Veterinary Microbiology, 24 (1990) 55-62 Elsevier Science Publishers B.V., Amsterdam
55
Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A T.G. Wijewardana and A.D. Sutherland Moredun Research Institute, 408 Gilrnerton Road, Edinburgh, EH17 7JH (Great Britain) (Accepted 4 December 1989 )
ABSTRACT Wijewardana, T.G. and Sutherland, A.D., 1990. Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A. Vet. Microbiol., 24: 55-62. The susceptibility ofPasteurella multocida to killing by serum and the ability of protective vaccines to stimulate this mechanism of immunity in mice were investigated. P. multocida type A of bovine origin was used to prepare a vaccine incorporating heat killed organisms and for homologous infection of mice. Bactericidal capacity and ELISA antibody titres were determined for individual mouse sera. Protection was clearly associated with bactericidal antibodies raised by vaccination. The bactericidal assay may be useful as a rapid, simple screening test of vaccinated mice for functional protective antibody levels.
INTRODUCTION
Pasteurella multocida is pathogenic for a wide variety of mammals and birds (Meyer, 1958). Capsular type A (Carter, 1955) isolates ofP. multocida are associated with important diseases such as bovine and porcine pneumonia, fowl cholera and rabbit septicaemia (Carter et al., 1977; Lu et al., 1983; Percy et al., 1984). Most capsulated strains recovered from acute or moderately acute natural infections are also virulent in mice (Carter, 1967). Mouse models of infection have therefore been used to test the efficacy of P. multocida vaccines (Murata et al., 1964; Heddleston and Rebers, 1969). Many experimental vaccines have incorporated heat-killed bacteria with or without adjuvants (Collins, 1973; Abdullahi, 1987). Protection in vaccinated mice seemed to be mediated by a humoral response (Carter, 1967; Woolcock and Collins, 1976; Collins and Woolcock, 1976), but the precise mechanism by which P. multocida was eliminated was not examined by these authors. One of the mechanisms of immunity implicated in protection against P. haemolytica infection in sheep is antibody- and complement-mediated bac0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
56
T.G. WIJEWARDANA AND A.D. SUTHERLAND
terial killing (Sutherland, 1988). Killing was demonstrated with antibodies found in both sera and lung washings of sheep recovered from injection. Mukkur (1980) also found that P. multocida was susceptible to killing by sera from vaccinated mice but did not examine the role of this i m m u n e mechanism in relation to protection. This paper therefore further examined the susceptibility ofP. multocida to killing by mouse serum, the correlation with ELISA titres, and the ability of protective vaccines to stimulate this mechanism of immunity in mice. MATERIALS AND METHODS
Mice Swiss white mice of either sex aged between 4 and 6 weeks were used.
Bacterial strain P. multocida type A (W674) was an isolate from a bovine lung (Abdullahi, 1987). Vaccine preparation Strain W674 was grown in 11 of nutrient broth on a shaker for 18 h at 37°C. The bacterial cells were harvested by centrifugation at 14 000 g for 20 min, washed once in phosphate buffered saline (PBS) and then re-suspended to 10 ml. The culture was tested for purity by spreading a loopful of suspension onto a 5% sheep blood agar (SBA) plate which was incubated at 37°C overnight before examination. The suspension was killed by heating at 60 °C for 60 min, tested for sterility by culture on SBA, and lyophilised. The vaccine was prepared by suspending the lyophilised organisms in PBS at a concentration of 5 m g / m l and emulsifying them in an equal volume of Bayol F (ESSO) containing 10% Arlacel (Sigma). P. multocida inoculum for infection of mice A 6-h broth culture of strain W674 was diluted with PBS to give 3.0 X 109 colony forming units (cfu) m l - 1 for infection of mice. This suspension contained approximately 10 LDso/0.1 ml which was the o p t i m u m mouse challenge dose determined for this strain previously by AbduUahi ( 1987 ).
BACTERICIDAL ACTIVITY IN SERA OF MICE VACCINATED WITH P. MULTOCIDA
57
Sera Standard positive serum. H y p e r i m m u n e serum to strain W674 was produced in two rabbits by modification of the m e t h o d of Abdullahi ( 1987 ), using formalin-killed bacteria in saline adjusted to 109 organisms m l - i. Following an initial inoculation of 1.0 ml of the bacterial suspension subcutaneously, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 3.0 ml and 5.0 ml volumes of suspension were inoculated intravenously (i.v.) at 4-day intervals. Seven days after the last inoculation 1.0 ml of live P. multocida ( 1 X 109 cFu m l - 1) in saline was given i.v. Rabbits were bled by cardiac puncture under anaesthesia 10 days after this final injection, and serum separated and stored. Standard negative serum. Fetal bovine serum (FBS) which was tested and proved to be free of bactericidal antibody to P. multocida was used as a negative control. Experimental mouse sera. Blood from each mouse was collected at necropsy from the posterior vena cava and serum was separated. Standard and test sera were heat-inactivated at 56 °C for 30 min before assay to destroy endogenous complement. Source o f complement. Sera from newborn calves or gnotobiotic lambs were tested for adequacy of complement activity and absence of bactericidal antibodies as described by Sutherland ( 1988 ). Complement was stored at - 70 ° C until used. Bactericidal assay Preparation of bacterial inoculum and determination of the optimal conditions for the demonstration of antibody-mediated, complement-dependent killing ofP. multocida were carried out using the methods described by Sutherland ( 1988 ). Standard sera (20/tl) were added in triplicate to wells of sterile tissue culture grade microtitre plates (Nunc, Denmark). P. multocida bacterial inoculum ( 100 #1) in modified barbitol buffer (MBB) (Oxoid, Great Britain) was added to each replicate and incubated at room temperature for 10 min. The complement source (80 #1) was then added to each well, except when evaluating the effect of antibody on P. multocida in the absence of complement and the effect of complement alone on P. multocida, when 80 pl of heat-inactivated complement and 100 /zl fresh complement were added respectively. Plates were sealed with plastic film (Flow Laboratories, Irvine, Great Britain) and incubated at 37°C for the required time. Volumes ( 10 #I) of each assay suspension were then removed with a multichannel pipette and inoculated onto 12X 12 cm square (Sterilin Ltd, Feltham, Great Britain) SBA
58
T.G. WIJEWARDANA AND A.D. SUTHERLAND
plates. When bacterial counts were too high for direct plate counting, as when the optimisation of the bacterial inoculum was being carried out, 1 in 10 and 1 in 100 dilutions of each suspension were made in MBB diluent in microtitre plates before direct plating. The SBA plates were allowed to dry and then incubated at 37°C overnight. The initial (T0) bacterial count ( c f u / m l ) in each assay suspension was calculated as 0.5 X the mean cfu ml-~ determined by counting the number of bacterial colonies in five 10/d volumes of the assay inoculum which were plated on SBA and incubated overnight. The mean cfu m l - ~ of assay suspension remaining after 30 min (T3o) was calculated from the mean number of colonies counted in each 10/d volume triplicate. The mean percentage of inoculum killed (%K) by each serum sample was then calculated from the formula: % K = 1 - (mean cfu ml-~ at T3o/mean cfu ml-~ at To) × 100.
Enzyme-linked immunosorbent assay (ELISA) Whole bacterial cells were used as antigen. A confluent growth of P. multocida from an overnight dextrose starch agar plate culture was removed into 3.0 ml of saline, the bacteria harvested by centrifugation at 5000 g for 15 rain and washed once in 10 ml of saline. The bacteria were resuspended in 3.0 ml of 0.5% formal saline and diluted 1 in 100 in 0.05 M carbonate/bicarbonate buffer pH 9.6. Wells of microtitre plates (type 129A, Dynatech Laboratories, Ltd), were coated with 100/d volumes of the antigen suspension, incubated at 4 °C overnight and washed three times with PBS containing 0.05% Tween 20 (PBS/Tween). The last wash was left on the plate for 3 min before it was shaken out. Serum samples were diluted 1 : 100 in PBS/Tween and 100/d volumes added to wells of the antigen-coated plates which were then incubated at 37°C for 3 h. Serum samples were removed and the plates washed three times with PBS/Tween. Sheep anti-mouse IgG conjugated with horse radish peroxidase (Scottish Antibody Production Unit, Lanarkshire, Great Britain) diluted 1 in 200 in P B S / T w e e n was added ( 100/d) to each well and incubated at 37 °C for 1 h. The plates were washed three times in PBS/Tween and finally 100/A of fresh substrate solution was added to each well. This consisted of 4 mg of orthophenylene diamine and 4/tl of H202 per 10 ml of 0.05 M sodium phosphate citric acid buffer at pH 5.0. The reaction was stopped with 2.3 M HzSO 4 after 5 min. The optical density of each well was read at 492 n m on a Titertek Multiskan (Flow Laboratories, Irvine, Ayrshire).
Experimen tal protocol Mice were allocated randomly into 4 groups of 15 (Table 1 ). Animals in groups 2 and 3 were vaccinated on days 0 and 14 with 0.2 ml of HKO vaccine subcutaneously. Mice in unvaccinated control group 1 and vaccinated group
59
BACTERICIDAL ACTIVITY IN SERA OF MICE VACCINATED WITH P. MULTOCIDA
TABLE 1 Vaccination, challenge a n d t i m e o f bleeding o f groups o f mice Group
Vaccinated
Infected
Bled
Day 0
D a y 14
Day 28
Day 30
1
_
_
..~
_1
2 3 4
+ + -
+ + -
+ -
+ + +
~All mice died within 24 h o f infection. + = treated; - = untreated.
3 were infected intraperitoneally with 0.1 ml ofP. multocida inoculum on day 28. Group 4 mice were included as untreated (unvaccinated and unchallenged) controls.
Statistical analysis Significance testing between groups was by the Mann-Whitney ranking test. Correlations of data were determined by the Spearman-Jackson rank correlation test. RESULTS
Optimisation of the bactericidal assay The standard positive serum gave 100% killing of bacterial suspension containing < 1.06 × 103 cfu ml-1. Killing dropped to 97.5% and 80.9% when the suspension titre was 1.06 X 104 and 1.06 X 105 cfu m1-1, respectively, and no killing was observed when the suspension titre was > 1.06 × 106 cfu m l - 1. The optimal bacterial inoculum was therefore selected as 2.0 X 103 cfu m l - ~. The 100% killing of P. multocida by the standard positive serum occurred only in the presence of complement. Any dilution or heat-inactivation of the complement source abolished its activity completely. The standard negative serum plus complement was not bactericidal at any dilution, while the standard positive serum remained upto 90.6% bactericidal from 1 in 2 to a 1 in 16 dilution. At higher dilution, activity dropped rapidly from 81.2% to 21.4% at 1 in 32 and 1 in 64 dilutions, respectively. After 2 min of incubation only 2.6% killing occurred in the presence of the standard positive serum. At 10 and 20 rain incubation 84.3 and 93.8% killing occurred. At and beyond 30 min the percentage killing was 100, and an incubation period of 30 min was therefore selected as optimal. Optimal conditions for assay of bacterial killing of P. multocida were therefore found to be
60
T.G.WIJEWARDANAANDA.D.SUTHERLAND
20/d o f undiluted test serum with 100/tl of 2 × l 0 3 cfu ml- ' of bacteria, mixed with 80/tl o f undiluted complement and incubation at 37 °C for 30 min.
Vaccination of mice In the mouse vaccination experiment all 15 mice in group 1 died within 24 h of infection. In contrast, none o f the vaccinated mice in group 3 died after infection. The HKO vaccine, therefore, gave 100% protection against a challenge producing 100% mortality.
Serum killing and serology of mouse sera Bactericidal capacities and ELISA titres o f sera from groups 2, 3 and 4 are shown in Table 2. The mean bactericidal capacities and ELISA titres o f groups 2 and 3 were not significantly different from each other. Both groups had raised serum bactericidal capacity and ELISA titres compared to the unvaccinated (group 4) mice which had no bactericidal capacity and significantly TABLE 2 Serology of experimental mice Animal no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Group mean (±S.E.)
Group 2'
Group 32
Group 43
%K 4
ELISA (OD)
%K
ELISA (OD)
%K
ELISA (OD)
NA 4 60 70 84 74 80 84 77 57 84 60 57 50 60 64.4 (+5.6)
NA 0.242 0.200 0.220 0.127 0.205 0.155 0.166 0.222 0.142 0.106 0.175 0.197 0.219 0.199 0.1839 (±0.04)
NA 70 44 44 74 80 60 84 94 77 74 87 54 97 70 72.1 (±4.5)
NA 0.148 0.181 0.148 0.259 0.222 NA 0.337 0.306 NA NA 0.249 0.113 0.119 0.151 0.2030 (±0.08)
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0.000 0.000 0.032 0.026 0.017 0.006 0.017 0.007 0.001 0.000 0.000 0.000 0.053 0.039 0.054 0.0168 (±0.02)
NA = not available. ~Group 2 was vaccinated and not challenged. 2Group 3 was vaccinated and challenged. 3Group 4 was untreated. 4%K = 1 - (mean cfu m l - ' at T3o/mean cfu ml-~ at To) X 100.
BACTERICIDAL ACTIVITY IN SERA OF MICE VACCINATED WITH P. MULTOCIDA
61
(P
55
Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A T.G. Wijewardana and A.D. Sutherland Moredun Research Institute, 408 Gilrnerton Road, Edinburgh, EH17 7JH (Great Britain) (Accepted 4 December 1989 )
ABSTRACT Wijewardana, T.G. and Sutherland, A.D., 1990. Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A. Vet. Microbiol., 24: 55-62. The susceptibility ofPasteurella multocida to killing by serum and the ability of protective vaccines to stimulate this mechanism of immunity in mice were investigated. P. multocida type A of bovine origin was used to prepare a vaccine incorporating heat killed organisms and for homologous infection of mice. Bactericidal capacity and ELISA antibody titres were determined for individual mouse sera. Protection was clearly associated with bactericidal antibodies raised by vaccination. The bactericidal assay may be useful as a rapid, simple screening test of vaccinated mice for functional protective antibody levels.
INTRODUCTION
Pasteurella multocida is pathogenic for a wide variety of mammals and birds (Meyer, 1958). Capsular type A (Carter, 1955) isolates ofP. multocida are associated with important diseases such as bovine and porcine pneumonia, fowl cholera and rabbit septicaemia (Carter et al., 1977; Lu et al., 1983; Percy et al., 1984). Most capsulated strains recovered from acute or moderately acute natural infections are also virulent in mice (Carter, 1967). Mouse models of infection have therefore been used to test the efficacy of P. multocida vaccines (Murata et al., 1964; Heddleston and Rebers, 1969). Many experimental vaccines have incorporated heat-killed bacteria with or without adjuvants (Collins, 1973; Abdullahi, 1987). Protection in vaccinated mice seemed to be mediated by a humoral response (Carter, 1967; Woolcock and Collins, 1976; Collins and Woolcock, 1976), but the precise mechanism by which P. multocida was eliminated was not examined by these authors. One of the mechanisms of immunity implicated in protection against P. haemolytica infection in sheep is antibody- and complement-mediated bac0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
56
T.G. WIJEWARDANA AND A.D. SUTHERLAND
terial killing (Sutherland, 1988). Killing was demonstrated with antibodies found in both sera and lung washings of sheep recovered from injection. Mukkur (1980) also found that P. multocida was susceptible to killing by sera from vaccinated mice but did not examine the role of this i m m u n e mechanism in relation to protection. This paper therefore further examined the susceptibility ofP. multocida to killing by mouse serum, the correlation with ELISA titres, and the ability of protective vaccines to stimulate this mechanism of immunity in mice. MATERIALS AND METHODS
Mice Swiss white mice of either sex aged between 4 and 6 weeks were used.
Bacterial strain P. multocida type A (W674) was an isolate from a bovine lung (Abdullahi, 1987). Vaccine preparation Strain W674 was grown in 11 of nutrient broth on a shaker for 18 h at 37°C. The bacterial cells were harvested by centrifugation at 14 000 g for 20 min, washed once in phosphate buffered saline (PBS) and then re-suspended to 10 ml. The culture was tested for purity by spreading a loopful of suspension onto a 5% sheep blood agar (SBA) plate which was incubated at 37°C overnight before examination. The suspension was killed by heating at 60 °C for 60 min, tested for sterility by culture on SBA, and lyophilised. The vaccine was prepared by suspending the lyophilised organisms in PBS at a concentration of 5 m g / m l and emulsifying them in an equal volume of Bayol F (ESSO) containing 10% Arlacel (Sigma). P. multocida inoculum for infection of mice A 6-h broth culture of strain W674 was diluted with PBS to give 3.0 X 109 colony forming units (cfu) m l - 1 for infection of mice. This suspension contained approximately 10 LDso/0.1 ml which was the o p t i m u m mouse challenge dose determined for this strain previously by AbduUahi ( 1987 ).
BACTERICIDAL ACTIVITY IN SERA OF MICE VACCINATED WITH P. MULTOCIDA
57
Sera Standard positive serum. H y p e r i m m u n e serum to strain W674 was produced in two rabbits by modification of the m e t h o d of Abdullahi ( 1987 ), using formalin-killed bacteria in saline adjusted to 109 organisms m l - i. Following an initial inoculation of 1.0 ml of the bacterial suspension subcutaneously, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 3.0 ml and 5.0 ml volumes of suspension were inoculated intravenously (i.v.) at 4-day intervals. Seven days after the last inoculation 1.0 ml of live P. multocida ( 1 X 109 cFu m l - 1) in saline was given i.v. Rabbits were bled by cardiac puncture under anaesthesia 10 days after this final injection, and serum separated and stored. Standard negative serum. Fetal bovine serum (FBS) which was tested and proved to be free of bactericidal antibody to P. multocida was used as a negative control. Experimental mouse sera. Blood from each mouse was collected at necropsy from the posterior vena cava and serum was separated. Standard and test sera were heat-inactivated at 56 °C for 30 min before assay to destroy endogenous complement. Source o f complement. Sera from newborn calves or gnotobiotic lambs were tested for adequacy of complement activity and absence of bactericidal antibodies as described by Sutherland ( 1988 ). Complement was stored at - 70 ° C until used. Bactericidal assay Preparation of bacterial inoculum and determination of the optimal conditions for the demonstration of antibody-mediated, complement-dependent killing ofP. multocida were carried out using the methods described by Sutherland ( 1988 ). Standard sera (20/tl) were added in triplicate to wells of sterile tissue culture grade microtitre plates (Nunc, Denmark). P. multocida bacterial inoculum ( 100 #1) in modified barbitol buffer (MBB) (Oxoid, Great Britain) was added to each replicate and incubated at room temperature for 10 min. The complement source (80 #1) was then added to each well, except when evaluating the effect of antibody on P. multocida in the absence of complement and the effect of complement alone on P. multocida, when 80 pl of heat-inactivated complement and 100 /zl fresh complement were added respectively. Plates were sealed with plastic film (Flow Laboratories, Irvine, Great Britain) and incubated at 37°C for the required time. Volumes ( 10 #I) of each assay suspension were then removed with a multichannel pipette and inoculated onto 12X 12 cm square (Sterilin Ltd, Feltham, Great Britain) SBA
58
T.G. WIJEWARDANA AND A.D. SUTHERLAND
plates. When bacterial counts were too high for direct plate counting, as when the optimisation of the bacterial inoculum was being carried out, 1 in 10 and 1 in 100 dilutions of each suspension were made in MBB diluent in microtitre plates before direct plating. The SBA plates were allowed to dry and then incubated at 37°C overnight. The initial (T0) bacterial count ( c f u / m l ) in each assay suspension was calculated as 0.5 X the mean cfu ml-~ determined by counting the number of bacterial colonies in five 10/d volumes of the assay inoculum which were plated on SBA and incubated overnight. The mean cfu m l - ~ of assay suspension remaining after 30 min (T3o) was calculated from the mean number of colonies counted in each 10/d volume triplicate. The mean percentage of inoculum killed (%K) by each serum sample was then calculated from the formula: % K = 1 - (mean cfu ml-~ at T3o/mean cfu ml-~ at To) × 100.
Enzyme-linked immunosorbent assay (ELISA) Whole bacterial cells were used as antigen. A confluent growth of P. multocida from an overnight dextrose starch agar plate culture was removed into 3.0 ml of saline, the bacteria harvested by centrifugation at 5000 g for 15 rain and washed once in 10 ml of saline. The bacteria were resuspended in 3.0 ml of 0.5% formal saline and diluted 1 in 100 in 0.05 M carbonate/bicarbonate buffer pH 9.6. Wells of microtitre plates (type 129A, Dynatech Laboratories, Ltd), were coated with 100/d volumes of the antigen suspension, incubated at 4 °C overnight and washed three times with PBS containing 0.05% Tween 20 (PBS/Tween). The last wash was left on the plate for 3 min before it was shaken out. Serum samples were diluted 1 : 100 in PBS/Tween and 100/d volumes added to wells of the antigen-coated plates which were then incubated at 37°C for 3 h. Serum samples were removed and the plates washed three times with PBS/Tween. Sheep anti-mouse IgG conjugated with horse radish peroxidase (Scottish Antibody Production Unit, Lanarkshire, Great Britain) diluted 1 in 200 in P B S / T w e e n was added ( 100/d) to each well and incubated at 37 °C for 1 h. The plates were washed three times in PBS/Tween and finally 100/A of fresh substrate solution was added to each well. This consisted of 4 mg of orthophenylene diamine and 4/tl of H202 per 10 ml of 0.05 M sodium phosphate citric acid buffer at pH 5.0. The reaction was stopped with 2.3 M HzSO 4 after 5 min. The optical density of each well was read at 492 n m on a Titertek Multiskan (Flow Laboratories, Irvine, Ayrshire).
Experimen tal protocol Mice were allocated randomly into 4 groups of 15 (Table 1 ). Animals in groups 2 and 3 were vaccinated on days 0 and 14 with 0.2 ml of HKO vaccine subcutaneously. Mice in unvaccinated control group 1 and vaccinated group
59
BACTERICIDAL ACTIVITY IN SERA OF MICE VACCINATED WITH P. MULTOCIDA
TABLE 1 Vaccination, challenge a n d t i m e o f bleeding o f groups o f mice Group
Vaccinated
Infected
Bled
Day 0
D a y 14
Day 28
Day 30
1
_
_
..~
_1
2 3 4
+ + -
+ + -
+ -
+ + +
~All mice died within 24 h o f infection. + = treated; - = untreated.
3 were infected intraperitoneally with 0.1 ml ofP. multocida inoculum on day 28. Group 4 mice were included as untreated (unvaccinated and unchallenged) controls.
Statistical analysis Significance testing between groups was by the Mann-Whitney ranking test. Correlations of data were determined by the Spearman-Jackson rank correlation test. RESULTS
Optimisation of the bactericidal assay The standard positive serum gave 100% killing of bacterial suspension containing < 1.06 × 103 cfu ml-1. Killing dropped to 97.5% and 80.9% when the suspension titre was 1.06 X 104 and 1.06 X 105 cfu m1-1, respectively, and no killing was observed when the suspension titre was > 1.06 × 106 cfu m l - 1. The optimal bacterial inoculum was therefore selected as 2.0 X 103 cfu m l - ~. The 100% killing of P. multocida by the standard positive serum occurred only in the presence of complement. Any dilution or heat-inactivation of the complement source abolished its activity completely. The standard negative serum plus complement was not bactericidal at any dilution, while the standard positive serum remained upto 90.6% bactericidal from 1 in 2 to a 1 in 16 dilution. At higher dilution, activity dropped rapidly from 81.2% to 21.4% at 1 in 32 and 1 in 64 dilutions, respectively. After 2 min of incubation only 2.6% killing occurred in the presence of the standard positive serum. At 10 and 20 rain incubation 84.3 and 93.8% killing occurred. At and beyond 30 min the percentage killing was 100, and an incubation period of 30 min was therefore selected as optimal. Optimal conditions for assay of bacterial killing of P. multocida were therefore found to be
60
T.G.WIJEWARDANAANDA.D.SUTHERLAND
20/d o f undiluted test serum with 100/tl of 2 × l 0 3 cfu ml- ' of bacteria, mixed with 80/tl o f undiluted complement and incubation at 37 °C for 30 min.
Vaccination of mice In the mouse vaccination experiment all 15 mice in group 1 died within 24 h of infection. In contrast, none o f the vaccinated mice in group 3 died after infection. The HKO vaccine, therefore, gave 100% protection against a challenge producing 100% mortality.
Serum killing and serology of mouse sera Bactericidal capacities and ELISA titres o f sera from groups 2, 3 and 4 are shown in Table 2. The mean bactericidal capacities and ELISA titres o f groups 2 and 3 were not significantly different from each other. Both groups had raised serum bactericidal capacity and ELISA titres compared to the unvaccinated (group 4) mice which had no bactericidal capacity and significantly TABLE 2 Serology of experimental mice Animal no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Group mean (±S.E.)
Group 2'
Group 32
Group 43
%K 4
ELISA (OD)
%K
ELISA (OD)
%K
ELISA (OD)
NA 4 60 70 84 74 80 84 77 57 84 60 57 50 60 64.4 (+5.6)
NA 0.242 0.200 0.220 0.127 0.205 0.155 0.166 0.222 0.142 0.106 0.175 0.197 0.219 0.199 0.1839 (±0.04)
NA 70 44 44 74 80 60 84 94 77 74 87 54 97 70 72.1 (±4.5)
NA 0.148 0.181 0.148 0.259 0.222 NA 0.337 0.306 NA NA 0.249 0.113 0.119 0.151 0.2030 (±0.08)
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0.000 0.000 0.032 0.026 0.017 0.006 0.017 0.007 0.001 0.000 0.000 0.000 0.053 0.039 0.054 0.0168 (±0.02)
NA = not available. ~Group 2 was vaccinated and not challenged. 2Group 3 was vaccinated and challenged. 3Group 4 was untreated. 4%K = 1 - (mean cfu m l - ' at T3o/mean cfu ml-~ at To) X 100.
BACTERICIDAL ACTIVITY IN SERA OF MICE VACCINATED WITH P. MULTOCIDA
61
(P
