective agar supplemented with ated from migration distances using NAD, as previously described (36). cubic spline algorithms with the RestrictoTyper program. A distance RAPD FINGERPRINTING matrix was calculated by the The primers used in this project were RestrictoTyper program with the fragnamed OPB1 to OPB20 (Oligo 10- ment length error tolerance set at 3%. mer Kit B, Operon Technologies Inc., A schematic representation of elecAlameda, California, USA). Primer trophoretic patterns was also pro#17 was kindly provided by Dr. R. duced with the RestrictoTyper proBrousseau (Biotechnology Research gram. The relationships between Institute, Montreal, Quebec). Poly- RAPD types were calculated by the merase chain reaction was conducted unweighted pair group method with under a layer of mineral oil in a 25-pL arithmetic mean (38) using the Adanvolume containing 10 mM Tris-HCl son clustering program (dissimilar(pH 8.3), 50 mM KCI, 2.5 mM MgCl2, ity). A dendrogram of the tree 100 ,uM (each) deoxynucleotide description file was drawn with the triphosphate (dNTP; Pharmacia, Upp- Dendrograf program. sala, Sweden), 0.2 pM primer (for OPB primers) or 8 pM primer (for PULSE FIELD GEL ELECTROPHORESIS primer #17), 0.5 U Taq DNA polyRelatedness of A. pleuropneumomerase (Pharmacia), and 25 ng of niae isolates was determined by comDNA extracted and purified as previ- paring their ApxI restriction digests of ously described (37). The cycling pro- chromosomal DNA separated by gram was: 1 cycle at 94°C for 4 min, PFGE as previously described (33). 36°C for 1 min, 72°C for 2 min; Briefly, restriction enzyme digestion 33 cycles at 94°C for 1 min, 36°C for of genomic DNA embedded in agarose 1 min, 72°C for 2 min; and 1 cycle at blocks was performed overnight with 94°C for 2 min, 36°C for 1 min, 72°C 40 U of ApxI. The PFGE was done at for 10 min (DNA Thermal Cycler 14°C with a DRII or DRIII CHEF sys480, Perkin-Elmer Cetus, Norwalk, tem (Biorad). The PFGE pattern analConnecticut, USA). Amplified prod- ysis was conducted from digitized gel ucts (20 pL) were separated by elec- pictures using the Taxotron package. trophoresis in a 1.4% agarose gel TOXIN GENE TYPING AND SPECIES(agarose LE, Boehringer-Mannheim, apx SPECIFIC PCR ASSAY Germany) and visualized by UV tranToxin gene typing was done as presillumination following ethidium bromide staining. A 1-kb DNA ladder viously described by Frey et al (28). (Life Technologies, Gaithersburg, This method allows the determination Maryland, USA) was used in each gel of the activator (C), the structural as molecular size standards. A negative gene (A), and the secretion genes control, consisting of the same reaction (B and D) of the 3 toxins ApxI, ApxII, mixture but with water instead of tem- and ApxIII (28). Briefly, the template plate DNA, was included in each run. DNA was obtained from bacterial cultures with a direct lysis method and RAPD PATTERN ANALYSIS multiplex-PCR was performed with Photographs of each gel were digi- specific oligonucleotide primer pairs tized with a video camera connected for the amplification of apxICA, 171
C.,I L
4
1
2
3
kb
4
L
1
Z
4
I,
OP1311
atnl 7
Figure 1. Illustration of the RAPD patterns most frequently generated with primers OPB7, OPB11, and #17. (A) Primer OPB7; lanes 1 to 4: patterns Al, A2, A3, and A4. (B) Primer OPB11; lanes 1 to 4: patterns Bi, B2, B3, and B4. (C) Primer #17; lanes 1 to 4: Cl, C2, C3, and C4. These primers were chosen because they generate patterns, which contain a large range of fragment sizes and a small number of minor fragments. L, 1-kb DNA ladder (DNA molecular size markers).
apxIICA, apxIIICA, apxIBD and apxIIIBD. The PCR products were separated on a 0.7% (w/v) agarose gel by electrophoresis. Species-specific PCR reaction was performed using the primers and method previously described by Sirois et al (24).
CATAG-3'). These primers were found between serotype 1 and selected to be used for all isolates. serotype 5. Genetic relatedness of The reproducibility of the RAPD pat- A. pleuropneumoniae isolates based terns was tested by using DNA prepa- on RAPD analysis is represented on rations of 5 strains made from sepa- the dendrogram shown in Figure 2. rate cultures on different days. This clustering analysis was clearly Identical strain-specific pattern was able to differentiate the strains of obtained from the paired DNA-prepa- serotype 1 (group A) from those of ration. In addition, isolates tested at serotype 5 (group B). Strains of least 3 times on the same DNA prepa- serotype 1 were distributed in RESULTS rations exhibited reproducible strain- 8 RAPD types. All the A. pleuropneuspecific patterns. However, slight moniae serotype 5 (5a and Sb) isolates GENETIC DIVERSITY OF ACTINOBACILLUS variations in faint bands were exhibit the same RAPD type. No comPLEUROPNEUMONIAE STRAINS AS observed. Therefore, low-intensity mon RAPD pattern was noticed DEFINED BY RAPD FINGERPRINTING. bands were not used for the analysis. between strains of serotype 1 isolated To identify primers that generate For the whole panel of 44 strains, from lungs of diseased pigs and those informative and discriminatory arrays 7 RAPD patterns composed of 4 to isolated from tonsils of healthy carrier of PCR products for A. pleuropneu- 6 bands with sizes between 0.7 kb and pigs. The 15 strains of serotype 1 moniae species, purified DNA from 4.1 kb were obtained with primer recovered from diseased pigs were 3 strains of serotype 1 (including the OPB7, 6 patterns containing 2 to assigned to 4 RAPD types with 10 out reference strain Shope 4074), 9 bands of 0.7 to 4.9 kb were achieved of 15 strains characterized by the 3 strains of serotype 5 (including the with primer OPB 11, and 7 patterns same RAPD type. By contrast, the reference strain K 17) and 1 strain of composed of 4 to 9 bands ranging 5 strains of serotype 1 isolated from serotype 2 (4226) were tested with from 0.5 to 2.4 kb resulted with healthy carriers were dispersed in 21 primers (primers OPB 1 to OPB20 primer #17. Each pattern contained 1 4 RAPD types, suggesting that and primer #17). The most discrimi- to 24 isolates. The most frequent pat- serotype 1 strains from healthy pig nant patterns with a limited number of terns obtained with each primer are were genetically more diverse than low-intensity bands were obtained presented in Figure 1. By combination those recovered from diseased pigs. using primers OPB7 (5'-GGT- of RAPD patterns obtained with these Finally, strains coming from different GACGCAG-3'), OPB 11 (5'-GTAGA- 3 primers, 9 RAPD types were identi- herds were identified in the same CCCGT-3'), and #17 (5'-ACGCG- fied. No common RAPD type was RAPD type (Fig. 2). 172
0.6
Dissimilarity 0.4 0.2
Strain name
Year of isolation
44
STF92-14352 ASS92-2596 Shy9l-1706
1992 1992 1991 1994 1993 1996 1995 1991 1991 1991 1991 1991 1992 1993 1992 1994 1991 1995 1991 1991 1991 1991 1981
H35 H34 H33 H32 H31 H30 H29 H28 H14 H27 H16 H16 H26 H25 H25 H24 H23 H22 H21 H21 H20 H19 H18
1996 1996 1991 1991 1992 1991 1991 1992 1991 1992 1992
H4 H4
43 42
B
41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 7
6 8 9 10 11 15 14 13 12 5 1 3 2 4
A
Herd site (pig nb. status)
Strain nb.
17 16 18 20 19
FMV94-3322 STF93-0320 FMV96-1900 FMV95-616 Arn91-0781NlOb Am910781H 102b Arn9107810 18a Am910781F lOc Am910781F 23a Shy92-4861 FMV93-0877 FMV92-1639 FMV94-3810
STP91-1064 FMV95-0822 ECN91-0781M 3a Am9l-0781M 21d Shy9l-1946 Am9l-0781D 22b 81750 K17-Ref Am96-S26a Am96-B26a Shy9l-4734 STF91-4098 Shy92-1357 FMV91-1333 FMV91-0405 FMV92-2515 Shy9l-4030 Shy92-2943 Shy92-1803 4074-Ref 95-8405 95-8997 FMV92-0339-8 Am91-0781C 3c
lung (D)
lung (D) lung (D) lung (D) lung (D)
lung (D) lung (D) tonsils (H) tonsils (H) tonsils (H) tonsils (H) tonsils (H) lung (D) lung (D) lung (D) lung (D) lung (D) lung (D) tonsils (H) tonsils lung (D) tonsils (H) lung (D)
sero- PFGE type pattern Sb 5b 5b 5b 5b 5b 5b 5b 5b 5b 5b 5b Sa Sa 5a 5a 5u Sa 5a 5a Su 5a 5a 5b
5S
H5 H6 H7 H8 H12
HI1 H10 H9 113
1995
H1
1995
Hl H2 H15 H13 H16 H17
1992 1991 Am9l-0781C 17e 1991 Arn9l-07811F26b 1991 Am9l-0781E 4d 1991 Am9l-0781E 8a 1991
1117
lung (D) lung (D) lung (D) lung (D) lung (D) lung (D) lung (D)
lung(D) lung (D) lung (D) lung (D) lung (D) lung (D) lung (D) lung (D) tonsils (H) tonsils (H) tonsils (H) tonsils (H) tonsils (H)
I
I I
RAPD type
Pi1
Pi
I
P1
k
P1 P1 P4 Pi
) ; ()
t}t) I
P8 P1 Pt Pi Pi P9
Al B1 C1
U
Y41)
PIO P1
P1 P1 P4 Pi P7 Pi Pi P3 P3 P2 P2 P2 P2 P12 P2 P2 P13 P2 P2 P2 P2 P18 1
P19
A2 B2 C2
13 _
A2 B4 C2 A3 B2 C7
~
~~81
A3 B2 C3
P51 j
A4 B3 C5
A5 B2 C6 1 A7 B5C4 P2 A6 B6 C4
P14
9-)]1
P6
Figure 2. Genetic relationship between 44 A. pleuropneumoniae isolates as estimated by clustering analysis of RAPD patterns obtained with 3 primers. The tree was generated by the unweighted pair group method with arithmetic means. H - healthy carrier pig; D - diseased pig. RELATION BETWEEN PFGE PATTERNS AND persed in 7 patterns with 12 out of RAPD TYPES FOR A. PLEUROPNEUMONIAE. 24 isolates exhibiting the same pat-
Genomic DNA of A. pleuropneumoniae was digested with ApaI, a restriction endonuclease with GC-rich recognition sequences. The 44 isolates previously studied by RAPD fingerprinting were distributed in 14 PFGE patterns composed of 8 to 16 bands ranging from 41 kb to 495 kb (Fig. 3). The genetic relatedness between the 44 isolates was evaluated by cluster analysis of the PFGE patterns (Fig. 4). Strains clearly clustered in 2 groups according to their serotype. Strains of serotype 5 were distributed in 7 patterns with 16 out of 24 strains sharing an identical pattern. Strains of serotype Sa shared common patterns with strains of serotype 5b. Strains of serotype 1 were also dis-
-
-)
9 |
W ev-;
Is
i7:
:
tern. Interestingly, one strain of serotype 1 (nb 18) was distant from the population defined by all other strains. The PFGE pattern of each strain is reported in the dendrogram obtained by RAPD fingerprinting (Fig. 2). The results obtained by Figure 3. Representative PFGE patterns PFGE were generally in agreement obtained for A. pleuropneumoniae strains serotype 1 or 5 with the enzyme Apal. with those generated by the RAPD from (A) Strains of serotype 1, Lanes 1 to 4: patfingerprinting analysis. However, the terns P14, P6, P5 and P2. (B) Strains of discriminatory power of PFGE was serotype 5; Lanes 1 to 4: patterns P1, P9, P1, higher than that of RAPD, especially and P8. Molecular sizes from polymers of X genomes are indicated on for strains of serotype 5 which were bacteriophage the left of the gel. assigned to 7 PFGE patterns. IDENTIFICATION OF A. PLEUROPNEUMOPCR reaction specific for the species. NIAE STRAINS BY PCR SPECIFIC FOR THE The reference strains of serotype 1 SPECIES AND apx TOXIN GENE TYPING and 5 both harbor genes encoding toxm resulting All the field strains tested gave the ins Apxl and Apxll The expected 985-bp fragment with the genotype obtained by PCR typing
173
of apx genes is apxICA apxIBD apxIICA. Thirty-nine field strains (93% of the total) showed the same apx genotype as their respective serotype reference strain. By contrast, 2 other strains of serotype 1 (number 19 and 20) possess only the apxI gene (apxICA apxIBD), and another strain (number 18) exhibit the genotype apxIBD apxIICA. Interestingly, these strains were also atypic regarding their RAPD and PFGE types (Figs. 2 and 4).
Strai
Dissimilarity 0.6
OA
ab.
O0
_44 23 26 28 36
35
-
DISCUSSION Rapid and accurate diagnosis and characterization of A. pleuropneumoniae strains potentially highly virulent in subclinically infected herds is important both for limiting the severity of the outbreak and for tracing the source of A. pleuropneumoniae entry into immunologically naive herds. As serotypes 1 and 5 of A. pleuropneumoniae are among the most frequent serotypes isolated in North America (39), reliable and fast methods to identify rapidly these strains are crucial in the control of swine pleuropneumonia. For instance, a multiplex PCR assay with primers specific to the biosynthesis regions of the capsular polysaccharide of serotype 5 has been recently proposed (27). The present study was conducted to assess the genetic relatedness of A. pleuropneumoniae among strains isolated from lungs of diseased pigs or tonsils from healthy carrier pigs and sharing the same serotype (1 or 5). The genetic diversity of the strains was evaluated with RAPD and PFGE methods. No common RAPD or PFGE patterns between strains of serotype 1 and serotype 5 were identified. A previous report indicated that RAPD can be used to differentiate currently recognized serotypes (32). However, it is important to note that strains of serotype 1 are not characterized by a single RAPD pattern. By contrast, a unique RAPD pattern, independently of the primer used, characterized all strains of serotype 5. Previous studies on genetic diversity of A. pleuropneumoniae species based on multilocus enzyme electrophoresis (22,23,40) revealed that A. pleuropneumoniae serotype 5 possesses a clonal structure. Interestingly, evaluation of the 174
A
-
40
P1
38
P1
2 37 21 22 39
P7 Pe
27 32 31
5 8
9 11 15 12 13 2
M -
Pi Pi
P1 P1 P1 P1 P1 P1
I
_ -
P1 P1 P1 P1 P1 P1
34 33 29 24 30 43 42 41
7 6
I
PFGE paem
Isolats of arotyp. 5
P3 P3 P4 P4 P9
P1O P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P2 P5 P5
3 17 16 4 10 14
P12 P13
19 20 18
P6 P14
Isolate of serotype 1
P1l
P6
Figure 4. Genetic relationship between 44 A. pleuropneumoniae isolates as estimated by clustering analysis of PFGE patterns obtained with the enzyme ApaI. The tree was generated by the unweighted pair group method with arithmetic means.
genetic diversity within geographic regions (22) indicated that all Canadian strains of serotype 5 tested clustered in the same electrophoretype. The entire identity of serotype 5 strains defined by RAPD fingerprinting strongly supports the idea that these strains originated from the same clone. Interestingly, these strains were isolated through a long period of time ranging from 1981 to 1995 (Fig. 2), suggesting the persistence of the same clone in Quebec for years. In addition, this suggests that the strains isolated from healthy carrier pigs probably possess the same virulence potential as strains recovered from pigs with clinical signs of pleuropneumonia. By contrast to RAPD, PFGE analysis revealed small variations among strains of serotype 5 (5a and Sb). These 2 methods do probably not target the same part of the genome. Therefore, it is possible that A. pleuropneumoniae serotype 5 strains orig-
inate from the same clone which has evolved by undergoing several genomic mutations detected by PFGE but not by RAPD. Interestingly, a limited heterogeneity among strains of serotype 5 has also been reported using restriction endonuclease fingerprinting or ribotyping (29,35). Serotype 5 strains from carriers as well as those from diseased pigs were assigned to several PFGE patterns, suggesting that the A. pleuropneumoniae population has not evolved to a specific subclone for strains isolated from diseased pigs. By contrast to strains of serotype 5, strains of serotype 1 exhibited a relative genetic diversity as defined by RAPD and PFGE analysis. Moreover, strains recovered from tonsils of carrier and those isolated from lungs of diseased pigs presented different RAPD and PFGE types. Interestingly, the strains recovered from tonsils were more genetically diverse than those
recovered from lungs. This finding may reflect the presence of different clonal populations of serotype 1 strains and only some of them are highly virulent. In that case, RAPD used in association with other typing methods can be very useful to recognize serotype 1 strains potentially responsible for severe outbreaks. The study included 6 cases of 2 or 3 strains isolated within the same herd. Interestingly, herd #16 was characterized by 2 serotype 5 strains and one serotype 1 isolate. In 5 cases, strains from the same herd were characterized by different PFGE patterns. Only the two serotype 1 strains from herd H17 represented different RAPD types. These findings suggest that the same herd can be contaminated by different strains. These can result from the introduction of new pigs from different genetic and geographic origins that potentially have been previously infected by A. pleuropneumoniae and become healthy carriers. Another possibility is the presence of mutations or deletions in the DNA sequences of A. pleuropneumoniae explored by RAPD and PFGE. However, since RAPD and PFGE methods cover different parts of bacterial DNA, the probability to get mutations in both sites is very low. Further molecular typing with additional strains of the 6 herds with 2 or 3 isolates included in the present study are needed to clearly address the genetic diversity of strains belonging to a same herd. Another valuable rapid tool for routine subtyping of A. pleuropneumoniae strains, complementary to serotyping is PCR toxin gene typing (28). Thirty-nine out the 42 field strains tested expressed the same apx genes as their serotype reference strains. Similar results have been obtained with other investigators (41). However, 3 strains of serotype 1 had certain apx genes missing compared with the genes present in the reference strain of serotype 1. One isolate exhibited the apx gene profile of the reference strains for serotypes 7 and 12. It has recently been shown that this isolate possess a LPS and a capsule antigenically identical to those of serotype 7 and 1, respectively. Consequently, this strain can be classified as Ki:07 based on serotyping (unpublished results). Two other strains pos-
rescent antibody technique. Can J Comp sessed only the apxl gene profile Med 1981; 45: 271-274. which is characteristic of serotype 10. 4. NIELSEN R. Serology of Haemophilus Although these 3 strains had atypic (Actinobacillus) pleuropneumoniae seroapx gene pattern, it is important to type 5 strains: establishment of subtypes a and b. Acta Vet Scand 1986; 27: 49-58. remember that detection of apx genes by PCR does not allow a determina- 5. NIELSEN R. Serological characterization of Actinobacillus pleuropneumoniae tion as to whether or not these strains strains and proposal of a new serotype: still harbor apx genes not detectable serotype 12. Acta Vet Scand 1986; 27: by this PCR because of gene deletions 453-455. and/or mutations. Interestingly, these 6. FODOR L, VARGA J, MOLNAR E, HAJTOS I. Biochemical and serological 3 strains with atypic apx gene profiles properties of Actinobacillus pleuropneualso exhibit atypic RAPD patterns but moniae biotype 2 strains isolated from were positive for the A. pleuropneuswine. Vet Microbiol 1989; 20: 173-180. moniae species-specific PCR assay. 7. NIELSEN R, ANDRESEN LO, PLAMBECK T, NIELSEN JP, KRARUP LT, Additional biochemical and genotypic SE. Serological characterization JORSAL characterization should be done to of Actinobacillus pleuropneumoniae biofurther characterize these atypic type 2 strains isolated from pigs in two Danish herds. Vet Microbiol 1997; 54: A. pleuropneumoniae strains. 35-46. In conclusion, RAPD alone with the KR, HIGGINS R, LARIprimers used in the present study was 8. MITTAL VIERE S, NADEAU M. Serological charnot discriminative for strains of acterization of Actinobacillus pleuropneuserotype 5. Other typing methods moniae strains isolated from pigs in Quebec. Vet Microbiol 1992; 32: 135-148. such as the PFGE may be needed to FREY J, BOSSE JT, CHANG YF, et al. allow discrimination between strains 9. Actinobacillus pleuropneumoniae RTXof this serotype. By contrast, RAPD toxins: uniform designation of haemoanalysis used alone for strains of lysins, cytolysins, pleurotoxin and their genes. J Gen Microbiol 1993: 1723-1728. serotype 1 is a valuable rapid tool for routine subtyping of strains and pro- 10. INZANA TJ, MATHISON B. Serotype specificity and immunogenicity of the capvide important information to estisular polymer of Haemophilus pleuropneumate the potential virulence of moniae serotype 5. Infect Immun 1987; 55: 1580-1587. A. pleuropneumoniae isolates.
ACKNOWLEDGMENTS We are in debt to Annie Gagne for her expert technical assistance in the RAPD study. We also thank Dr. K.R. Mittal for serotyping the A. pleuropneumoniae strains and to Dr. J. Frey and Dr. A. Broes for critical reading of the manuscript. This research was supported by the grant (STRO 181154) from Natural Sciences and Engineering Research Council of Canada and a postdoctoral fellowship offered by the AUPELF.UREF to S.C.
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