Veterinary Microbiology, 22 (1990) 249-257 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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Isolation and C h a r a c t e r i z a t i o n of T e m p e r a t u r e S e n s i t i v e M u t a n t s of Streptococcus suis: E f f i c a c y Trial of the M u t a n t Vaccine in Mice MIZANU KEBEDE 1, M.M. CHENGAPPA2 and JAMES G. STUART 1'*
1Department o/Biological Sciences, Murray State University, Murray, K Y 42071 (U.S.A.) 2Department o/Laboratory Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (U.S.A.) (Accepted 12 October 1989)
ABSTRACT Kebede, M., Chengappa, M.M. and Stuart, J.G., 1990. Isolation and characterization of temperature-sensitive mutants of Streptococcus suis: efficacy trial of the mutant vaccine in mice. Vet. Microbiol., 22: 249-257. A model of experimental Streptococcus suis infection was developed in young mice. Minimum lethal dose (MLD) values were calculated for four virulent serotypes (1/2, 1, 2, 3) of S. suis using this model. Temperature-sensitive (ts) mutants ofS. suis serotypes 1/2 and 1-8 were isolated and characterized on the basis of their growth kinetics and reversion rates. Ts mutants of S. suis 1/2, 1, 2, and 3 were tested as vaccines against the virulent homologous and heterologous challenges in mice. The protection provided was evaluated by analyzing the clinical signs, death or survival. Homologous but not heterologous protection was noted in all mice vaccinated with the mutant strains. Ts mutants of S. suis 1/2 provided 100% protection against challenge by virulent strains ofS. suis 1/2, 1, and2.
INTRODUCTION
Streptococcus suis has been associated with contagious disease of swine, which is characterized by arthritis, septicemia, meningitis, pneumonia, and high morbidity in piglets 3-12 weeks old (Windsor and Elliot, 1975; Guise et al., 1985; Hoffman and Henderson, 1985). Although swine of all ages are susceptible to this bacterial agent, the mortality in swine over 14 weeks old is very low (Guise et al., 1985; Hoffman and Henderson, 1985). Since S. suis disease was first reported by DeMoor (1963) from the Netherlands, other researchers have reported outbreaks in additional European countries Canada and the United *To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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States (Sanford et al., 1982; Perch et al., 1983; Larson and Kott, 1983; Guise et al., 1985; Hommez et al., 1986). Until now nine different serotypes of a S. suis have been identified on the basis of their capsular polysaccharide antigen (Koehne et al., 1979; Perch et al., 1983 ). These serotypes are designated as S. suis 1/2 and 1-8. One study on vaccination of swine with S. suis has previously been reported. This report by Elliott et al. (1980) showed that of 17 young pigs vaccinated with S. suis type 2 capsular polysaccharide plus Freund's incomplete adjuvant, all developed opsonizing antibodies to the parent bacterial strain. Pigs which were vaccinated with capsular polysaccharide alone were much less likely to develop an opsonizing antibody response. Only four of 14 pigs responded to this challenge with antibody. Given the poor response of young pigs to capsular polysaccharide alone, it seemed reasonable to develop an attenuated bacterial strain and test the efficacy as a vaccine. Thus temperature-sensitive (ts) mutants of nine serotypes of S. suis ( 1/2 and 1-8) were isolated and characterized on the basis of their growth kinetics. Also, vaccine trials were conducted with ts mutants of S. suis 1/2, 1, 2, and 3 respectively. MATERIALS AND METHODS
Mice Six-week-old Swiss-Webster ICR Albino mice were used in this study. Bacterial strains All serotypes of S. suis used in the study were obtained from Char' H. Armstrong, Animal Disease Diagnostic Laboratory, Purdue University, Lafayette, Indiana 47907. The serotypes include S. suis 1/2 and 1-8. Ts m u t a n t strains (MK-1 through MK-9) for these serotypes were isolated in this laboratory. Strain MK-1 was isolated from serotype 1/2, MK-2 from serotype 1, MK-3 from serotype 2, and so on to MK-9 which was isolated from serotype 8. Stock cultures were made for both wild strains and ts m u t a n t strains using 1 ml horse serum and 0.2 ml glycerol. The suspensions were held in sterile screw cap vials at - 80 ° C. Media Serum Yeast Todd-Hewitt (SYTH) broth consisting of 0.6% yeast extract, 3% Todd-Hewitt broth, and 0.038% K2H PO4 buffer was used. Five per cent horse serum (Gibco) was added immediately before use. Yeast Todd-Hewitt (YTH) agar was prepared by adding 1.5% bacteriological agar (Difco) to YTH broth. Sheep blood agar (SBA) combined YTH agar and 5 % sterile sheep blood.
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Determination of the minimum lethal dose (MLD) A 10-ml overnight S Y T H broth culture of each wild S. suis serotype (1, 2, 3, and 1/2) was pelleted and resuspended in physiologically sterile saline ( P S S ) to a standard optical density at 550 nm. From this suspension a viable count titration was made in triplicate and four twofold dilutions in P S S were made. Eight-week-old mice were divided into six groups, with six per group and injected intraperitoneally with i ml each of the above concentrations. Each group of six mice received a different concentration of bacteria; one group served as a saline control and received 1 ml each of P S S intraperitoneally. The experiment proceeded for 1 week following injection. The lowest concentration of bacteria which killed all the mice in one group was considered the MLD.
Isolation of ts mutants Ts m u t a n t s of S. suis were isolated by a penicillin double selection method. Cells treated with the mutagen N-methyl-N'nitro-N-nitrosoguanidine (Sigma) at a concentration of 100/~g/ml were grown overnight at 30°C in S Y T H broth. A 0.5-ml aliquot of this culture was inoculated into 9.5 ml of S Y T H broth and incubated at 30°C until the mid-log phase of growth. The culture was transferred to a 37 °C water bath and incubated for 10 rain. Penicillin (Sigma) was then added to the culture at a final concentration of 0.5 mg m l - 1, and incubation continued for 2 h. The cells were then centrifuged, washed three times in P S S and incubated overnight at 30 °C in 10 ml of S Y T H broth. The selection procedure was then repeated. Upon completion of the second selection, dilutions of the cells were spread on Y T H agar and incubated overnight at 30 ° C. Colonies were picked and streaked on replica plates. One of the plates was incubated at 30 ° C while the other one was incubated at 37 ° C. Those colonies that grew only at 30 °C b u t failed to survive at 37 °C were considered to be ts mutants.
Characterization of ts sensitive mutants Ts m u t a n t s of each serotype were characterized on the basis of their growth kinetics. The growth kinetics were determined by inoculating i ml of an overnight culture into 19 ml of S Y T H broth. The culture was allowed to grow at 30 ° C and turbidity was measured every hour beginning at 0 time using a K l e t t Summerson Photoelectric Colorimeter (Klett) with a green filter. W h e n the growth reached 20 Klett units, half of the culture (10 ml) was transferred to an incubator at 37°C while the remaining culture was maintained at 30°C. Turbidity was measured until the growth in both cultures reached stationary phase.
Determination of reversion frequency Ts m u t a n t s were grown overnight at 30 °C in S Y T H broth. From the overnight culture, a 0.1-ml aliquot was spread on Y T H agar and incubated over-
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night at 37 oC; a viable count at 30 °C was also made. The reversion frequency was determined by multiplying the number of revertants that grew at 37 ° C by 10 and dividing by the titer at 30 ° C.
Vaccine preparation and challenge Ts m u t a n t s of S. suis type 1/2, 1, 2, and 3 were employed as vaccines in this study. The m u t a n t s were grown in S Y T H broth to a standard optical density (550 nm), pelleted, and resuspended in P S S at the desired concentrations before injection. A 1 × concentration was made by resuspending the bacteria in an equal volume of PSS. Viable count titrations were made from the 1 X concentration of each m u t a n t vaccine. Prior to vaccination, 6-week-old mice were divided into two groups of 18 for each vaccine. One group was injected with 1 ml each of ts vaccine intraperitoneally, while the second group was injected with 1 ml each of sterile saline. Clinical signs of both vaccinated and nonvaccinated groups were observed daily following vaccination. The amount of vaccine administered was measured by viable counts. Two weeks following the vaccination, each group was subdivided into three groups of six mice each, and each subgroup was challenged with wild type S. suis serotypes 1/2, 1, and 2 respectively. T h u s two groups, vaccinated and nonvaccinated, were challenged with each strain. The challenge inoculum (1 ml) was given to all mice intraperitoneally. Challenge doses were chosen to be equivalent to the M L D for each virulent strain. The results of the challenge tests were evaluated on the basis of clinical signs, mortality, or survival of the mice for a 7-day post-challenged period. A final vaccine experiment was performed with a similar design using a ts m u t a n t of S. suis 3 (MK-4) as a vaccine. The challenge strains in this experiment were S. suis serotypes 1, 2, and 3. RESULTS
Several ts m u t a n t s for each serotype of S. suis were isolated. However, only those ts m u t a n t s with the lowest reversion frequency were utilized in the study. Table 1 shows reversion frequencies for the ts m u t a n t s employed. Ts m u t a n t s were characterized both in the context of their growth kinetics and maximum growth temperature. The growth kinetics were determined for each m u t a n t by measuring turbidity of growth every hour in Klett units. Fig. 1 indicates the growth kinetics for the m u t a n t MK-1. As indicated in the graph, the m u t a n t strain showed little or no growth when switched to 37 ° C. This growth curve is representative of all m u t a n t s isolated since very little variation from this kinetic pattern was observed. The M L D titrations were necessary for estimating the virulence of the challenge bacteria in mice. M L D values for the wild S. suis serotypes are given in Table 2, which are determined on the basis of viable counts and optical density. Table 3 includes results of the vaccine trials performed. In the initial exper-
253
TEMPERATURE-SENSITIVE MUTANTS OF STREPTOCOCCUS SUIS TABLE 1
Reversion frequencies Mutant
Average revertants per ml
Titer at
strain MK-1 MK-2 MK-3 MK-4 MK-5 MK-6 MK-7 MK-8 MK-9
20 190 20 0 0 20 0 20 600
7.2 X 1.0 X 6.5 X 9.7 X 8.5 X 8.6X 8.1X 6.9 X 1.0 X
120
[]
30C
o •
37C
30 ° C 10 s 10 s l0 s 10 v 107 10 s 10 s 108 10 o
Reversion frequency 2.8 X 1 0 - s 1.9 X 1 0 - s 3.1 X 10 - s < 1.0 X 10 - s < 1.0 X 10 - s 2.3X 10 - s < 1.0X 10 -9 3.0 X 10 8 6.0 X 10-7
Switched to 37 C
100 8O 6O 40
~2o ~10 M 8 6 4
2
I
1
2
3
4
5
6
7
8
9
Hours Fig. 1. T h e grow curve of MK-1 as determined on the basis of turbidity.
iment using MK-1, the vaccine was used at a dose equivalent to the minimum lethal dose (MLD) of the wild type. The viable count for the vaccine was calculated as 2 X 10 ° colony forming units (CFU) ml-1 after an overnight incubation at 30 ° C. Vaccinated mice showed no clinical signs or side-effects during the 2-weeks period following vaccination. Following the challenge, all but one mouse in the non-vaccinated group died within 24 h after infection and the
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TABLE 2 Minimum lethal dose of S. suis in mice
Serotype
Optical density a
Viable count
( S. suis ) 1/2 1 2 3
1.8 2.5 1.3 5.8
4.8X 109 C F U X m l 1 2.3 X 101° CFU X m l 5.4 × 109 CFU X m l 7.4 X 109 CFU X m l - t
aOD measured at 550 nm. Challenge doses were injected in 1 ml volumes.
TABLE 3 Vaccine trials with ts mutants of S. suis Vaccine a
MK-1 MK-1 MK-1 MK-2 MK-2 MK-2 MK-3 MK-3 MK-3 MK-4 MK-4 MK-4 Saline Saline Saline Saline
Challenge b
Survival
(S. suis)
(%)
1/2 1 2 1/2 1 2 1/2 1 2 1 2 3 1/2 1 2 3
100 100 100 100 100 0 83 0 100 0 0 100 0 0 0 0
aVaccine doses are provided in the text. bChallenge doses are equivalent to M L D values given in Table 2.
latter died within 48 h. A few mice in the vaccinated group initially exhibited clinical signs of illness, however, their symptoms disappeared gradually. The vaccinated groups showed survival rates between 83 and 100%, while the nonvaccinated groups showed 0% survival. Attempts were made to define the m i n i m u m dose of MK-1 cells that could protect the test animals. Accordingly, the vaccine concentration was reduced from 2 X 109 CFU m l - 1 to viable counts of 1 X 109, 5 X l0 s, and 2.0 X l0 s CFU
TEMPERATURE-SENSITIVE MUTANTS OF STREPTOCOCCUS SUIS
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ml-1, respectively. Complete protection was seen in all trials except the one with the lowest concentration of vaccine. No clinical illness was produced in mice by vaccinating with MK-2 (viable count of 4.3 × 109 CFU m1-1). Vaccinated mice challenged with S. suis 1/2 and S. suis 1 were completely protected without any clinical symptoms after 2 weeks, while non-vaccinated controls challenged with S. suis 2 died within 24 h of the challenge. Following vaccination with MK-3 (2X 109 CFU ml-1), all mice remained healthy with no symptoms before challenge. As shown in Table 3 the vaccine provided protection against the virulent strains of S. suis 1/2 (83% survival) and S. suis 2 (100% survival). In contrast, a 0% survival rate was noted in the group challenged with a virulent strain of S. suis 1. In the final vaccine trial, MK-4 containing 1.3 X 101° CFU ml-1 was used. During the first few days following vaccination, over 90% of mice showed clinical signs of partial hair loss and weight loss that was not observed in nonvaccinated groups. As indicated in Table 3, the vaccine provided protection only to those mice challenged with virulent S. suis 3. No heterologous protection against S. suis 1 or 2 was observed. DISCUSSION
The isolation of ts mutants of S. suis was time-consuming; the isolation of MK-5 was the most difficult, requiring the screening of over 2000 mutagenized colonies to isolate only two ts mutants. Once the ts mutants were isolated, the reversion frequency for each isolate was determined (Table 1 ). The primary reason for this procedure was to avoid the chance of picking those strains that revert frequently to growth at 37 oC. As a result, only those ts mutants with lower or no detectable reversion rate were utilized. Ts mutants were also characterized on the basis of growth kinetics. During the growth kinetic study, each serotype showed an accelerated growth rate at 30 oC. However, upon switching to 37 oC, bacterial growth quickly ceased. The growth curves, in addition to showing the growth pattern of the strains, also provided further evidence that the mutants were temperature sensitive. Although experimental infection has been reproduced in swine (DeMoor, 1963; Chengappa et al., 1986), this report is the first observation of S. suis infection in mice. In order to develop the S. suis/mouse model, adult mice were chosen first. However, adults were able to tolerate extremely high doses of the organism without clinical signs. Therefore, younger mice, 8 weeks old, were employed to establish the lethal dose. Once the mouse model was developed, the MLD values served as a guide to the proper challenge concentration after vaccination. The minimal lethal dose (MLD) that killed all test mice in a particular group was utilized as the challenge dose in this study. Therefore, a
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concentration of ts mutants that gave full protection against the specific challenge dose was considered to be a protective dosage of vaccine. In Table 2, MLD data show that there were some differences in virulence among the challenge strains. S. suis serotypes 1/2 and 2 were roughly equal in virulence, whereas both strains appeared to be more virulent that either S. suis 1 or 3. It should be noted that although MLD levels were high, the results were very reproducible. This is probably due to the fact that all challenge doses were also quantitated by optical density as well as viable count. Thus, even though the chaining morphology of the organism made accurate viable counts difficult to obtain, the challenge cells were injected at a standard optical density which gave consistent results. The results of the present study showed that ts mutants of S. suis afford protection to mice exposed to the homologous virulent strain. As indicated in Table 3, mice vaccinated with MK-1 showed 100% survival after being exposed to the virulent strains of S. suis 1/2, 1, and 2 respectively. In contrast, no survival was noted in the non-vaccinated groups. The protection of MK-1 vaccine against infection by S. suis 1 and 2 strains relies on the fact that the vaccine possesses common antigens of both serotypes. Early studies by DeMoor (1963) provided evidence that this bacterium contains antigens that are found in both serotypes S. suis 1 and 2. Therefore, MK-1 provides homologous protection to all three serotypes, S. suis 1/2, 1, and 2 (Table 3). The results of the resistance to homologous and heterologous challenge in mice vaccinated with MK-2 are also indicated in Table 3. From these results only homologous protection was observed among mice vaccinated with MK-2. For example, only those mice challenged with S. suis 1/2 and 1 survived while those challenged with the heterologous serotype S. suis 2 died within 24 h. The failure to protect against the heterologous challenge most likely resulted from the absence of a similar capsular antigen between S. suis I and 2. Repeated vaccine trials with MK-3 and MK-4 also showed similar results that supported the above observation. Thus, MK-3 and MK-4 protected only their homologous virulent counterpart strains of S. suis 2 and 3 respectively. It was shown earlier by Elliott et al. (1980) that capsular antigen of S. suis induces opsonizing antibodies in pigs. Also, the primary difference between these streptococcal serotypes is the capsular polysaccharide antigen (Perch et al., 1983 ). It is suggested that the capsular antigen may be the most important antigen for stimulating protective antibody in mice against an experimental challenge with S. suis. Therefore, the most effective vaccine tested was strain MK-1 because it protected mice from lethal doses of all three virulent strains of S. suis 1/2, 1 and 2. REFERENCES Chengappa, M.M., Maddux, R.L., Kadel, W.L., Greer, S.G. and Herren, C.E., 1986. Streptococcus suis infection in pigs: Incidence and experimental reproduction of the syndrome. Am. Assoc. Vet. Lab. Diag. 29th Annual Proc.
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DeMoor, C.E., 1963. Septicaemic infections in pigs, caused by haemolytic streptococci of new lancefield groups designated R, S, and T. Antonie van Leeuwenhoek, 29: 272-280. Elliott, S.D., Clifton Hadley, F. and Tai, J., 1980. Streptococcal infection in young pigs. V. An immunologic polysaccharide from Strepococcus suis type 2 with particular reference to vaccination against streptococcal meningitis in pigs. J. Hyg. Camb., 85: 275-285. Guise, H.J., Penny, R.H.C., Duthic, A.N.S., 1985. Streptococcal meningitis in pigs. Vet. Rec., 117: 43-44. Hoffman, L.J. and Henderson, L.M., 1985. The significance of Streptococcus suis in swine disease: clinical, pathologic and bacteriologic data from a two-year study. Am. Assoc. Vet. Lab. Diagnosticians 28th Ann. Proc., pp. 201-210. Hommez, J., DeVriese, L.A., Henrichsen, J. and Castryck, F., 1986. Identification and characterization of Streptococcus suis. Vet. Microbiol., 11: 349-355. Koehne, G., Maddux, R.L. and Cornell, W.D., 1979. Lancefield group R streptococci associated with pneumonia in swine. Am. J. Vet. Res., 40: 1640-1641. Larson, D.J. and Kott, B., 1983. Iowa State University cases of swine pneumonia and meningitis associated with Streptococcus suis. Am. Assoc. Vet. Lab. Diagnosticals 26th Ann. Proc., pp. 121-130. Perch, B., Pedersen, K.B. and Henrichsen, J., 1983. Serology of capsulated streptococci pathogenic for pigs: six new serotypes of Streptococcus suis. J. Clin. Microbiol. 17: 993-996. Sanford, E. and Tilker, M.E., 1982. Streptococcus suis type II-associated disease in swine: observations of a one-year study. J. Am. Vet. Med. Assoc., 23: 5-97. Windsor, R.S. and Elliott, S.D., 1975. Streptococcal infection in young pigs. J. Hyg. Camb., 75: 69-78.
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Isolation and C h a r a c t e r i z a t i o n of T e m p e r a t u r e S e n s i t i v e M u t a n t s of Streptococcus suis: E f f i c a c y Trial of the M u t a n t Vaccine in Mice MIZANU KEBEDE 1, M.M. CHENGAPPA2 and JAMES G. STUART 1'*
1Department o/Biological Sciences, Murray State University, Murray, K Y 42071 (U.S.A.) 2Department o/Laboratory Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (U.S.A.) (Accepted 12 October 1989)
ABSTRACT Kebede, M., Chengappa, M.M. and Stuart, J.G., 1990. Isolation and characterization of temperature-sensitive mutants of Streptococcus suis: efficacy trial of the mutant vaccine in mice. Vet. Microbiol., 22: 249-257. A model of experimental Streptococcus suis infection was developed in young mice. Minimum lethal dose (MLD) values were calculated for four virulent serotypes (1/2, 1, 2, 3) of S. suis using this model. Temperature-sensitive (ts) mutants ofS. suis serotypes 1/2 and 1-8 were isolated and characterized on the basis of their growth kinetics and reversion rates. Ts mutants of S. suis 1/2, 1, 2, and 3 were tested as vaccines against the virulent homologous and heterologous challenges in mice. The protection provided was evaluated by analyzing the clinical signs, death or survival. Homologous but not heterologous protection was noted in all mice vaccinated with the mutant strains. Ts mutants of S. suis 1/2 provided 100% protection against challenge by virulent strains ofS. suis 1/2, 1, and2.
INTRODUCTION
Streptococcus suis has been associated with contagious disease of swine, which is characterized by arthritis, septicemia, meningitis, pneumonia, and high morbidity in piglets 3-12 weeks old (Windsor and Elliot, 1975; Guise et al., 1985; Hoffman and Henderson, 1985). Although swine of all ages are susceptible to this bacterial agent, the mortality in swine over 14 weeks old is very low (Guise et al., 1985; Hoffman and Henderson, 1985). Since S. suis disease was first reported by DeMoor (1963) from the Netherlands, other researchers have reported outbreaks in additional European countries Canada and the United *To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
250
M. KEBEDE ET AL.
States (Sanford et al., 1982; Perch et al., 1983; Larson and Kott, 1983; Guise et al., 1985; Hommez et al., 1986). Until now nine different serotypes of a S. suis have been identified on the basis of their capsular polysaccharide antigen (Koehne et al., 1979; Perch et al., 1983 ). These serotypes are designated as S. suis 1/2 and 1-8. One study on vaccination of swine with S. suis has previously been reported. This report by Elliott et al. (1980) showed that of 17 young pigs vaccinated with S. suis type 2 capsular polysaccharide plus Freund's incomplete adjuvant, all developed opsonizing antibodies to the parent bacterial strain. Pigs which were vaccinated with capsular polysaccharide alone were much less likely to develop an opsonizing antibody response. Only four of 14 pigs responded to this challenge with antibody. Given the poor response of young pigs to capsular polysaccharide alone, it seemed reasonable to develop an attenuated bacterial strain and test the efficacy as a vaccine. Thus temperature-sensitive (ts) mutants of nine serotypes of S. suis ( 1/2 and 1-8) were isolated and characterized on the basis of their growth kinetics. Also, vaccine trials were conducted with ts mutants of S. suis 1/2, 1, 2, and 3 respectively. MATERIALS AND METHODS
Mice Six-week-old Swiss-Webster ICR Albino mice were used in this study. Bacterial strains All serotypes of S. suis used in the study were obtained from Char' H. Armstrong, Animal Disease Diagnostic Laboratory, Purdue University, Lafayette, Indiana 47907. The serotypes include S. suis 1/2 and 1-8. Ts m u t a n t strains (MK-1 through MK-9) for these serotypes were isolated in this laboratory. Strain MK-1 was isolated from serotype 1/2, MK-2 from serotype 1, MK-3 from serotype 2, and so on to MK-9 which was isolated from serotype 8. Stock cultures were made for both wild strains and ts m u t a n t strains using 1 ml horse serum and 0.2 ml glycerol. The suspensions were held in sterile screw cap vials at - 80 ° C. Media Serum Yeast Todd-Hewitt (SYTH) broth consisting of 0.6% yeast extract, 3% Todd-Hewitt broth, and 0.038% K2H PO4 buffer was used. Five per cent horse serum (Gibco) was added immediately before use. Yeast Todd-Hewitt (YTH) agar was prepared by adding 1.5% bacteriological agar (Difco) to YTH broth. Sheep blood agar (SBA) combined YTH agar and 5 % sterile sheep blood.
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Determination of the minimum lethal dose (MLD) A 10-ml overnight S Y T H broth culture of each wild S. suis serotype (1, 2, 3, and 1/2) was pelleted and resuspended in physiologically sterile saline ( P S S ) to a standard optical density at 550 nm. From this suspension a viable count titration was made in triplicate and four twofold dilutions in P S S were made. Eight-week-old mice were divided into six groups, with six per group and injected intraperitoneally with i ml each of the above concentrations. Each group of six mice received a different concentration of bacteria; one group served as a saline control and received 1 ml each of P S S intraperitoneally. The experiment proceeded for 1 week following injection. The lowest concentration of bacteria which killed all the mice in one group was considered the MLD.
Isolation of ts mutants Ts m u t a n t s of S. suis were isolated by a penicillin double selection method. Cells treated with the mutagen N-methyl-N'nitro-N-nitrosoguanidine (Sigma) at a concentration of 100/~g/ml were grown overnight at 30°C in S Y T H broth. A 0.5-ml aliquot of this culture was inoculated into 9.5 ml of S Y T H broth and incubated at 30°C until the mid-log phase of growth. The culture was transferred to a 37 °C water bath and incubated for 10 rain. Penicillin (Sigma) was then added to the culture at a final concentration of 0.5 mg m l - 1, and incubation continued for 2 h. The cells were then centrifuged, washed three times in P S S and incubated overnight at 30 °C in 10 ml of S Y T H broth. The selection procedure was then repeated. Upon completion of the second selection, dilutions of the cells were spread on Y T H agar and incubated overnight at 30 ° C. Colonies were picked and streaked on replica plates. One of the plates was incubated at 30 ° C while the other one was incubated at 37 ° C. Those colonies that grew only at 30 °C b u t failed to survive at 37 °C were considered to be ts mutants.
Characterization of ts sensitive mutants Ts m u t a n t s of each serotype were characterized on the basis of their growth kinetics. The growth kinetics were determined by inoculating i ml of an overnight culture into 19 ml of S Y T H broth. The culture was allowed to grow at 30 ° C and turbidity was measured every hour beginning at 0 time using a K l e t t Summerson Photoelectric Colorimeter (Klett) with a green filter. W h e n the growth reached 20 Klett units, half of the culture (10 ml) was transferred to an incubator at 37°C while the remaining culture was maintained at 30°C. Turbidity was measured until the growth in both cultures reached stationary phase.
Determination of reversion frequency Ts m u t a n t s were grown overnight at 30 °C in S Y T H broth. From the overnight culture, a 0.1-ml aliquot was spread on Y T H agar and incubated over-
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night at 37 oC; a viable count at 30 °C was also made. The reversion frequency was determined by multiplying the number of revertants that grew at 37 ° C by 10 and dividing by the titer at 30 ° C.
Vaccine preparation and challenge Ts m u t a n t s of S. suis type 1/2, 1, 2, and 3 were employed as vaccines in this study. The m u t a n t s were grown in S Y T H broth to a standard optical density (550 nm), pelleted, and resuspended in P S S at the desired concentrations before injection. A 1 × concentration was made by resuspending the bacteria in an equal volume of PSS. Viable count titrations were made from the 1 X concentration of each m u t a n t vaccine. Prior to vaccination, 6-week-old mice were divided into two groups of 18 for each vaccine. One group was injected with 1 ml each of ts vaccine intraperitoneally, while the second group was injected with 1 ml each of sterile saline. Clinical signs of both vaccinated and nonvaccinated groups were observed daily following vaccination. The amount of vaccine administered was measured by viable counts. Two weeks following the vaccination, each group was subdivided into three groups of six mice each, and each subgroup was challenged with wild type S. suis serotypes 1/2, 1, and 2 respectively. T h u s two groups, vaccinated and nonvaccinated, were challenged with each strain. The challenge inoculum (1 ml) was given to all mice intraperitoneally. Challenge doses were chosen to be equivalent to the M L D for each virulent strain. The results of the challenge tests were evaluated on the basis of clinical signs, mortality, or survival of the mice for a 7-day post-challenged period. A final vaccine experiment was performed with a similar design using a ts m u t a n t of S. suis 3 (MK-4) as a vaccine. The challenge strains in this experiment were S. suis serotypes 1, 2, and 3. RESULTS
Several ts m u t a n t s for each serotype of S. suis were isolated. However, only those ts m u t a n t s with the lowest reversion frequency were utilized in the study. Table 1 shows reversion frequencies for the ts m u t a n t s employed. Ts m u t a n t s were characterized both in the context of their growth kinetics and maximum growth temperature. The growth kinetics were determined for each m u t a n t by measuring turbidity of growth every hour in Klett units. Fig. 1 indicates the growth kinetics for the m u t a n t MK-1. As indicated in the graph, the m u t a n t strain showed little or no growth when switched to 37 ° C. This growth curve is representative of all m u t a n t s isolated since very little variation from this kinetic pattern was observed. The M L D titrations were necessary for estimating the virulence of the challenge bacteria in mice. M L D values for the wild S. suis serotypes are given in Table 2, which are determined on the basis of viable counts and optical density. Table 3 includes results of the vaccine trials performed. In the initial exper-
253
TEMPERATURE-SENSITIVE MUTANTS OF STREPTOCOCCUS SUIS TABLE 1
Reversion frequencies Mutant
Average revertants per ml
Titer at
strain MK-1 MK-2 MK-3 MK-4 MK-5 MK-6 MK-7 MK-8 MK-9
20 190 20 0 0 20 0 20 600
7.2 X 1.0 X 6.5 X 9.7 X 8.5 X 8.6X 8.1X 6.9 X 1.0 X
120
[]
30C
o •
37C
30 ° C 10 s 10 s l0 s 10 v 107 10 s 10 s 108 10 o
Reversion frequency 2.8 X 1 0 - s 1.9 X 1 0 - s 3.1 X 10 - s < 1.0 X 10 - s < 1.0 X 10 - s 2.3X 10 - s < 1.0X 10 -9 3.0 X 10 8 6.0 X 10-7
Switched to 37 C
100 8O 6O 40
~2o ~10 M 8 6 4
2
I
1
2
3
4
5
6
7
8
9
Hours Fig. 1. T h e grow curve of MK-1 as determined on the basis of turbidity.
iment using MK-1, the vaccine was used at a dose equivalent to the minimum lethal dose (MLD) of the wild type. The viable count for the vaccine was calculated as 2 X 10 ° colony forming units (CFU) ml-1 after an overnight incubation at 30 ° C. Vaccinated mice showed no clinical signs or side-effects during the 2-weeks period following vaccination. Following the challenge, all but one mouse in the non-vaccinated group died within 24 h after infection and the
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TABLE 2 Minimum lethal dose of S. suis in mice
Serotype
Optical density a
Viable count
( S. suis ) 1/2 1 2 3
1.8 2.5 1.3 5.8
4.8X 109 C F U X m l 1 2.3 X 101° CFU X m l 5.4 × 109 CFU X m l 7.4 X 109 CFU X m l - t
aOD measured at 550 nm. Challenge doses were injected in 1 ml volumes.
TABLE 3 Vaccine trials with ts mutants of S. suis Vaccine a
MK-1 MK-1 MK-1 MK-2 MK-2 MK-2 MK-3 MK-3 MK-3 MK-4 MK-4 MK-4 Saline Saline Saline Saline
Challenge b
Survival
(S. suis)
(%)
1/2 1 2 1/2 1 2 1/2 1 2 1 2 3 1/2 1 2 3
100 100 100 100 100 0 83 0 100 0 0 100 0 0 0 0
aVaccine doses are provided in the text. bChallenge doses are equivalent to M L D values given in Table 2.
latter died within 48 h. A few mice in the vaccinated group initially exhibited clinical signs of illness, however, their symptoms disappeared gradually. The vaccinated groups showed survival rates between 83 and 100%, while the nonvaccinated groups showed 0% survival. Attempts were made to define the m i n i m u m dose of MK-1 cells that could protect the test animals. Accordingly, the vaccine concentration was reduced from 2 X 109 CFU m l - 1 to viable counts of 1 X 109, 5 X l0 s, and 2.0 X l0 s CFU
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ml-1, respectively. Complete protection was seen in all trials except the one with the lowest concentration of vaccine. No clinical illness was produced in mice by vaccinating with MK-2 (viable count of 4.3 × 109 CFU m1-1). Vaccinated mice challenged with S. suis 1/2 and S. suis 1 were completely protected without any clinical symptoms after 2 weeks, while non-vaccinated controls challenged with S. suis 2 died within 24 h of the challenge. Following vaccination with MK-3 (2X 109 CFU ml-1), all mice remained healthy with no symptoms before challenge. As shown in Table 3 the vaccine provided protection against the virulent strains of S. suis 1/2 (83% survival) and S. suis 2 (100% survival). In contrast, a 0% survival rate was noted in the group challenged with a virulent strain of S. suis 1. In the final vaccine trial, MK-4 containing 1.3 X 101° CFU ml-1 was used. During the first few days following vaccination, over 90% of mice showed clinical signs of partial hair loss and weight loss that was not observed in nonvaccinated groups. As indicated in Table 3, the vaccine provided protection only to those mice challenged with virulent S. suis 3. No heterologous protection against S. suis 1 or 2 was observed. DISCUSSION
The isolation of ts mutants of S. suis was time-consuming; the isolation of MK-5 was the most difficult, requiring the screening of over 2000 mutagenized colonies to isolate only two ts mutants. Once the ts mutants were isolated, the reversion frequency for each isolate was determined (Table 1 ). The primary reason for this procedure was to avoid the chance of picking those strains that revert frequently to growth at 37 oC. As a result, only those ts mutants with lower or no detectable reversion rate were utilized. Ts mutants were also characterized on the basis of growth kinetics. During the growth kinetic study, each serotype showed an accelerated growth rate at 30 oC. However, upon switching to 37 oC, bacterial growth quickly ceased. The growth curves, in addition to showing the growth pattern of the strains, also provided further evidence that the mutants were temperature sensitive. Although experimental infection has been reproduced in swine (DeMoor, 1963; Chengappa et al., 1986), this report is the first observation of S. suis infection in mice. In order to develop the S. suis/mouse model, adult mice were chosen first. However, adults were able to tolerate extremely high doses of the organism without clinical signs. Therefore, younger mice, 8 weeks old, were employed to establish the lethal dose. Once the mouse model was developed, the MLD values served as a guide to the proper challenge concentration after vaccination. The minimal lethal dose (MLD) that killed all test mice in a particular group was utilized as the challenge dose in this study. Therefore, a
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concentration of ts mutants that gave full protection against the specific challenge dose was considered to be a protective dosage of vaccine. In Table 2, MLD data show that there were some differences in virulence among the challenge strains. S. suis serotypes 1/2 and 2 were roughly equal in virulence, whereas both strains appeared to be more virulent that either S. suis 1 or 3. It should be noted that although MLD levels were high, the results were very reproducible. This is probably due to the fact that all challenge doses were also quantitated by optical density as well as viable count. Thus, even though the chaining morphology of the organism made accurate viable counts difficult to obtain, the challenge cells were injected at a standard optical density which gave consistent results. The results of the present study showed that ts mutants of S. suis afford protection to mice exposed to the homologous virulent strain. As indicated in Table 3, mice vaccinated with MK-1 showed 100% survival after being exposed to the virulent strains of S. suis 1/2, 1, and 2 respectively. In contrast, no survival was noted in the non-vaccinated groups. The protection of MK-1 vaccine against infection by S. suis 1 and 2 strains relies on the fact that the vaccine possesses common antigens of both serotypes. Early studies by DeMoor (1963) provided evidence that this bacterium contains antigens that are found in both serotypes S. suis 1 and 2. Therefore, MK-1 provides homologous protection to all three serotypes, S. suis 1/2, 1, and 2 (Table 3). The results of the resistance to homologous and heterologous challenge in mice vaccinated with MK-2 are also indicated in Table 3. From these results only homologous protection was observed among mice vaccinated with MK-2. For example, only those mice challenged with S. suis 1/2 and 1 survived while those challenged with the heterologous serotype S. suis 2 died within 24 h. The failure to protect against the heterologous challenge most likely resulted from the absence of a similar capsular antigen between S. suis I and 2. Repeated vaccine trials with MK-3 and MK-4 also showed similar results that supported the above observation. Thus, MK-3 and MK-4 protected only their homologous virulent counterpart strains of S. suis 2 and 3 respectively. It was shown earlier by Elliott et al. (1980) that capsular antigen of S. suis induces opsonizing antibodies in pigs. Also, the primary difference between these streptococcal serotypes is the capsular polysaccharide antigen (Perch et al., 1983 ). It is suggested that the capsular antigen may be the most important antigen for stimulating protective antibody in mice against an experimental challenge with S. suis. Therefore, the most effective vaccine tested was strain MK-1 because it protected mice from lethal doses of all three virulent strains of S. suis 1/2, 1 and 2. REFERENCES Chengappa, M.M., Maddux, R.L., Kadel, W.L., Greer, S.G. and Herren, C.E., 1986. Streptococcus suis infection in pigs: Incidence and experimental reproduction of the syndrome. Am. Assoc. Vet. Lab. Diag. 29th Annual Proc.
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DeMoor, C.E., 1963. Septicaemic infections in pigs, caused by haemolytic streptococci of new lancefield groups designated R, S, and T. Antonie van Leeuwenhoek, 29: 272-280. Elliott, S.D., Clifton Hadley, F. and Tai, J., 1980. Streptococcal infection in young pigs. V. An immunologic polysaccharide from Strepococcus suis type 2 with particular reference to vaccination against streptococcal meningitis in pigs. J. Hyg. Camb., 85: 275-285. Guise, H.J., Penny, R.H.C., Duthic, A.N.S., 1985. Streptococcal meningitis in pigs. Vet. Rec., 117: 43-44. Hoffman, L.J. and Henderson, L.M., 1985. The significance of Streptococcus suis in swine disease: clinical, pathologic and bacteriologic data from a two-year study. Am. Assoc. Vet. Lab. Diagnosticians 28th Ann. Proc., pp. 201-210. Hommez, J., DeVriese, L.A., Henrichsen, J. and Castryck, F., 1986. Identification and characterization of Streptococcus suis. Vet. Microbiol., 11: 349-355. Koehne, G., Maddux, R.L. and Cornell, W.D., 1979. Lancefield group R streptococci associated with pneumonia in swine. Am. J. Vet. Res., 40: 1640-1641. Larson, D.J. and Kott, B., 1983. Iowa State University cases of swine pneumonia and meningitis associated with Streptococcus suis. Am. Assoc. Vet. Lab. Diagnosticals 26th Ann. Proc., pp. 121-130. Perch, B., Pedersen, K.B. and Henrichsen, J., 1983. Serology of capsulated streptococci pathogenic for pigs: six new serotypes of Streptococcus suis. J. Clin. Microbiol. 17: 993-996. Sanford, E. and Tilker, M.E., 1982. Streptococcus suis type II-associated disease in swine: observations of a one-year study. J. Am. Vet. Med. Assoc., 23: 5-97. Windsor, R.S. and Elliott, S.D., 1975. Streptococcal infection in young pigs. J. Hyg. Camb., 75: 69-78.
