BACKGROUND
haemophilus parasuis causes glässer’s disease in pigs, with symptoms of fibrinous polyserositis, pericarditis, polyarthritis, and meningitis
<cit> .
h. parasuis also causes septicemia and pneumonia without polyserositis and can be isolated from nasal passages of healthy swine.
introduction of conventionally raised pigs into segregated early weaning herds may result in infection and high economic losses because the latter lack immunity to h. parasuis <cit> .
h. parasuis also remains a problem in many high health status herds.
economic losses in  <dig> in the united states were estimated at $ <dig> million dollars ;
<cit> .
haemophilus parasuis strains are classified into  <dig> serovars based on immunodiffusion of heat-stable polysaccharide antigens
<cit> .
however, reagents for serotyping field isolates are not readily available, and a large number of isolates cannot be identified by serotyping and are designated as nontypeable
<cit> .
other serotyping methods, such as the indirect hemagglutination test
<cit>  have been employed to identify nt isolates.
nonetheless, there are still nt isolates that do not have serovar-specific reagents and cannot be characterized.
the virulence of each serovar was determined in specific pathogen free pigs
<cit> .
molecular typing techniques are increasingly used to identify field isolates including nt isolates.
these methods include polymerase chain reaction-restriction fragment length polymorphism
<cit> , enterobacterial repetitive intergenic concensus-polymerase chain reaction
<cit> , restriction endonuclease analysis
<cit> , multilocus enzyme electrophoresis
<cit> , and multilocus sequence typing  analysis
<cit> .
the molecular typing methods have shown that considerable genetic diversity exists among strains of isolates of a particular serotype and that the genotyping techniques were more discriminating compared to conventional serotyping, especially for use in epidemiological studies.
each of these molecular typing techniques offers advantages and disadvantages.
for example, restriction endonuclease experiments
<cit>  found distinct patterns of isolates from animals with systemic disease compared to respiratory isolates from healthy animals but restriction enzymes are expensive.
the pcr-rflp method uses restriction enzymes and sometimes does not generate multiple bands
<cit> .
multilocus sequence typing  is a technique that studies housekeeping genes
<cit> .
however, the latter procedure requires isolation of genomic dna, performing pcr, and sequencing of pcr products.
both eric-pcr
<cit>  and mslt analysis
<cit>  could detect strain variation but not all strains were classified as virulent or avirulent.
although eric-pcr has recently been extensively used to study the epidemiology of h. parasuis isolates
<cit> , the random amplified polymorphic dna  technique has not been utilized for this purpose.
however, rapd has been used to distinguish other gamma-proteobacteria, including salmonella spp.
<cit> , e. coli o <dig>
<cit> , and klebsiella pneumonia <cit> .
both eric-pcr and random amplified polymorphic dna  are global techniques since known primers can be easily synthesized, reagents are affordable and readily obtained, and the techniques have high levels of reproducibility.
in the pcr-based rapd method, dna does not have to be double-stranded, highly purified, or of high molecular weight
<cit> .
both eric-pcr and rapd can utilize dna from crude lysates
<cit>  which shortens the time needed for completing the assays.
however, most laboratories prefer to purify genomic dna before it is used in genome-based techniques, especially if it is to also be used in assays other than the eric-pcr technique
<cit> .
the eric-pcr technique uses higher annealing temperatures  and longer primers  than the rapd method.
these primers are specific for areas of the genome that are highly conserved and include an inverted repeat.
the rapd assay uses low stringency conditions of approximately 30–36°c annealing temperatures and short  primers.
one or more of these arbitrarily chosen rapd primers can anneal at multiple locations throughout the genome and amplify many products of the template dna.
in addition to genomic-based methods, protein-based methods offer a different and complementary approach.
whole cell protein  technique have been described.
these studies suggested that isolates from systemic sites had unique protein profiles.
isolates from respiratory sites had different protein profiles than the systemic isolates had.
the 36– <dig>  kda proteins were described as virulence markers based on the isolation site of the strain
<cit> .
this work analyzed the dna and protein profiles of 46 h. parasuis reference and field isolates.
random amplified polymorphic dna is a molecular typing technique that is often used to differentiate closely related strains.
it is especially sensitive to strain variation when three optimized primers are employed
<cit> .
random amplified polymorphic dna may detect single base changes in genomic dna and genetic maps consisting of rapd markers can be generated more efficiently than by using rflp targeted pcr-based methods
<cit> .
intra-specific variation in the rapd patterns can be observed for each primer and the sequence complexity of small plasmids is unlikely to contribute to the patterns
<cit> .
however, bacteriophage and larger plasmids with transposons could possibly mediate horizontal gene transfer between strains and increase rapd heterogeneity
<cit> .
by using the relatively simple and economical rapd technique, known primer sequences can be utilized by different laboratories, making it a standardized technique and amenable to epidemiological studies.
however, interpretation of gel electrophoresis results could introduce some variability between laboratories.
the objectives of this study were to compare the relatedness of the reference strains and field isolates based on the rapd and wcp lysate profiles and to determine if clustering that occurred was related to the site of isolation or to the pathogenicity of the strain.
RESULTS
comparison of rapd profiles and pattern analysis
of the three primers used for genotyping, primer  <dig> had an intermediate number of bands; primer  <dig> had the most polymorphic dna bands; and primer  <dig> had the least number of polymorphic dna bands .
identical patterns were obtained for each isolate for each primer when the assays were performed in triplicate.
band sizes of dna ranged between 220– <dig> base pairs .
there were bands that were more densely stained than others, but all bands were treated identically.
four outgroup strains that were in the same family as h. parasuis but from different genera were included in the analysis.
fingerprints of dna were unique for each outgroup isolate and different from the fingerprint of h. parasuis for each primer .
<dig>  reference strains were obtained between  <dig> and  <dig>  field strains 1– <dig> are described in table
<dig>  field strains 1– <dig>  25– <dig>  30– <dig> were obtained in  <dig>   <dig>  and  <dig>  respectively.
each lane was loaded with 10 μl of pcr amplification product containing approximately 30 ng of dna.
a dna control  was included in lanes marked “no”.
the standard  was a 1 kb dna ladder.
h. parasuis
reference strains
a
b
aoriginally published by kielstein and rapp-gabrielson  and adapted by zehr and tabatabai .
bh, highly virulent, death of pig within 96 h post-inoculation; l+, polyserositis and arthritis at necropsy; l-, mild clinical symptoms; a, avirulent, no clinical symptoms at necropsy as described by kielstein and rapp-gabrielson .
ch <dig>  is a field strain with the same characteristics as the original h <dig>  reference strain h <dig> was lost during culture passage prior to our acquisition of the reference strains above.
h. parasuis
field isolates
a
aoriginally published by zehr and tabatabai .
bunk, did not serotype to serovars  <dig>   <dig>   <dig>   <dig>   <dig>  or  <dig>
cisolated from same farm site.
disolated from same animal.
e”old” field strains obtained in  <dig>
fsick animal.
goriginally serotype  <dig> in  <dig>
a composite dendrogram prepared from the rapd data obtained by using three primers is shown in figure
<dig>  at  <dig> % similarity, this dendrogram showed three clades  and two unique reference serovars, one virulent isolate  from germany which caused polyserositis and one avirulent nasal isolate  from switzerland from a healthy animal.
all except one of the isolates in clade a were systemic.
clade b contained three avirulent reference strains from sweden , japan , and switzerland .
the remaining isolates in clade b were isolated from “healthy” animals but were virulent  reference strains or systemic  field isolates.
clade c isolates were systemic h. parasuis with the exception of one avirulent reference strain isolated from the trachea of a pig diagnosed with pneumonia from germany  as well as all outgroup strains, m. haemolytica, p. trehalosi, a. pleuropneumoniae, and p. multocida.
duplicate cultures of h. parasuis ia84- <dig>  and two outgroup strains , which are closely related phylogenetically, were grouped within one branch of clade c. duplicate cultures of h. parasuis field isolate  <dig>  were included as controls and gave identical fingerprints when analyzed with primers  <dig>   <dig> and  <dig>
both of the recent field isolates in clade a  could be serotyped and 79% of the recent field isolates in clade c  were typeable, whereas 72% of the recent field isolates  in clade b were classified as “unk”.
three isolates  from the same animal but with two different serotypes  clustered in the same clonal grouping .
comparison of sds-page protein profiles and pattern analysis
protein bands between  <dig> and  <dig> kilodalton  were present in all of the reference strains and field isolates , as well as a few bands higher than 180 kda in four of the reference strains c, f, h, and i, respectively.
the latter reference strains corresponded to serovars  <dig>   <dig>   <dig>  and  <dig>  respectively, which all designated as avirulent.
isolates gave identical patterns when the analysis was performed in triplicate.
each serovar showed unique band patterns, but there were also common protein bands among the reference serovars  and field isolates .
for example, reference strains c and f showed a common protein at 253 kda; and reference strains h and i showed a common band at 217 kda.
all reference strains  and field isolates 25– <dig> expressed prominent bands at 140 kda and 70 kda and all strains except reference strains b and h  showed prominent bands at approximately 40 kda.
visual inspection of the protein profiles of the field strains 25– <dig>  showed that these were similar to but not identical to reference strains k and l. field strains 1– <dig> protein profiles were more heterogeneous than the reference strains or field isolates 25– <dig> protein profiles.
field isolates  <dig>   <dig>   <dig>   <dig>  and  <dig> all had major protein bands at approximately 50 kda, which were not apparent in the other protein profiles.
outgroup strains  had unique wcp lysate patterns, which differed from the h. parasuis pattern, on an sds-page gel.
<dig>  reference strains were obtained between  <dig> and  <dig>  field strains 1– <dig> are described in table
<dig>  field strains 1– <dig>  25– <dig>  30– <dig> were obtained in  <dig>   <dig>  and  <dig>  respectively.
each lane was loaded with 10 μg of protein.
molecular weights  are indicated in kilodaltons.
the neighbor joining dendrogram showing phylogenetic analysis of wcp lysates  used a band optimization of  <dig> % and a band position tolerance of  <dig> % and had one unique isolate .
three clades  at  <dig> % similarity were generated and three subclades of clade a at 63% similarity were produced.
subclade a <dig> contained all systemic field isolates .
subclade a <dig> contained eleven of the fifteen original reference strains of various pathogenicities and isolation sites .
subclade a <dig> contained four of the fifteen original reference strains of varied diagnosis as well as the duplicate systemic field strains h. parasuis  and all of the outgroup strains.
clade b contained field isolate  <dig> from  <dig> and eight systemic field isolates  from  <dig> and clade c contained  <dig> systemic field isolates  from  <dig> .
isolates in clades b and c clustered all of the systemic type and subclade a <dig> strains were entirely of the reference type, including four  of the five avirulent strains.
the majority  of field isolates from  <dig>  were clustered in subclade a <dig> .
additionally, all three of the north carolina isolates  grouped in subclade a <dig>  there appeared to be some discrimination as to state of origin between isolates in clades b and c because there were three north carolina , one illinois , and one oklahoma  isolates among the nine clade b isolates whereas there were only one north carolina , one missouri , and one minnesota  isolates among fifteen clade c isolates.
as with the rapd neighbor joining analysis , recent field isolates seemed to group by serotype with 56% and 27% of the isolates in clades b and c, respectively, not being serotyped to serovars  <dig>   <dig>   <dig>   <dig>   <dig>  or  <dig>
discrimination of isolates using simpson’s index of diversity
simpson’s index of diversity estimates the discriminatory ability of typing systems by calculating the discrimination index .
d is the probability that two unrelated strains randomly selected from the test population are in two different typing groups.
the only rapd with a single primer that gave a significant index level of discrimination above 90% was rapd <dig> .
groups and singletons were determined by using 55% similarity for the composite rapd  and 63% similarity for the wcp lysate .
combining the results of all  <dig> primers gave an index of  <dig> %.
while the wcp lysate index was less than 90%, combining it with the composite rapd gave an index of diversity of  <dig> %.
a
aresults of various characterization methods with the respective simpson’s index of diversity value, 95% confidence interval, and the number of samples in the largest group produced by the method.
brapd bands using only primer  <dig>
crapd bands using only primer  <dig>
drapd bands using only primer  <dig>
ecomposite rapd combining bands from all three primers.
fwhole cell protein lysate.
gcombination bands from whole cell protein lysate and composite rapd.
discussion
this study was undertaken to utilize the rapd technique and sds-page protein profiles in order to compare  <dig> reference strains and  <dig> field isolates of h. parasuis to establish if a relationship existed between a particular clustering profile or if there was a relationship to the site of isolation or to the pathogenicity of the strain.
the clinical origin and pathogenesis of a strain is an indication of its virulence, but conclusions as to its virulence cannot be made in our study because pathogenesis studies were not conducted in specific pathogen free pigs
<cit> .
however, the virulence potential of h. parasuis strains, based on their serotype classification, isolation sites and the presence or absence of major proteins with molecular weights between  <dig> and  <dig>  kda, has been investigated
<cit> .
some of the expressed proteins in our recent field isolates may be called virulence markers but no direct association of the 40 kda proteins could be made.
few laboratories have the ability to serotype h. parasuis isolates because of the lack of reagents.
therefore, a genome-based method and a phenotypic analysis of the reference strains and field isolates were emphasized in our study.
neighbor joining analysis based on dice coefficients of similarity was used to compare rapd and protein  profiles of the reference strains and field isolates.
the samples were statistically discriminated better by using the composite rapd technique than by using the wcp lysate technique but combining the results of both techniques gave a high discrimination index.
outgroups were included to compare the presence or absence of bands in these isolates to the bands in the more closely related h. parasuis isolates.
the only monophyletic ingroup with the four “outgroups” was the sds-page dendrogram as determined by the neighbor joining analysis .
the results suggest that the four outgroup species selected may have been too closely related to h. parasuis to act as a true outgroup.
dijkman et al.
<cit>  were also unable to discriminate a. minor and a. porcinus strains from h. parasuis strains in an eric-pcr technique.
additionally, olvera et al.
<cit>  could not demonstrate that a. indolicus and a. minor strains were outgroups to h. parasuis strains when they used the variation of the partial hsp <dig> sequence of h. parasuis as a classification tool.
others have shown that the geographic distribution or age of the isolate may cause the “outgroup” to act as an ingroup
<cit>  and that if the isolates in the study were too closely related, then the outgroups could be rerooted to locations within phylogenetic trees
<cit> .
a fourth possibility for the lack of outgroup observance in the dendrograms could be that horizontal gene transfer has occurred between the outgroup species and h. parasuis <cit> , which would cause unexpected similarities and unusual phyletic patterns
<cit> .
this theory is supported by the presence of bacteriophages in h. parasuis <cit> , e. coli <cit> , p. multocida <cit> , m. haemolytica <cit> , and p. trehalosi <cit> , plasmids in h. parasuis <cit>  and a. pleuropneumoniae <cit> , and a dna uptake sequence in h. parasuis <cit> .
although isolates from known systemic sites
<cit>   were able to be separated into groups by the rapd technique described here, the composite diagram of the three individual primers ultimately showed a limited degree of relatedness based on pathogenicity among the reference strains and the  <dig> field strains.
the strains showed high heterogeneity with the rapd method which indicated possible horizontal transfer of genes or chromosomal recombination between unrelated and potentially transient strains.
wang et al.
<cit>  compared rapd and mee and found that rapd data that combined five primers was more discriminatory than mee tests that used  <dig> enzymes.
the eric-pcr technique is a comparable method to rapd.
zulkifli et al.
<cit>  found rapd to be more discriminatory than eric-pcr.
some h. parasuis isolates were not able to be assayed by using the eric-pcr
<cit>  because they gave no or very poor results.
recent studies have found a high diversity of h. parasuis strains isolated in various geographic areas but have not been able to assign a clear correlation between virulence or serovar and eric-pcr clusters
<cit> .
this conclusion agrees with other h. parasuis eric-pcr studies
<cit> .
macedo et al.
<cit>  reported that the highest diversity was in the nt isolates which discriminated  <dig> genotypes.
our rapd dendrogram also indicated high diversity of the h. parasuis strains, with only field isolates  <dig> and  <dig> being identical.
although there was no definite correlation between serovar and pathogenicity, most of the isolates that were serotypeable and from diseased animals clustered in clade c.
other genomic methods such as mee and mlst
<cit> , also did not completely discriminate field isolates of h. parasuis.
blackall et al.
<cit>  found  <dig> different electrophoretic types from  <dig> field isolates and  <dig> reference serovars, which clustered into  <dig> major subdivisions, which were not associated with virulence.
olvera et al.
<cit>  concluded that subgroups of  <dig> field isolates and  <dig> reference serovars clustered into branches containing avirulent, nasal isolates and virulent, systemic isolates.
however,  <dig> additional clinical isolates did not cluster within the virulent branch.
two different studies
<cit>  combined serotyping and iha methods and concluded that isolates of serovars  <dig>   <dig>   <dig>  and nt isolates were the most prevalent in  <dig> and  <dig>  with serovar  <dig> the most frequently isolated from the respiratory tract while nt isolates were usually systemic isolates.
this study’s field isolates were known to be systemic except for isolates  <dig> and  <dig>  and included serovars  <dig>   <dig>   <dig>   <dig>  and  <dig>  identified by available serotyping reagents.
the serovars used in this study were the six most prevalent in the united states and canada
<cit> .
the range of nt  to the frequency of identification of serovars  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> , respectively, by immunodiffusion
<cit>  compares to the frequencies of our “unk”  and six identified serovars .
some of our field isolates may have lost the expression of their polysaccharide capsule in vitro and may not be able to be serotyped presently
<cit>  as can be inferred from field isolate  <dig>  which was serotype  <dig> in  <dig> but “unk” in our study.
field isolate  <dig> may have lost an enzyme involved in the polysaccharide capsule synthesis.
all of our field isolates of known serotype were associated with animals with systemic disease.
the majority of field isolates of known serotype were in clade c of the rapd experiment except for isolates  <dig>   <dig>  and  <dig> and in clades b and c of the wcl experiment.
rapp-gabrielson and gabrielson
<cit>  and olvera et al.
<cit>  noted that the distribution of h. parasuis serovars isolated from healthy animals may differ from that found in diseased animals and that more than one serovar could be isolated from the same animal or same isolation site.
our study also identified isolates with different serovars within the same farm site  and in from the same isolation sites in the same animal .
“unk” isolates with septicemia were the most prevalent, indicating that nt isolates harboring bacteriophages, plasmids, or repetitive elements may influence genotyping methods
<cit> .
there may not have been a correlation between serotype and rapd because only a small number of genes is involved in serotyping while the entire genome is analyzed with the rapd technique
<cit> .
our sds-page results agree with those of oliviera and pijoan
<cit>  who reported that isolates from systemic sites were usually virulent and clustered together as shown by using a computer-based analysis of protein profiles from serovars  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and nontypeable  isolates.
their results are similar to protein profiles described in our study for field isolates and their isolation sites and pathogenesis as shown in the wcp lysate dendrogram of figure
<dig> and table
<dig>  the field strains clustered in subclade a <dig> and clades b and c were primarily systemic.
ruiz et al.
<cit>  found different omp profiles between isolates from healthy pigs and those from diseased pigs.
however, they concluded that respiratory isolates were more heterogeneous than systemic isolates.
four studies have stated that a protein of approximately 36– <dig>  kda may be associated with glässer’s disease
<cit> .
in this work, a protein band was observed at approximately 40 kda in all of the field isolates and thirteen of fifteen of the reference strains .
the results shown for the wcp lysate dendrogram  imply that protein expression may be related to age or number of passages of the isolatein vitro, because reference strains clustered together, as did the “old” field strains  isolated in  <dig> , a <dig> , and a <dig> , respectively).
the phenotypic change of an isolate after serial passage was also reported by rapp-gabrielson and gabrielson and oliviera et al.
<cit> .
although we had only seven samples from north carolina, three isolates  from  <dig> grouped together in subclade a <dig> of the sds-page neighbor joining dendrogram .
our wcp lysate patterns easily discriminated between a. pleuropneumoniae serotype  <dig> and h. parasuis as well as the other three outgroup strains .
identical h. parasuis field isolates  , bands did not match sufficiently to obtain identity in the protein profile computer analysis.
this may have been because the bands were not fully “matched” in the gel compar ii program.
they were, however, in the same clonal branch of subclade a <dig>
oliviera and pijoan
<cit> , kielstein and rapp-gabrielson
<cit> , rosner et al.
<cit>  and blackall et al.
<cit>  did not find any correlation between virulence and serotype of the isolate.
however, the results reported in this study seem to indicate an association of virulence with isolates of clade c in the wcp lysate analysis.
there also seemed to be more serotypeable isolates among the recent field isolates of clade c. this work also found that the genome-based rapd composite analysis method  showed more heterogeneity than the protein-based wcp lysate analysis, which grouped isolates according to length of passage in vitro and to possible geographic origin .
the underlying genome did not change as much as the protein expression did over time
<cit> .
the recent field isolates from this study were obtained from swine diagnosed mostly with septicemia caused by serovars  <dig>   <dig>   <dig>   <dig>  and  <dig>  all of the isolates from diseased animals grouped into clades in the rapd neighbor joining dendrogram containing systemic isolates  or subclade or clades  in the wcl neighbor joining dendrogram containing systemic isolates .
bootstrap values were low for both dendrograms.
we did not raise bootstrap cut-off values because others have reported that gains and losses of genes may not be reflected when higher cut-off values are used in the analysis
<cit> .
in order to estimate the discriminatory ability of the primers in the rapd typing system and of the protein profiles, we used simpson’s index of diversity.
the simpson’s index of diversity calculation assumes that samples are randomly selected from the population and that all groups are equally represented in the population.
samples in this study were from a few respiratory sites and mostly from diseased animals.
additionally, certain strains may be overrepresented because of their increased pathogenicity in diseased animals.
however, if simpson’s assumptions were not met, a decrease in discrimination would be expected.
this was not the case in our study because differences between strains and isolates were seen in both the composite rapd or wcp lysate results as shown in table
<dig>
CONCLUSIONS
the results of this study suggested that reference strains, “old” strains isolated in  <dig>  and recent field strains isolated in  <dig> clustered by age of isolate when using wcl methods but not by using rapd methods.
both the rapd and the sds-page methods clustered strains from systemic sites.
there was no strong correlation between site of isolation and genotype or between the rapd and wcl techniques in this study.
the rapd technique showed the high heterogeneity of the h. parasuis isolates, whereas the protein profiles indicated that the number of passages in vitro of an isolate may affect its protein expression.
the protein profiles of h. parasuis and a. pleuropneumoniae were unique and this wcp lysate technique may be useful as a tool to differentiate the two nad-dependent swine respiratory organisms.
the protein studies suggested that expressed genes of the organism may help to elucidate the virulence factors involved in the infection.
moreover, the relatively low cost, including supplies and equipment and relatively short amount of time required to perform the rapd and wcp lysate methods are more advantageous when compared to other genomic or protein methods.

 haemophilus parasuis causes glässer’s disease in pigs, with symptoms of fibrinous polyserositis, pericarditis, polyarthritis, and meningitis. h. parasuis also causes septicemia and pneumonia without polyserositis and can be isolated from nasal passages of healthy swine. introduction of conventionally raised pigs into segregated early weaning herds may result in infection and high economic losses because the latter lack immunity to h. parasuis. h. parasuis also remains a problem in many high health status herds. economic losses in in the united states were estimated at $ million dollars;. haemophilus parasuis strains are classified into serovars based on immunodiffusion of heat-stable polysaccharide antigens. however, reagents for serotyping field isolates are not readily available, and a large number of isolates cannot be identified by serotyping and are designated as nontypeable. other serotyping methods, such as the indirect hemagglutination test have been employed to identify nt isolates. nonetheless, there are still nt isolates that do not have serovar-specific reagents and cannot be characterized. the virulence of each serovar was determined in specific pathogen free pigs. molecular typing techniques are increasingly used to identify field isolates including nt isolates. these methods include polymerase chain reaction-restriction fragment length polymorphism, enterobacterial repetitive intergenic concensus-polymerase chain reaction, restriction endonuclease analysis, multilocus enzyme electrophoresis, and multilocus sequence typing analysis. the molecular typing methods have shown that considerable genetic diversity exists among strains of isolates of a particular serotype and that the genotyping techniques were more discriminating compared to conventional serotyping, especially for use in epidemiological studies. each of these molecular typing techniques offers advantages and disadvantages. for example, restriction endonuclease experiments found distinct patterns of isolates from animals with systemic disease compared to respiratory isolates from healthy animals but restriction enzymes are expensive. the pcr-rflp method uses restriction enzymes and sometimes does not generate multiple bands. multilocus sequence typing is a technique that studies housekeeping genes. however, the latter procedure requires isolation of genomic dna, performing pcr, and sequencing of pcr products. both eric-pcr and mslt analysis could detect strain variation but not all strains were classified as virulent or avirulent. although eric-pcr has recently been extensively used to study the epidemiology of h. parasuis isolates, the random amplified polymorphic dna technique has not been utilized for this purpose. however, rapd has been used to distinguish other gamma-proteobacteria, including salmonella spp., e. coli o, and klebsiella pneumonia. both eric-pcr and random amplified polymorphic dna are global techniques since known primers can be easily synthesized, reagents are affordable and readily obtained, and the techniques have high levels of reproducibility. in the pcr-based rapd method, dna does not have to be double-stranded, highly purified, or of high molecular weight. both eric-pcr and rapd can utilize dna from crude lysates which shortens the time needed for completing the assays. however, most laboratories prefer to purify genomic dna before it is used in genome-based techniques, especially if it is to also be used in assays other than the eric-pcr technique. the eric-pcr technique uses higher annealing temperatures and longer primers than the rapd method. these primers are specific for areas of the genome that are highly conserved and include an inverted repeat. the rapd assay uses low stringency conditions of approximately 30–36°c annealing temperatures and short primers. one or more of these arbitrarily chosen rapd primers can anneal at multiple locations throughout the genome and amplify many products of the template dna. in addition to genomic-based methods, protein-based methods offer a different and complementary approach. whole cell protein technique have been described. these studies suggested that isolates from systemic sites had unique protein profiles. isolates from respiratory sites had different protein profiles than the systemic isolates had. the 36–  kda proteins were described as virulence markers based on the isolation site of the strain. this work analyzed the dna and protein profiles of 46 h. parasuis reference and field isolates. random amplified polymorphic dna is a molecular typing technique that is often used to differentiate closely related strains. it is especially sensitive to strain variation when three optimized primers are employed. random amplified polymorphic dna may detect single base changes in genomic dna and genetic maps consisting of rapd markers can be generated more efficiently than by using rflp targeted pcr-based methods. intra-specific variation in the rapd patterns can be observed for each primer and the sequence complexity of small plasmids is unlikely to contribute to the patterns. however, bacteriophage and larger plasmids with transposons could possibly mediate horizontal gene transfer between strains and increase rapd heterogeneity. by using the relatively simple and economical rapd technique, known primer sequences can be utilized by different laboratories, making it a standardized technique and amenable to epidemiological studies. however, interpretation of gel electrophoresis results could introduce some variability between laboratories. the objectives of this study were to compare the relatedness of the reference strains and field isolates based on the rapd and wcp lysate profiles and to determine if clustering that occurred was related to the site of isolation or to the pathogenicity of the strain.  comparison of rapd profiles and pattern analysis of the three primers used for genotyping, primer had an intermediate number of bands; primer had the most polymorphic dna bands; and primer had the least number of polymorphic dna bands. identical patterns were obtained for each isolate for each primer when the assays were performed in triplicate. band sizes of dna ranged between 220– base pairs. there were bands that were more densely stained than others, but all bands were treated identically. four outgroup strains that were in the same family as h. parasuis but from different genera were included in the analysis. fingerprints of dna were unique for each outgroup isolate and different from the fingerprint of h. parasuis for each primer. reference strains were obtained between and field strains 1– are described in table  field strains 1– 25– 30– were obtained in and respectively. each lane was loaded with 10 μl of pcr amplification product containing approximately 30 ng of dna. a dna control was included in lanes marked “no”. the standard was a 1 kb dna ladder. h. parasuis reference strains a b aoriginally published by kielstein and rapp-gabrielson and adapted by zehr and tabatabai. bh, highly virulent, death of pig within 96 h post-inoculation; l+, polyserositis and arthritis at necropsy; l-, mild clinical symptoms; a, avirulent, no clinical symptoms at necropsy as described by kielstein and rapp-gabrielson. ch is a field strain with the same characteristics as the original h reference strain h was lost during culture passage prior to our acquisition of the reference strains above. h. parasuis field isolates a aoriginally published by zehr and tabatabai. bunk, did not serotype to serovars or cisolated from same farm site. disolated from same animal. e”old” field strains obtained in fsick animal. goriginally serotype in a composite dendrogram prepared from the rapd data obtained by using three primers is shown in figure  at % similarity, this dendrogram showed three clades and two unique reference serovars, one virulent isolate from germany which caused polyserositis and one avirulent nasal isolate from switzerland from a healthy animal. all except one of the isolates in clade a were systemic. clade b contained three avirulent reference strains from sweden, japan, and switzerland. the remaining isolates in clade b were isolated from “healthy” animals but were virulent reference strains or systemic field isolates. clade c isolates were systemic h. parasuis with the exception of one avirulent reference strain isolated from the trachea of a pig diagnosed with pneumonia from germany as well as all outgroup strains, m. haemolytica, p. trehalosi, a. pleuropneumoniae, and p. multocida. duplicate cultures of h. parasuis ia84- and two outgroup strains, which are closely related phylogenetically, were grouped within one branch of clade c. duplicate cultures of h. parasuis field isolate were included as controls and gave identical fingerprints when analyzed with primers and both of the recent field isolates in clade a could be serotyped and 79% of the recent field isolates in clade c were typeable, whereas 72% of the recent field isolates in clade b were classified as “unk”. three isolates from the same animal but with two different serotypes clustered in the same clonal grouping. comparison of sds-page protein profiles and pattern analysis protein bands between and kilodalton were present in all of the reference strains and field isolates, as well as a few bands higher than 180 kda in four of the reference strains c, f, h, and i, respectively. the latter reference strains corresponded to serovars and respectively, which all designated as avirulent. isolates gave identical patterns when the analysis was performed in triplicate. each serovar showed unique band patterns, but there were also common protein bands among the reference serovars and field isolates. for example, reference strains c and f showed a common protein at 253 kda; and reference strains h and i showed a common band at 217 kda. all reference strains and field isolates 25– expressed prominent bands at 140 kda and 70 kda and all strains except reference strains b and h showed prominent bands at approximately 40 kda. visual inspection of the protein profiles of the field strains 25– showed that these were similar to but not identical to reference strains k and l. field strains 1– protein profiles were more heterogeneous than the reference strains or field isolates 25– protein profiles. field isolates and all had major protein bands at approximately 50 kda, which were not apparent in the other protein profiles. outgroup strains had unique wcp lysate patterns, which differed from the h. parasuis pattern, on an sds-page gel. reference strains were obtained between and field strains 1– are described in table  field strains 1– 25– 30– were obtained in and respectively. each lane was loaded with 10 μg of protein. molecular weights are indicated in kilodaltons. the neighbor joining dendrogram showing phylogenetic analysis of wcp lysates used a band optimization of % and a band position tolerance of % and had one unique isolate. three clades at % similarity were generated and three subclades of clade a at 63% similarity were produced. subclade a contained all systemic field isolates. subclade a contained eleven of the fifteen original reference strains of various pathogenicities and isolation sites. subclade a contained four of the fifteen original reference strains of varied diagnosis as well as the duplicate systemic field strains h. parasuis and all of the outgroup strains. clade b contained field isolate from and eight systemic field isolates from and clade c contained systemic field isolates from. isolates in clades b and c clustered all of the systemic type and subclade a strains were entirely of the reference type, including four of the five avirulent strains. the majority of field isolates from were clustered in subclade a. additionally, all three of the north carolina isolates grouped in subclade a there appeared to be some discrimination as to state of origin between isolates in clades b and c because there were three north carolina, one illinois, and one oklahoma isolates among the nine clade b isolates whereas there were only one north carolina, one missouri, and one minnesota isolates among fifteen clade c isolates. as with the rapd neighbor joining analysis, recent field isolates seemed to group by serotype with 56% and 27% of the isolates in clades b and c, respectively, not being serotyped to serovars or discrimination of isolates using simpson’s index of diversity simpson’s index of diversity estimates the discriminatory ability of typing systems by calculating the discrimination index. d is the probability that two unrelated strains randomly selected from the test population are in two different typing groups. the only rapd with a single primer that gave a significant index level of discrimination above 90% was rapd. groups and singletons were determined by using 55% similarity for the composite rapd and 63% similarity for the wcp lysate. combining the results of all primers gave an index of %. while the wcp lysate index was less than 90%, combining it with the composite rapd gave an index of diversity of %. a aresults of various characterization methods with the respective simpson’s index of diversity value, 95% confidence interval, and the number of samples in the largest group produced by the method. brapd bands using only primer crapd bands using only primer drapd bands using only primer ecomposite rapd combining bands from all three primers. fwhole cell protein lysate. gcombination bands from whole cell protein lysate and composite rapd. discussion this study was undertaken to utilize the rapd technique and sds-page protein profiles in order to compare reference strains and field isolates of h. parasuis to establish if a relationship existed between a particular clustering profile or if there was a relationship to the site of isolation or to the pathogenicity of the strain. the clinical origin and pathogenesis of a strain is an indication of its virulence, but conclusions as to its virulence cannot be made in our study because pathogenesis studies were not conducted in specific pathogen free pigs. however, the virulence potential of h. parasuis strains, based on their serotype classification, isolation sites and the presence or absence of major proteins with molecular weights between and  kda, has been investigated. some of the expressed proteins in our recent field isolates may be called virulence markers but no direct association of the 40 kda proteins could be made. few laboratories have the ability to serotype h. parasuis isolates because of the lack of reagents. therefore, a genome-based method and a phenotypic analysis of the reference strains and field isolates were emphasized in our study. neighbor joining analysis based on dice coefficients of similarity was used to compare rapd and protein profiles of the reference strains and field isolates. the samples were statistically discriminated better by using the composite rapd technique than by using the wcp lysate technique but combining the results of both techniques gave a high discrimination index. outgroups were included to compare the presence or absence of bands in these isolates to the bands in the more closely related h. parasuis isolates. the only monophyletic ingroup with the four “outgroups” was the sds-page dendrogram as determined by the neighbor joining analysis. the results suggest that the four outgroup species selected may have been too closely related to h. parasuis to act as a true outgroup. dijkman et al. were also unable to discriminate a. minor and a. porcinus strains from h. parasuis strains in an eric-pcr technique. additionally, olvera et al. could not demonstrate that a. indolicus and a. minor strains were outgroups to h. parasuis strains when they used the variation of the partial hsp sequence of h. parasuis as a classification tool. others have shown that the geographic distribution or age of the isolate may cause the “outgroup” to act as an ingroup and that if the isolates in the study were too closely related, then the outgroups could be rerooted to locations within phylogenetic trees. a fourth possibility for the lack of outgroup observance in the dendrograms could be that horizontal gene transfer has occurred between the outgroup species and h. parasuis, which would cause unexpected similarities and unusual phyletic patterns. this theory is supported by the presence of bacteriophages in h. parasuis, e. coli, p. multocida, m. haemolytica, and p. trehalosi, plasmids in h. parasuis and a. pleuropneumoniae, and a dna uptake sequence in h. parasuis. although isolates from known systemic sites were able to be separated into groups by the rapd technique described here, the composite diagram of the three individual primers ultimately showed a limited degree of relatedness based on pathogenicity among the reference strains and the field strains. the strains showed high heterogeneity with the rapd method which indicated possible horizontal transfer of genes or chromosomal recombination between unrelated and potentially transient strains. wang et al. compared rapd and mee and found that rapd data that combined five primers was more discriminatory than mee tests that used enzymes. the eric-pcr technique is a comparable method to rapd. zulkifli et al. found rapd to be more discriminatory than eric-pcr. some h. parasuis isolates were not able to be assayed by using the eric-pcr because they gave no or very poor results. recent studies have found a high diversity of h. parasuis strains isolated in various geographic areas but have not been able to assign a clear correlation between virulence or serovar and eric-pcr clusters. this conclusion agrees with other h. parasuis eric-pcr studies. macedo et al. reported that the highest diversity was in the nt isolates which discriminated genotypes. our rapd dendrogram also indicated high diversity of the h. parasuis strains, with only field isolates and being identical. although there was no definite correlation between serovar and pathogenicity, most of the isolates that were serotypeable and from diseased animals clustered in clade c. other genomic methods such as mee and mlst, also did not completely discriminate field isolates of h. parasuis. blackall et al. found different electrophoretic types from field isolates and reference serovars, which clustered into major subdivisions, which were not associated with virulence. olvera et al. concluded that subgroups of field isolates and reference serovars clustered into branches containing avirulent, nasal isolates and virulent, systemic isolates. however, additional clinical isolates did not cluster within the virulent branch. two different studies combined serotyping and iha methods and concluded that isolates of serovars and nt isolates were the most prevalent in and with serovar the most frequently isolated from the respiratory tract while nt isolates were usually systemic isolates. this study’s field isolates were known to be systemic except for isolates and and included serovars and identified by available serotyping reagents. the serovars used in this study were the six most prevalent in the united states and canada. the range of nt to the frequency of identification of serovars and, respectively, by immunodiffusion compares to the frequencies of our “unk” and six identified serovars. some of our field isolates may have lost the expression of their polysaccharide capsule in vitro and may not be able to be serotyped presently as can be inferred from field isolate which was serotype in but “unk” in our study. field isolate may have lost an enzyme involved in the polysaccharide capsule synthesis. all of our field isolates of known serotype were associated with animals with systemic disease. the majority of field isolates of known serotype were in clade c of the rapd experiment except for isolates and and in clades b and c of the wcl experiment. rapp-gabrielson and gabrielson and olvera et al. noted that the distribution of h. parasuis serovars isolated from healthy animals may differ from that found in diseased animals and that more than one serovar could be isolated from the same animal or same isolation site. our study also identified isolates with different serovars within the same farm site and in from the same isolation sites in the same animal. “unk” isolates with septicemia were the most prevalent, indicating that nt isolates harboring bacteriophages, plasmids, or repetitive elements may influence genotyping methods. there may not have been a correlation between serotype and rapd because only a small number of genes is involved in serotyping while the entire genome is analyzed with the rapd technique. our sds-page results agree with those of oliviera and pijoan who reported that isolates from systemic sites were usually virulent and clustered together as shown by using a computer-based analysis of protein profiles from serovars and nontypeable isolates. their results are similar to protein profiles described in our study for field isolates and their isolation sites and pathogenesis as shown in the wcp lysate dendrogram of figure  and table  the field strains clustered in subclade a and clades b and c were primarily systemic. ruiz et al. found different omp profiles between isolates from healthy pigs and those from diseased pigs. however, they concluded that respiratory isolates were more heterogeneous than systemic isolates. four studies have stated that a protein of approximately 36–  kda may be associated with glässer’s disease. in this work, a protein band was observed at approximately 40 kda in all of the field isolates and thirteen of fifteen of the reference strains. the results shown for the wcp lysate dendrogram imply that protein expression may be related to age or number of passages of the isolatein vitro, because reference strains clustered together, as did the “old” field strains isolated in, a, and a, respectively). the phenotypic change of an isolate after serial passage was also reported by rapp-gabrielson and gabrielson and oliviera et al.. although we had only seven samples from north carolina, three isolates from grouped together in subclade a of the sds-page neighbor joining dendrogram. our wcp lysate patterns easily discriminated between a. pleuropneumoniae serotype and h. parasuis as well as the other three outgroup strains. identical h. parasuis field isolates, bands did not match sufficiently to obtain identity in the protein profile computer analysis. this may have been because the bands were not fully “matched” in the gel compar ii program. they were, however, in the same clonal branch of subclade a oliviera and pijoan, kielstein and rapp-gabrielson, rosner et al. and blackall et al. did not find any correlation between virulence and serotype of the isolate. however, the results reported in this study seem to indicate an association of virulence with isolates of clade c in the wcp lysate analysis. there also seemed to be more serotypeable isolates among the recent field isolates of clade c. this work also found that the genome-based rapd composite analysis method showed more heterogeneity than the protein-based wcp lysate analysis, which grouped isolates according to length of passage in vitro and to possible geographic origin. the underlying genome did not change as much as the protein expression did over time. the recent field isolates from this study were obtained from swine diagnosed mostly with septicemia caused by serovars and all of the isolates from diseased animals grouped into clades in the rapd neighbor joining dendrogram containing systemic isolates or subclade or clades in the wcl neighbor joining dendrogram containing systemic isolates. bootstrap values were low for both dendrograms. we did not raise bootstrap cut-off values because others have reported that gains and losses of genes may not be reflected when higher cut-off values are used in the analysis. in order to estimate the discriminatory ability of the primers in the rapd typing system and of the protein profiles, we used simpson’s index of diversity. the simpson’s index of diversity calculation assumes that samples are randomly selected from the population and that all groups are equally represented in the population. samples in this study were from a few respiratory sites and mostly from diseased animals. additionally, certain strains may be overrepresented because of their increased pathogenicity in diseased animals. however, if simpson’s assumptions were not met, a decrease in discrimination would be expected. this was not the case in our study because differences between strains and isolates were seen in both the composite rapd or wcp lysate results as shown in table   the results of this study suggested that reference strains, “old” strains isolated in and recent field strains isolated in clustered by age of isolate when using wcl methods but not by using rapd methods. both the rapd and the sds-page methods clustered strains from systemic sites. there was no strong correlation between site of isolation and genotype or between the rapd and wcl techniques in this study. the rapd technique showed the high heterogeneity of the h. parasuis isolates, whereas the protein profiles indicated that the number of passages in vitro of an isolate may affect its protein expression. the protein profiles of h. parasuis and a. pleuropneumoniae were unique and this wcp lysate technique may be useful as a tool to differentiate the two nad-dependent swine respiratory organisms. the protein studies suggested that expressed genes of the organism may help to elucidate the virulence factors involved in the infection. moreover, the relatively low cost, including supplies and equipment and relatively short amount of time required to perform the rapd and wcp lysate methods are more advantageous when compared to other genomic or protein methods. 
this study was done to compare random amplified polymorphic dna profiles and whole cell protein lysate profiles as methods for distinguishing h. parasuis reference strains and field isolates. the dna and wcp lysate profiles of reference strains and field isolates of h. parasuis were analyzed using the dice and neighbor joining algorithms. the results revealed unique and reproducible dna and protein profiles among the reference strains and field isolates studied. the rapd profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of dna mutations in the genes of the samples. the recent field isolates had different wcp lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the rapd method and also when both the rapd and wcp lysate typing methods were combined. haemophilus parasuis is the causative agent of glässer’s disease and is a pathogen of swine in high-health status herds.
 haemophilus parasuis is the causative agent of glässer’s disease and is a pathogen of swine in high-health status herds. reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. molecular typing methods have been used as alternatives to serotyping. this study was done to compare random amplified polymorphic dna profiles and whole cell protein lysate profiles as methods for distinguishing h. parasuis reference strains and field isolates.  the dna and wcp lysate profiles of reference strains and field isolates of h. parasuis were analyzed using the dice and neighbor joining algorithms. the results revealed unique and reproducible dna and protein profiles among the reference strains and field isolates studied. simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the rapd method and also when both the rapd and wcp lysate typing methods were combined.  the rapd profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of dna mutations in the genes of the samples. the recent field isolates had different wcp lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. haemophilus parasuisrapdsds-page 