Dynamics of Staphylococcus aureus infections during vaccination with an autogenous bacterin in dairy cattle

The effect of an autogenous vaccine against Staphylococcus aureus on S. aureus prevalence and mastitis, as well as on somatic cell count (SCC), was studied in a dairy herd with a high prevalence of S. aureus. The vaccination group (n = 35; 22 cows and 13 heifers) and the control group (n = 36; 23 cows and 13 heifers) received the vaccine or a placebo, respectively, according to the following protocol: all animals: basic immunization (twice, 3 weeks apart); cows: booster dose at the time of drying off, 5 and 2 weeks before calculated calving date; heifers: booster dose 2 and 5 weeks before calculated calving date. The vaccine or the placebo was administered subcutaneously in the area of the supramammary lymph nodes. Quarter milk samples were collected monthly and subjected to SCC and bacteriological evaluation. At this time, the animals were also checked for signs of clinical mastitis. Non-clinical S. aureus mastitis diagnoses were based on udder quarter SCC and a positive S. aureus culture. In order to compare the SCC in individual whole milk samples, records from the monthly milk quality testing were evaluated. Cow and udder quarter prevalence of S. aureus intramammary infections calculated for the experimental animals and quarters, respectively, did not differ between groups. However, during the lactation period following the boostcr dose, the prevalence of S. aureus increased in both groups (P < 0.05). The cumulative incidence of various mastitis diagnoses (clinical, subclinical, latent infection) due to S. aureus on an animal basis did not differ between groups. On an udder quarter basis, the cumulative incidence of subclinical mastitis was higher in vaccinated animals than in control animals (33.8 versus 26.0%; P < 0.05). This was mainly due to a higher cumulative incidence of subclinical mastitis in vaccinated than control heifers. The SCC in composite milk samples did not differ between groups, but increased as lactation progressed. The herd prevalence of S. aureus differed considerably throughout the study period, but declined consistently to below 10% at the end of the study period. Recent herd checks revealed a prevalence of S aureus infections of < 5%. It is concluded that the autogenous bacterin tested in this study did not have the desired effect on the prevalence of S. aureus infections and mastitis or SCC. The decline in S. aureus prevalence was very probably due to other factors than specific immunization against S. aureus.     

金黄色葡萄球菌性奶牛乳房炎多价疫苗的研制和免疫效果评价

为研制用于预防金黄色葡萄球菌性奶牛乳房炎的免疫疫苗,使用4株血清型为外膜多糖336型(336PS)、荚膜多糖5型(CP5)和荚膜多糖8型(CP8)强毒力金黄色葡萄球菌试制出金黄色葡萄球菌多价疫苗,并通过抗体水平监测、攻毒保护试验以及两个牧场的田间试验对免疫效果进行了评价。抗体水平监测显示,首免后30 d即可产生较高的抗体水平,二次免疫后有效抗体水平可持续到产后210 d;人工攻毒保护试验显示,疫苗对金黄色葡萄球菌引起的临床型乳房炎的保护率为89%;田间试验结果表明,该疫苗对奶牛临床型乳房炎的保护率在75.2%～83.3%之间,对隐性乳房炎的保护率在49.9%～52.2%之间,免疫组日产奶量较对照组平均提高1.26 kg以上。上述结果表明,所试制的疫苗可以显著降低泌乳牛金黄色葡萄球菌引起的临床型和隐性乳房炎发病率并提高产奶量。     

Incidence of clinical mastitis in a cohort of British dairy herds

A three-year survey to determine the incidence of clinical mastitis and the associated bacteria in dairy herds in England and Wales is described. Escherichia coli was the predominant organism in each year. Streptococcus uberis and Staphylococcus aureus were important for part for each year. The annual incidence of mastitis declined from 54.6 cases per 100 cows in 1980 to 41.2 in 1982. The incidence increased with age and declined with increasing herd size. The culling rate due solely to mastitis was 3 per cent. Strep uberis was the pathogen most frequently isolated from clinical cases which occurred in the dry period. Thirty per cent of all cases recurred at least once and staphylococcal cases exhibited the highest frequency of recurrence.     

Experimental trial in heifers vaccinated with Staphylococcus aureus avirulent mutant against bovine mastitis

Staphylococcus aureus, is the most frequently isolated pathogen from cases of bovine mastitis. Vaccination against S. aureus seems to be a rational approach for the control of staphylococcal mastitis. In the present work we evaluate the response of heifers vaccinated with a S. aureus avirulent mutant to the intramammary challenge with a S. aureus virulent strain. Clinical signs, production of milk, shedding of S. aureus cells, somatic cell count (SCC) and antigen-specific IgG in blood and milk, were determined. Two subcutaneous doses of a culture of the mutant, used as vaccine, was administered to four pregnant heifers 30 and 10 days before calving. The vaccinated heifers and four non-vaccinated were challenged 10 days after calving with the homologous virulent S. aureus strain, which was inoculated by intramammary route into two quarters of each animal. No local tissue damage was observed due to the administration of the vaccine. A significantly increase of specific IgG to S. aureus RC122 was detected in blood and milk of vaccinate heifers as well as a slight increase in daily milk yield during the trial. No significant difference on shedding of bacteria in milk and SCC were found among groups. In conclusion, vaccination of heifers before calving by an avirulent mutant vaccine of S. aureus, induced specific and significant antibody responses and provide better post-challenge conditions in vaccinated heifers.     

Vaccination against experimental staphylococcal mastitis in ewes

Experiments were carried out in ewes using a new vaccine developed for the prevention of mastitis caused by Staphylococcus aureus. The vaccine comprised three major components: (i) killed S aureus cells which had been cultured to induce synthesis of pseudocapsule; (ii) toxoided staphylococcal beta haemolysin and (iii) the adjuvant dextran sulphate. Ewes systemically vaccinated twice during pregnancy developed significantly elevated circulating levels of IgG1 and IgG2 anti-pseudocapsule antibody, as well as increased serum titres of anti-beta haemolysin. Five different strains of S aureus were used to challenge both vaccinated and control ewes by the intramammary route during the ensuing lactation. The incidence of acute gangrenous mastitis and nonacute, clinical mastitis was significantly lower in vaccinated than in control groups after challenge with each strain. Vaccinated ewes produced significantly more milk than control ewes after challenge with four of the five strains of S aureus.     

Efficacy of a herd-specific vaccine against Staphylococcus aureus to prevent post-partum mastitis in dairy heifers

A placebo-controlled field study was performed to evaluate the effect of a herd-specific vaccine against Staphylococcus aureus on intramammary infection (IMI), somatic cell count (SCC) and clinical mastitis. Three hundred and twenty-one heifers were assigned to two groups. Heifers in the vaccination group (n = 164) were vaccinated twice, i.e. 5 and 2 weeks before their expected calving date. Animals of the control group (n = 157) received the same treatment with a placebo containing no bacterial antigen. Quarter milk samples were collected immediately after parturition prior to the first machine milking, 3-4 weeks after calving and before the onset of treatment in animals with signs of clinical mastitis during the first 3 months after calving. For comparison of SCC the data from the monthly milk test records were evaluated. The prevalence of S. aureus in quarter milk samples taken at calving and 3-4 weeks post-partum did not differ significantly between the vaccine and control group. Incidence of clinical mastitis during the first 3 months after calving and the prevalence of S. aureus in quarter milk samples taken before the onset of treatment did not differ significantly between the groups. The SCC was lower in vaccinated than in control heifers. However, the difference was only significant on the third milk test day. Regarding prevalence of IMI with S. aureus and incidence of clinical mastitis the use of a herd-specific vaccine against S. aureus did not prove to be efficient on this farm.     

Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine.

A prospective cohort study was undertaken in two commercial California dairies. The treatment group, 246 cows, received three doses of a whole cell bacterin of J5 Escherichia coli (mutant of E. coli O111:B4) plus Freund's incomplete adjuvant vaccine (two in the dry period and one after calving) while 240 unvaccinated cows served as controls. Thirty-five cases of clinical coliform mastitis were diagnosed, six in vaccinated cows and 29 in unvaccinated cows. Bacteria isolated from the clinical cases included 15 E. coli five Klebsiella pneumoniae, three K. oxytoca, three K. ozaenae, five Enterobacter aerogenes, three Serratia marcescens and one Serratia spp. Four control cows were culled, three of them because of chronic coliform mastitis and one because of postcoliform infection agalactia. Incidence rate of clinical gram-negative mastitis was 2.57% in vaccinated cows and 12.77% in unvaccinated cows. The estimated risk ratio, the measure of risk of having clinical gram-negative mastitis for vaccinated cows to unvaccinated cows, was 0.20 (p less than 0.005), indicating a strong relationship between vaccination and lack of clinical gram-negative mastitis. The results of this trial indicate that the administration of the E. coli J5 vaccine is protective against natural challenge to gram-negative bacteria, and reduces the incidence of clinical gram-negative mastitis in dairy cows during the first three months of lactation.     

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

ABSTRACT: Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme

The objective was to investigate the spread of S. aureus in seven dairy herds. Milk samples were taken to determine mastitis pathogens and somatic cell count. An approved hygiene programme was established to control the spread of S. aureus from quarter to quarter. S. aureus isolates were differentiated by geno- and phenotyping to trace their spread within a herd.In two herds S. aureus showed a high prevalence, but were eliminated from the herds by the control programme. In these herds and a third herd typing results identified a particular type that was found much more frequently than all other types. The frequent types were repeatedly detectable during the study. In three other herds with a lower S. aureus prevalence and also in one herd with a long history of S. aureus and high prevalence subclinical mastitis was caused by several distinguishable S. aureus types. These types occurred simultaneously in the particular herd and showed little or no tendency to spread from quarter to quarter. They seemed to circumvent the control procedures resulting in a relatively high rate of new infected animals and therefore were hardly eliminated. The typing results and the clinical observations indicated that strains differed in their tendency to spread and their ability to infect udder quarters. In three herds typing methods identified a predominant type with the common epidemiological features of a contagious mastitis pathogen, while in the other herds the S. aureus type patterns were similar to that of environmental pathogens.     

A benefit to cost analysis of the effect of premilking teat hygiene on somatic cell count and intramammary infections in a commercial dairy herd.

A field trial was conducted to determine the effect of premilking teat disinfection (predipping) on several measures of mastitis in a commercial dairy farm where the predominant organisms isolated from intramammary infections were coagulase negative Staphylococcus spp. Cows were randomly assigned to a treated (predipped with 0.5% iodine germicide plus "good udder preparation") or a control group ("good udder preparation" alone). Sterile composite milk samples were collected at the initiation of the trial and on an approximately bimonthly basis throughout the duration of the trial. There was no difference in the prevalence of isolation of coagulase-negative Staphylococcus spp. from composite milk samples obtained during the 6 herd cultures. The incidence rate for clinical mastitis in the control group was 1.38 cases per 1000 cow days. The incidence rate for clinical mastitis in the treatment group was 1.06 cases per 1000 cow days. The ratio of these 2 was 1.3, suggesting a higher rate in the control group, but the ratio was not statistically significant (P = 0.34). Logistic regression analysis indicated that the effect of treatment group was not significant, although the coefficient suggested that predipping reduced the risk of clinical mastitis. The benefit to cost ratio of 0.37 indicated that the benefit of reduced incidence of clinical cases of mastitis would not have justified the added expense required to predip the herd.     

Occurrence of clinical and sub-clinical mastitis in dairy herds in the West Littoral Region in Uruguay

Twenty-nine dairy farms were selected to determine the incidence of clinical mastitis, prevalence of sub-clinical mastitis and bacterial aetiology in the West Littoral Region of Uruguay. In samples taken by the owner and frozen at -20 degrees C during a week the incidence rate of clinical mastitis was determined as 1.2 cases per 100 cow-months at risk. Staphylococcus aureus was the most common isolated pathogen in 37.5% of 40 milk samples from clinical cases obtained in 1 month. No bacteria grew in the 32.5% of the total samples. A sub-sample including 1077 dairy cows from randomly selected farms was used to determine the prevalence of sub-clinical mastitis. These samples were taken on one visit to each farm. The prevalence was 52.4% on a cow basis and 26.7% on an udder quarter basis. In 55.1% of the quarters of the selected animals with more than 300,000 cells/ml there was no growth. The isolated pathogens from sub-clinical cases and their relative frequencies were: Staphylococcus aureus 62.8%, Streptococcus agalactiae 11.3%, Enterococcus sp. 8%, coagulase-negative staphylococci 7.4%, Streptococus uberis 6.4%, Streptococcus dysgalactiae 1.8%, Escherichia coli 1.5% and Staphylococcus hyicus coagulase-positive 0.6%.     

Development of a Staphylococcus aureus vaccine against mastitis in dairy cows. I. Challenge trials

A vaccine composed of three field isolates of Staphylococcus aureus (S. aureus) derived from cases of mastitis in cows was developed. The vaccine was administered to nine uninfected cows while 10 other cows were used as controls. All cows were challenged with a highly virulent S. aureus strain administered into two quarters of each cow. Quarters were tested for clinical signs, secretion of S. aureus, and somatic cell count (SCC). No systemic effects were observed in any of the cows, vaccinated or control. Vaccinated cows had 70% protection from infection compared with fewer than 10% in the controls. Moreover, all quarters challenged in the vaccinated cows, regardless of whether they were successfully infected or not with S. aureus, exhibited very mild inflammatory reactions, identified by their low SCCs (<100,000).     

Within herd comparison of teat dipping and dry cow therapy with only selective dry cow therapy in six herds

A within herd comparison of teat dipping and dry cow therapy (full treatment) with only selective dry cow therapy (partial treatment) was carried out in six commercial dairy herds for a two year period. In four herds, the incidence of clinical mastitis was 2 to 12 per cent higher in the partial treatment group. In another herd, in which the pattern of clinical mastitis isolates was unusual in that minor pathogens were isolated from 30 per cent of mastitis cases, the incidence was 43 per cent higher in the partial treatment group. In the remaining herd the incidence was 10 percent higher in the full treatment group. Streptococcus uberis mastitis was more common in the partial treatment groups of five herds; coliform mastitis was more common in the full treatment groups of two herds and similar in both groups in the other herds. High rates of coliform mastitis were associated with poor herd environmental conditions but this was not true for Strep uberis mastitis. Rates of staphylococcus aureus and Strep dysgalactiae mastitis were low in all herds. The level of major pathogen infection in cows completing the trial in all herds increased in the partial treatment group from 5 per cent of quarters at the start to 12 per cent at the finish of the trial. In the full treatment group, however, there was only a small increase in this level. In contrast, levels of Corynebacterium bovis infection increased by 17 per cent in both treatment groups. Continued use of teat dipping and dry cow therapy was associated with a higher rate of coliform mastitis in two of the three herds where there were poor standards of hygiene and husbandry.     

Prevalence of staphylococcal species in four dairy herds

The prevalence of staphylococcal species isolated from bovine mammary glands was determined in four dairy herds. Staphylococcus aureus and S hyicus were the predominant organisms isolated from cows in a herd with a bulk milk somatic cell count (SCC) greater than 900 X 10(3). One herd with a bulk milk SCC of 565 X 10(3) had a high incidence of S aureus while the predominant coagulase-negative species were S epidermidis and S hyicus. S hyicus predominated in two herds with bulk milk SCC less than 200 X 10(3); prevalence of S aureus was low. The impact of herd management practices such as post-milking teat antisepsis on distribution of staphylococcal species is discussed.     

Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales

A survey of clinical and subclinical mastitis was carried out on 97 dairy farms in England and Wales, selected at random from members of a national milk recording scheme. The farmers were asked to collect aseptic milk samples from five consecutive cases of clinical mastitis and from five quarters with high somatic cell counts using a defined protocol, and they completed a questionnaire that included information on the cows sampled, the herd and the history of mastitis in the herd. The samples were collected throughout the year. The mean incidence of clinical mastitis was 47 cases per 100 cows per year (estimated from historic farm records) and 71 cases per 100 cows per year (estimated from the samples collected). Streptococcus uberis and Escherichia coli were isolated in pure culture from 23.5 per cent and 19.8 per cent, respectively, of the clinical samples; 26.5 per cent of the clinical samples produced no growth. The most common isolates from the samples with high cell counts were coagulase-negative staphylococci (15 per cent), S uberis (14 per cent) and Corynebacterium species (10 per cent). Staphylococcus aureus and coagulase-positive staphylococci together accounted for 10 per cent of the samples with high somatic cell counts; 39 per cent produced no bacterial growth.     

Management strategies to decrease the prevalence of mastitis caused by one strain of Staphylococcus aureus in a dairy herd

The dairy herd at Washington State University had an outbreak of mastitis caused by a single strain of Staphylococcus aureus. The outbreak strain, termed novel, could not be controlled with routine contagious mastitis pathogen control procedures (incidence, 3.4 infections/100 cow months; peak prevalence > 22%). Our objective was to implement mastitis control measures that would decrease the incidence and prevalence of intramammary infection (IMI) caused by S aureus in the herd. The following intervention strategies were successfully implemented: strict segregation of cattle with IMI caused by S aureus, intensified culling of cattle with multiple-quarter IMI caused by S aureus, and inducing cessation of lactation of infected quarters in single-mammary-quarter infected cattle. One year after implementation of these control measures, incidence of IMI caused by S aureus was 0.35 infections/100 cow months, and prevalence had decreased from 20 to 8%.     

Effect of application of an external teat sealant and/or oral treatment with a monensin capsule pre-calving on the prevalence and incidence of subclinical and clinical mastitis in dairy heifers

AIM: To assess the effect of applying an external teat sealant or a slow-release intraruminal monensin capsule pre-calving on the prevalence and incidence of subclinical or clinical mastitis in dairy heifers post-calving. METHODS: The studies were undertaken in 13 herds. In two herds application of an external teat sealant was compared with a negative control; in nine herds treatment with monensin was compared with controls, and in two herds treatments were applied in a 2 x 2 factorial design. Heifers were randomly assigned within herd to be treated with a slow-release intraruminal device containing 32 g monensin (n=383), or left as untreated controls (n=389), approximately 30 days before the start of the spring calving period; or to be treated with a latex external teat sealant (n=206) when calving was impending, or be left as untreated controls (n=205). Milk samples were collected from each gland between 0 and 5 days after calving and from any gland diagnosed with clinical mastitis. The effect of the treatments on the prevalence of intramammary infection (IMI), i.e. subclinical mastitis, and clinical mastitis were initially analysed at the univariate level, with associated variables (p<0.2) being used in construction of multivariable models. RESULTS: The prevalence of IMI was 18.4% at the gland level, and coagulase-negative staphylococci followed by Streptococcus uberis were the most common isolates. Treatment with an external teat sealant reduced the prevalence, compared with controls, of any IMI (12.1 (SE 1.7)% vs 16.5 (SE 2.1)%, respectively; p=0.05) or IMI caused by a major pathogen (3.8 (SE 0.8)% vs 6.0 (SE 1.1)%, respectively; p=0.05). Monensin treatment did not affect the prevalence of any IMI (p=0.68) or IMI due to a major pathogen (p=0.11). The cumulative incidence of clinical mastitis was 14.2%. Neither monensin treatment (p=0.47) nor application of an external teat sealant (p=0.71) altered the risk of clinical mastitis. There was no interaction between the monensin and external teat sealant in the two herds where the treatments were applied in a 2 x 2 factorial design. CONCLUSIONS: Application of an external teat sealant resulted in a lower prevalence of any IMI and of an IMI caused by a major pathogen. Despite monensin reducing the mobilisation of body tissue and the risk of subclinical ketosis, it had no effect on the prevalence or incidence of IMI or clinical mastitis. CLINICAL RELEVANCE: Application of an external teat sealant is an option for reducing the prevalence of IMI in pasture fed dairy heifers.     

Staphylococcus aureus proteins differentially recognized by the ovine immune response in mastitis or nasal carriage

Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is found in healthy carriage and can be a major cause of mastitis. A better knowledge of the host-pathogen interactions is needed to tackle this serious animal health problem. This study aimed at identifying S. aureus proteins differentially expressed by S. aureus in nasal colonization versus mastitis. Serological proteome analysis (SERPA) was used to examine protein samples prepared from culture supernatants of S. aureus strains originally isolated from gangrenous mastitis and nasal carriage (O11) or subclinical mastitis (O46) and to compare patterns of immune-reactive proteins. These staphylococcal proteins were revealed by sera obtained from ewes suffering from S. aureus mastitis and by sera obtained from healthy nulliparous ewes (i.e. no lactation and no mastitis or other symptoms) that were nasally colonized by S. aureus. Altogether 49 staphylococcal immune-reactive proteins were identified in this study. Patterns of proteins revealed by sera from infected- or healthy carrier- animals were comparable and analysis singled out one immune-reactive protein, N-acetylmuramyl-L-alanine amidase, which was recognized by each of the 6 sera from infected animals, when tested individually, and not by the sera of healthy carriers. This is the first study that compares the S. aureus seroproteome in colonization versus mastitis context in ruminants. These results open avenues for studies aiming at a better understanding of the balance between infection and commensal lifestyle in this opportunistic pathogen and at new prevention strategies.     

Effects of adjuvants on the immune response of staphylococcal alpha toxin and capsular polysaccharide (CPS) in rabbit

This study was performed to isolate a vaccine strain of S. aureus from clinical or subclinical mastitis and to choose the most optimal adjuvant for immune response of alpha toxin and capsular polysaccharide (CPS) of field strain. Of thirty strains of S. aureus isolated from milk of clinical or subclinical mastitis, V112 strain isolated from milk of gangrenous mastitis was used in this vaccine. Twenty one of rabbits were allocated into 5 groups based on adjuvants and immunized twice every 2 weeks for 8 weeks. This vaccine was composed of alpha toxin (10 hemolytic units) and formalinized whole cells (1 x 10(11) cells/ml. Five rabbits received PBS solution as a control group. The highest antibody titers against alpha toxin and CPS were observed in dextran sulfate- and aluminium hydroxide-adjuvant group at 8 weeks after immunization, respectively. These results of the study showed that one adjuvant could not induce strong and long-term immune response of alpha toxin and CPS antigens. Therefore, the use of combined adjuvants in subunit vaccine may be useful and feasible.