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Protection of pigs against challenge withvirulent Streptococcus suis serotype 2 strainsby a muramidase-released protein andextracellular factor vaccineH. J. WISSELINK, U. VECHT, N. STOCKHOFE-ZURWIEDEN, H. E. SMITH

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventinginfection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suisserotype 2 strain (MRP+EF+) was compared with the efficacy of a vaccine containing formalin-killed bacterinof S suis serotype 2 (MRP+EF+). The enhancement of the immune response by different adjuvants (a water-in-oil emulsion [wo] and an aluminium hydroxide-based adjuvant [AHJ) and their side effects were also studied.The MRP and EF were purified by affinity chromatography. Pigs were vaccinated twice at three weeks and sixweeks of age and challenged intravenously with virulent S suis serotype 2 strains (MRP+EF+) at eight weeksof age. At challenge, the pigs vaccinated with MRP+EF/WO had high anti-MRP and anti-EF titres and wereprotected as effectively as pigs vaccinated with wo-formulated vaccines with bacterin. Eight of the nine pigssurvived the challenge and almost no clinical signs of disease were observed. The titres obtained with theMRP+EF/AH vaccine were low and only two of the five pigs were protected. Pigs vaccinated with either MRP orEF were less well protected; three of the four pigs died after challenge but the clinical signs of disease weresignificantly less severe than those observed in the placebo-vaccinated pigs. The protective capacity of thebacterin/AH vaccine was very low, and the mortality among these pigs was as high as in the placebo-vaccinated pigs (80 per cent). Postmortem histological examination revealed meningitis, polyserositis andarthritis in the clinically affected pigs. The results demonstrate that a subunit vaccine containing both MRPand EF, formulated with the wo adjuvant, protected pigs against challenge with virulent S suis type 2 strains.

Veterinary Record (2001)148, 473-477

H. J. Wisselink, BSc,U. Vecht, DVM, PhD,H. E. Smith, PhD,N. Stockhofe-Zurwieden,DVM, Division ofInfectious Diseases andFood Chain Quality,Institute for AnimalScience and Health,PO Box 65, 8200 ABLelystad, The Netherlands

Dr Vecht's present addressis Inspectorate for HealthProtection, Commoditiesand Veterinary PublicHealth, East District,PO Box 202, 7200 AEZutphen, The Netherlands

STREPTOCOCCAL meningitis, polyserositis and polyarthri-tis, is a severe, often fatal disease ofyoung pigs at weaning, andis usually caused by Streptococcus suis serotype 2 (Vecht andothers 1985, Higgins and others 1992, Reams and others1994). S suis strains can be differentiated by serotype on thebasis of their capsular polysaccharides. At present, 35serotypes are known (Perch and others 1983, Gottschalk andothers 1989, 1991, Higgins and others 1995). The virulence ofmost serotypes is unclear. Strains ofserotypes 2 and 1 are con-sidered to be the most virulent serotypes, but strains of someother serotypes, such as 7, 9 and 14 have also been associatedwith disease (Sihovenen and others 1988, Jacobs and others1995, MacLennan and others 1996).

The economic impact of S suis infections on the pig indus-try is substantial (Chengappa and others 1990), and theprophylactic use of antibiotics in food and drinking waterhas so far been unsuccessful in controlling the disease.Furthermore, antibiotics are becoming less effective becauseof an increase in resistance among S suis isolates and their useis unpopular because of the public awareness of antimicro-bial residues (Aarestrup and others 1998).

Little is known about the protective antigens of S suis.Whole-cell vaccines with live avirulent or killed virulent S suisserotype 2 cells protected pigs against challenge with a strainof a homologous serotype (Holt and others 1988, 1990,Busque and others 1997). However, vaccination with wholecells did not induce protection against challenge with a strainof a heterologous serotype (Kebede and others 1990). Jacobsand others (1996) showed that a vaccine based on the puri-fied 54 kDa suilysin protected against a homologous chal-lenge, but the absence of this haemolysin from a substantialnumber of isolates obtained from diseased pigs limits theusefulness of this vaccine (Segers and others 1998).

In previous work, the authors identified two proteins, the136 kDa muramidase-released protein (MRP) and the 110 kDaextracellular factor protein (EF), as markers of virulent S suisserotype 1 and 2 strains (Vecht and others 1991). The MRP iSa membrane-associated protein and EF is an extracellular pro-

tein. Both are transported across the bacterial membrane andare major antigens recognised by convalescent sera of infectedpigs (Vecht and others 1991).

This paper describes the results of studies of the efficacyof MRP and EF vaccines in preventing infection and disease inpigs challenged with a homologous or a heterologous S suisserotype 2 strain (MRP+EF+), in comparison with the protec-tive capacity of a vaccine containing formalin-killed bacterinof S suis serotype 2 (MRP+EF+).

MATERIALS AND METHODS

Bacterial strainsStrains 4005 (3) and 3881 (10) ofSsuis serotype 2, both belong-ing to the phenotype MRP+EF+, were used. Both strains were fieldisolates, isolated from pigs suffering from meningitis and werevirulent for newborn germ-free pigs (Vecht and others 1992).

AdjuvantsTwo adjuvants, a water-in-oil emulsion (Specol; ID-Lelystad)(wo) and a 2 per cent aluminium hydroxide gel (Alhydrogel;Superfos Bisosector) (AH) were used.

AntigensFor the preparation of the vaccine, MRP and EF were purifiedby affinity chromatography. A two litre culture of strain 4005,grown in Todd Hewitt broth (CM 189; Oxoid) was centrifugedat 4000 g for 15 minutes. The supernatant was cleared fromremaining cells by filtration under air pressure through 0-2pm filters (Millipore). Monoclonal antibodies MRP3, or EF2(ID-Lelystad) were coupled separately to cyanogen bromide-activated Sepharose 4B in accordance with the instructions ofthe supplier (Pharmacia). After appropriate washing thebound proteins were eluted with glycine-hydrochloric acidbuffer (0-IM, pH 2.8), and the pH of the fractions wasimmediately increased to 7-0 with 1M sodium hydroxide. Thefractions were measured with the MRP or EF double antibody

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sandwich (DAS) ELISA for the detection of MRP and EF proteinsas described byVecht and others (1993). Fractions positive forMRP and EF were pooled and dialysed overnight against phys-iological saline. The purified proteins gave single bands in sil-ver-stained sodium dodecyl sulphate-polyacrylamide gels.The quantities of proteins were measured with the bicin-choninic acid protein assay reagent (Pierce), using bovineserum albumin (BSA) as standard. Approximately 100 [ig ofMRP and 240 jIg of EF were obtained per litre of culture.

For the preparation of the whole-cell vaccine, strain 4005was incubated overnight at 37°C in 100 ml Todd-Hewittbroth. The culture contained about 1 x 109 colony-formingunits (cfu)/ml. Fifty ml was centrifuged at 4000 g for 15 min-utes. Pellets were washed twice in phosphate-buffered saline(PBS: 136-89 mM sodium chloride, 2-68 mM potassium chlo-ride, 8-1 mM sodium hydrogen phosphate, 2-79 mM potas-sium hydrogen phosphate, pH 7-2) and resuspended in 2-5 mlPBS. To this suspension, 250 V113 per cent formalin was added,and it was maintained at 4°C overnight. The next day, thesuspension was checked for the absence of live bacteria byplating on 6 per cent Columbia horse blood agar base (cM331; Oxoid). To remove formalin, the cells were washed twicewith physiological saline and resuspended in physiologicalsaline to a final count of approximately 1 x 109 cells/ml.

Vaccine preparationwo or AH was used as the adjuvant. For the preparation ofemulsions in the wo adjuvant, four parts of the water phasecontaining the antigen were mixed with five parts of wo(Bokhout and others 1981).

For the vaccinations with the AH adjuvant, 1 25 mg metal-lic aluminium was used per dose, according to the manufac-turer's instructions. The antigens and AH were stirred for fourhours at 4°C. To control adsorption, 1 ml of the mixture wascentrifuged in an Eppendorf centrifuge at 10,000 rpm forthree minutes, and the supernatants were analysed for theabsence of the antigens either by MRP and EF DAS ELISA (Vechtand others 1993) or spectrophotometrically by using anUltrospec 3000 spectrophotometer (Pharmacia Biotech) atwavelengths of 250 to 650 nm.

For the preparation of the placebo-vaccines the antigensolutions were replaced by a physiological saline solution.

VaccinationFifty-five three-week-old pigs, crossbreeds of Yorkshire andDutch landrace, were obtained from the specified pathogen-free herd of the ID-Lelystad for two experiments. In bothexperiments the pigs were alloted to six treatment groups eachconsisting of four or five pigs. The pigs were separated andhoused in boxes at the animal facilities of the ID-Lelystad.

Priming vaccinations were given at the age of three weeks.Each dose contained 50 jtg MRP, 50 jig EF or 1 x 109 formalin-killed whole cells, either separately or in combination, andthey were administered intramuscularly, divided over twoinjection sites, in both upper hind legs. Three weeks later thepigs were boosted intramuscularly in the neck with the samedose of the vaccines without adjuvants.

ChallengeTwo weeks after the second vaccination, the pigs were chal-lenged intravenously in the ear vein with 1 x 107 cfu of thehomologous S suis serotype 2 strain 4005 (experiment 1) orthe heterologous S suis serotype 2 strain 3881 (experiment 2).The inocula were prepared as described by Vecht and others(1992). The pigs were monitored twice daily for the followingclinical signs of disease: fever, disorders of the nervous sys-tem, lameness, inappetence and depression. Blood sampleswere taken once a week before the challenge and three timesa week after the challenge to monitor the immune response.White blood cells were counted with a semi-cell blood

counter (Sysmex, model F 800; Charles Goffin MedicalSystems). The number of neutrophils was calculated after adifferential count of Giemsa-stained blood smears. For ani-mal welfare reasons, pigs that were moribund or showed ner-vous signs were killed by an intravenous injection ofpentobarbiturate, exsanguinated and examined postmortem.Tissue specimens taken from the central nervous system(CNS), serosae, and joints were examined bacteriologically andhistologically as described byVecht and others (1992). Lesionsresulting from the injections of the vaccines were recorded.

The experiments were approved by the ethical committeeof the Institute for Animal Science and Health in accordancewith Dutch law on animal experiments.

Antibodies against MRP and EFThe sera were tested for antibodies against MRP and EF by anindirect ELISA. Each well of the polystyrene microtitre plateswas coated for 18 hours at 37°C with 30 ng of MRP for theindirect MRP ELISA or with 25 ng of EF for the indirect EF ELISA.Pig sera in two-fold dilutions from 1:5 to 1:5120 in PBS con-taining 0.05 per cent Tween 80 were added and the plates wereincubated for one hour at 37°C. Serum from a gnotobiotic pigwhich had survived an infection with the virulent S suisserotype 2 strain 4005 (MRP+EF+) was used as a positive con-trol. As a conjugate, monoclonal antibody mouse anti-swineimmunoglobulin G labelled with horseradish peroxidase (ID-Lelystad) was used. After incubation for one hour at 37°C, thesubstrate 5-aminosalycylic acid with hydrogen peroxide wasadded. After incubation for two hours at room temperature,the absorbance at 450 nm was read. Titres were expressed asthe reciprocal of the log2 of the highest dilution showing anabsorbance of more than 50 per cent of the positive control.

Statistical analysisData concerning the mortality and lesions of the various groupswere analysed simultaneously by the non-parametric Fisher-Freeman-Halton exact test.When there was overall significance,Fisher's exact test was used to make pair-wise comparisonsbetween the various groups. In a similar manner, the antibodytitres against MRP and EF, the clinical signs of disease, fever andthe number of leucocytes were subjected to exact median testsfor simultaneous comparisons, followed by exact pair-wise per-mutation tests. The last test was only used if there was overallsignificance. The significance level was set at 95 per cent.

RESULTS

Antibody titres against MRP and EFAll the pigs vaccinated with the MRP and EF vaccines with woas adjuvant developed high anti-MRP and anti-EF titres (Table1). At the time of challenge the average antibody titres againstMRP ranged from 6208 to 23,170 and against EF from 7131 to13,308. The titres obtained after vaccination with MRP+EF/AHwere much lower; at the time of challenge, the averageantibody titre against MRP was 388 and against EF it was 1522.

Pigs vaccinated with the bacterin vaccines developed lowanti-MRP titres only. In the pigs vaccinated with bacterin/wothey started to develop after the booster administration to anaverage anti-MRP titre of 158 at the time of challenge. No anti-EF titres could be detected in this group either before or afterchallenge. In the bacterin/AH group, none of the pigs hadanti-MRP or anti-EF titres at the time of challenge.

As expected, none of the pigs vaccinated with the placebovaccines had developed antibodies against MRP or EF at thetime of challenge.

ProtectionTwo weeks after the second vaccination, the pigs were chal-lenged intravenously in the ear vein with 1 x 107 cfu of the

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.V_1:1gm.[&.tE. _- S S. .. . S. ~ 5 5 g - - - - - -

Experiment* AntigensAntibody titre Antibody titre

Adjuvant against MRP Antilog against EF Antilog

MRP WO 13-8a (1.1) 14,263 0O0 0EF WO 0.0 0 12.88 (1-3) 7131MRP+EF WO 13.58 (0.9) 11,585 13.3a (0.8) 10,086MRP+EF+bacterin WO 12.68 (1.7) 6208 13.18 (0.8) 8780Placebo wo 0-0 0 0-0 0Placebo No adjuvant 0-0 0 0-0 0

2 MRP+EF WO 14.5b (1t3) 23,170 13.7b (1-8) 13,308MRP+EF AH 8.6C (1.3) 388 10.6c (1.5) 1552Bacterin wo 7-3b (1.6) 158 0-0 0Bacterin AH 0.0 0 0-0 0Placebo wo 0.0 0 0-0 0Placebo AH 0.0 0 0-0 0

* In experiment 1, pigs were challenged with the homologous S suis serotype 2 strain 4005(MRP+EF+), and in experiment 2 with the heterologous S suis serotype 2 strain 3881 (MRP+EF+)a Significantly different from group placebo/wo, experiment 1b Significantly different from group placebo/wo, experiment 2c Significantly different from group placebo/AH, experiment 2wo Water-in-oil emulsion, AH Aluminium hydroxide-based adjuvant

homologous S suis serotype 2 strain 4005 (experiment 1) orthe heterologous S suis serotype 2 strain 3881 (experiment 2).Seventeen of the 19 pigs vaccinated with the placebo vaccinesdied one to four days after the challenge as a result of the infec-tion or had to be killed for animal welfare reasons (Table 2).In these groups, specific clinical signs of disease, such as lame-ness and nervous signs, were frequently recorded. Non-spe-cific clinical signs of disease, such as depression, recumbencyand lack of appetite were also frequently observed. The pigs'body temperatures and leucocyte counts were also increased.In contrast, the wo-formulated vaccines containing both MRPand EF conferred a high degree of protection. Compared withthe placebo-vaccinated pigs, mortality was significantly(P<0.05) lower, and 11 of the 13 pigs survived the challenge,both with the homologous and with the heterologous serotype

2 strain. The clinical signs of disease and the increase in bodytemperatures were also significantly (P<0.05) reduced.Vaccines containing either MRP or EF were less protective thanthe vaccine containing both proteins (Table 2, experiment 1),and most of the pigs in these groups did not survive the chal-lenge. However, compared with the pigs in the placebo-vac-cinated groups, the pigs vaccinated with either MRP or EFshowed significantly (P<0.05) fewer specific clinical signs ofdisease and had lower fever and lower leucocyte counts. TheMRP+EF/AH vaccine conferred a low degree of protection; onlythree of the five pigs survived the challenge with the heterol-ogous S suis type 2 strain, but, compared with the placebo-vac-cinated pigs, the specific clinical signs of disease and the feverwere significantly (P<0.05) lower (Table 2, experiment 2).Eight of the nine pigs vaccinated with wo-formulated vaccinescontaining bacterin were protected against a challenge withthe homologous or heterologous serotype 2 strain; mortalityin these groups was significantly (P<0 05) lower than in theplacebo-vaccinated groups, and there were fewer specific ornon-specific signs of disease (P<0.05) and a lower level offever. In contrast, a bacterin/AH vaccine conferred less protec-tion; four of the five pigs vaccinated with this vaccine died twoto four days after challenge, and specific signs of disease wereobserved as often as in the placebo-vaccinated pigs.

Postmortem results confirmed the clinical findings.Histological examination revealed meningitis, polyserositisand arthritis in all 19 of the placebo-vaccinated pigs, in fourof the five pigs vaccinated with bacterin/AH, and in three ofthe five vaccinated with EF/WO (Table 3). In contrast, the pigsvaccinated with MRP+EF/WO, bacterin/wo and MRP+EF+bac-terin/wo had significantly (P<0.05) fewer lesions, andbacteria were isolated from the lesion sites less frequently.

Lesions were observed at the injection site in the pigs of allgroups, except for those vaccinated with the AH-formulatedplacebo vaccine. They ranged in severity from being less than1 cm3 in size and involving connective tissue only, to beingmore than 1 cm3, with necrosis, microabscesses or granulo-mas (Table 3).

Mortality$ Clinical signsNumber Number Mean number of of disease (%)v Fever Leucocytosis

Experiment* Antigenst Adjuvant of pigs of pigs days after challenge§ Specific# Non-specific* index (%)¶ index (%)A

1 MRP WO 4 3 5.0 15a 21 a 38a 65EF WO 4 3 2-6 38a 43 65 28MRP+EF WO 4 1 3 16a 11a1g 31MRP+EF+bacterin WO 4 1 2 13a 17a 16a 25Placebo wo 5 5 2 60 70 75 80Placebo No adjuvant 5 5 2-4 46 84 77 100

2 MRP+EF WO 5 ob NA 1 b 4b 7b 28bMRP+EF AH 5 2 3.5 13C 31 36C 65Bacterin wo 5 ob A ob 3b bBacterin AH 5 4 2-8 37 56 86 90Placebo wo 4 3 3.3 31 54 76 88Placebo AH 5 4 3.3 34 57 78 90

* In experiment 1, pigs were challenged with the homologous S suis serotype 2 strain 4005 (MRP+EF+), and in experiment 2 with theheterologous S suis serotype 2 strain 3881 (MRP+EF+)t MRP and EF were purified from S suis serotype 2 strain 4005 (MRP+EF+). Bacterin was prepared from S suis serotype 2 strain 4005(MRP+EF+)* Number of pigs that died owing to infection or had to be killed for animal welfare reasons§ Mean number of days after challenge on which the pigs died owing to infection or had to be killed for animal welfare reasonsv Individual mean percentages were calculated as the number of observations/number of all observations per animal. Group means werethen calculated by total of individual means/number of pigs per group# Nervous signs and/or lameness on at least one joint* Inappetence and/or depression1 Percentage of observations of pigs with a body temperature above 40CA Percentage of observations of pigs in which the concentration of granulocytes was greater than 1010/litrea Significantly different from group placebo/wo, experiment 1bSignificantly different from group placebo/wo, experiment 2c Significantly different fromgroup placebo/AH, experiment 2NA Not applicable, wo Water-in-oil emulsion, AH Aluminium hydroxide-based adjuvant

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Number of pigsNumber Number of pigs with lesions with lesions at

Experiment* Antigens Adjuvant of pigs Meningitist Polyserositist Polyarthritis§ Total* site of injection

1 MRP wO 4 2 0 2 3 2EF WO 4 3 3 2 3 2

MRP+EF WO 4 1 1 la 2 4MRP+EF+bacterin wo 4 1 1 1a 1a 2

Placebo wo 5 4 5 5 5 5Placebo No adjuvant 5 4 4 5 5 2

2 MRP+EF WO 5 0 ob 0 ob 3MRP+EF AH 5 1 1 1 2 3Bacterin wo 5 0 ob 0 ob 3Bacterin AH 5 2 4 2 4 4Placebo wo 4 3 3 3 4 4Placebo AH 5 1 5 3 5 0

* In experiment 1, pigs were challenged with the homologous S suis serotype 2 strain 4005 (MRP+EF+), and in experiment 2 with theheterologous S suis serotype 2 strain 3881 (MRP+EF+)t Meningitis was characterised by inflammation of cerebrum, cerebellum, pons, mesencephalon and medulla oblongatat Polyserositis was characterised by inflammation of peri- and epicardium, thoracic pleura and peritoneum§ Polyarthritis was characterised by inflammation of carpal, tarsal, knee, elbow, shoulder and hip jointsv Total number of pigs with lesions; some pigs developed more than one lesiona Significantly different from group placebo/wo, experiment 1b Significantly different from group placebo/wo, experiment 2wo Water-in-oil emulsion, AH Aluminium hydroxide-based adjuvant

DISCUSSION

These result show that vaccines containing MRP+EF/WO pro-tected pigs against challenge with either a homologous or het-erologous S suis serotype 2 strain with the phenotypeMRP+EF+. The MRP+EF/WO vaccine was as protective as a bac-terin/wo vaccine. All but one of the pigs vaccinated withMRP+EF/WO survived the challenge and few specific clinicalsigns of disease were observed. The MRP/WO or EF/WO vaccineswere much less protective; three of the four vaccinated pigsdied after challenge. However, compared with the placebo-vaccinated pigs, the pigs vaccinated with MRP/WO or EF/WOshowed significantly fewer clinical signs of disease. These dataare in accordance with the results of Jacobs and others ( 1996)who described a vaccine which contained most of the extra-cellular antigens produced by an S suis serotype 2 strain, withEF being the most abundant protein. However, the vaccineonly partially protected pigs against challenge with a virulentS suis type 2 strain.

The protection observed with the MRP and/or EF vaccineswas associated with the levels of anti-MRP and anti-EF anti-bodies. The MRP+EF/WO vaccine induced high antibody titresand protected pigs effectively against challenge with eitherhomologous or heterologous S suis serotype 2 strains. On theother hand, the vaccine with MRP+EF/AH induced lower anti-body titres and the pigs were less well protected. In contrast, noassociation between anti-MRP and anti-EF titres and protectionwas observed with the bacterin vaccines. Pigs vaccinated withbacterin/wo had low antibody titres against MRP and EF butnevertheless appeared to be completely protected against chal-lenge. It seems likely that other antigens than MRP and EF areresponsible for this protection, for example capsular antigens,as has been suggested by Kebede and others (1990).wo adjuvant was superior to AH adjuvant in its capacity

to stimulate an immune response after vaccination with MRPand EF and to confer protection against challenge with viru-lent S suis serotype 2 strains. Similarly, Ripley (1983) showedthat an oil-based adjuvant produced a significant antibodyresponse with killed bacterins, whereas only a transientincrease in antibodies was observed after vaccination with anAH-formulated vaccine. However, it appeared that both woand AH adjuvants caused serious lesions at the injection sites,and a suitable alternative adjuvant or a refinement of wo istherefore desirable.

Holt and others (1990) found that a vaccine containingbacterin without an adjuvant protected as well as bacterin for-mulated either with Freund's incomplete adjuvant or withaluminium hydroxide gel as an adjuvant. However, the sameexperiments showed that the protective response to bacterinwas stimulated when the size of the inoculum was increasedfrom 1010 to 1011 killed organisms. In this study, the bacterincontained only 109 killed cells, and a strong potentiatingadjuvant like wo seemed to be necessary to obtain protection.

Whole-cell vaccines are probably serotype-specific,because protection was achieved only against a strain of ahomologous serotype, and the vaccines failed to protectagainst other serotypes (Kebede and others 1990, Foster andothers 1994). Subunit vaccines based on proteins conservedamong serotypes may be more useful in veterinary practiceif they protect against challenge with strains of heterologousserotypes. Jacobs and others ( 1996) suggested that suilysin, athiol-activated haemolysin from S suis serotype 2, could besuch a cross-protection factor. Vaccination challenge experi-ments in pigs indicated that this vaccine protected againstchallenge with a homologous serotype. However, thehaemolysin is absent from quite a number of strains of S suisisolated from diseased pigs in the field. This implies that othervaccine components will be necessary to provide protectionagainst all field strains (Jacobs and others 1996, Segers andothers 1998). A 52 kDa immunoglobulin-binding proteinwhich has recently been shown to be identical to a 60 kDaheat-shock protein that is produced by various serotypes,could be another candidate for a subunit vaccine (Serhirand others 1993, Benkirane and others 1997, 1998). Theprotective value of this protein has not been tested.

In Europe, the USA and Australia, most of the S suisserotype 2 strains isolated from diseased pigs produce MRPand EF (Mwaniki and others 1994, Galina and others 1996,Wisselink and others 2000). In these countries an MRP+EF vac-cine could therefore be of great value. However, most of theS suis serotype 2 strains isolated from diseased pigs in Canadaappeared to be MRP and EF negative (Gottschalk and others1998). Apparently, Canadian strains differ from strains iso-lated in other countries. The proteins MRP and/or EF are notonly produced by serotype 2 strains. High percentages ofEuropean S suis serotype 1, 1/2 and 14 strains, isolated fromtissues associated with S suis infections such as brain, serosa,joint, heart and other organs of diseased pigs, expressed MRP

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and EF, and more than 80 per cent of the S suis serotype 9strains produced an MRP+ protein, a high molecular weightvariant of the 136 kDa MRP (Wisselink and others 2000). Inaddition to serotype 2 strains, strains of serotypes 1, 1/2, 7, 9and 14 are frequently isolated from diseased pigs. Furtherwork is needed to determine whether MRP and EF are involvedin the protection of pigs infected with strains of otherserotypes producing MRP and/or EF.

ACKNOWLEDGEMENTS

This work was partly supported by Vetoquinol Biotech-nologie, Lure, France.

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_ ABSTRACT

Measurements of postoperativepain in catsTHREE groups of six cats were subjected to either tenectomyor onychectomy, or to no surgical interference. Manual pal-pation and simple descriptive scales and visual analogue scaleswere then applied to assess and record the level of pain suf-fered by the cats, and the results were compared with mea-surements of the plasma concentrations of beta-endorphinand cortisol. Only the scores on the visual analogue scales andthe responses of the cats to palpation differed significantlybetween the control group and the groups subjected tosurgery; the physiological measurements failed to differenti-ate between them.

CAMBRIDGE, A. J., TOBIAS, K. M., NEWBERRY, R. C. & SARKAR, D. K.(2000) Subjective and objective measurements of postoperative pain in cats.Journal of the American Veterinary Medical Association 217, 685-690

The Veterinary Record, April 14, 2001 477

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doi: 10.1136/vr.148.15.473 2001 148: 473-477Veterinary Record

 H. J. Wisselink, U. Vecht, H. E. Smith, et al. and extracellular factor vaccinestrains by a muramidase-released protein

serotype 2Streptococcus suisvirulent Protection of pigs against challenge with

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