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Epidemiology of Streptococcus suis serotype 5 infection in a pig herd with and without clinical disease
Veterinary Microbiology 97 (2003) 135–151
Epidemiology of Streptococcus suis serotype 5 infection in a pig herd with and without clinical disease G. Cloutier a , S. D’Allaire a , G. Martinez a , C. Surprenant b , S. Lacouture a , M. Gottschalk a,∗ a
Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de pathologie et microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, St-Hyacinthe, Que., Canada J2S 7C6 b F. Ménard Inc., 251 Route 235, Ange Gardien, Que., Canada J0E 1E0 Received 1 April 2003; received in revised form 26 August 2003; accepted 25 September 2003
Abstract The aim of this study was to describe the transmission and the kinetics of the infection caused by Streptococcus suis serotype 5 in a multisite farrow-to-finish pig herd. Most sows carried S. suis serotype 5 in their vaginal tract, but not in their nasal cavities, as demonstrated by immunomagnetic separation (IMS) technique. Their offspring became infected during farrowing, confirming vertical transmission. During the first 4 weeks of life, a low number of piglets were carriers of S. suis serotype 5 in their nasal cavities. However, when clinical signs appeared, the carrier rate significantly increased, suggesting that isolation from nasal cavities is a better indication of active transmission than of a carrier state. Clinical cases were present in animals between 4 and 8 weeks of age, when maternal antibodies were at their lowest level. Up to six different genotypes of the same serotype could be identified by random amplified polymorphic DNA; however, a single clone was responsible for all clinical cases studied. This clone could only be isolated from a single sow, indicating that its prevalence in breeding animals was low. Interestingly, 1 year later, clinical disease associated with S. suis serotype 5 spontaneously disappeared. At that time, the genotype responsible for the clinical signs was not detected in the herd and the levels of antibodies in sows and maternal antibodies in piglets were not higher than those of the previous year. © 2003 Elsevier B.V. All rights reserved. Keywords: Streptococcus suis; Vertical transmission; Maternal antibodies; Genotypes
∗
Corresponding author. Tel.: +1-450-773-8521x8374; fax: +1-450-778-8108. E-mail address: [email protected] (M. Gottschalk). 0378-1135/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2003.09.018
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1. Introduction Streptococcus suis is one of the most important swine pathogens worldwide. It can cause septicemia, meningitis, arthritis, endocarditis, polyserositis and pneumonia (Higgins and Gottschalk, 1999). Mortality due to S. suis is frequently observed 2 to 4 weeks after weaning; however, a herd often has its own age profile. In some herds, severe clinical disease which has lasted for a time may disappear without any known reason (Mogollon et al., 1991; Lapointe et al., 2002). A total of 35 capsular types have been described so far (Higgins and Gottschalk, 1999), with serotype 2 being the most frequently recovered from diseased pigs in most countries (Higgins and Gottschalk, 2000). However, other serotypes can also be associated with severe disease (Reams et al., 1996; Higgins and Gottschalk, 2000). In fact, the percentage of serotype 2 strains isolated from diseased animals in Canada has decreased in recent years (Higgins and Gottschalk, 2001). In addition, several genotypes within the same serotype have been reported using different methods such as restriction endonuclease analysis (Mogollon et al., 1990; Beaudoin et al., 1992), ribotyping (Okwumabua et al., 1995; Staats et al., 1998), multilocus enzyme electrophoresis (Hampson et al., 1993; Mwaniki et al., 1994), macrorestriction analysis (Allgaier et al., 2001; Berthelot-Hérault et al., 2002), and randomly amplified polymorphic DNA (RAPD) (Chatellier et al., 1999; Martinez et al., 2002). Although different mechanisms of S. suis transmission have been investigated, the most important features of the epidemiology of the infection remain unclear. It is well known that most pigs are carriers of multiple serotypes in their upper respiratory tract (Monter Flores et al., 1993; Higgins and Gottschalk, 1999) and that sows can harbour different S. suis in their vaginal secretions (Amass et al., 1996). Failure to eliminate the infection in medicated early weaned piglets led to the hypothesis that animals are colonized very early in their life and that dam-to-pig transmission would be important. Although early data suggested a lack of vertical transmission (Mogollon et al., 1991), contamination of piglets during their transit through the vaginal canal has been demonstrated (Amass et al., 1995, 1996, 1997). It has been proposed that caesarean section is probably the only method that can be used to produce S. suis-free animals (Amass et al., 1996). In addition to the vertical transmission, direct and indirect contamination of animals has also been demonstrated (Robertson et al., 1991; Dee et al., 1993; Higgins and Gottschalk, 1999) as well as airborne transmission of S. suis over a distance of at least 40 cm, without nose-to-nose contact (Berthelot-Hérault et al., 2001). All studies demonstrating vertical transmission of S. suis were carried out in herds where no clinical signs associated with S. suis infection were present. In addition, bacterial isolation was carried out using standard bacterial isolation, the only technique available at the time of these studies. Isolation of specific serotypes of S. suis from carrier animals using this technique offers a very low sensitivity when compared to the serotype-specific immunomagnetic separation (IMS) technique recently described (Gottschalk et al., 1999). In the present work, we studied the vertical transmission of S. suis serotype 5 in a clinically affected herd, using the IMS technique. Levels of antibodies against S. suis serotype 5 in sows and piglets were evaluated. In addition, the presence of different genotypes within this same serotype was investigated. Results were compared to those obtained 1 year later from the same herd, following the disappearance of clinical signs due to S. suis.
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2. Material and methods 2.1. Herd selection A multisite farrow-to-finish herd of 2400 sows with high level of mortality associated with S. suis in the nurseries was selected. This PRRS-free herd was a newly established farm with an average weaning age of 16 days. Most of the mortalities occurred between 5 and 8 weeks of age and were attributed to S. suis meningitis. The second part of the study was carried out on the same herd one year later, following the disappearance of clinical signs due to S. suis. Vaccines against S. suis infections were never used in this herd. 2.2. Sampling To confirm that the pigs in the nursery stage were affected mainly by a single serotype of S. suis, a total of 30 pigs with signs of meningitis were examined post-mortem over a 2-week period. Swab samples in transport medium (Culturette, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) were collected from the brain immediately after death and sent to the laboratory to be cultured within the same day. In the first part of the study (in the presence of clinical signs), a total of 40 sows were sampled 2 weeks before parturition. Blood samples and nasal and vaginal swabs were collected to establish their carrier status regarding S. suis serotype 5. A total of eight sows (six positive and two negative for S. suis serotype 5 isolation) as well as their respective offspring (94 piglets) were further studied. Vaginal swabs of these sows as well as swabs of the nasal surface of their piglets were taken at parturition. Nasal swabs were taken from all piglets once a week until 14 weeks of age. Blood samples from piglets were taken 24 h after birth and once every other week until 14 weeks of age. Tonsils were collected from 42 of these animals at slaughter. Piglets included in this study that died during the follow-up period were necropsied and tissue samples (brain, liver, spleen, tonsils) were sent for bacteriological examination (Higgins and Gottschalk, 1990; Devriese et al., 1991). No antibiotic treatment was allowed for the control of S. suis infections during the study. In the second part of the study (one year later, in the absence of clinical signs), vaginal swabs were collected from 40 gestating sows to verify their status regarding S. suis serotype 5. In addition, 10 sows were sampled (vaginal swabs and blood) at parturition and blood samples were taken from 20 piglets 24 h after birth (two piglets per sow). Cross-sectional blood samples of 20 pigs were obtained from each of the following age groups: 2, 4, 6, 8, 10, 12 and 14 weeks of age. In addition, nasal swabs were taken from 20 piglets at 4, 6 and 8 weeks of age, which corresponded to the age at which isolation from carrier piglets was maximal a year before, in the presence of clinical signs (see results). Finally, a total of 60 tonsils were collected at slaughterhouse. 2.3. Bacterial isolation Bacterial isolation and serotyping of S. suis isolated from diseased or dead animals were carried out as described elsewhere (Higgins and Gottschalk, 1990). Isolation of S. suis serotype 5 from carrier animals was performed using the IMS technique previously
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reported for selective isolation of S. suis serotypes 2 and 1/2 from tonsils (Gottschalk et al., 1999). The technique was adapted for isolation of S. suis serotype 5 from swabs. Production of rabbit polyclonal antibodies against serotype 5 of S. suis was carried out as previously described (Higgins and Gottschalk, 1990). Immunoglobulins G (IgG) fractions were purified by using a protein A column and the protein concentration was measured as described previously (Markwell et al., 1978). Coating of beads (Dynabeads M-280; Dynal, Oslo, Norway) was carried out as described by Gottschalk et al. (1999). Swabs were put into suspension in 1 ml of phosphate-buffered saline (PBS) with 0.1% of bovine serum albumin (BSA) (PBS-0.1%–BSA). A volume of 40 l of IgG-coated beads was added to 1 ml of each suspension. After incubation and two washings of 10 min each in PBS-0.5% Tween, (PBS-T20; Sigma Chemical Co., St. Louis, MO, USA), the immunomagnetic beads were plated on blood agar plates supplemented with the selective reagent SR-126E (Oxoid Canada, Nepean, Ont., Canada) (Monter Flores et al., 1993). After 24 h of incubation at 37 ◦ C, with 5% of CO2 , a maximum of five 0.5–1.0 mm flat, alpha-hemolytic colonies were selected from each sample for further characterization. All selected isolates underwent coagglutination testing using serum against serotype 5, as well as a negative serum (negative control) (Gottschalk et al., 1993). Isolates of serotype 5 were biochemically confirmed as being S. suis using standard procedures (Higgins and Gottschalk, 1990). Isolation of S. suis serotype 5 from tonsils was also carried out by the IMS technique (Gottschalk et al., 1999). When available, a minimum of two isolates of S. suis serotype 5 from the same animal were kept for further studies. All isolates were stored at −80 ◦ C until being studied. 2.4. Serologic evaluation An indirect ELISA, adapted for S. suis serotype 5, was developed in this study, using a methodology similar to that recently described (Lapointe et al., 2002). A representative clinical isolate (isolate 4B) recovered from a case of meningitis in the same herd was used as the source of antigen. This isolate was first inoculated on sheep blood agar, incubated for 24 h at 37 ◦ C, then inoculated into 5 ml of Todd-Hewitt broth (Difco Laboratories, Detroit, MI, USA) and incubated for 18 h at 37 ◦ C. Subsequently, bacteria were lysed by sonication by seven pulse cycles of 5 min each, at 80% duty cycle (Sonics & Materials, New Town, CT, USA). After sonication, unlysed cells were removed by centrifugation at 3000 × g for 30 min. The supernatant was collected and dialysed for 24 h in filtered water with a porous regenerated cellulose membrane with a molecular weight cut-off of 12,000–14,000 (Spectrum Medical Industries, Houston, TX, USA). The solution was stored at −80 ◦ C until used. The antigen was suspended in a carbonate buffer (pH 9.6) at a standardized final concentration of 15 g/ml of protein. Each well of flat-bottomed plates (Polysorb, Nunc-Immunoplates, Rochester, NY, USA) was coated with 100 l of the antigen solution to give a final concentration of 1.5 g of protein/well. The plates were incubated overnight at 4 ◦ C, drained and washed three times with PBS containing 0.05% Tween 20 (PBS-T20; Sigma Chemical Co.). Sera from piglets were tested at a dilution of 1/1600 which was established as the best dilution that allowed differentiation between positive and negative sera (data not shown). Sera were added in 100 l amounts to appropriate wells (in duplicate, on the same plate). The positive control was obtained by repetitive vaccination of a piglet with an autogenous
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bacterin containing the isolate 4B used for the production of the antigen. The negative control was a serum of an animal obtained from a herd that did not present any clinical signs related to S. suis during at least the two previous years. After 1 h incubation, plates were washed three times with PBS-T20 and then 100 l of peroxydase-conjugated goat anti-swine IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA) were added to each well for 1 h. Plates were washed and 100 l of the substrate 3,3 ,5,5 -tetramethylbenzidine (TM Blue, Intergen, St. Milford, MA, USA) were added to each well, and the blue color was allowed to develop at room temperature. The enzyme reaction was stopped with the addition of 50 l/well of 1 N H2 SO4 . The absorbance was read at 450 nm with an ELISA plate reader (UVmax, Molecular Devices, Menlo Park, CA, USA). Results were reported as S/P ratios which are the optical density obtained for serum divided by the optical density of the positive control. Each serum sample was tested at least three times. The average optical density obtained was only accepted when the coefficient of variation was below 15%; otherwise, the serum was tested again. In addition, plates were only accepted when sera from positive and negative controls presented an optical density of 1.0 ± 0.15 and 0.2 ± 0.10, respectively. 2.5. Statistical analyses Mixed linear models for repeated measures with litter treated as a random effect were used to evaluate the effect of age and status of the sow on the S/P ratios of piglets. Post-hoc contrasts were performed with the Tukey–Kramer test to determine differences between pairs of sampling points. For the study of the second year in which a cross-sectional sampling was used, the effect of age on S/P ratios was evaluated with a one-way analysis of variance (ANOVA). For comparison of S/P ratios obtained in the first and second year, a two-way ANOVA with year, time and the interaction between them was used. For this latter analysis, two piglets per litter were randomly selected at each sampling point for each of the eight sows involved in the study of the first year. Homogeneity of variance of the S/P ratios of sows and of piglets at birth and at 2 weeks of age was tested by a modified-Levene equal variance test. Level for statistical significance was set at 0.05. 2.6. Randomly amplified polymorphic DNA (RAPD) fingerprinting RAPD for S. suis serotype 5 was performed based on the technique previously described (Chatellier et al., 1999; Martinez et al., 2002). Since primers used for serotypes 2 and 1/2 strains did not clearly differentiate serotype 5 strains (data not shown), the standardization of RAPD for this serotype was carried out in our study. Twenty-seven primers of 10 nucleotides in length were randomly chosen (Table 1) to amplify the DNA of 14 epidemiologically not-related S. suis serotype 5 strains. Ten of these strains, including the isolate 4B, were from unrelated herds in Canada; three strains (A-6165, B-92/97 and B-26/22) originated from Germany (kindly provided by Dr. Peter Valentin-Weigand, University of Hannover) and presented different genotypes as previously tested by pulsed-field gel electrophoresis (Allgaier et al., 2001); the remaining strain originated from The Netherlands (reference strain 11538). The PCR mixture consisted of buffer (10 mM Tris–HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl2 ), 100 M each of the four deoxynucleotide triphosphates (Amersham
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Table 1 List of primers tested by RAPD for the study of Streptococcus suis serotype 5 isolates Primers
Sequence, 5 –3
GC (%)
OPB-01 OPB-02 OPB-03 OPB-04 OPB-05 OPB-06∗ OPB-07 OPB-08 OPB-09 OPB-10∗ OPB-11∗ OPB-12 OPB-13 OPB-14 OPB-15 OPB-16 OPB-17 OPB-18 OPB-19 OPB-20 Primer 1 Primer 2 Primer 3 Primer 4 OPS-11 OPS-16 CLAU
GTT TCG CTC C TGA TCC CTG G CAT CCC CCT G GGA CTG GAG T TGC GCC CTT C TGC TCT GCC C GGT GAC GCA G GTC CAC ACG G TGG GGG ACT C CTG CTG GGA C GTA GAC CCG T CCT TGA CGC A TTC CCC CGC T TCC GCT CTG G GGA GGG TGT T TTT GCC CGG A AGG GAA CGA G CCA CAG CAG T ACC CCC GAA G GGA CCC TTA C TCA CGA TGC A ACG TAT CTG C GTG ACG TAG G GCA TCA ATC T AGT CGG GTG G AGG GGG TTC C AAA GGA TGC T
60 60 70 60 70 70 70 70 70 70 60 60 70 70 60 60 60 60 70 60 50 50 60 40 70 70 40
Primers selected to perform the analysis are followed by an asterisk (*).
Biosciences Inc., Baie d’Urfé, Que., Canada), 1.5 M primer, 30 ng of DNA extracted and purified as described (Pitcher et al., 1989) and 2.5 U of Taq DNA polymerase (Amersham Biosciences Inc.) in a total volume of 25 l. Primers used were synthesized by Invitrogen (Burlington, Ont., Canada). Each sample was subjected to an initial cycle of denaturation (5 min at 94 ◦ C) in a DNA Thermal Cycler 480 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Each of the 35 subsequent cycles consisted of denaturing at 94 ◦ C for 30 s, annealing at 35 ◦ C for 30 s, and extension at 72 ◦ C for 1 min. The last cycle included an extension at 72 ◦ C for 5 min. Amplified products were separated by electrophoresis in a 1.2% agarose gel (Sigma–Aldrich, Oakville, Ont., Canada) and were visualized by UV transillumination following ethidium bromide staining. A 1 kb DNA ladder (Invitrogen) was used in each gel as a molecular size standard, and a negative control, consisting of the same reaction mixture but with water instead of template DNA, was included in each run. When testing field isolates, a positive control, containing the same reaction mixture and template DNA from the strain 4B was also included. A total of 350 individual field isolates recovered from the study were tested under identical conditions at least twice with the three selected primers. Photographs of each gel were digitized with a video camera connected to
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a microcomputer (Alpha EaseTM ; Alpha Innotech Corporation, San Leandro, CA, USA). The genetic diversity was studied by comparing the different patterns among all the tested isolates of the study.
3. Results 3.1. Determination of the serotype of S. suis responsible for the mortality in the nursery Pure cultures of S. suis serotype 5 were isolated from the meninges of 23 out of 30 piglets that died indicating that this serotype was the main pathogen associated with meningitis in the nursery pigs of this farm. These 23 strains were studied by RAPD and only one clone was demonstrated to be responsible for these clinical signs (see the following sections). 3.2. Study in the presence of clinical signs 3.2.1. Bacterial isolation In the first part of the study, of the 40 sows that were pre-selected, 43 and 28% were positive for S. suis serotype 5 isolation from vaginal and nasal cavities, respectively. Nasal cavities of sows were highly contaminated with other streptococci, making difficult the isolation of S. suis serotype 5 even though IMS was used. A lower number of investigated colonies from nasal swabs (36 out of 529: 6.8%) were positive for S. suis serotype 5, when compared to vaginal swabs (147 out of 441: 33.3%). Eight sows (six positive for vaginal isolation and two negative for isolation from both sites) were then selected to further study the pattern of infection in their respective offspring. A total of 94 piglets born from the eight sows were included in the study. At birth, 15% of the piglets from positive sows were positive for S. suis serotype 5 isolation at the nasal surface, but these animals were negative at 1 week of age. Some of them (2.8%) became positive 1 week later (2 weeks of age). No piglets from the negative sows were carriers of the bacterium at birth, but 15% of them became carriers at 1 week of age. The level of nasal carriage of S. suis serotype 5 by piglets was low (less than 15%) until week 6 (Fig. 1). The isolation rate reached 35% between weeks 5 and 8, a period corresponding to the presence of clinical signs due to S. suis in the nursery (Figs. 1 and 2). A total of 20 animals (21%) died during the experiment, and S. suis serotype 5 could be isolated in pure culture or as a predominant bacterium in 11 of them (55%). After week 9, the isolation rate from nasal cavities dropped again to low values (Fig. 1). Ten of the 42 tonsils (24%) collected at slaughterhouse were positive for S. suis serotype 5 isolation. 3.2.2. Genotyping of S. suis serotype 5 isolates by RAPD Among the 27 different primers tested with total DNA from 14 unrelated S. suis serotype 5 isolates, three primers named OPB-06, OPB-10 and OPB-11 were selected (Table 1) because they gave different and reproducible patterns containing fragments with large size range and a small number of low-intensity bands. These primers were used to study more
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50% 45%
Positive animals (%)
35% 30% 25% 20% 15% 10% 5% 0% 0
1
2
3
4
5
6
7
8
9
10
Age (weeks) Fig. 1. Isolation of Streptococcus suis serotype 5 from nasal cavities of carrier piglets using the immunomagnetic separation technique. (䊊), Piglets from S. suis serotype 5 carrier sows; (䊏), piglets from S. suis serotype 5 negative sows; (䉱), mean.
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40%
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Fig. 2. Mortality due to Streptococcus suis serotype 5 according to the age of dead piglets.
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Fig. 3. Illustration of RAPD patterns generated with primers OPB 06, OPB 10 and OPB 11 from different strains of Streptococcus suis serotype 5. Lane 1: Pattern A; Lane 2: Pattern B; Lane 3: Pattern C; Lane 4: Pattern D (strain responsible for clinical cases); Lane 5: Pattern E; Lane 6: Pattern F.
than 350 selected isolates recovered in this study. Representative isolates from each animal were included (at least two isolates per animal when available). A total of six RAPD patterns (Patterns A–F), each composed of three to seven bands with sizes of between 0.5 and 3 kbp, could be differentiated using the three primers (Fig. 3). Only the Pattern D was recovered from diseased animals throughout the study. The 22 vaginal isolates recovered from positive sows were assigned to two RAPD patterns (Patterns A and C), whereas the 25 isolates from nasal cavities of these animals presented five different patterns (Patterns A, C, D, E, and F). The Pattern D was only observed in one nasal isolate of a single sow. However, this sow was not included among the eight selected for the study. Patterns A, B and C were detected in the piglets at farrowing. On the other hand, in presence of clinical signs (weeks 4–8), all the piglets that were carriers presented
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Fig. 4. Distribution of the different RAPD patterns of Streptococcus suis serotype 5 isolates recovered from piglets, according to their age. (䊐), Pattern A; ( ), Pattern B; ( ), Pattern C; (䊏), Pattern D.
the Pattern D (Fig. 4). At slaughter, 7 of 10 positive animals were carriers of the Pattern D. The remaining isolates were related to Pattern A (two isolates) and C (one isolate). 3.2.3. Serology The average level of antibodies against S. suis serotype 5 in the 40 sows was high (mean S/P ratio = 1.169). After birth, a heterogeneous amount of maternal antibodies was observed in the piglets (Fig. 5); however, there was no significant difference between piglets from positive or negative sows (1.16 versus 1.21). The maternal immunity declined and reached the lowest level at weeks 4 and 6. Then, the level of antibodies increased until week 14, as a result of active immunity to infection. This immune response was also very heterogeneous, with some animals producing high level of antibodies and others not presenting any detectable antibodies to S. suis serotype 5. Most of the mortalities occurred at the time when the antibodies were at their lowest level. 3.3. Study in the absence of clinical signs One year later, following the spontaneous disappearance of clinical disease related to S. suis infection in this herd, 40 sows were tested for their carriage of S. suis serotype 5 at the vaginal level. Similarly to the previous year, 53% of them were positive. Nasal swabs of 20 piglets per age group were taken at 4, 6 and 8 weeks of age, corresponding to the high prevalence of nasal carriage in the previous study. However, no piglets were positive at
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1.8 Average S/P ratio
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
2
4
6 8 Age (weeks)
10
12
14
Fig. 5. Kinetics of antibodies against Streptococcus suis serotype 5 according to the age of the piglets in the presence (䊉) or absence (䊊) of clinical signs.
isolation by the IMS technique during the second period of the study. On the other hand, 32% of the 60 tonsils collected at slaughterhouse were positive for S. suis serotype 5 isolation. RAPD analysis of these isolates revealed that a single pattern (Pattern C) was present. In absence of clinical signs in the herd, sows did not present a higher level of antibodies when compared to the previous year (0.929 versus 1.169). The level of maternal antibodies of the piglets at birth and 2 weeks of age were also lower than those of the previous year (Fig. 5). Moreover, an active immune response in the absence of clinical signs could not be detected.
4. Discussion The herd included in this study was affected by a single serotype of S. suis which was considered as the primary pathogen responsible for most clinical cases observed. In this PRRSV-free herd, more than 75% of the samples received from dead animals in the post-weaning section (during the herd selection study) and more than 50% of the samples during the transmission study were attributed to S. suis serotype 5. Other samples were either completely negative or too contaminated (different bacterial species retrieved in the same sample with no main pathogen isolated). Sudden death caused by S. suis without bacterial isolation is sometimes observed, as death may be the result of a septic shock caused by the up-regulation of pro-inflammatory cytokines (Gottschalk and Segura, 2000). A high percentage of sows were infected by S. suis serotype 5, independent of the presence (first year) or absence (second year) of clinical signs in post-weaned piglets. A higher level of carriage was identified in vaginal than in nasal cavities from sows. There are two possible explanations for that observation. First, and although the nasal cavities have been extensively used as sampling sites for S. suis isolation (Brisebois et al., 1990; Dee et al., 1993; Monter Flores et al., 1993), it is possible that bacteria are mainly located at the tonsils rather than in the nostrils in the upper respiratory tract of carrier pigs (Gottschalk et al., 2001). Secondly,
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the contaminating flora present in nasal cavities might have precluded the specific isolation of S. suis serotype 5, even with the use of IMS. Indeed, in positive carrier animals, a higher number of positive colonies was recovered from vaginal samples where, in some cases, five out of five colonies tested belonged to S. suis serotype 5. With the nasal samples, a lower percentage of positive colonies was usually obtained. Results from our study confirm that serotyping is not discriminative enough to differentiate strains. A more sensitive technique such as the RAPD has been shown to be more useful to genotype S. suis isolates of serotype 2 (Chatellier et al., 1999; Martinez et al., 2002). However, a standardization of the technique needs to be carried out when testing for a different serotype, as recently reported for serotypes 2 and 1/2 (Chatellier et al., 1999; Martinez et al., 2002). In this regard, the RAPD for S. suis serotype 5 was standardized in this study. Surprisingly, up to six different genotypic patterns of the same serotype could be observed in carrier sows. A considerably higher heterogeneity was observed among the lower number of isolates recovered from nasal cavities compared to those from vaginal origin. Only one clone (clone D) was associated with the clinical cases, as all isolates recovered from diseased or dead animals (previously and during the study) belonged to this genotype. In this regard, the molecular epidemiology of the serotype 5 infection in this herd seems to be similar to that already described for serotype 2. Indeed, it was previously reported for the latter serotype that, in closed infected herds, a single clone of S. suis was responsible for the disease (Mogollon et al., 1991; Reams et al., 1996; Torremorell and Pijoan, 1998; Torremorell et al., 1998; Martinez et al., 2002). The situation for other serotypes, such as serotype 1/2, may be different, as recently reported (Martinez et al., 2002). Distinctive features of this particular Pattern D that may explain its increased virulence are not known. Interestingly, Pattern D was not isolated from the eight sows included in this study, probably due to its very low prevalence. It has been previously hypothesized that strains of S. suis serotype 2 isolated from the upper respiratory tract are part of the normal flora and tend to be very heterogeneous, even in closed populations (Mogollon et al., 1991; Reams et al., 1996; Torremorell and Pijoan, 1998; Torremorell et al., 1998; Martinez et al., 2002), whereas, in contrast, pathogenic strains are found at a very low prevalence in healthy carrier animals (Torremorell and Pijoan, 1998). It has already been suggested that this low prevalence could be a predisposing factor for the latter onset of disease in a herd (Pijoan, 1995). The situation may be similar for S. suis serotype 5 for the herd participating in the present study. Although a low prevalence for the Pattern D is an interesting hypothesis, the possibility that this genotype might be present as a minor population among other serotype 5 genotypes in the same animal cannot be excluded, since in some cases, a single sow may carry different serotype 5 strains. If the genotype D is present in lower concentrations than other serotype 5 genotypes, the probability of isolating it would be lower, and results may be biased. The genotype D could be detected neither in sows nor in piglets 1 year later in the absence of clinical signs. A vertical transmission of S. suis serotype 5 isolates could be observed in this study. In general, similar genotypes were found in vaginal cavities of the sows and on nasal surfaces of 15% of the piglets. Vertical transmission was also supported by other studies (Robertson et al., 1991; Reed et al., 1998). In this study, Pattern B was isolated from the nasal surface of some piglets but not from sows. It might be concluded that this pattern was in fact present in the vagina of sows, but we failed to isolate it prior to or at farrowing. A real colonization of
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the oropharynx could not be demonstrated in our study, since S. suis serotype 5 could not be detected 1 week after birth in the nasal cavities of the piglets. However, it may be assumed that animals were colonized when they swallowed vaginal secretions from the sows but bacteria were mainly located at the tonsils that were not sampled in this study. Indeed, 24% of the tonsils collected cultured positive for S. suis serotype 5 at slaughter. In general, the proportion of detected carrier piglets was low, especially during the weeks where no clinical disease was present. This is not necessarily an indication of the absence of S. suis serotype 5, since bacteria were probably present at the tonsils. It may rather indicate a slower transmission of the infection due to the lack of clinical illness. Although the Pattern D was isolated from only two piglets at 2 weeks of age, it was the only genotype isolated from carrier animals from the time of the manifestation of clinical signs until the end of the experiment. This would suggest a higher number of bacteria in the environment that would increase transmission either by aerosol or direct contact. Furthermore, most isolates recovered from tonsils of these animals at slaughter were also of genotype D. In the absence of S. suis-associated disease in the herd (1 year later), no isolation of S. suis serotype 5 from piglets could be achieved. Interestingly, the rate of isolation of S. suis at slaughter at that moment was similar to that obtained in the previous year, in the presence of clinical signs. However, the genotype D could not be detected in the absence of disease, and all isolates from tonsils belonged to the genotype C. It is not clear whether this is due to either its real absence from the herd or a too low aerosol transmission. Results obtained by serological examination showed that concentration of antibodies against S. suis serotype 5 were high in sows, suggesting that animals had already been in contact with S. suis. The concentration of maternal antibodies detected at 24 h in piglets was similar to those detected in sows; however, a high individual variation among animals was observed, even within the same litter (data not shown). This may indicate a variation in intestinal absorption of maternal IgG or different levels of colostrum intake. Results from this study showed that clinical signs appeared when maternal immunity was at its lowest level, which is in agreement with other studies (Torremorell et al., 1998; Lapointe et al., 2002). However, the sole absence of maternal antibodies does not necessarily trigger the clinical disease, since similar low amount of antibodies were also detected 1 year later in piglets at 4 weeks of age and older, in the complete absence of disease associated with S. suis. In the presence of clinical cases (first year), an active antibody response to S. suis serotype 5 infection could be observed. Some animals produced high levels of antibodies whereas others did not produce any detectable levels of antibodies against S. suis. This confirms the fact that the humoral response to S. suis infections and vaccination is usually erratic (Blouin et al., 1994; del Campo Sepulveda et al., 1996; Lapointe et al., 2002). As expected, in the absence of clinical signs, no marked antibody level increase was observed in pigs. The disappearance of clinical signs in the nursery pigs 1 year later, with no isolation of S. suis from diseased animals, is difficult to explain. No major changes in the management of the farm or vaccination program had been implemented. For both years, the carrier rate for S. suis serotype 5 in adult animals was similar. It might be hypothesized that the virulent strain was no longer present in the herd, but no explanation for such an event could be found. Since the rate of isolation of Pattern D from sows in the presence of clinical signs in the nursery pigs was extremely low (one sow), the negative result obtained 1 year later may
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only reflect this very low prevalence. Moreover, the absence of this genotype from tonsils of pigs at slaughter may be the consequence of a low transmission due to the lack of disease. For several infectious diseases, the overall level of immunity of a herd and the homogeneity among individuals are important factors in the development of clinical signs. However, in our study, the level of antibodies in sows and piglets at birth was slightly lower in absence of clinical signs and the variation in antibodies was similar during the two periods. In conclusion, serotypes other than serotype 2 of S. suis can cause severe clinical signs. Different strains of the same serotype of S. suis may be present in a herd. However, only one of these strains may be responsible for clinical signs. Hence, genotypic characterization (by the use of RAPD or other genotyping methods) is indispensable to understand the epidemiology of the infection. Although piglets were infected at birth by strains present in the vaginal tract of the sows, we were unable to determine whether the virulent strain was transmitted from the sows to their offspring at birth. Additional studies should be carried out to investigate the vertical transmission of the virulent strain. It appears that nasal cavities are not the most appropriate site for studying the asymptomatic carriage of S. suis, since positive results are mainly observed in presence of clinical signs when there is active transmission of the infection.
Acknowledgements The authors want to thank Julie-Mélanie Trudel, Marcelo Ribotta, Marcel Roy, Avila Croisetière and Guy Beauchamp for their excellent technical assistance. This study was supported by the Conseil de Recherche en Pˆeche et en Agroalimentaire du Québec grant # 4778 and by the Canadian Research Network on Bacterial Diseases of Swine.
References Allgaier, A., Goethe, R., Wisselink, H.J., Smith, H.E., Valentin-Weigand, P., 2001. Relatedness of Streptococcus suis isolates of various serotypes and clinical background as evaluated by macrorestriction analysis and expression of potential virulence traits. J. Clin. Microbiol. 39, 445–453. Amass, S.F., Clark, L.K., Wu, C.C., 1995. Source of timing of Streptococcus suis infection in neonatal pigs: implications for early weaning procedures. Swine Health Prod. 3, 189–193. Amass, S.F., Clark, L.K., Knox, K.E., Wu, C.C., Hill, M.A., 1996. Streptococcus suis colonization of piglets during parturition. Swine Health Prod. 4, 269–272. Amass, S.F., SanMiguel, P., Clark, L.K., 1997. Demonstration of vertical transmission of Streptococcus suis in swine by genomic fingerprinting. J. Clin. Microbiol. 35, 1595–1959. Beaudoin, M., Harel, J., Higgins, R., Gottschalk, M., Frenette, M., MacInnes, J.I., 1992. Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS–PAGE and by hybridization with an rDNA probe. J. Gen. Microbiol. 138, 2639–2645. Berthelot-Hérault, F., Gottschalk, M., Labbé, A., Cariolet, R., Kobisch, M., 2001. Experimental airborne transmission of Streptococcus suis capsular type 2 in pigs. Vet. Microbiol. 82, 69–80. Berthelot-Hérault, F., Marois, C., Gottschalk, M., Kobisch, M., 2002. Genetic diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis. J. Clin. Microbiol. 40, 615–619. Blouin, C., Higgins, R., Gottschalk, M., Simard, J., 1994. Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich ELISA. Can. J. Vet. Res. 58, 49–54.
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Brisebois, L.M., Charlebois, R., Higgins, R., Nadeau, M., 1990. Prevalence of Streptococcus suis in four to eight week old clinically healthy piglets. Can. J. Vet. Res. 54, 174–177. Chatellier, S., Gottschalk, M., Higgins, R., Brousseau, R., Harel, J., 1999. Relatedness of Streptococcus suis serotype 2 isolates from different geographic origins as evaluated by molecular fingerprinting and phenotyping. J. Clin. Microbiol. 37, 362–366. Dee, S.A., Carlson, A.R., Winkelman, N.L., Corey, M.M., 1993. Effect of management practices on the Streptococcus suis carrier rate in nursery swine. J. Am. Vet. Med. Assoc. 203, 295–299. del Campo Sepulveda, E.M., Altman, E., Kobisch, M., D’Allaire, S., Gottschalk, M., 1996. Detection of antibodies against Streptococcus suis capsular type 2 using a purified capsular polysaccharide antigen-based indirect ELISA. Vet. Microbiol. 52, 113–125. Devriese, L.A., Ceyssens, K., Hommez, J., Kilpper-Balz, R., Schleifer, K.H., 1991. Characteristics of different Streptococcus suis serovars and description of a simplified identification method. Vet. Microbiol. 26, 141–150. Gottschalk, M., Segura, M., 2000. The pathogenesis of the meningitis caused by Streptococcus suis: the unresolved questions. Vet. Microbiol. 76, 259–272. Gottschalk, M., Higgins, R., Boudreau, M., 1993. Use of polyvalent coagglutination reagents for serotyping of Streptococcus suis. J. Clin. Microbiol. 31, 2192–2194. Gottschalk, M., Lacouture, S., Odierno, L., 1999. Immunomagnetic isolation of Streptococcus suis serotypes 2 and 1/2 from swine tonsils. J. Clin. Microbiol. 37, 2877–2881. Gottschalk, M., Kobisch, M., Berthelot-Hérault, F., 2001. L’infection à Streptococcus suis chez le porc. J. Rech. Porcine France 33, 269–276. Hampson, D.J., Trott, D.J., Clarke, I.L., Mwaniki, C.G., Robertson, I.D., 1993. Population structure of Australian isolates of Streptococcus suis. J. Clin. Microbiol. 31, 2895–2900. Higgins, R., Gottschalk, M., 1990. An update on Streptococcus suis identification. J. Vet. Diagn. Investig. 2, 249–252. Higgins, R., Gottschalk, M., 1999. Streptococcal diseases. In: Straw, B., D’Allaire, S., Mengeling, W., Taylor, D. (Eds.), Diseases of Swine, eighth ed. Iowa State University Press, Ames, IA, pp. 563–578. Higgins, R., Gottschalk, M., 2000. Distribution of Streptococcus suis capsular types in 1999. Can. Vet. J. 41, 414. Higgins, R., Gottschalk, M., 2001. Distribution of Streptococcus suis capsular types in 2000. Can. Vet. J. 42, 223. Lapointe, L., D’Allaire, S., Lebrun, A., Lacouture, S., Gottschalk, M., 2002. Antibody response to an autogenous vaccine and serological profile for Streptococcus suis capsular type 1/2. Can. J. Vet. Res. 66, 8–14. Markwell, M.A., Haas, S.M., Bieber, L.L., Tolbert, N.E., 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 206–210. Martinez, G., Harel, J., Lacouture, S., Gottschalk, M., 2002. Genetic diversity of Streptococcus suis serotypes 2 and 1/2 isolates recovered from carrier pigs in closed herds. Can. J. Vet. Res. 66, 240–248. Mogollon, J.D., Pijoan, C., Murtaugh, M.P., Kaplan, E.L., Collins, J.E., Cleary, P.P., 1990. Characterization of prototype and clinically defined strains of Streptococcus suis by genomic fingerprinting. J. Clin. Microbiol. 28, 2462–2466. Mogollon, J.D., Pijoan, C., Murtaugh, M.P., Collins, J.E., Cleary, P.P., 1991. Identification of epidemic strains of Streptococcus suis by genomic fingerprinting. J. Clin. Microbiol. 29, 782–787. Monter Flores, J.L., Higgins, R., D’Allaire, S., Charette, R., Boudreau, M., Gottschalk, M., 1993. Distribution of the different capsular types of Streptococcus suis in 19 swine nurseries. Can. Vet. J. 34, 170–171. Mwaniki, C.G., Robertson, I.D., Trott, D.J., Atyeo, R.F., Lee, B.J., Hampson, D.J., 1994. Clonal analysis and virulence of Australian isolates of Streptococcus suis type 2. Epidemiol. Infect. 113, 321–334. Okwumabua, O., Staats, J., Chengappa, M.M., 1995. Detection of genomic heterogeneity in Streptococcus suis isolates by DNA restriction fragment length polymorphisms of rRNA genes (ribotyping). J. Clin. Microbiol. 33, 968–972. Pijoan, C., 1995. Diseases of high health pigs: some ideas on pathogenesis. In: Proceedings of A.D. Leman Conference, St. Paul, Minnesota, USA, pp. 16–17. Pitcher, D.G., Saunders, N.A., Owen, R.J., 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151–156. Reams, R.Y., Harrington, D.D., Glickman, L.T., Thacker, H.L., Bowersock, T.L., 1996. Multiple serotypes and strains of Streptococcus suis in naturally infected swine herds. J. Vet. Diagn. Investig. 8, 119–121. Reed, A.J., Amass, S.F., Stevenson, G.W., 1998. Evidence of vertical transmission of pathogenic Streptococcus suis resulting in meningitis in nursery pig. In: Proceedings of the 15th International Pig Veterinary Society Congress, Birmingham, UK, pp. 5–9.
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Robertson, I.D., Blackmore, D.K., Hampson, D.J., Fu, Z.F., 1991. A longitudinal study of natural infection of piglets with Streptococcus suis types 1 and 2. Epidemiol. Infect. 107, 119–126. Staats, J.J., Brandon, L., Plattner, B.L., Nietfeld, J., Dritz, S., Chengappa, M.M., 1998. Use of ribotyping and hemolysin activity to identify highly virulent Streptococcus suis type 2 isolates. J. Clin. Microbiol. 36, 15–19. Torremorell, M., Pijoan, C., 1998. Prolonged persistence of an epidemic Streptococcus suis strain in a closed pig population. Vet. Rec. 143, 394–395. Torremorell, M., Calsamiglia, M., Pijoan, C., 1998. Colonization of suckling pigs by Streptococcus suis with particular reference to pathogenic serotype 2 strains. Can. J. Vet. Res. 62, 21–26.
Epidemiology of Streptococcus suis serotype 5 infection in a pig herd with and without clinical disease G. Cloutier a , S. D’Allaire a , G. Martinez a , C. Surprenant b , S. Lacouture a , M. Gottschalk a,∗ a
Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de pathologie et microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, St-Hyacinthe, Que., Canada J2S 7C6 b F. Ménard Inc., 251 Route 235, Ange Gardien, Que., Canada J0E 1E0 Received 1 April 2003; received in revised form 26 August 2003; accepted 25 September 2003
Abstract The aim of this study was to describe the transmission and the kinetics of the infection caused by Streptococcus suis serotype 5 in a multisite farrow-to-finish pig herd. Most sows carried S. suis serotype 5 in their vaginal tract, but not in their nasal cavities, as demonstrated by immunomagnetic separation (IMS) technique. Their offspring became infected during farrowing, confirming vertical transmission. During the first 4 weeks of life, a low number of piglets were carriers of S. suis serotype 5 in their nasal cavities. However, when clinical signs appeared, the carrier rate significantly increased, suggesting that isolation from nasal cavities is a better indication of active transmission than of a carrier state. Clinical cases were present in animals between 4 and 8 weeks of age, when maternal antibodies were at their lowest level. Up to six different genotypes of the same serotype could be identified by random amplified polymorphic DNA; however, a single clone was responsible for all clinical cases studied. This clone could only be isolated from a single sow, indicating that its prevalence in breeding animals was low. Interestingly, 1 year later, clinical disease associated with S. suis serotype 5 spontaneously disappeared. At that time, the genotype responsible for the clinical signs was not detected in the herd and the levels of antibodies in sows and maternal antibodies in piglets were not higher than those of the previous year. © 2003 Elsevier B.V. All rights reserved. Keywords: Streptococcus suis; Vertical transmission; Maternal antibodies; Genotypes
∗
Corresponding author. Tel.: +1-450-773-8521x8374; fax: +1-450-778-8108. E-mail address: [email protected] (M. Gottschalk). 0378-1135/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2003.09.018
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1. Introduction Streptococcus suis is one of the most important swine pathogens worldwide. It can cause septicemia, meningitis, arthritis, endocarditis, polyserositis and pneumonia (Higgins and Gottschalk, 1999). Mortality due to S. suis is frequently observed 2 to 4 weeks after weaning; however, a herd often has its own age profile. In some herds, severe clinical disease which has lasted for a time may disappear without any known reason (Mogollon et al., 1991; Lapointe et al., 2002). A total of 35 capsular types have been described so far (Higgins and Gottschalk, 1999), with serotype 2 being the most frequently recovered from diseased pigs in most countries (Higgins and Gottschalk, 2000). However, other serotypes can also be associated with severe disease (Reams et al., 1996; Higgins and Gottschalk, 2000). In fact, the percentage of serotype 2 strains isolated from diseased animals in Canada has decreased in recent years (Higgins and Gottschalk, 2001). In addition, several genotypes within the same serotype have been reported using different methods such as restriction endonuclease analysis (Mogollon et al., 1990; Beaudoin et al., 1992), ribotyping (Okwumabua et al., 1995; Staats et al., 1998), multilocus enzyme electrophoresis (Hampson et al., 1993; Mwaniki et al., 1994), macrorestriction analysis (Allgaier et al., 2001; Berthelot-Hérault et al., 2002), and randomly amplified polymorphic DNA (RAPD) (Chatellier et al., 1999; Martinez et al., 2002). Although different mechanisms of S. suis transmission have been investigated, the most important features of the epidemiology of the infection remain unclear. It is well known that most pigs are carriers of multiple serotypes in their upper respiratory tract (Monter Flores et al., 1993; Higgins and Gottschalk, 1999) and that sows can harbour different S. suis in their vaginal secretions (Amass et al., 1996). Failure to eliminate the infection in medicated early weaned piglets led to the hypothesis that animals are colonized very early in their life and that dam-to-pig transmission would be important. Although early data suggested a lack of vertical transmission (Mogollon et al., 1991), contamination of piglets during their transit through the vaginal canal has been demonstrated (Amass et al., 1995, 1996, 1997). It has been proposed that caesarean section is probably the only method that can be used to produce S. suis-free animals (Amass et al., 1996). In addition to the vertical transmission, direct and indirect contamination of animals has also been demonstrated (Robertson et al., 1991; Dee et al., 1993; Higgins and Gottschalk, 1999) as well as airborne transmission of S. suis over a distance of at least 40 cm, without nose-to-nose contact (Berthelot-Hérault et al., 2001). All studies demonstrating vertical transmission of S. suis were carried out in herds where no clinical signs associated with S. suis infection were present. In addition, bacterial isolation was carried out using standard bacterial isolation, the only technique available at the time of these studies. Isolation of specific serotypes of S. suis from carrier animals using this technique offers a very low sensitivity when compared to the serotype-specific immunomagnetic separation (IMS) technique recently described (Gottschalk et al., 1999). In the present work, we studied the vertical transmission of S. suis serotype 5 in a clinically affected herd, using the IMS technique. Levels of antibodies against S. suis serotype 5 in sows and piglets were evaluated. In addition, the presence of different genotypes within this same serotype was investigated. Results were compared to those obtained 1 year later from the same herd, following the disappearance of clinical signs due to S. suis.
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2. Material and methods 2.1. Herd selection A multisite farrow-to-finish herd of 2400 sows with high level of mortality associated with S. suis in the nurseries was selected. This PRRS-free herd was a newly established farm with an average weaning age of 16 days. Most of the mortalities occurred between 5 and 8 weeks of age and were attributed to S. suis meningitis. The second part of the study was carried out on the same herd one year later, following the disappearance of clinical signs due to S. suis. Vaccines against S. suis infections were never used in this herd. 2.2. Sampling To confirm that the pigs in the nursery stage were affected mainly by a single serotype of S. suis, a total of 30 pigs with signs of meningitis were examined post-mortem over a 2-week period. Swab samples in transport medium (Culturette, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) were collected from the brain immediately after death and sent to the laboratory to be cultured within the same day. In the first part of the study (in the presence of clinical signs), a total of 40 sows were sampled 2 weeks before parturition. Blood samples and nasal and vaginal swabs were collected to establish their carrier status regarding S. suis serotype 5. A total of eight sows (six positive and two negative for S. suis serotype 5 isolation) as well as their respective offspring (94 piglets) were further studied. Vaginal swabs of these sows as well as swabs of the nasal surface of their piglets were taken at parturition. Nasal swabs were taken from all piglets once a week until 14 weeks of age. Blood samples from piglets were taken 24 h after birth and once every other week until 14 weeks of age. Tonsils were collected from 42 of these animals at slaughter. Piglets included in this study that died during the follow-up period were necropsied and tissue samples (brain, liver, spleen, tonsils) were sent for bacteriological examination (Higgins and Gottschalk, 1990; Devriese et al., 1991). No antibiotic treatment was allowed for the control of S. suis infections during the study. In the second part of the study (one year later, in the absence of clinical signs), vaginal swabs were collected from 40 gestating sows to verify their status regarding S. suis serotype 5. In addition, 10 sows were sampled (vaginal swabs and blood) at parturition and blood samples were taken from 20 piglets 24 h after birth (two piglets per sow). Cross-sectional blood samples of 20 pigs were obtained from each of the following age groups: 2, 4, 6, 8, 10, 12 and 14 weeks of age. In addition, nasal swabs were taken from 20 piglets at 4, 6 and 8 weeks of age, which corresponded to the age at which isolation from carrier piglets was maximal a year before, in the presence of clinical signs (see results). Finally, a total of 60 tonsils were collected at slaughterhouse. 2.3. Bacterial isolation Bacterial isolation and serotyping of S. suis isolated from diseased or dead animals were carried out as described elsewhere (Higgins and Gottschalk, 1990). Isolation of S. suis serotype 5 from carrier animals was performed using the IMS technique previously
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reported for selective isolation of S. suis serotypes 2 and 1/2 from tonsils (Gottschalk et al., 1999). The technique was adapted for isolation of S. suis serotype 5 from swabs. Production of rabbit polyclonal antibodies against serotype 5 of S. suis was carried out as previously described (Higgins and Gottschalk, 1990). Immunoglobulins G (IgG) fractions were purified by using a protein A column and the protein concentration was measured as described previously (Markwell et al., 1978). Coating of beads (Dynabeads M-280; Dynal, Oslo, Norway) was carried out as described by Gottschalk et al. (1999). Swabs were put into suspension in 1 ml of phosphate-buffered saline (PBS) with 0.1% of bovine serum albumin (BSA) (PBS-0.1%–BSA). A volume of 40 l of IgG-coated beads was added to 1 ml of each suspension. After incubation and two washings of 10 min each in PBS-0.5% Tween, (PBS-T20; Sigma Chemical Co., St. Louis, MO, USA), the immunomagnetic beads were plated on blood agar plates supplemented with the selective reagent SR-126E (Oxoid Canada, Nepean, Ont., Canada) (Monter Flores et al., 1993). After 24 h of incubation at 37 ◦ C, with 5% of CO2 , a maximum of five 0.5–1.0 mm flat, alpha-hemolytic colonies were selected from each sample for further characterization. All selected isolates underwent coagglutination testing using serum against serotype 5, as well as a negative serum (negative control) (Gottschalk et al., 1993). Isolates of serotype 5 were biochemically confirmed as being S. suis using standard procedures (Higgins and Gottschalk, 1990). Isolation of S. suis serotype 5 from tonsils was also carried out by the IMS technique (Gottschalk et al., 1999). When available, a minimum of two isolates of S. suis serotype 5 from the same animal were kept for further studies. All isolates were stored at −80 ◦ C until being studied. 2.4. Serologic evaluation An indirect ELISA, adapted for S. suis serotype 5, was developed in this study, using a methodology similar to that recently described (Lapointe et al., 2002). A representative clinical isolate (isolate 4B) recovered from a case of meningitis in the same herd was used as the source of antigen. This isolate was first inoculated on sheep blood agar, incubated for 24 h at 37 ◦ C, then inoculated into 5 ml of Todd-Hewitt broth (Difco Laboratories, Detroit, MI, USA) and incubated for 18 h at 37 ◦ C. Subsequently, bacteria were lysed by sonication by seven pulse cycles of 5 min each, at 80% duty cycle (Sonics & Materials, New Town, CT, USA). After sonication, unlysed cells were removed by centrifugation at 3000 × g for 30 min. The supernatant was collected and dialysed for 24 h in filtered water with a porous regenerated cellulose membrane with a molecular weight cut-off of 12,000–14,000 (Spectrum Medical Industries, Houston, TX, USA). The solution was stored at −80 ◦ C until used. The antigen was suspended in a carbonate buffer (pH 9.6) at a standardized final concentration of 15 g/ml of protein. Each well of flat-bottomed plates (Polysorb, Nunc-Immunoplates, Rochester, NY, USA) was coated with 100 l of the antigen solution to give a final concentration of 1.5 g of protein/well. The plates were incubated overnight at 4 ◦ C, drained and washed three times with PBS containing 0.05% Tween 20 (PBS-T20; Sigma Chemical Co.). Sera from piglets were tested at a dilution of 1/1600 which was established as the best dilution that allowed differentiation between positive and negative sera (data not shown). Sera were added in 100 l amounts to appropriate wells (in duplicate, on the same plate). The positive control was obtained by repetitive vaccination of a piglet with an autogenous
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bacterin containing the isolate 4B used for the production of the antigen. The negative control was a serum of an animal obtained from a herd that did not present any clinical signs related to S. suis during at least the two previous years. After 1 h incubation, plates were washed three times with PBS-T20 and then 100 l of peroxydase-conjugated goat anti-swine IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA) were added to each well for 1 h. Plates were washed and 100 l of the substrate 3,3 ,5,5 -tetramethylbenzidine (TM Blue, Intergen, St. Milford, MA, USA) were added to each well, and the blue color was allowed to develop at room temperature. The enzyme reaction was stopped with the addition of 50 l/well of 1 N H2 SO4 . The absorbance was read at 450 nm with an ELISA plate reader (UVmax, Molecular Devices, Menlo Park, CA, USA). Results were reported as S/P ratios which are the optical density obtained for serum divided by the optical density of the positive control. Each serum sample was tested at least three times. The average optical density obtained was only accepted when the coefficient of variation was below 15%; otherwise, the serum was tested again. In addition, plates were only accepted when sera from positive and negative controls presented an optical density of 1.0 ± 0.15 and 0.2 ± 0.10, respectively. 2.5. Statistical analyses Mixed linear models for repeated measures with litter treated as a random effect were used to evaluate the effect of age and status of the sow on the S/P ratios of piglets. Post-hoc contrasts were performed with the Tukey–Kramer test to determine differences between pairs of sampling points. For the study of the second year in which a cross-sectional sampling was used, the effect of age on S/P ratios was evaluated with a one-way analysis of variance (ANOVA). For comparison of S/P ratios obtained in the first and second year, a two-way ANOVA with year, time and the interaction between them was used. For this latter analysis, two piglets per litter were randomly selected at each sampling point for each of the eight sows involved in the study of the first year. Homogeneity of variance of the S/P ratios of sows and of piglets at birth and at 2 weeks of age was tested by a modified-Levene equal variance test. Level for statistical significance was set at 0.05. 2.6. Randomly amplified polymorphic DNA (RAPD) fingerprinting RAPD for S. suis serotype 5 was performed based on the technique previously described (Chatellier et al., 1999; Martinez et al., 2002). Since primers used for serotypes 2 and 1/2 strains did not clearly differentiate serotype 5 strains (data not shown), the standardization of RAPD for this serotype was carried out in our study. Twenty-seven primers of 10 nucleotides in length were randomly chosen (Table 1) to amplify the DNA of 14 epidemiologically not-related S. suis serotype 5 strains. Ten of these strains, including the isolate 4B, were from unrelated herds in Canada; three strains (A-6165, B-92/97 and B-26/22) originated from Germany (kindly provided by Dr. Peter Valentin-Weigand, University of Hannover) and presented different genotypes as previously tested by pulsed-field gel electrophoresis (Allgaier et al., 2001); the remaining strain originated from The Netherlands (reference strain 11538). The PCR mixture consisted of buffer (10 mM Tris–HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl2 ), 100 M each of the four deoxynucleotide triphosphates (Amersham
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Table 1 List of primers tested by RAPD for the study of Streptococcus suis serotype 5 isolates Primers
Sequence, 5 –3
GC (%)
OPB-01 OPB-02 OPB-03 OPB-04 OPB-05 OPB-06∗ OPB-07 OPB-08 OPB-09 OPB-10∗ OPB-11∗ OPB-12 OPB-13 OPB-14 OPB-15 OPB-16 OPB-17 OPB-18 OPB-19 OPB-20 Primer 1 Primer 2 Primer 3 Primer 4 OPS-11 OPS-16 CLAU
GTT TCG CTC C TGA TCC CTG G CAT CCC CCT G GGA CTG GAG T TGC GCC CTT C TGC TCT GCC C GGT GAC GCA G GTC CAC ACG G TGG GGG ACT C CTG CTG GGA C GTA GAC CCG T CCT TGA CGC A TTC CCC CGC T TCC GCT CTG G GGA GGG TGT T TTT GCC CGG A AGG GAA CGA G CCA CAG CAG T ACC CCC GAA G GGA CCC TTA C TCA CGA TGC A ACG TAT CTG C GTG ACG TAG G GCA TCA ATC T AGT CGG GTG G AGG GGG TTC C AAA GGA TGC T
60 60 70 60 70 70 70 70 70 70 60 60 70 70 60 60 60 60 70 60 50 50 60 40 70 70 40
Primers selected to perform the analysis are followed by an asterisk (*).
Biosciences Inc., Baie d’Urfé, Que., Canada), 1.5 M primer, 30 ng of DNA extracted and purified as described (Pitcher et al., 1989) and 2.5 U of Taq DNA polymerase (Amersham Biosciences Inc.) in a total volume of 25 l. Primers used were synthesized by Invitrogen (Burlington, Ont., Canada). Each sample was subjected to an initial cycle of denaturation (5 min at 94 ◦ C) in a DNA Thermal Cycler 480 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). Each of the 35 subsequent cycles consisted of denaturing at 94 ◦ C for 30 s, annealing at 35 ◦ C for 30 s, and extension at 72 ◦ C for 1 min. The last cycle included an extension at 72 ◦ C for 5 min. Amplified products were separated by electrophoresis in a 1.2% agarose gel (Sigma–Aldrich, Oakville, Ont., Canada) and were visualized by UV transillumination following ethidium bromide staining. A 1 kb DNA ladder (Invitrogen) was used in each gel as a molecular size standard, and a negative control, consisting of the same reaction mixture but with water instead of template DNA, was included in each run. When testing field isolates, a positive control, containing the same reaction mixture and template DNA from the strain 4B was also included. A total of 350 individual field isolates recovered from the study were tested under identical conditions at least twice with the three selected primers. Photographs of each gel were digitized with a video camera connected to
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a microcomputer (Alpha EaseTM ; Alpha Innotech Corporation, San Leandro, CA, USA). The genetic diversity was studied by comparing the different patterns among all the tested isolates of the study.
3. Results 3.1. Determination of the serotype of S. suis responsible for the mortality in the nursery Pure cultures of S. suis serotype 5 were isolated from the meninges of 23 out of 30 piglets that died indicating that this serotype was the main pathogen associated with meningitis in the nursery pigs of this farm. These 23 strains were studied by RAPD and only one clone was demonstrated to be responsible for these clinical signs (see the following sections). 3.2. Study in the presence of clinical signs 3.2.1. Bacterial isolation In the first part of the study, of the 40 sows that were pre-selected, 43 and 28% were positive for S. suis serotype 5 isolation from vaginal and nasal cavities, respectively. Nasal cavities of sows were highly contaminated with other streptococci, making difficult the isolation of S. suis serotype 5 even though IMS was used. A lower number of investigated colonies from nasal swabs (36 out of 529: 6.8%) were positive for S. suis serotype 5, when compared to vaginal swabs (147 out of 441: 33.3%). Eight sows (six positive for vaginal isolation and two negative for isolation from both sites) were then selected to further study the pattern of infection in their respective offspring. A total of 94 piglets born from the eight sows were included in the study. At birth, 15% of the piglets from positive sows were positive for S. suis serotype 5 isolation at the nasal surface, but these animals were negative at 1 week of age. Some of them (2.8%) became positive 1 week later (2 weeks of age). No piglets from the negative sows were carriers of the bacterium at birth, but 15% of them became carriers at 1 week of age. The level of nasal carriage of S. suis serotype 5 by piglets was low (less than 15%) until week 6 (Fig. 1). The isolation rate reached 35% between weeks 5 and 8, a period corresponding to the presence of clinical signs due to S. suis in the nursery (Figs. 1 and 2). A total of 20 animals (21%) died during the experiment, and S. suis serotype 5 could be isolated in pure culture or as a predominant bacterium in 11 of them (55%). After week 9, the isolation rate from nasal cavities dropped again to low values (Fig. 1). Ten of the 42 tonsils (24%) collected at slaughterhouse were positive for S. suis serotype 5 isolation. 3.2.2. Genotyping of S. suis serotype 5 isolates by RAPD Among the 27 different primers tested with total DNA from 14 unrelated S. suis serotype 5 isolates, three primers named OPB-06, OPB-10 and OPB-11 were selected (Table 1) because they gave different and reproducible patterns containing fragments with large size range and a small number of low-intensity bands. These primers were used to study more
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50% 45%
Positive animals (%)
35% 30% 25% 20% 15% 10% 5% 0% 0
1
2
3
4
5
6
7
8
9
10
Age (weeks) Fig. 1. Isolation of Streptococcus suis serotype 5 from nasal cavities of carrier piglets using the immunomagnetic separation technique. (䊊), Piglets from S. suis serotype 5 carrier sows; (䊏), piglets from S. suis serotype 5 negative sows; (䉱), mean.
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40%
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Fig. 2. Mortality due to Streptococcus suis serotype 5 according to the age of dead piglets.
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Fig. 3. Illustration of RAPD patterns generated with primers OPB 06, OPB 10 and OPB 11 from different strains of Streptococcus suis serotype 5. Lane 1: Pattern A; Lane 2: Pattern B; Lane 3: Pattern C; Lane 4: Pattern D (strain responsible for clinical cases); Lane 5: Pattern E; Lane 6: Pattern F.
than 350 selected isolates recovered in this study. Representative isolates from each animal were included (at least two isolates per animal when available). A total of six RAPD patterns (Patterns A–F), each composed of three to seven bands with sizes of between 0.5 and 3 kbp, could be differentiated using the three primers (Fig. 3). Only the Pattern D was recovered from diseased animals throughout the study. The 22 vaginal isolates recovered from positive sows were assigned to two RAPD patterns (Patterns A and C), whereas the 25 isolates from nasal cavities of these animals presented five different patterns (Patterns A, C, D, E, and F). The Pattern D was only observed in one nasal isolate of a single sow. However, this sow was not included among the eight selected for the study. Patterns A, B and C were detected in the piglets at farrowing. On the other hand, in presence of clinical signs (weeks 4–8), all the piglets that were carriers presented
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Fig. 4. Distribution of the different RAPD patterns of Streptococcus suis serotype 5 isolates recovered from piglets, according to their age. (䊐), Pattern A; ( ), Pattern B; ( ), Pattern C; (䊏), Pattern D.
the Pattern D (Fig. 4). At slaughter, 7 of 10 positive animals were carriers of the Pattern D. The remaining isolates were related to Pattern A (two isolates) and C (one isolate). 3.2.3. Serology The average level of antibodies against S. suis serotype 5 in the 40 sows was high (mean S/P ratio = 1.169). After birth, a heterogeneous amount of maternal antibodies was observed in the piglets (Fig. 5); however, there was no significant difference between piglets from positive or negative sows (1.16 versus 1.21). The maternal immunity declined and reached the lowest level at weeks 4 and 6. Then, the level of antibodies increased until week 14, as a result of active immunity to infection. This immune response was also very heterogeneous, with some animals producing high level of antibodies and others not presenting any detectable antibodies to S. suis serotype 5. Most of the mortalities occurred at the time when the antibodies were at their lowest level. 3.3. Study in the absence of clinical signs One year later, following the spontaneous disappearance of clinical disease related to S. suis infection in this herd, 40 sows were tested for their carriage of S. suis serotype 5 at the vaginal level. Similarly to the previous year, 53% of them were positive. Nasal swabs of 20 piglets per age group were taken at 4, 6 and 8 weeks of age, corresponding to the high prevalence of nasal carriage in the previous study. However, no piglets were positive at
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1.8 Average S/P ratio
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
2
4
6 8 Age (weeks)
10
12
14
Fig. 5. Kinetics of antibodies against Streptococcus suis serotype 5 according to the age of the piglets in the presence (䊉) or absence (䊊) of clinical signs.
isolation by the IMS technique during the second period of the study. On the other hand, 32% of the 60 tonsils collected at slaughterhouse were positive for S. suis serotype 5 isolation. RAPD analysis of these isolates revealed that a single pattern (Pattern C) was present. In absence of clinical signs in the herd, sows did not present a higher level of antibodies when compared to the previous year (0.929 versus 1.169). The level of maternal antibodies of the piglets at birth and 2 weeks of age were also lower than those of the previous year (Fig. 5). Moreover, an active immune response in the absence of clinical signs could not be detected.
4. Discussion The herd included in this study was affected by a single serotype of S. suis which was considered as the primary pathogen responsible for most clinical cases observed. In this PRRSV-free herd, more than 75% of the samples received from dead animals in the post-weaning section (during the herd selection study) and more than 50% of the samples during the transmission study were attributed to S. suis serotype 5. Other samples were either completely negative or too contaminated (different bacterial species retrieved in the same sample with no main pathogen isolated). Sudden death caused by S. suis without bacterial isolation is sometimes observed, as death may be the result of a septic shock caused by the up-regulation of pro-inflammatory cytokines (Gottschalk and Segura, 2000). A high percentage of sows were infected by S. suis serotype 5, independent of the presence (first year) or absence (second year) of clinical signs in post-weaned piglets. A higher level of carriage was identified in vaginal than in nasal cavities from sows. There are two possible explanations for that observation. First, and although the nasal cavities have been extensively used as sampling sites for S. suis isolation (Brisebois et al., 1990; Dee et al., 1993; Monter Flores et al., 1993), it is possible that bacteria are mainly located at the tonsils rather than in the nostrils in the upper respiratory tract of carrier pigs (Gottschalk et al., 2001). Secondly,
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the contaminating flora present in nasal cavities might have precluded the specific isolation of S. suis serotype 5, even with the use of IMS. Indeed, in positive carrier animals, a higher number of positive colonies was recovered from vaginal samples where, in some cases, five out of five colonies tested belonged to S. suis serotype 5. With the nasal samples, a lower percentage of positive colonies was usually obtained. Results from our study confirm that serotyping is not discriminative enough to differentiate strains. A more sensitive technique such as the RAPD has been shown to be more useful to genotype S. suis isolates of serotype 2 (Chatellier et al., 1999; Martinez et al., 2002). However, a standardization of the technique needs to be carried out when testing for a different serotype, as recently reported for serotypes 2 and 1/2 (Chatellier et al., 1999; Martinez et al., 2002). In this regard, the RAPD for S. suis serotype 5 was standardized in this study. Surprisingly, up to six different genotypic patterns of the same serotype could be observed in carrier sows. A considerably higher heterogeneity was observed among the lower number of isolates recovered from nasal cavities compared to those from vaginal origin. Only one clone (clone D) was associated with the clinical cases, as all isolates recovered from diseased or dead animals (previously and during the study) belonged to this genotype. In this regard, the molecular epidemiology of the serotype 5 infection in this herd seems to be similar to that already described for serotype 2. Indeed, it was previously reported for the latter serotype that, in closed infected herds, a single clone of S. suis was responsible for the disease (Mogollon et al., 1991; Reams et al., 1996; Torremorell and Pijoan, 1998; Torremorell et al., 1998; Martinez et al., 2002). The situation for other serotypes, such as serotype 1/2, may be different, as recently reported (Martinez et al., 2002). Distinctive features of this particular Pattern D that may explain its increased virulence are not known. Interestingly, Pattern D was not isolated from the eight sows included in this study, probably due to its very low prevalence. It has been previously hypothesized that strains of S. suis serotype 2 isolated from the upper respiratory tract are part of the normal flora and tend to be very heterogeneous, even in closed populations (Mogollon et al., 1991; Reams et al., 1996; Torremorell and Pijoan, 1998; Torremorell et al., 1998; Martinez et al., 2002), whereas, in contrast, pathogenic strains are found at a very low prevalence in healthy carrier animals (Torremorell and Pijoan, 1998). It has already been suggested that this low prevalence could be a predisposing factor for the latter onset of disease in a herd (Pijoan, 1995). The situation may be similar for S. suis serotype 5 for the herd participating in the present study. Although a low prevalence for the Pattern D is an interesting hypothesis, the possibility that this genotype might be present as a minor population among other serotype 5 genotypes in the same animal cannot be excluded, since in some cases, a single sow may carry different serotype 5 strains. If the genotype D is present in lower concentrations than other serotype 5 genotypes, the probability of isolating it would be lower, and results may be biased. The genotype D could be detected neither in sows nor in piglets 1 year later in the absence of clinical signs. A vertical transmission of S. suis serotype 5 isolates could be observed in this study. In general, similar genotypes were found in vaginal cavities of the sows and on nasal surfaces of 15% of the piglets. Vertical transmission was also supported by other studies (Robertson et al., 1991; Reed et al., 1998). In this study, Pattern B was isolated from the nasal surface of some piglets but not from sows. It might be concluded that this pattern was in fact present in the vagina of sows, but we failed to isolate it prior to or at farrowing. A real colonization of
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the oropharynx could not be demonstrated in our study, since S. suis serotype 5 could not be detected 1 week after birth in the nasal cavities of the piglets. However, it may be assumed that animals were colonized when they swallowed vaginal secretions from the sows but bacteria were mainly located at the tonsils that were not sampled in this study. Indeed, 24% of the tonsils collected cultured positive for S. suis serotype 5 at slaughter. In general, the proportion of detected carrier piglets was low, especially during the weeks where no clinical disease was present. This is not necessarily an indication of the absence of S. suis serotype 5, since bacteria were probably present at the tonsils. It may rather indicate a slower transmission of the infection due to the lack of clinical illness. Although the Pattern D was isolated from only two piglets at 2 weeks of age, it was the only genotype isolated from carrier animals from the time of the manifestation of clinical signs until the end of the experiment. This would suggest a higher number of bacteria in the environment that would increase transmission either by aerosol or direct contact. Furthermore, most isolates recovered from tonsils of these animals at slaughter were also of genotype D. In the absence of S. suis-associated disease in the herd (1 year later), no isolation of S. suis serotype 5 from piglets could be achieved. Interestingly, the rate of isolation of S. suis at slaughter at that moment was similar to that obtained in the previous year, in the presence of clinical signs. However, the genotype D could not be detected in the absence of disease, and all isolates from tonsils belonged to the genotype C. It is not clear whether this is due to either its real absence from the herd or a too low aerosol transmission. Results obtained by serological examination showed that concentration of antibodies against S. suis serotype 5 were high in sows, suggesting that animals had already been in contact with S. suis. The concentration of maternal antibodies detected at 24 h in piglets was similar to those detected in sows; however, a high individual variation among animals was observed, even within the same litter (data not shown). This may indicate a variation in intestinal absorption of maternal IgG or different levels of colostrum intake. Results from this study showed that clinical signs appeared when maternal immunity was at its lowest level, which is in agreement with other studies (Torremorell et al., 1998; Lapointe et al., 2002). However, the sole absence of maternal antibodies does not necessarily trigger the clinical disease, since similar low amount of antibodies were also detected 1 year later in piglets at 4 weeks of age and older, in the complete absence of disease associated with S. suis. In the presence of clinical cases (first year), an active antibody response to S. suis serotype 5 infection could be observed. Some animals produced high levels of antibodies whereas others did not produce any detectable levels of antibodies against S. suis. This confirms the fact that the humoral response to S. suis infections and vaccination is usually erratic (Blouin et al., 1994; del Campo Sepulveda et al., 1996; Lapointe et al., 2002). As expected, in the absence of clinical signs, no marked antibody level increase was observed in pigs. The disappearance of clinical signs in the nursery pigs 1 year later, with no isolation of S. suis from diseased animals, is difficult to explain. No major changes in the management of the farm or vaccination program had been implemented. For both years, the carrier rate for S. suis serotype 5 in adult animals was similar. It might be hypothesized that the virulent strain was no longer present in the herd, but no explanation for such an event could be found. Since the rate of isolation of Pattern D from sows in the presence of clinical signs in the nursery pigs was extremely low (one sow), the negative result obtained 1 year later may
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only reflect this very low prevalence. Moreover, the absence of this genotype from tonsils of pigs at slaughter may be the consequence of a low transmission due to the lack of disease. For several infectious diseases, the overall level of immunity of a herd and the homogeneity among individuals are important factors in the development of clinical signs. However, in our study, the level of antibodies in sows and piglets at birth was slightly lower in absence of clinical signs and the variation in antibodies was similar during the two periods. In conclusion, serotypes other than serotype 2 of S. suis can cause severe clinical signs. Different strains of the same serotype of S. suis may be present in a herd. However, only one of these strains may be responsible for clinical signs. Hence, genotypic characterization (by the use of RAPD or other genotyping methods) is indispensable to understand the epidemiology of the infection. Although piglets were infected at birth by strains present in the vaginal tract of the sows, we were unable to determine whether the virulent strain was transmitted from the sows to their offspring at birth. Additional studies should be carried out to investigate the vertical transmission of the virulent strain. It appears that nasal cavities are not the most appropriate site for studying the asymptomatic carriage of S. suis, since positive results are mainly observed in presence of clinical signs when there is active transmission of the infection.
Acknowledgements The authors want to thank Julie-Mélanie Trudel, Marcelo Ribotta, Marcel Roy, Avila Croisetière and Guy Beauchamp for their excellent technical assistance. This study was supported by the Conseil de Recherche en Pˆeche et en Agroalimentaire du Québec grant # 4778 and by the Canadian Research Network on Bacterial Diseases of Swine.
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Robertson, I.D., Blackmore, D.K., Hampson, D.J., Fu, Z.F., 1991. A longitudinal study of natural infection of piglets with Streptococcus suis types 1 and 2. Epidemiol. Infect. 107, 119–126. Staats, J.J., Brandon, L., Plattner, B.L., Nietfeld, J., Dritz, S., Chengappa, M.M., 1998. Use of ribotyping and hemolysin activity to identify highly virulent Streptococcus suis type 2 isolates. J. Clin. Microbiol. 36, 15–19. Torremorell, M., Pijoan, C., 1998. Prolonged persistence of an epidemic Streptococcus suis strain in a closed pig population. Vet. Rec. 143, 394–395. Torremorell, M., Calsamiglia, M., Pijoan, C., 1998. Colonization of suckling pigs by Streptococcus suis with particular reference to pathogenic serotype 2 strains. Can. J. Vet. Res. 62, 21–26.