Groupe de Recherche sur les Maladies
Infectieuses du Porc (GREMIP), Faculté de médecine
vétérinaire, Université de Montréal,
St-Hyacinthe, Québec, Canada J2S 7C6
Received 24 February 1999/Returned for modification 13 April
1999/Accepted 20 May 1999
 |
INTRODUCTION |
Streptococcus suis is an
important swine pathogen and is a causative agent of many pathological
conditions, such as meningitis, endocarditis, arthritis, polyserositis,
and pneumonia (10). It has been isolated from a large
variety of animal species, and it is also an important zoonotic agent
for people in contact with swine or pig by-products (8).
Thirty-five capsular types have been described, with 2 and 1/2 being
among the most prevalent serotypes recovered from diseased animals
(7). Pigs carrying pathogenic S. suis serotypes
and/or strains are known to be the source of infection for naive herds.
Piglets born to sows with uterine and/or vaginal infections are either
born infected or become infected at, or soon after, birth
(22).
Isolation of specific S. suis serotypes from the tonsils,
nasal cavities, and genital tract is difficult, since low-pathogenic serotypes and untypeable strains also inhabit these sites
(13). Traditional microbiological techniques present low
sensitivity, since the colony morphologies of different S. suis serotypes and of untypeable strains and other streptococcal
species are very similar. Selective isolation of S. suis
serotype 2 with antibody-containing selective media has been described;
however, results obtained with this technique may vary depending on the
concentration of antibodies used and, in addition, cross-reactions with
other serotypes complicate the diagnosis (14, 16). An
indirect immunofluorescence test has also been used to visualize
S. suis serotype 2 on tonsilar smears (20, 21),
but its specificity is probably low, since several antigens are common
to all known S. suis serotypes (24). Moreover,
the latter technique cannot differentiate serotype 2 from serotype 1/2.
The lack of reliable methods is probably responsible for reports of
tonsillar carrier rates varying from 0 to 100% (3).
Identification of infected animals or herds by serology has also been
disappointing (4).
The immunomagnetic separation (IMS) method allows the specific recovery
of target bacteria from highly heterogeneous suspensions (23). This method has recently been used for the selective
isolation of Actinobacillus pleuropneumoniae serotype 1 from
swine tonsils (5). The aim of this study was to develop and
to standardize an immunomagnetic separation technique for the selective
isolation of S. suis serotypes 2 and 1/2 from tonsils of
carrier animals.
| |
MATERIALS AND METHODS |
Bacterial strains and antibodies.
S. suis reference
strains (18) of serotypes 2 (S735), 1/2 (2651), 3 (4961), 7 (8074), and 8 (14636), used to standardize the IMS technique, were from
our collection. Growth conditions on blood agar, Todd-Hewitt broth
(THB), or Todd-Hewitt agar (THA) plates (Difco Laboratories, Detroit,
Mich.) have been described elsewhere (9). Production of
anti-S. suis serotype 2 rabbit polyclonal antibodies (PAb)
was carried out as previously reported (9). These antibodies
are specific for S. suis serotype 2, are mainly directed
against the capsular polysaccharide, and are routinely used for
serotyping of field strains (9). They also recognize common
capsular epitopes presented by serotype 1/2 strains (9).
Monoclonal antibody (MAb) Z3, an immunoglobulin G2b (IgG2b) directed to
a sialic acid-containing epitope which is shared by serotypes 2 and
1/2, was also used (2). IgG fractions were purified by using
protein A (PAb) or G (MAb) columns and were measured as described
previously (11).
Standardization of the IMS technique.
Two IMS techniques,
one with a PAb and another with an MAb, were standardized.
Superparamagnetic polystyrene beads or immunomagnetic beads (IMB)
precoated with sheep anti-rabbit or sheep anti-mouse IgG (Dynabeads
M-280; Dynal, Oslo, Norway) were used. The optimal concentration of
S. suis serotype 2-specific PAb or MAb IgG antibodies to be
used to coat the IMB was determined with different concentrations of
IgG incubated with 6 × 107 to 7 × 107 IMB/ml for 3 h at room temperature on the Dynal
sample mixer (Dynal) to avoid settling of the beads. Using a particle
concentrator (MDC-M; Dynal), the beads were magnetized and retained on
one side of the tube and were then washed twice in 1 ml of
phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA)
for 30 min each time with agitation at room temperature. The coated IMB
were then resuspended to obtain the original concentration in 100 µl
of PBS-0.1% BSA at 4°C. A volume of 20 µl of each of the
different IgG/bead ratios was added to 1 ml of a suspension of low
(103) or high (106) numbers of S. suis serotype 2 or 1/2 bacteria. An incubation period of 30 min at
room temperature with agitation was followed by two washings of 10 min
each in PBS-0.05% Tween. The IMB were plated on THB-agar, and viable
counts were determined. Temperature of incubation and number of
washings were previously standardized to optimize target recovery.
To evaluate the optimal IMB concentration, different numbers of coated
IMB were added to 1 ml of 103 S. suis serotype 2 or 1/2 bacteria and a procedure similar to that described above was
done. Since tonsils are commonly colonized by more than one serotype of
S. suis, the carryover effect (recovery of nontargeted
microorganisms) was verified by testing the PAb and MAb-coated IMB with
a suspension of 106 CFU of S. suis serotypes 3, 7, and 8 per ml in THB.
Sensitivity of the IMS technique.
The effect of S. suis serotype 1/2 on the recovery rate of serotype 2 (and vice
versa) was evaluated. To better identify, in these experiments, the
recovery of the targeted S. suis (serotype 2 or 1/2),
streptomycin-resistant (Sr) variants of the reference strains of these
serotypes were used (2). Tenfold dilutions of S. suis serotype 2Sr or 1/2Sr (from
105 to 101) were added individually to
different tubes. A suspension of a streptomycin-sensitive strain of
serotype 1/2 or 2 (105 to 106 CFU/ml),
depending on the experiment, was added to each tube. To evaluate the
effect of other serotypes of S. suis (normally present in
tonsils) on the recovery rate of serotype 2 or 1/2, similar experiments
were done with the streptomycin-sensitive reference strains of S. suis serotypes 3, 7, and 8. The IMS protocol described above,
using the PAb or MAb and the optimal IgG and IMB concentrations (see
Results), was used. Viable counts were determined on THA plates
supplemented with 0.9 g of streptomycin per ml to isolate only
targeted serotype 2 or 1/2, depending on the experiment. To evaluate
total bacterial growth, samples were also cultured on nonsupplemented
THA plates.
To evaluate the sensitivity of the IMS technique with tonsils, S. suis serotype 2- and 1/2-negative tonsils were used. Tonsil pieces
were cut and processed as described previously (5), with the
modification of avoiding searing the surface with a hot spatula. The
supernatants of the vortex-mixed S. suis tonsils were
filtered (5), and 10-fold dilutions of S. suis
serotype 2 or 1/2 (depending on the experiment, with concentrations of 104 to 101) were added to the different tubes.
The artificially inoculated tonsil supernatants were then processed by
using the IMS protocol presented above. Viable counts were determined
on blood agar plates. Colonies recovered were confirmed as being
S. suis serotype 2 or 1/2 by serotyping using the
coagglutination test (6).
Validation of the IMS technique.
For the validation of the
IMS technique, 24 and 168 tonsils from herds which presented clinical
disease due to S. suis serotypes 1/2 and 2, respectively,
were randomly collected at the slaughter house and stored at 
20°C.
Isolation of S. suis serotypes 2 and 1/2 from each tonsil
was carried out by the IMS technique and the standard procedure. For
the IMS technique, 0.3 g of each tonsil was taken and then reduced
to small pieces with a scalpel and added to 3 ml of PBS-0.1% BSA.
After vortex mixing and filtration of the supernatants, IMS was
performed by using the standardized protocol presented above. For the
standard procedure, parallel incisions were made and samples were taken
by using cotton swabs, which were then inoculated on blood agar plates
supplemented with the selective reagent SR126 (Oxoid Canada, Nepean,
Ontario) (13). For both methods, the count and a description
of the types of colonies on each plate were noted. A maximum of five
alpha-hemolytic colonies per plate were randomly selected and tested by
the coagglutination test (6) using sera against serotypes 1 and 2, as well as a negative serum (negative control). Serotype 2- or
1/2-positive strains were biochemically confirmed as being S. suis by using the following tests (9): absence of
growth in 6.5% NaCl, a negative Voges-Proskauer test, and production
of amylase.
| |
RESULTS |
Standardization of the IMS technique.
For each step, the mean
of at least three independent assays is presented. Unless otherwise
specified, the results presented are those obtained with serotype 2 since similar results were obtained with S. suis serotype
1/2 (data not shown). The highest number of bound S. suis
serotype 2 bacteria was observed from concentrations of 1.5 and 25 µg/ml for the PAb and MAb, respectively (Fig.
1A and B). A good recovery rate was
obtained with both high and low numbers of bacteria.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 1.
Recovery of S. suis serotype 2 with an
initial count of 103 CFU ( ) or 106 CFU (×)
using IMB coated with different concentrations of S. suis
serotype 2- and 1/2-specific MAb Z3 (A) or PAb IgG (B).
|
|
Optimum bead concentrations for the serotype 2 strain were 1.4 × 107 and 5.6 × 105 when MAb- and
PAb-coated IMB were used, respectively (Fig.
2).
View larger version (10K):
[in this window]
[in a new window]
|
FIG. 2.
Recovery of S. suis serotype 2 (initial count
of 103 CFU) using different concentrations of IMB coated
with serotype 2- and 1/2-specific MAb Z3 () or PAb IgG (×).
|
|
A higher carryover was observed with MAb-coated IMB than with
PAb-coated IMB, probably due to nonspecific binding of bacteria to the
beads, especially for serotypes 3 and 8 (Table
1). This was confirmed when
experimentally contaminated tonsils were tested (see below). The
nonspecific carryover observed with the PAb-coated beads can also be
explained by recognition of noncapsular epitopes by the PAb.
Sensitivity of the IMS technique.
Since both PAb and MAb are
able to recognize serotypes 2 and 1/2, the effect of interference of
one serotype on the recovery rate of the other serotype using the IMS
technique was investigated. To differentiate serotype 2 from serotype
1/2 colonies, streptomycin-resistant strains were used as targeted
strains and streptomycin-containing medium was used for bacterial
isolation. The presence of high numbers of serotype 1/2 bacteria did
not affect the recovery rate of S. suis serotype 2 when
PAb-coated (Table 2; P > 0.1, Student's unpaired t test) or MAb-coated (data
not shown) IMB were used. Even when a low number of serotype 2 (101) and a high number of serotype 1/2 (106)
CFU was used, no significant difference in the recovery of serotype 2 bacteria was observed in the presence or in the absence of the contaminants. However, in medium without streptomycin, colonies belonging to both serotypes could be recovered. Since both colonies are
present on the plates and a restrictive number of colonies are
routinely subcultured, an overgrowth of colonies of serotype 1/2 would
statistically prevent the isolation of those of serotype 2. Similar
results were obtained for the isolation of serotype 1/2 in a high
concentration of serotype 2 strains (data not shown). The presence of
other serotypes did not affect the recovery of the targeted serotype of
S. suis (Table 2; P > 0.1, Student's unpaired t test), independently of the antibody used.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Effect of the presence of high concentrations of
different contaminantsa on the immunomagnetic
isolation of S. suis serotype 2 using PAb IgG-coated beads
|
|
When experimentally contaminated tonsils were tested, the number of
contaminants (bacteria different from S. suis or S. suis not belonging to serotype 2 or 1/2) was between 10 and 100 times higher with MAb-coated IMB than with PAb-coated IMB and this was true for both serotype 2- (Table 3) and
1/2-infected tonsils (data not shown). This confirmed previous results
showing a higher carryover of other serotypes by MAb-coated IMB. For
artificially serotype 2-inoculated tonsils, the detection limit of the
IMS technique was at least 101 CFU/0.1 g of tonsil with
PAb- and MAb-coated IMB. In fact, 100% of the original inoculum could
be recovered at this concentration (Table 3).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Sensitivity of the IMS isolation technique obtained with
artificially S. suis serotype 2-inoculated tonsils using PAb
and MAb IgG-coated IMB
|
|
Validation of the IMS technique.
Since higher concentrations
of IgG and beads are needed and the number of contaminants (carryover)
is considerably higher, there was no advantage in using MAb-coated IMB
for validation of the technique. Tonsils from infected herds were
therefore processed by using the standardized IMS technique (with
PAb-coated IMB and isolation on blood agar plates) and the standard
procedure using isolation on selective media.
Of the 24 tonsils from the S. suis serotype 1/2-infected
herd, 46% were positive and none was negative by both the IMS
technique and the standard procedure (Table
4). In fact, all of the tonsils were
positive by the IMS technique. Fifty-four percent of tonsils were
positive by the IMS technique alone. The total percentage of S. suis serotype 1/2-positive tonsils detected by the IMS technique (100%) was significantly higher than that obtained with the standard procedure (46%) (P < 0.001; chi-square test).
S. suis serotype 2 was also isolated more frequently
with the IMS technique alone than with the standard procedure alone
(three and one positive tonsils, respectively). In fact, the single
tonsil which was positive only by the standard procedure was heavily
infected with S. suis serotype 2 and most of the colonies
selected with the IMS technique in this particular tonsil belonged to
this serotype.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Recovery of S. suis serotypes 1/2 and 2 from
tonsils by the IMS method with PAb IgG-coated beads and the
standard procedurea
|
|
When 168 tonsils from a herd infected with S. suis serotype
2 were tested, 8% were positive and 23% were negative by both the IMS
technique and the standard procedure for the isolation of serotype 2 (Table 4). Sixty-eight percent of tonsils were positive by the IMS
technique alone, whereas only 1% (two tonsils) were positive by the
standard procedure but negative by the IMS technique. In this herd, the
total percentage of S. suis serotype 2-positive tonsils
detected by the IMS technique (76%) was also significantly higher than
that obtained with the standard procedure (9%) (P < 0.001; chi-square test). Finally, more serotype 1/2-positive tonsils were also detected in this herd by the IMS technique (27%) than with the standard procedure (5%).
The number of nonrelated microorganisms isolated by the IMS technique
in nonselective medium was considerably reduced compared to that
isolated in selective medium by the standard procedure with tonsils
from both of the herds tested (Table 5).
In addition, in positive tonsils, the ratio of the number of positive
colonies to the total number of colonies tested was significantly
higher with the IMS technique than with the standard procedure. For
example, in the serotype 2-infected herd, 65% of the colonies tested
(492 positive colonies among 759 colonies tested) and 3% (16 positive colonies among 539 colonies tested) belonged to serotype 2 according to
the IMS technique and the standard procedure, respectively. In the case
of the serotype 1/2-infected herd, all of the colonies recovered from
19 of the 24 serotype 1/2 IMS-positive tonsils were identified as
S. suis serotype 1/2.
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Distribution of contaminants (normal flora) recovered
from tonsils of infected herds by the IMS method with PAb
IgG-coated beads and the standard procedure
|
|
| |
DISCUSSION |
The IMS technique described previously for isolation of A. pleuropneumoniae from tonsils uses PAb-coated beads
(5). Since PAb often exhibit unwanted cross-reactions and
are more difficult to prepare in a reproducible form and, on the other
hand, MAbs are more specific, PAb- and MAb-coated IMB were compared.
The amount of antibodies required for optimum coating of the beads was
20 times higher with purified MAb than PAb IgG. Antibodies did not seem
to aggregate, since no decrease in the recovery rate was observed with
a high concentration of antibodies, as observed for Listeria
monocytogenes (25). Surprisingly, MAb-coated IMB presented a significantly higher carryover than PAb-coated beads when
tested with pure cultures of heterologous serotypes of S. suis and with artificially inoculated tonsils (Tables 1 and 2). Since MAb Z3 is highly specific for capsular epitopes of S. suis serotypes 2 and 1/2, this carryover can be mainly related to
nonspecific binding of bacteria to the beads. Interestingly, the IMS
technique with MAb-coated IMB required 100 times more magnetic beads
than the PAb-IMS technique to keep the same sensitivity (Fig. 2). A higher number of beads would lead to a greater surface area to which
nonspecific bacteria would be able to bind. The fact that such a high
concentration of beads is needed to maintain an acceptable recovery
rate is probably a consequence of the sparse distribution of the
recognized epitope. The low carryover obtained with PAb-coated IMB is
probably due to the fact that the rabbit antibody response was mainly
directed to the capsule. The same antibodies are routinely used for
capsular serotyping, and a low level of cross-reaction with other
serotypes is observed (6).
Since the serotype 2 capsular antigenic fraction of serotype 1/2 is
indistinguishable from that presented by serotype 2, immunomagnetic isolation of serotype 2 S. suis without the simultaneous
isolation of serotype 1/2 strains would be unexpected. In fact,
serotype 1/2 strains contain a type 2 antigen fraction identical to
that of serotype 2 strains since all antibody activity against the type
2 antigen was removed from anti-serotype 2 and anti-serotype 1/2 sera
by absorption with serotype 1/2 and 2 strains, respectively (17). Both of the antibodies used in this study strongly
recognized serotype 2 as well as serotype 1/2 strains. However, enough
antibodies seem to coat the beads since the presence of serotype 1/2
does not significantly affect the recovery of a serotype 2 strain. Although both serotypes would be recovered if present in the samples, it is not possible to differentiate them on a primary culture plate.
Since a limited number of colonies are subcultured, the predominant
serotype would have more chances to be isolated from the plates. This
was confirmed in the validation, since in the few cases where the
targeted serotype (for example, serotype 2) was isolated by the
standard procedure but not by the IMS technique, tonsils were heavily
infected by the other serotype (in this case, serotype 1/2). This led
to a concentration of the predominant serotype by the IMS technique,
whereas in the standard procedure the positive colony was randomly
selected. In contrast to the work of Mortlock (15) and in
agreement with that of Gagné et al. (5), no
differences in IMS sensitivity were found when pure cultures and
artificially inoculated samples were used (data not shown).
The validation with tonsils from infected herds showed that the IMS
technique is significantly more sensitive than the standard procedure.
In addition, nonselective medium can be used and subculture of colonies
consumes little time since the number of contaminants per plate is
considerably lower with the IMS technique. Results showed that herds
with clinical disease due to S. suis present a very high
prevalence of the concerned serotype (100 and 77% of the positive
animals in serotype 1/2- and 2-infected herds, respectively). The
reported prevalence of carrier animals in the S. suis
serotype 2-infected herd would have been significantly lower if only
the standard procedure (9%) instead of the IMS technique (76%) had
been used. The relatively low prevalence of the heterologous serotype
(for example, that of serotype 2 in the serotype 1/2-affected herd)
should not be taken into consideration, since the high prevalence of
the target serotype probably prevented the isolation of its counterpart
by the IMS technique, as mentioned before. In another study and using
the same IMS technique, the prevalence of serotype 2 strains in a herd
without a history of clinical cases due to this serotype was 30%
(unpublished data).
One of the main concerns in modern swine production is to possess
reliable tools for the detection of specific infectious agents to
prevent their entry into a naive herd through the introduction of
carrier animals. Lack of these reliable methods for important S. suis serotypes led researchers to get contradictory results. For
example, pathogenic S. suis was first considered to spread only horizontally among nursery pigs, with no evidence of vertical transmission (12). However, vertical transmission of the
infection has recently been reported (1, 19). The IMS
technique developed in this study will allow more reliable
epidemiological studies of colonization by and transmission of this
pathogen. Moreover, viable bacteria are recovered with this technique,
which may also facilitate testing of antimicrobial sensitivity and
virulence, as well as molecular epidemiological studies. It would also
be possible to adapt the technique to the recovery of other important serotypes of S. suis by changing only the specificity of the
antibody used.
We acknowledge C. Moore for providing some of the samples, R. Ethier for taking the samples at the slaughterhouse, and R. Higgins for
critically reviewing the manuscript. We also thank Julie-Mélanie
Trudel, Michèle Matton, and Marcelo Ribotta for invaluable
technical assistance.
This work was supported by a grant from the Fonds pour la Formation de
Chercheurs et l'Aide à la Recherche (grant 98-NC-1037) and the
Natural Sciences and Engineering Research Council of Canada (grant OGP0154280).
| 1.
|
Amass, S. F.,
P. SanMiguel, and L. K. Clark.
1997.
Demonstration of vertical transmission of Streptococcus suis by genomic fingerprinting.
J. Clin. Microbiol.
35:1595-1596[Abstract].
|
| 2.
|
Charland, N.,
M. Jacques,
S. Lacouture, and M. Gottschalk.
1997.
Characterization and protective activity of a monoclonal antibody against a capsular epitope shared by Streptococcus suis serotypes 1, 2 and 1/2.
Microbiology
143:3607-3614[Abstract].
|
| 3.
|
Clifton-Hadley, F.-A.,
T. J. L. Alexander,
M. R. Enright, and J. Guise.
1984.
Monitoring herds for Streptococcus suis type 2 by sampling tonsils of slaughter pigs.
Vet. Rec.
115:562-564[Abstract].
|
| 4.
|
del Campo Sepulveda, E.,
E. Altman,
M. Kobisch,
S. D'Allaire, and M. Gottschalk.
1996.
Detection of antibodies against Streptococcus suis capsular type 2 using a purified capsular polysaccharide antigen-based indirect ELISA.
Vet. Microbiol.
52:113-125[Medline].
|
| 5.
|
Gagné, A.,
S. Lacouture,
A. Broes,
S. D'Allaire, and M. Gottschalk.
1998.
Development of an immunomagnetic method for selective isolation of Actinobacillus pleuropneumoniae serotype 1 from tonsils.
J. Clin. Microbiol.
36:251-254[Abstract/Free Full Text].
|
| 6.
|
Gottschalk, M.,
R. Higgins, and M. Boudreau.
1993.
Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis.
J. Clin. Microbiol.
31:2192-2194[Abstract/Free Full Text].
|
| 7.
|
Higgins, M., and M. Gottschalk.
1999.
Distribution of Streptococcus suis capsular types in 1998.
Can. Vet. J.
40:277[Medline].
|
| 8.
|
Higgins, M., and M. Gottschalk.
1999.
Streptococcal diseases, p. 563-578.
In
B. Straw, S. D'Allaire, W. Mengeling, and D. Taylor (ed.), Diseases of swine, 8th ed. Iowa State University Press, Ames.
|
| 9.
|
Higgins, R., and M. Gottschalk.
1990.
An update on Streptococcus suis identification.
J. Vet. Diagn. Investig.
2:249-252[Free Full Text].
|
| 10.
|
Higgins, R.,
M. Gottschalk,
M. Beaudoin, and S. Rawluk.
1992.
Distribution of Streptococcus suis capsular types in Quebec and western Canada.
Can. Vet. J.
33:27-30[Medline].
|
| 11.
|
Markwell, M. A.,
S. M. Haas,
L. L. Bieber, and N. E. Tolbert.
1978.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.
Anal. Biochem.
87:206-210[Medline].
|
| 12.
|
Mogollon, J. D.,
C. Pijoan,
M. P. Murtaugh,
J. E. Collins, and P. P. Cleary.
1991.
Identification of epidemic strains of Streptococcus suis by genomic fingerprinting.
J. Clin. Microbiol.
29:782-787[Abstract/Free Full Text].
|
| 13.
|
Monter Flores, J. L.,
R. Higgins,
S. D'Allaire,
R. Charette,
M. Boudreau, and M. Gottschalk.
1993.
Distribution of the different capsular types of Streptococcus suis in 19 swine nurseries.
Can. Vet. J.
34:170-171[Medline].
|
| 14.
|
Moreau, A.,
R. Higgins,
M. Bigras-Poulin, and M. Nadeau.
1989.
Rapid detection of Streptococcus suis serotype 2 in weaned pigs.
Am. J. Vet. Res.
50:1667-1671[Medline].
|
| 15.
|
Mortlock, S.
1994.
Recovery of Escherichia coli O157:H7 from mixed suspensions: evaluation and comparison of pre-coated immunomagnetic beads and direct plating.
Br. J. Biomed. Sci.
51:207-214[Medline].
|
| 16.
|
Mwaniki, C. G.,
I. D. Robertson, and D. J. Hampson.
1994.
The prevalence of Streptococcus suis type 2 in Western Australian piggeries.
Aust. Vet. J.
71:385-386[Medline].
|
| 17.
|
Perch, B.,
E. Kjems,
P. Slot, and K. B. Pedersen.
1981.
Biochemical and serological properties of R, S and RS streptococci.
Acta Pathol. Microbiol. Scand. Sect. B
89:167-171[Medline].
|
| 18.
|
Perch, B.,
K. B. Pedersen, and J. Henrichsen.
1983.
Serology of capsulated streptococci pathogenic for pigs: six new serotypes of Streptococcus suis.
J. Clin. Microbiol.
17:993-996[Abstract/Free Full Text].
|
| 19.
|
Reed, A. J.,
S. F. Amass, and G. W. Stevenson.
1998.
Evidence of vertical transmission of pathogenic Streptococcus suis resulting in meningitis in nursery pigs.
Proc. Int. Pig Vet. Soc. Congr.
15:89.
|
| 20.
|
Robertson, I. D.
1985.
An indirect fluorescent antibody technique for the detection of Streptococcus suis type 2.
N. Z. J. Med. Lab. Technol.
39:171-172.
|
| 21.
|
Robertson, I. D., and D. K. Blackmore.
1987.
The detection of pigs carrying Streptococcus suis type 2.
N. Z. Vet. Res.
35:1-4.
|
| 22.
|
Robertson, I. D., and D. K. Blackmore.
1989.
Prevalence of Streptococcus suis types 1 and 2 in domestic pigs in Australia and New Zealand.
Vet. Rec.
124:391-394[Abstract].
|
| 23.
|
Safarik, I.,
M. Safarikova, and S. J. Forsythe.
1995.
The application of magnetic separations in applied microbiology.
J. Appl. Bacteriol.
78:575-585[Medline].
|
| 24.
|
Serhir, B.,
R. Higgins,
B. Foiry, and M. Jacques.
1993.
Detection of immunoglobulin-G-binding proteins in Streptococcus suis.
J. Gen. Microbiol.
139:2953-2958[Medline].
|
| 25.
|
Skjerve, E.,
L. M. Rorvik, and O. Olsvik.
1990.
Detection of Listeria monocytogenes in foods by immunomagnetic separation.
Appl. Environ. Microbiol.
56:3478-3481[Abstract/Free Full Text].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
